WO2006043701A1 - Method of inhibiting phosphorylation of transcriptional factor for gluconeogenesis-associated gene and phosphorylation inhibitor - Google Patents

Method of inhibiting phosphorylation of transcriptional factor for gluconeogenesis-associated gene and phosphorylation inhibitor Download PDF

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Publication number
WO2006043701A1
WO2006043701A1 PCT/JP2005/019518 JP2005019518W WO2006043701A1 WO 2006043701 A1 WO2006043701 A1 WO 2006043701A1 JP 2005019518 W JP2005019518 W JP 2005019518W WO 2006043701 A1 WO2006043701 A1 WO 2006043701A1
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rskb
hnf
gene
phosphorylation
kinase
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PCT/JP2005/019518
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French (fr)
Japanese (ja)
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Hirofumi Doi
Yuka Shozaki
Gen Kudo
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Daiichi Pharmaceutical Co., Ltd.
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Publication of WO2006043701A1 publication Critical patent/WO2006043701A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value

Definitions

  • the present invention relates to a method for phosphorylating hepatocyte nuclear factor 4a (hereinafter referred to as HNF-4a), a phosphorylating agent, a method for inhibiting phosphorylation, and phosphoric acid Relates to inhibitors. More specifically, the present invention relates to a method for phosphorylating HNF-4 ⁇ and a phosphorylating agent characterized by using ribosome 1 S6 kinase B (abbreviated as RSKB hereinafter). The present invention also relates to a method for inhibiting HNF-4 Q; Furthermore, the present invention relates to a method for promoting the production of a gene product of a gene in which HNF-4 ⁇ acts as a transcription factor.
  • HNF-4a hepatocyte nuclear factor 4a
  • RSKB ribosome 1 S6 kinase B
  • the present invention also relates to a method for inhibiting the production of a gene product of a gene for which HNF-4a acts as a transcription factor and a production inhibitor. Further, the present invention relates to prevention and Z or treatment methods and prevention and Z or treatment agents for diseases caused by phosphate lees from HNF-4. Further, the present invention relates to a method for identifying a compound that inhibits the interaction between RSKB and HNF-4 ⁇ , the binding of RSKB and HNF-4a, or phosphorylation of HNF-4a by RSKB, and the compound identified by the identification method. The Furthermore, the present invention relates to a polynucleotide encoding RSKB, HNF-4a, and a reagent kit comprising a vector containing the polynucleotide.
  • the liver is an important organ for maintaining glucose homeostasis, and maintains the balance of glucose in the living body by gluconeogenesis that produces glucose and glycogen that produces glycogen from glucose. Yes. In diabetic patients, excessive glucose production occurs in the liver, which is considered to be one of the causes of hyperglycemia (Non-patent Document 1).
  • Glucogenesis is a pathway in which pyruvate also synthesizes glucose, most of which is performed in the liver.
  • phosphoenol pyruvate carboxy kinase hereinafter abbreviated as PEPCK.
  • PEPCK phosphoenol pyruvate carboxy kinase
  • Non-patent Document 8 This transcription is regulated by hormones, darcocorticoids promote the transcription of the PEPCK gene (Non-patent Document 8), and insulin represses the transcription of the PEPCK gene (Non-patent Documents 4, 6, and 8).
  • the half-life of PEPCK gene mRNA is as short as 40 minutes, and the amount of transcription dramatically increases or decreases in units of several hours (Non-patent Document 3).
  • PE PCK enzyme activity, gluconeogenesis, and blood glucose level are regulated by the amount of transcription from the PEPCK gene (Non-patent Document 3).
  • PEPCK gene expression In the liver in the diabetic state, PEPCK gene expression is increased by darcocorticoids, and as a result, gluconeogenesis is enhanced (Non-patent Documents 8 and 9).
  • Type 1 diabetes insulin-dependent diabetes mellitus, IDDM
  • type 2 diabetes non-insulin-dependent diabetes mellitus, NIDDM
  • IDDM insulin-dependent diabetes mellitus
  • NIDDM non-insulin-dependent diabetes mellitus
  • Type 2 diabetes is further divided into two types. One is diabetes mainly due to a decrease in insulin secretion, and the other is diabetes where insulin is secreted but the insulin sensitivity to glucose in target cells is mainly reduced. The latter is particularly said to be insulin resistant.
  • the target tissues of insulin are the liver, fat, and muscle, and blood glucose levels are lowered by suppressing gluconeogenesis and sugar release in the liver. Sustained hyperglycemia reduces the sensitivity of the liver to insulin, thereby making it impossible to expect an insulin-dependent inhibitory effect on gluconeogenesis.
  • the mechanism that lowers insulin sensitivity in the liver due to persistent hyperglycemia has not been clarified yet, such as the potential for insulin receptor down-regulation.
  • HNF-4a is one of the nuclear receptors and is expressed in ⁇ cells, kidney and small intestine of liver and spleen. HNF-4 ⁇ is known to act as a transcription factor, and is involved in the expression of various genes encoding proteins involved in metabolism and liver development and differentiation, such as cholesterol, fatty acids and glucose. In gluconeogenesis, HNF-4a binds to the AF1 site present in the promoter of the PEPCK gene and is involved in the expression of the PEPCK gene (Non-patent Document 16). Transcription factors have been shown to change their DNA-binding ability by phosphorylation (Non-patent Document 17).
  • HNF-4 ⁇ is phosphorylated by PKA (cAMP-dependent protein kinase), and as a result, the ability of L-type pyruvate kinase to bind to the promoter decreases. 1 is reported (Non-patent Document 10).
  • RSKB is a kinase activated by the p38 kinase family, which is a stress-responsive MAPK (mitogen activated kinase), and is distributed in the nucleus (Non-patent Document 11).
  • MAPK mitogen activated kinase
  • P38 kinase is activated in various pathological conditions (Non-patent Document 12).
  • P38 kinase is also activated by oxidative stress caused by active oxygen in cells that are continuously exposed to hyperglycemia under diabetic conditions (Non-patent Documents 13 and 14).
  • Non-patent Document 15 vascular smooth muscle cells cultured under hyperglycemia (16.5 mM)
  • Non-patent Document 29 obZob mouse livers of diabetes model animals
  • Patent Document 1 International Publication No. WO01Z67299 pamphlet.
  • Non-patent literature l “Nature” 2001, No. 413, No. 13, p. 131-138.
  • Non-Patent Document 2 Japanese Clinical” 2002, 60th, Special Issue 7, p. 121-128.
  • Non-Patent Document 3 “J. Biol. Chem.” 1982, No. 257, No. 13, p. 7629-7636 0
  • Non-patent Reference 4 1983, No. 305, No. 5934, p. 549— 551.
  • Non-Patent Document 5 “Biochem.” 1974, 138th, p. 387-394.
  • Non-Patent Document 6 “Mol. Endocrinol.” 1993, No. 7, No. 11, p. 1456–1462.
  • Non-Patent Document 7 “Proc. Natl. Acad. Sci. USA” 1994, 91st pp. 9151-915
  • Non-Patent Document 8 “J. Biol. Chem.” 1993, No. 268, No. 17, p.
  • Non-Patent Document 9 “Lab. Anim. Sci.” 1993, 42nd pp. 473-477.
  • Non-Patent Document 10 “Mol. Cell. Biol.” 1997, Vol. 17, No. 8, p. 4208-4219.
  • Non-patent document 11 “J. Biol. Chem.” 1998, 273, 45, p. 29661-2967
  • Non-patent document 12 “Crit. Care Med.” 2000, 28, N67— 77.
  • Non-Patent Document 13 “Japanese Clinical” 2002, 60th, Special Issue 7, p. 395-398.
  • Non-Patent Document 14 “Endocr. Rev.” 2002, No. 23, No. 5, p. 599-622.
  • Non-Patent Document 15 “J. Clin. Invest.” 1999, No. 103, p. 185-195.
  • Non-Patent Document 16 “Proc. Natl. Acad. Sci. USA” 1995, 92nd, p. 412-416
  • Non-patent document 17 -Cell, 1992, 70th, No. 3, p. 375 — 387.
  • Non-Patent Document 18 "Mol. Cell. Biol.” 1988, VIII, p. 3467-3475.
  • Non-Patent Document 19 J. Biol. Chem., 1995, 270, p. 8225-8232.
  • Non-Patent Document 20 “Proc. Natl. Acad. Sci. USA” 1994, 91st, p.
  • Non-Patent Document 21 -Science "1995, 269th, p. 1108-1112.
  • Non-Patent Document 22 J. Biol. Chem., 1997, 272, p. 26306-26312.
  • Non-Patent Document 23 J. Biol. Chem., 2000, Vol. 275, p. 5804-5809.
  • Non-Patent Document 24 “Mol. Endocrinol.” 1999, 13th, p. 604-618.
  • Non-Patent Document 25 J. Biol. Chem., 1999, No. 274, No. 9, p. 5880-5887.
  • Non-Patent Document 26 -Diabetes ", 1989, 38th page, p. 550-557.
  • Non-Patent Document 27 "Annu Rev Biochem.” 1997, 66th, p. 581-611.
  • Non-Patent Document 28 J. Biol. Chem., 2002, No. 277, No. 35, p.
  • Non-patent document 29 “Mol. Endocrinol.” 2003, Vol. 17, No. 6, p. 1131— 1143
  • Non-patent document 30 “J. Biol. Chem.” 2004, [Epub ahead of pront].
  • Non-Patent Document 31 Ulmer, K. M. “Science”, 1983, 219, p. 666-671.
  • Non-Patent Document 32 “Peptide Synthesis” (Japan), Maruzen Co., Ltd., 1975.
  • Non-Patent Document 33 “Peptide Synthesis” (USA), Interscience, 1996.
  • Non-Patent Document 34 “J. Biol. Chem.” 2000, No. 275, No. 31, p.
  • Non-Patent Document 35 “Mol. Endocrinol.” 1999, 13th, p. 604-618.
  • Non-Patent Document 36 “DNA”, 1989, VIII, p. 127-133.
  • Non-Patent Document 37 “Mol. Endocrinol.” 1990, No. 4, No. 9, pl302-1310.
  • Non-Patent Document 38 “Diabetes” 2001, 50th, p. 131-138.
  • Non-Patent Document 39 “Arch Biochem Biophys.” 1995, No. 323, No. 2, p477 483.
  • Non-Patent Document 40 “Diabetes” 2000, 49th, p. 1165–1168.
  • Non-Patent Document 41 “: Biol. Pharm. Bull.” 2005, Vol. 28, No. 4, p. 565-568. Disclosure of the invention
  • An object of the present invention is to phosphorylate HNF-4a involved in transcription of a gene encoding PEPCK, which is considered to be a gluconeogenic rate-determining enzyme, and to bind the PEPCK gene promoter to a region.
  • a gene encoding PEPCK which is considered to be a gluconeogenic rate-determining enzyme
  • bind the PEPCK gene promoter to a region.
  • HNF-4a and RSKB are combined, HKB-4a is cleaved by RSKB, HNF-4a is phosphorylated by RSKB, and the PEPCK gene promoter It was demonstrated that the ability to bind to AF1 (accessory factor binding site 1, accessory factor binding sitel) within the region is promoted, and that the transcriptional activity of the PEPCK gene is enhanced by vigorous promotion. It was also clarified that RSKB activity is required for phosphorylation of HNF-4a by RSKB.
  • the PEPCK gene is a gene encoding PEPCK which is considered to be a gluconeogenic rate-determining enzyme. From this, phosphorylation by RSKB promotes the ability of HNF-4 ⁇ to bind to PE1 in the motor region of PEPCK gene, and as a result, the transcriptional activity of PEPCK gene is enhanced, thereby It can be considered that expression is promoted and gluconeogenesis is enhanced.
  • RSKB is known to be activated by p38 kinase!
  • p38 kinase is It is activated under various pathological conditions.
  • p38 kinase such as oxidative stress caused by active oxygen, is activated in cells that are continuously exposed to hyperglycemia under diabetic conditions. Therefore, it can be considered that p38 kinase is activated and thereby RSKB is activated under diabetic conditions.
  • RSKB the active I spoon has been P 38 kinase is active I spoon, can be considered to be further HNF- 4 a mosquito ⁇ phosphorylated by RSKB. It can be considered that HNF-4Q; phosphorylated by RSKB enhances gluconeogenesis and worsens hyperglycemia by promoting the transcriptional activity of the PEPCK gene.
  • HNF-4a is known to act as a transcription factor.
  • HNF-4a is also involved in the metabolism of various genes, such as cholesterol, fatty acids and glucose, and the development and differentiation of the liver. It is involved in the expression of genes that code for the proteins involved. Therefore, the ability of HNF-4a to bind to the promoter region of a gene other than the PEPCK gene, where HNF-4 ⁇ acts as a transcription factor, is promoted by the phosphate of HNF-4 ⁇ by RSKB, As a result, it can be considered that expression of the gene may be promoted.
  • HNF-4a phosphorylates HNF-4a by interacting RSKB with HNF-4a, thereby promoting a gene for which HNF-4 ⁇ acts as a transcription factor.
  • the ability to bind HNF-4a to the region can be promoted, and further the production of the gene product of the gene can be promoted.
  • HNF-4 ⁇ can be phosphorylated by interacting with RSKB and HNF-4, thereby promoting a promoter region of a gluconeogenesis-related gene, such as the promoter region of the PEPCK gene.
  • the ability to bind HNF4a to the AF1 site of can be promoted, and further the production of the gene product of the gene can be promoted.
  • HNF-4a by inhibiting phosphorylation of HNF-4a by RSKB, the ability of HNF-4a to bind to the promoter region of a gene in which HNF-4a acts as a transcription factor can be reduced. As a result, production of the gene product of the gene can be inhibited. Specifically, by inhibiting the phosphorylation of HNF-4a by RSKB, the ability of HNF4 ⁇ to bind to AF1 in the promoter region of a gluconeogenesis-related gene, such as the promoter region of the PEPCK gene, is reduced. Production of the gene product of the gene And can be inhibited, and can inhibit gluconeogenesis.
  • HNF-4a can inhibit the production of a gene product of a gene that acts as a transcription factor
  • prevention and Z or treatment of a disease caused by an increase in the gene product can be performed.
  • diseases caused by an increase in gene products of gluconeogenesis-related genes, more specifically diseases caused by an increase in gene products of PEPCK genes such as diabetes can be prevented and Z or treated.
  • the present invention is characterized in that ribosome S6 kinase B (RSKB) and hematocytonuclear factor 4 a (HNF-4 a) are allowed to coexist under conditions that allow interaction. It relates to the phosphorylation method of HNF-4a by RSKB.
  • RSKB ribosome S6 kinase B
  • HNF-4 a hematocytonuclear factor 4 a
  • the present invention also relates to a method for inhibiting phosphorylation of hepatocyte nuclear factor 4a (HNF-4a) by RKSB, characterized by inhibiting the activity of ribosomal S6 kinase B (RSKB) .
  • HNF-4a hepatocyte nuclear factor 4a
  • RSKB ribosomal S6 kinase B
  • the present invention relates to HNF-4a by RSKB, characterized in that it inhibits the binding of ribosomal S6 kinase B (RSKB) to hetocytonuclea factor 1a (HNF-4a).
  • RSKB ribosomal S6 kinase B
  • HNF-4a hetocytonuclea factor 1a
  • the present invention relates to a method for inhibiting phosphorylation.
  • the present invention provides treatment of a cell expressing at least ribosomal S6 kinase B (RSKB) and henocytonuclear factor 4a (HNF-4a) with an inhibitor of RSKB activity. It relates to a method for inhibiting HNF-4a phosphorylation by RSKB.
  • RSKB ribosomal S6 kinase B
  • HNF-4a henocytonuclear factor 4a
  • the present invention also selects an antibody that recognizes RSKB, an inhibitory force of ribosomal S6 kinase B (RSKB) activity, and an antibody that recognizes hetocytonuclea factor 4 ⁇ (HNF-4) 1
  • RSKB ribosomal S6 kinase B
  • HNF-4a hetocytonuclea factor 4 ⁇
  • the present invention relates to a method for inhibiting phosphorylation of HNF-4a by RSKB, which is one or more antibodies.
  • the present invention relates to a phosphorylation inhibitor of hepatocyte nuclear factor 4a (HNF-4a) by RSKB, characterized by inhibiting ribosomal S6 kinase B (RSKB) activity.
  • HNF-4a hepatocyte nuclear factor 4a
  • RSKB ribosomal S6 kinase B
  • the present invention relates to HNF-4 by RSKB, characterized by inhibiting the binding of ribosomal S6 kinase B (RSKB) to hetocytonuclea factor 4a (HNF-4a). It relates to a phosphate inhibitor of a.
  • RSKB ribosomal S6 kinase B
  • HNF-4a hetocytonuclea factor 4a
  • the present invention also includes an effective amount of an inhibitor of ribosomal S6 kinase B (RSKB) activity, and the phosphorylation of hepatocyte nuclear factor 4a (HNF-4a) by RSKB. It relates to an anther inhibitor.
  • RSKB ribosomal S6 kinase B
  • HNF-4a hepatocyte nuclear factor 4a
  • the present invention is selected from an antibody that recognizes the inhibitory power RSKB of ribosomal S6 kinase B (RSKB) activity and an antibody that recognizes hetocytonuclea factor 4 ⁇ (HNF-4).
  • RSKB ribosomal S6 kinase B
  • HNF-4 hetocytonuclea factor 4 ⁇
  • the present invention relates to a phosphate inhibitor of HNF-4a by RSKB, which is one or more antibodies.
  • the present invention relates to a method for promoting gene product production of a gene involved in gluconeogenesis, which comprises phosphorylating hepatocyte nuclear factor 4a using ribosome S6 kinase B.
  • the present invention also relates to a method for promoting gene product production of the gluconeogenesis-related gene, wherein the gluconeogenesis-related gene is a phosphoenolpyruvate carboxykinase gene.
  • the present invention relates to a method for inhibiting the production of a gene product of a gluconeogenesis-related gene, which comprises inhibiting phosphorylation of hepatocyte nuclease factor 4a by ribosomal S6 kinase B.
  • the present invention relates to a method for inhibiting the production of a gene product of a phosphoenolpyruvate carboxykinase gene, which comprises inhibiting phosphorylation of henocytocytolase factor 14a by ribosome S6 kinase B. About.
  • the present invention also relates to a method for inhibiting the production of a glycoprotein-related gene product, characterized by using any one of the methods for inhibiting phosphate kneading.
  • the present invention relates to a method for inhibiting the production of a gene product of a gene that acts as a transcription factor, which is characterized in that any one of the above-described methods for inhibiting phosphate kneading is used. .
  • the present invention relates to a method for inhibiting the gene product production of a phosphoenolpyruvate carboxykinase gene, characterized in that any one of the above-described methods for inhibiting phosphate kine is used.
  • the present invention provides a ribozyme characterized by using any one of the methods for inhibiting phosphorylation.
  • the present invention relates to a method for preventing and / or treating a disease caused by phosphorylation of hetocytonuclea factor 4a by S6 kinase B.
  • the present invention relates to a method for the prevention and Z or treatment of a disease caused by an increase in the gene product of a gluconeogenesis-related gene, characterized by using any one of the above-described methods for inhibiting phosphorylation.
  • the present invention provides a method for preventing and / or treating a disease caused by an increase in the gene product of the phosphoenolpyruvate carboxykinase gene, characterized by using any one of the methods for inhibiting phosphate kneading. About.
  • the present invention also relates to prevention and / or treatment of diabetes, characterized by using the method for inhibiting hepatocyte nuclear factor 1a phosphorylation by any one of the above-mentioned ribosome S6 kinase B. Regarding the method.
  • the present invention uses a phosphate inhibitor of hepatocyte nuclear factor 4a and an inhibitor of Z or RSKB activity by any one of the above-mentioned ribosomal S6 kinase B. Prevention and Z or treatment methods.
  • the present invention provides a phosphoprotein comprising an effective amount of any one of the above-mentioned phosphosomal S6 kinase B phosphorylation inhibitors of henocytonuclear factor 4a and an inhibitor of Z or RSKB activity.
  • the present invention relates to an inhibitor of production of the gene product of the enolpyruvate carboxykinase gene.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of a phosphorylation inhibitor of hepatocyte nuclear factor 1a by any one of the ribosome S6 kinase B and an inhibitor of Z or RSKB activity. About.
  • the present invention provides a phosphoenolpyruvate comprising an effective amount of any one of the aforementioned ribosomal S6 kinase B hepatocyte nuclear factor 4a phosphate inhibitors and inhibitors of Z or RSKB activity.
  • the present invention relates to a preventive and Z or therapeutic agent for diseases caused by an increase in the gene product of a carboxykinase gene.
  • the present invention comprises a diabetic comprising an effective amount of any one of the above-mentioned phosphosomal S6 kinase B phosphoric acid phosphatase inhibitors of cytoplasmic factor 1a 4a and inhibitors of Z or RSKB activity. Prevention and Z or treatment.
  • the present invention also relates to a method for identifying a compound that inhibits the interaction between ribosomal S6 kinase B (RSKB) and hetocytonuclea factor 4a (HNF-4a), wherein RSKB and HNF A system that uses a signal and Z or marker to detect the binding of RSKB and HNF-4a by contacting RSKB and Z or HNF-4a with the test compound under conditions that allow -4a interaction Use to determine whether the test compound inhibits the interaction between RSKB and HNF-4a by detecting the presence or absence of this signal and the Z or marker and the Z or change.
  • the present invention relates to an identification method.
  • the present invention provides a method for identifying a compound that inhibits the binding of ribosomal S6 kinase B (RSKB) to hetocytonuclea factor 1a (HNF-4a), comprising RSKB and HNF4 Using a system using RSKB and Z or HNF-4a in contact with a test compound under conditions that allow binding of a, and using a signal that detects the binding of RSKB and HNF-4a and Z or marker
  • the present invention relates to an identification method comprising determining whether or not a compound has a test compound strength 3 ⁇ 4 SKB and a binding to HNF-4a by detecting the presence or absence of this signal and Z or a marker and Z or a change.
  • the present invention provides a method for identifying a compound that inhibits phosphorylation of hepatocyte nuclear factor 1a (HNF-4 ⁇ ) by ribosomal S6 kinase B (RSKB), comprising: — RSKB and Z or HNF-4a in contact with the test compound under conditions that allow phosphorylation of 4a, and HNF by RSKB — Uses signal and Z or marker to detect phosphorylation of 4a To determine whether the test compound inhibits phosphorylation of HNF-4a by RSKB by detecting the presence or absence and Z or change of this signal and Z or marker. It relates to identification methods including
  • the present invention also relates to at least one of ribosomal S6 kinase B (RSKB), a polynucleotide encoding RSKB, and a vector containing a polynucleotide encoding RSKB, and hepatocyte nuclear factor 4
  • RSKB ribosomal S6 kinase B
  • the present invention relates to a kit comprising at least one of a (HNF-4a), a polynucleotide encoding HNF-4a, and a vector containing the polynucleotide encoding HNF-4a.
  • the invention's effect it is possible to provide a method for phosphorylating HNF-4a by RSKB, a method for inhibiting phosphorylation, and a phosphoric acid inhibitor. Further, according to the present invention, it is possible to provide a productivity method, a production inhibition method, and a productivity inhibitor of a gene product of a gene on which HNF-4a acts.
  • a method for inhibiting the production of a gene product of a gene on which HNF-4a acts can be carried out by using a phosphoric acid inhibitor of HNF-4a and a Z or phosphoric acid inhibitor method by RSKB.
  • a preventive and Z or therapeutic method and preventive and Z or therapeutic agent for diseases caused by an increase in the gene product of a gene on which HNF-4a acts.
  • diseases caused by an increase in gene products of gluconeogenesis-related genes more specifically diseases caused by an increase in the gene product of PEPCK gene diabetes, such as diabetes prevention and Z or treatment methods and prevention and Z
  • a therapeutic agent can be provided.
  • the present invention is thus very useful for the prevention and Z or treatment of diseases caused by excessive phosphorylation of HNF-4a involved in the expression of gluconeogenesis-related genes such as PEPCK gene.
  • diseases caused by excessive phosphorylation of HNF-4a involved in the expression of gluconeogenesis-related genes such as PEPCK gene.
  • Preferred examples of such diseases include diseases caused by increased gluconeogenesis due to expression of the PEPCK gene, specifically diabetes.
  • PEPCK gene expression is repressively controlled by insulin, and gene expression occurs in units of several hours as necessary.
  • the PE PCK gene is overexpressed continuously for some reason, gluconeogenesis in the liver is increased and hyperglycemia occurs, causing insulin-resistant diabetes.
  • ⁇ -cells in the spleen have collapsed and the amount of insulin in the blood has decreased, so that it is considered that the inhibitory effect of insulin on the PEPCK gene is decreased in end-stage patients with diabetes.
  • the expression of the PEPCK gene can be inhibited in an insulin-independent manner by the phosphate inhibitor of HNF-4a and Z or phosphate inhibitory method by RSKB according to the present invention, insulin resistant diabetes
  • gluconeogenesis involving the PEPCK gene can be suppressed. That is, in the liver of an insulin resistant patient with type 2 diabetes who can not be expected to have an insulin effect by the phosphate inhibitor of HNF-4 ⁇ and the method of inhibiting phosphate or phosphate by RSKB according to the present invention
  • gluconeogenesis can be suppressed, and as a result, hyperglycemia can be suppressed.
  • thiazolidinedione derivatives are used in clinical settings.
  • the thiazolidinedione derivative has an action of changing large adipocytes having low insulin sensitivity in adipose tissue to small adipocytes having high insulin sensitivity.
  • the phosphate inhibitor of HNF-4a by RSKB according to the present invention has an action of inhibiting the expression of PEPCK gene in the liver, and as a result, can suppress gluconeogenesis.
  • the phosphorylation inhibitor of HNF-4a by RSK B according to the present invention and the thiazolidinedione derivative differ in the target organ and the mechanism of action.
  • FIG. 1 is a diagram showing the results of in silico prediction of the interaction between RSKB and HNF-4a.
  • FIG. 1 and 2 show the elution fraction 1 and elution fraction 2 by FLAG, respectively. Elution fraction 1 was used for the experiment. Arrowheads indicate purified FLAG-RSKB.
  • the numerical values shown in the left column of the figure are the molecular weights of the molecular weight markers (indicated as M in the figure).
  • FIG. 3 shows that HNF-4a was phosphorylated by RSKB as a result of in vitro immunoprecipitation phosphorylation test of HNF-4a.
  • Panel A shows the results of the phosphate control test with the positive control PKA
  • Panel B shows the results of the phosphate test with RSKB.
  • CREB1 cyclic AMP responsive element binding protein 1, cAMP responsive element binding protein 1
  • phosphate by RSKB indicates that the purified RSKB has activity.
  • Figure Inside + and one indicate the presence or absence of each protein.
  • the arrowhead indicates HNF-4 ⁇ , and the star (*) indicates CREB1 phosphate.
  • the numbers listed in the left column of the figure are the molecular weights of the molecular weight markers. (Example 2)
  • FIG. 4 shows the nucleotide sequence of the promoter region of the human PEPCK gene (NCBI accession number U31519).
  • the AF1 sequence used in the gel shift assembly is shown in bold.
  • PEPCK AF1 promoter ZpGL3 is the 882 to 140 6th base sequence (bold display) separated by arrows 1 and 3
  • PEPCK ⁇ AF1 promoter ZpGL3 is the 934 to 1406 bases separated by arrows 2 and 3 Each sequence was used.
  • the numbers in the left column indicate the base numbers in U31519. (Example 3)
  • FIG. 5 FLAG-HNF-4a (DC BB stained image, transiently expressed in HEK293T cells using an N-terminal FLAG-tagged animal cell expression plasmid and purified using FLAG M2 affinity gel.
  • 1 and 2 indicate FLAG elution fraction 1 and elution fraction 2, respectively, and elution fraction 1 was used in the experiment, and arrowheads indicate purified FLAG-HNF-4a, as shown in the right column of the figure.
  • the numerical value is the molecular weight of the molecular weight marker (shown as M in the figure) (Example 3)
  • FIG. 6 is a diagram showing the results of examining the binding ability of HNF-4a to the AF1 sequence in the PEPCK gene promoter region using EMSA (electrophoretic mobility shift assay).
  • Panel A shows that HNF-4a bound to the AF1 sequence. By adding anti-HNF-4 ⁇ antibody, a supershift was observed in which the mobility of the HNF-4a 'DNA complex was reduced.
  • Panel B shows the effect of phosphorylation by RSKB on the ability of HNF-4 ⁇ to bind DNA to the A F 1 sequence. Phosphate treatment with RSKB enhanced the ability of HNF-4a to bind to the AF1 sequence. Phosphate treatment with PKA showed no such enhancement.
  • the amount of HNF-4 ⁇ used in Panel B is about one-tenth that of Panel A. In the figure, + and — indicate the presence or absence of anti-HNF-4 ⁇ antibody supplements. (Example 3)
  • FIG. 7 shows that the binding of HNF-4a to the AF1 sequence is enhanced in a dose-dependent manner by the phosphorylation of HNF-4a by RSKB.
  • RSKB used for HNF-4a phosphate treatment
  • the binding activity of HNF-4 ⁇ to AF1 decreased.
  • ⁇ In the A treatment indicated as PKA in the figure
  • IX, 1/2, and 1Z4 show the EMS results of the phosphorylated samples with RSKB diluted 2 and 4 times, respectively.
  • the star mark (*) indicates the addition of anti-HNF-4a antibody.
  • FIG. 8 shows the results of reporter assembly using PEPCK AF1 promoter. Transcriptional activity increased depending on the amount of HNF-4a introduced. This result indicates that HNF-4a force SPEPCK gene is positively controlled. Even when RSKB was expressed, the transcriptional activity of HNF-4a was not affected.
  • the vertical axis is a multiple when the transcriptional activity of the control group (introducing only PEPCK AF1 promoter ZpGL3) is 1.00.
  • the number next to each group of proteins is the amount of DNA introduced (eg, HNF-4a 0.05 was introduced with 0.05 ⁇ g of F-4a expression plasmid).
  • FIG. 9 shows the results of reporter assembly using PEPCK AF1 promoter.
  • Transcriptional activity by HNF-4a Significantly enhanced by RSKB activated by MAPK11. Since only the expression of MAPK11 except RSKB had no effect on the transcriptional activity of HNF-4a, this action can be attributed to RSKB.
  • the vertical axis is the multiple when the transcriptional activity of the control group (introducing only PEPCK AF1 promoter ZpGL3) is 1.00.
  • the number next to each group of proteins is the amount of DNA introduced (eg, HNF-4a 0.05 was introduced with 0. g of HNF-4 ⁇ expression plasmid).
  • FIG. 10 is a diagram showing the results of reporter assembly using PEPCK AF1 promoter.
  • the Transcriptional activity by HNF-4 ⁇ was significantly enhanced by RSKB activated by MAPK11.
  • dominant negative RSKB S343A
  • the vertical axis is a multiple of the transcriptional activity of the control group (only PEPCK AF1 promoter ZpGL3 introduced) is assumed to be 1.00.
  • the number next to each group of proteins is the amount of DNA introduced (eg, HNF-4a 0.05 introduced 0.05 g of HNF-4 ⁇ expression plasmid). *: Significantly different from control ( ⁇ ⁇ 0. 05), #: HNF—4 Significantly different from 4 ⁇ 0.05 ( ⁇ ⁇ 0.05), $: HNF-4 a 0. 05 + Significantly different from RSKB 0.25 + MAPK11 0. 05 (p ⁇ 0. 05). (Example 4)
  • FIG. 11 shows the results of reporter assembly using PEPCK A AF1 promoter.
  • HNF-4a promotes transcriptional activity by co-expressing HNF-4a, RS KB and MAPK11 even in cells into which HNF-4a has been introduced. It was not recognized even in the cells!
  • HNF-4 ⁇ is dependent on AF1 within the promoter region of the SPEPCK gene promoter.
  • the vertical axis is a multiple when the transcriptional activity of the control group (in which only PEPCK A AF1 promoter ZpGL3 is introduced) is 1.00.
  • the number next to each group of proteins is the amount of DNA introduced (eg, HNF-4 ⁇ 0.05 was introduced with 0.05 g of HNF-4 ⁇ expression plasmid). *: Significantly different from the control ( ⁇ 0. 05), #: Significantly different from HNF— 4 a 0. 05 (p ⁇ 0. 05), $: HNF — 4 ⁇ 0.2 There is a significant difference ( ⁇ ⁇ 0.05). (Example 4)
  • FIG. 12 shows the results of reporter assembly using the PEPCK AF1 promoter in HepG2 cells. This result was obtained under the condition that the darcocorticoid receptor was expressed by transfecting pMMGR into HepG2 cells. Transcriptional activity by HNF-4a was promoted by RSKB activated with MAPK11. When the MAPK11 inhibitor SB203580 was added, co-expression of MAPK11 and RSKB did not promote the transcriptional activity of HNF-4a. This result shows that the activity of RSKB is HNF-4 ⁇ It is important for transcriptional activity.
  • the vertical axis is a multiple of the transfer activity of the HNF-4 and 0.5 treatment groups as 1.00.
  • the number next to each group of proteins is the amount of DNA introduced (eg, HNF-4a 0.5 introduced 0.5 g of HNF-4a expression plasmid). *: Significantly different from HNF— 4 ⁇ 0.5 ( ⁇ 0. 05), #: Significantly different from HNF— 4 ⁇ 0.5 + R SKB 0.25 (p 0. 05) ), $: HNF— 4 ⁇ 0.5 + RSKB 0.5 (p ⁇ 0.05), +: RSKB 0.5 (p ⁇ 0.05) ⁇ : HNF-4 a 0.
  • FIG. 13 is a drawing showing that expression of PEPCK gene is enhanced by RSKB depending on its kinase activity.
  • RSKB wild-type RSKB
  • MAPK11 MAPK11
  • the expression level of endogenous PE PCK gene increased compared to cells that did not express them.
  • an inactive mutant RSKB RSKB (S 196A / S343A / T568A)
  • RSKB S 196A / S343A / T568A
  • MAPK11 co-expressing cells showed an increase in the expression level of endogenous PEPCK gene. I was helped.
  • the expression level of the endogenous control GAPDH gene was almost the same in all cells.
  • each gene was detected by RT-PCR using the total RNA prepared by the cells.
  • the PCR product derived from the PEPCK gene is 575 bp
  • the PCR product derived from the GAPDH gene is 209 bp.
  • the 50 bp DNA ladder (1 adder) shown in the left lane in the figure is a DNA size marker during electrophoresis. (Example 5)
  • FIG. 14 HepG2 cells co-expressed with wild-type RSKB (shown as RSKB (WT)) and MAPK11, inactive mutant RSKB (RSKB (S196AZS343AZT568A)) and HepG2 co-expressed with M APK11
  • FIG. 3 is a view showing the expression level of each protein in cells and cells in which these were not expressed.
  • the upper panel shows the results of detecting the expression of FLAG-RSK B and MAPKll-FLAG in each cell by immunoblotting (ib) using an anti-FLAG antibody.
  • N-terminal FLAG tag wild type RSKB and N-terminal FLAG tag inactivation detected by immunoblotting (ib) using anti-FLAG antibody The band indicating the sexual variant RSKB! Is indicated by the FLAG-RSKB display on the right of the panel.
  • the middle panel shows the results of detecting phosphorylated RSKB with anti-phosphorylated RSKB antibody. This anti-phosphorylated RSKB antibody recognizes the 360th serine phosphate that is important for the kinase activity of RSKB.
  • FIG. 15 is a diagram showing that HNF-4a and RSKB bind in cells.
  • Cell force in which FLAG—HNF-4a and FLAG—RSKB were transiently co-expressed Prepared cell lysate was prepared by immunoprecipitation using anti-HNF-4a antibody and detection using anti-FLAG M2 antibody. A band indicating FLAG-HNF-4a and a band indicating FLAG—RSKB were both detected (Panel A). This result indicates that RSKB co-precipitated in a HNF-4 ⁇ -dependent manner.
  • Panel A shows the results of immunoblotting (i. B.) Of an immunoprecipitation sample (i. P.) With anti-HNF-4 ⁇ antibody with anti-FLA G M2 antibody.
  • Panel B shows the results of immunoblotting (i.b.) the cell lysate with anti-FLAG M2 antibody.
  • + and 1 indicate the presence or absence of each expression plasmid, respectively.
  • amino acids may be represented by one letter or three letters.
  • Peptide also means any peptide comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds.
  • Peptide Short-chain peptides such as isolated or synthetic full-length oligopeptides, referred to as oligomers, and long chains such as isolated or synthetic full-length polypeptides and isolated or synthetic full-length proteins Also means peptide.
  • HNF-4a and RSKB bind, HNF-4a cation is converted by RSKB, HNF-4a is phosphorylated by RSKB, and PEPCK gene promoter It was demonstrated that the ability to bind to AF1 within the region was promoted, and that the transcriptional activity of the PEPCK gene was enhanced by vigorous promotion. It was also clarified that the activity of RSKB is required for phosphorylation of HNF-4a by RSKB.
  • the phosphorylation power of HNF-4a by RSKB can be used to positively regulate the transcriptional activity of the PEPCK gene through promoting the binding of HNF-4a to AF1 in the PEPCK gene promoter region. Turned out to be involved.
  • the PEPCK gene is a gene encoding PEPCK which is considered to be a gluconeogenic rate-determining enzyme. From this, phosphorylation by RSKB promotes the ability of HNF-4 ⁇ to bind to PE1 in the motor region of PEPCK gene, and as a result, the transcriptional activity of PEPCK gene is enhanced, thereby It can be considered that expression is promoted and gluconeogenesis is enhanced.
  • Non-patent Documents 8 and 27 Transcription of the PEPCK gene is regulated by hormones, promoted by darcocorticoids, and repressed by insulin. Insulin regulates gluconeogenesis in the liver by suppressing transcription of PEPCK gene through LIP (liver-enriched transcriptional inhibitory protein), a transcriptional regulator in PEPC K gene promoter. (Non-patent document 28). There are also reports that insulin stimulation acts directly on the PEPCK gene promoter to suppress transcriptional activity (Non-patent Documents 6 and 37).
  • PEPCK gene is repressively controlled by insulin. For some reason, if overexpression of PEPCK gene persists, It is thought that diabetes develops when gluconeogenesis is increased and hyperglycemia occurs and insulin resistance is further developed. In fact, gluconeogenesis by PEPCK in non-insulin-dependent diabetic patients is about 3 times higher than that in normal individuals, and this gluconeogenesis is correlated with the fasting plasma glucose concentration in patients. (Non-patent Document 26).
  • Overexpression of the PEPCK gene includes phosphorylation by RSKB, which promotes the ability of HNF-4a to bind to AF1 in the PEPC K gene promoter region and thereby increases the transcriptional activity of the PEP CK gene. It is thought to be involved.
  • the PEPCK gene promoter has a region called GRU (Glucocorticoid Response Unit), and AF1, AF2 (accessory factor binding site 2, accessory factor binding site 2) in the region , GR1, 2 (glucocorticoreticoid receptor binding ⁇
  • GRU Glucocorticoid Response Unit
  • AF1, AF2 accessory factor binding site 2, accessory factor binding site 2
  • GR1, 2 glucocorticoreticoid receptor binding ⁇
  • CRE cyclic AMP response element, cAMP responive element
  • CREB cyclic AMP response element binding protein, cAMP responsive element binding protein
  • CREB is known as a transcription factor that is phosphorylated by RSKB and binds to the promoter.
  • CREB is a transcription factor that binds to CRE of PEPCK gene promoter (Non-patent Document 19).
  • CRE is a region that exists in the PEPCK gene promoter and positively regulates PEPCK gene expression (Non-patent Document 18).
  • CZEBP CCAATZenhancer binding protein
  • CZEBP CCAATZenhancer binding protein
  • RSKB phosphorylates CREB, and phosphorylated CREB enhances the transcriptional activity of a promoter having CRE (Non-patent Document 11). Therefore, when RSKB is activated, CREB is phosphorylated without passing through HNF-4a, and CREB binds to CRE of PEPCK gene promoter, which may promote PEPCK gene expression.
  • AF1 to which HNF-4a binds has a greater effect on the expression of the PEPCK gene than CRE. Specifically, substitution or deletion of AF1 reduces the transcriptional activity of the PEPCK gene promoter to about one-fourth that of the wild-type PEPCK gene promoter (Non-patent Document 24). On the other hand, depending on CRE substitution and deletion, the transcriptional activity of the PEPCK gene promoter can be reduced to about half that of the wild-type PEPCK gene promoter! (Non-patent Document 25).
  • AF1 to which HNF-4a binds can be considered to be a more important factor in the regulation of PEPCK gene expression compared to CRE.
  • RSKB does not promote the transcriptional activity of the PEPCK gene promoter.
  • a reporter gene having RSKB, MAPK11 that activates RSKB, and PEPCK A AF1 promoter lacking AF1 in the PEPCK gene promoter region was expressed in cells, the reporter gene Transcriptional activity was hardly promoted (see Example 4 and FIG. 11).
  • AFNF of the PEPCK gene promoter region involved in transcription binds to HNF-4a by being converted to HNF-4a by RSKB, resulting in transcription of the PE PCK gene promoter. Activity is promoted.
  • HNF-4a is phosphorylated by RSKB, HNF-4a cannot bind to the PEPCK gene promoter. Therefore, it can be considered that the transcription activity of the PEPCK gene promoter by RSKB was not promoted.
  • RSKB is activated by p38, an upstream kinase, and exhibits kinase activity.
  • Activity RSKB is known to phosphorylate CREB. Therefore, the activity of RSKB can be determined using the activity of P38 and the phosphate of CREB as indicators.
  • the p38 phosphoric acid type active p38
  • the p38 phosphoric acid type is about 2.5 to 3 times higher than that of normal individuals, and the phosphorylated form of CREB is also about 2 times higher. Have been reported (Non-Patent Documents 29 and 30).
  • PEPCK activity is regulated at the mRNA level, and PEPCK mRNA level, enzyme activity, gluconeogenic potential, and blood glucose level increase are well correlated. Specifically, in experiments using hepatocytes, it was reported that when the amount of PEPCK gene mRNA was increased 2-fold, enzyme activity increased 2.8-fold and gluconeogenesis increased 2.1-fold. (Non-patent document 6). In mice, when the gluconeogenic potential in the liver is increased by about 2.5 times, the blood glucose level rises by a factor of 2 (Non-patent Document 7), and in humans, the glucose gluconeogenic potential by PEPCK is 2.5 times higher in humans. Is 3 times higher than normal (Non-patent Document 26).
  • the present inventors considered the degree of increase in blood glucose level due to RSKB activity as follows. Assuming that the RSNF HNF-4a phosphate is similar to the CREB phosphate, the liver KBN HNF-4a phosphate is normal in the pathogenesis of diabetes It can be estimated that it is about 2 times higher. This is because, as described above, the phosphorylated form of CREB is increased about 2-fold in the liver of diabetic model animals, and RSKB activated by p38 is involved in phosphorylation of CREB. (Non-Patent Documents 29 and 30).
  • phosphorylation of HNF-4 ⁇ by RSKB increased the PEPCK gene promoter activity involving HNF-4 ⁇ force S by about 2-3 times (Example 4). And Figures 9, 10 and 12). If the promoter activity of the PEPCK gene is replaced with the amount of mRNA, it can be estimated that the amount of mRNA of the PEPCK gene increases by about 2 to 3 times due to the activity of RSKB in diabetic conditions. The increase in the amount of PEPCK gene mRNA is as described above. It is directly reflected in the enzyme activity, leading to increased gluconeogenic potential and increased blood glucose level.
  • One embodiment of the present invention achieved by these findings relates to a method for phosphorylating HNF-4a, characterized by using RSKB.
  • the HNF-4a phosphate method according to the present invention can be carried out by allowing RS KB and HNF-4a to coexist under conditions that allow their interaction.
  • an HNF-a phosphate inhibitor containing an effective amount of RSKB can be provided.
  • RSKB and HNF-4a interaction means that RSKB and HNF-4a are directly related.
  • the direct association between RSKB and HNF-4a includes a reaction in which RSKB and HNF-4a bind, and a reaction in which HNF-4 ⁇ is phosphorylated by RSKB as a result of the binding.
  • the reaction of RSKB first binds to HNF4a, and as a result, the reaction of HNF-4a cation is caused by the action of RSKB.
  • reaction of RSKB and HNF-4a binding can be called the interaction of RSKB and HNF-4 ⁇ , and as a result of the binding reaction, the reaction of RSKB causes HNF-4a to be oxidized. This reaction can be called the interaction between RSKB and HNF-4a.
  • the bond between RSKB and HNF-4a is closer to non-covalent bonds such as hydrogen bonds, hydrophobic bonds, or electrostatic interactions to form RSKB and HNF-4a force complexes. Means that. In this case, the bond is sufficient if RSKB and HNF-4a are bonded in part. For example, it means a bond between RSKB or part thereof and HNF-4 ⁇ or part thereof. In addition, the complex formed by RSKB and HNF-4 ⁇ contains a different type of protein! /, Or even! /.
  • phosphorylation of HNF-4a by RSKB refers to the transfer of the ⁇ -phosphate group of ATP to the hydroxyl group of the serine, threonine or tyrosine residue of HNF-4a by the kinase activity of RSKB. As a result, it means a reaction in which an HNF-4 ⁇ protein having a phosphate group bound thereto is produced.
  • RSKB kinase activity refers to an RSKB force that binds to another protein (hereinafter sometimes referred to as a substrate protein) and binds to the hydroxyl group of an amino acid residue in the substrate protein with adenosine triphosphate ( It means a function of phosphorylating the substrate protein by catalyzing a reaction of transferring the ⁇ -phosphate group of (ATP).
  • substrate means a compound or molecule that is catalyzed by an enzyme.
  • the conditions allowing the interaction between RSKB and HNF-4a may be any of in vitro and in vivo conditions. That is, the HNF-4 ⁇ phosphate method according to the present invention is used in vitro and in vivo. However, it can also be implemented.
  • the HNF-4a phosphate method according to the present invention can be preferably performed in an in vitro sample or a non-human mammal.
  • “In vitro sample” refers to cells and tissues prepared from animals such as mammals, cultured cells derived from animals, and solutions containing proteins and genes prepared from the cells, tissues and cultured cells. Means sample. Preferred examples of cells include liver cells and liver cell lines.
  • “Non-human mammal” means mammals other than human, for example, mammals such as mouse, rat, rabbit, nu, goat and the like. Preferably a mouse is used.
  • the method for phosphorylating HNF-4a according to the present invention specifically includes expressing RSKB in a cell expressing HNF-4a, or coexisting RSKB and HNF-4a in the cell. It can be implemented by expressing. Alternatively, the phosphorylation method of HNF-4a can be performed by coexisting RSKB and HNF-4Q; for example, in a test tube or a multiwell plate.
  • HNF-4a phosphate system characterized by coexistence of RSKB and HNF-4a.
  • the phosphorylation system of HNF-4a according to the present invention may be either in vitro or in vivo.
  • the HNF-4 phosphate system by RSKB can preferably be a system utilizing in vitro samples or non-human mammals.
  • the phosphorylation system of HNF-4a according to the present invention specifically allows RSKB to be expressed in cells expressing HNF-4a, or allows RSKB and HNF-4a to be co-expressed in cells. It can be constructed by expressing it.
  • the phosphorylation system of HNF-4a can be constructed by phosphorylating RSKB and HNF-4Q; for example, in a test tube or a multiwell plate.
  • RSKB expression and HNF-4a expression can be performed by a conventional genetic engineering technique using an appropriate vector containing a gene encoding RSKB and an appropriate vector containing a gene encoding HNF-4a, respectively. These can be achieved by transfecting the cells. Phosphorylation of HNF-4a by RSKB is achieved by the interaction of RSKB and HNF-4a in the cell.
  • a cell used for expression a cell generally used for protein expression can be used.
  • eukaryotic cells are used.
  • Eukaryotic cells can be any of cells prepared from eukaryotes, primary cultured cells, and cultured cell lines.
  • a mammalian cell line more preferably a human cell line is used.
  • a cultured cell line derived from a mammalian liver more preferably a cultured cell line derived from a human liver is used.
  • human-derived cultured cell lines include HeLa cells (cervical cancer-derived cell lines) and HepG2 cells (human liver cancer-derived cell lines).
  • Detection of HNF-4 ⁇ phosphate by RSKB was carried out by contacting RSKB with HNF-4a using a well-known protein phosphate assay method. After that, it can be carried out by measuring phosphoric acid type HNF-4.
  • the detection of phosphate-type HNF-4 can be performed, for example, by Western blotting using an antibody against phosphate-type HNF-4.
  • detection of phosphoric acid-type HNF-4 was performed using ATP labeled with a radioisotope for phosphorylation, such as [y 32 P] ATP.
  • amino acid residues of HNF-4 a It can be carried out by measuring the radioactivity of [ ⁇ - 32 P] transferred to. Specifically, with reference to Example 2 described later, detection of HNF-4a phosphate by RSKB can be performed.
  • RSKB and HNF-4 ⁇ are preferably human-derived proteins, but mammal-derived proteins having the same functions as the human-derived proteins and having sequence homology, eg, For example, it can be a protein derived from mouse, horse, hidge, ushi, nu, monkey, cat, rat or rabbit.
  • the genes encoding RSKB and HNF-4a are preferably human-derived genes, but mammal-derived proteins having the same function and sequence homology as the human-derived proteins.
  • Examples of RSKB functions include kinase activity.
  • functions of HNF-4 ⁇ include a function of binding to AF1 and a function of promoting the transcriptional activity of the PEPCK gene promoter.
  • RSKB-encoding gene and RSKB include the human-derived gene represented by the nucleotide sequence set forth in SEQ ID NO: 1 and the human-derived protein represented by the amino acid sequence set forth in SEQ ID NO: 2, respectively.
  • the gene encoding RSKB is not limited to the above-exemplified gene, and any gene may be used as long as it is a gene encoding a protein having sequence homology with the gene and having the same function as the protein encoding the gene. Denko is also included.
  • RSKB is not limited to the above-exemplified proteins, and any protein is included as long as it has a sequence homology with the protein and has the same function as the protein.
  • sequence homology is usually 50% or more, preferably at least 70% of the entire amino acid sequence or base sequence. More preferably, it is 70% or more, more preferably 80% or more, still more preferably 90% or more, and even more preferably 95% or more.
  • RSKB functions include kinase activity.
  • HNF-4a and HNF-4a a human-derived gene represented by the nucleotide sequence set forth in SEQ ID NO: 3 and the human represented by the amino acid sequence set forth in SEQ ID NO: 4, respectively Preferred examples are derived proteins.
  • the gene encoding HNF-4a is not limited to the gene exemplified above, as long as it is a gene encoding a protein having sequence homology with the gene and having the same function as the protein encoded by the gene. In this regard, any gene is included.
  • HNF-4Q is not limited to the above-exemplified proteins, and any protein is included as long as it is a protein having sequence homology with the protein and having the same function as the protein.
  • the Sequence homology is usually amino acids It is appropriate that the total sequence or base sequence is 50% or more, preferably at least 70%. More preferably, it is 70% or more, more preferably 80% or more, still more preferably 90% or more, and even more preferably 95% or more.
  • Examples of the function of HNF-4a include a function of binding to AF1 and a function of promoting the transcriptional activity of the PEPCK gene promoter.
  • RSKB and HNF-4a may each be a cell sample prepared by genetic engineering techniques, or may be a cell-free synthetic product or a chemical synthesis product. It may be further purified. In addition, cells in which at least one of RSKB and HNF-4a is expressed by a genetic engineering technique can also be used.
  • RSKB and HNF-4a can be directly or phosphorylated with other types of labeling substances such as proteins and polypeptides on the N-terminal side and C-terminal side, as long as their properties and functions are not affected. It can be added indirectly via a carpeptide or the like using genetic engineering techniques. By measuring the labeling substance itself or its function, it is possible to easily detect the binding of RSKB and HNF-4 and the detection of phosphate of HNF-4a by RSKB.
  • labeling substances enzymes (Dartathione S-transferase, horseradish peroxidase, anolekali phosphatase or 13 galactosidase), tag peptides (His-tag, Myc-tag, HA-tag, FLAG) — Tag or Xpress— tag), fluorescent proteins (green fluorescent protein, fluoresceinis othiocyanate or phycoerythrin, etc.), maltose binding protein, immunoglobulin Fc fragment, Alternatively, piotin can be exemplified, but not limited thereto. Alternatively, it can be labeled with a radioisotope. When labeling is performed, one type of labeling substance may be added, or a plurality of labeling substances may be added in combination.
  • the genes encoding RSKB and HNF-4a are, for example, from an appropriate origin (for example, liver tissue and cells derived from liver tissue) from which each gene is expressed, or from a human cDNA library. It can be easily obtained using a known clawing method or the like. Proteins encoded by these genes can be obtained by, for example, a known genetic engineering technique using each gene. Specifically, each gene An appropriate expression vector DNA, for example, a vector derived from a bacterial plasmid is introduced by a known genetic engineering technique to obtain a vector containing the gene, and the vector is introduced into an appropriate host cell. A cell expressing the gene can be obtained, and a protein encoded by the gene can be obtained from the cell.
  • an appropriate expression vector DNA for example, a vector derived from a bacterial plasmid is introduced by a known genetic engineering technique to obtain a vector containing the gene, and the vector is introduced into an appropriate host cell. A cell expressing the gene can be obtained, and a protein encoded by the gene can be obtained
  • RSKB can be prepared as an immunoprecipitate obtained from a cell lysate of a cell expressing a gene encoding RSKB using an anti-RSKB antibody or the like.
  • HNF-4a can be similarly prepared using a gene encoding HNF-4a and an anti-HNF-4 ⁇ antibody.
  • the phosphorylating agent of HNF-4a, the phosphorylating method, and the phosphoric acid salt system according to the present invention include
  • a method for identifying a compound that inhibits phosphorylation of HNF-4a by RSKB can be constructed using the phosphorylation method and the phosphoric acid-containing system.
  • Another embodiment of the present invention relates to a phosphorylation inhibitor and a method for inhibiting HNF-4a by RSKB.
  • the phosphorylation inhibitor and inhibition method of HNF-4a according to the present invention inhibits RSKB activity or inhibits the interaction between RSKB and HNF-4 ⁇ , that is, RSKB and HNF-4a. By inhibiting the binding of or by inhibiting the phosphate of HNF-4a by RSKB.
  • Examples of the target to which the inhibitor and the inhibition method according to the present invention are applied include a target including at least RSKB and HNF-4a, for example, an in vitro sample including at least these.
  • Examples thereof include cells expressing at least RSKB and HNF-4 ⁇ , such as liver cells, and non-human mammals carrying such cells.
  • RSKB activity means a function of RSKB, a function to bind to other proteins, for example, a function to bind to HNF-4a, and a function to phosphorylate other proteins as kinases. For example, a function of phosphorylating HNF-4a is included.
  • “Inhibiting RSKB activity” means inhibiting the function of RSKB, for example, inhibiting the function of binding to HNF-4a and phosphorylating HNF-4a Inhibiting function, ie kinase activity against HNF4a.
  • the phosphate inhibitor of HNF-4a by RSKB according to the present invention contains at least one compound (RSKB activity inhibitor) having an effect of inhibiting RSKB activity in one aspect thereof.
  • RSKB activity inhibitor means a compound having a function of inhibiting RSKB activity. That is, “RSKB activity inhibitors” include RSKB kinase activity inhibitors and RSKBH NF-4a binding inhibitors. The “RSKB activity inhibitor” may be a composition comprising one or more compounds having a function of inhibiting RSKB activity.
  • Examples of RSKB activity inhibitors include antibodies having a competitive inhibitory effect and low molecular weight compounds.
  • Examples of the antibody include an antibody that recognizes and binds to RSKB or HNF-4a and inhibits phosphorylation of HNF-4 ⁇ by RSKB.
  • the antibody can be obtained by an antibody production method known per se using RSKB or HNF-4a itself, a partial peptide derived therefrom, or a peptide having an amino acid sequence at the site where they interact as an antigen.
  • Low molecular weight compounds include peptides, peptide-like substances, polypeptides, polynucleotides, organic compounds, and inorganic compounds, and the molecular weight is preferably 10,000 or less, more preferably 5000 or less, and even more preferably 1000. Hereinafter, even more preferably 500 or less compounds are meant.
  • Low molecular weight compounds include compounds that inhibit the kinase activity of RSKB, preferably compounds that specifically inhibit the kinase activity.
  • the compound to be obtained can be identified, for example, by determining whether it can inhibit the phosphorylation of HNF-4a by RSKB using the phosphorylation method or phosphorylation system according to the present invention.
  • To specifically inhibit the kinase activity of RSKB means to strongly inhibit the kinase activity of RSKB, but not or weakly inhibit the activity of other enzymes.
  • a peptide having an amino acid sequence at the site where RSKB and HNF-4a interact can be exemplified.
  • peptides include peptides containing the amino acid sequences of the sites to which these proteins bind in the amino acid sequences of RSKB and HNF-4a.
  • An example of such a peptide is a peptide containing the amino acid sequence of the site phosphorylated by RSKB in the amino acid sequence of HNF-4a.
  • Such peptides compete for phosphorylation of HNF-4 ⁇ by RSKB It is thought to inhibit.
  • peptides were designed from the amino acid sequence of RSKB or HNF-4Q; and synthesized by a peptide synthesis method known per se, phosphorylation of HNF-4a by RSKB and Z or RSKB and HNF-4a It can be obtained by selecting those that inhibit the binding of.
  • the peptide having a mutation may be a naturally occurring peptide or a mutation introduced.
  • Means for introducing mutation such as deletion, substitution, addition or insertion are known per se, and for example, Ulmer's technology (Non-patent Document 31) can be used.
  • Ulmer's technology Ulmer's technology
  • homologous amino acids polar amino acids, nonpolar amino acids, hydrophobic amino acids, Mutual substitution between hydrophilic amino acids, positively charged amino acids, negatively charged amino acids and aromatic amino acids, etc.
  • a peptide that inhibits the interaction between RSKB and HNF-4a can be altered to such an extent that it does not undergo a significant change in function, such as modification of its constituent amino group or carboxyl group, for example, by amido.
  • modifications generally used to stabilize the interaction between peptides and other proteins and make it difficult to dissociate peptides such as C-terminal aldehydes or N-terminal acetylation Is useful to increase the effectiveness of peptides that inhibit the interaction between RSKB and HNF-4a.
  • the peptide can be produced by a general method known in peptide science.
  • methods Non-Patent Documents 32 and 33
  • known literatures can be mentioned, but the known methods can be widely used without being limited thereto.
  • HNF-4a is known to be involved in gluconeogenesis-related gene expression as a transcription factor in the liver in one of its functions.
  • the “gluconeogenesis-related gene” is a gene that encodes a substance that regulates gluconeogenesis in a living body, and preferably includes, for example, the PEPCK gene. Activation of HNF-4a during diabetic conditions is thought to further increase gluconeogenesis and cause exacerbation of hyperglycemia.
  • Another aspect of the present invention relates to a method for regulating the production of a gene product of a gluconeogenesis-related gene.
  • the method for regulating the production of gene products of gluconeogenesis-related genes regulates the phosphorylation by RSKB of HNF-4a related to the expression of genes encoding them, thereby regulating the production of the gene products of the genes. It is characterized by adjusting.
  • One aspect of the method for regulating gene product production of a gluconeogenesis-related gene is a method for promoting the production of a gene product of a gluconeogenesis-related gene, and phosphorylating HNF-4a related to the gene expression using RSKB It is characterized by.
  • Another aspect of the method for regulating gene product production of a gluconeogenesis-related gene is a method for inhibiting the production of a gene product of a gluconeogenesis-related gene, and the phosphorylation by RSK B of HNF-4a related to the gene expression. It is characterized by inhibiting oxidation.
  • the production inhibition method can be achieved by using a phosphorylation inhibitor of HNF-4a by RS KB or a phosphorylation inhibition method of HNF-4a by RSKB.
  • Examples of a gene product of a gene on which HNF-4a acts on gluconeogenesis-related gene expression include a gene product of a gene having a binding site of HNF-4 ⁇ in a promoter or enhancer.
  • a gene on which HNF-4 ⁇ acts includes a gene having AF1 which is a binding site of HNF-4a phosphorylated by RSKB in a promoter or enhancer, for example, PEPCK gene.
  • Yet another embodiment of the present invention relates to a production inhibitor of a gene product of a gluconeogenesis-related gene.
  • the gene product production inhibitor comprises the phosphorylation inhibitor as an active ingredient in an effective amount thereof.
  • the production inhibitor of this gene product is preferably a PEPCK gene product production inhibitor comprising the above phosphorylation inhibitor as an active ingredient and an effective amount thereof.
  • Another embodiment of the present invention relates to a method for identifying a compound that inhibits the interaction between RSKB and HNF-4a.
  • This identification method can be constructed using a pharmaceutical screening system known per se. Moreover, this identification method can be implemented using the phosphorylation system or phosphorylation method according to the present invention.
  • a condition that enables interaction between RSKB and HNF-4a is selected, and a compound (test compound) with RSKB and Z or HNF-4a is brought into contact under these conditions.
  • R Using a system that uses a signal and Z or marker to detect the interaction between SKB and HNF-4a, by detecting the presence or absence or change of this signal and Z or marker, RSKB and HNF-4a Identify compounds that inhibit the interaction.
  • whether a test compound inhibits the interaction between RSKB and HNF-4a is determined by the binding of RSKB and HNF-4a in the presence of the test compound and Z or RSKB.
  • the signal generated by phosphorylation of HNF-4a or the binding and Z or phosphate marker and the signal generated by the binding and Z or phosphate in the absence of the test compound are This can be done by comparing the binding and Z or phosphorylation markers. In the absence of a test compound, there is a signal generated by the binding and Z or the phosphoric acid, and the binding and Z in the presence of the test compound are compared with the binding and Z or the phosphorylation marker. Alternatively, when the signal generated by the phosphorylation or the binding and Z or phosphate markers are reduced or eliminated, the test compound can be determined to inhibit the interaction between RSKB and HNF-4a. .
  • the conditions allowing the interaction between RSKB and HNF-4a may be any of in vitro and in vivo conditions.
  • cells in which RSKB and HNF-4 ⁇ are co-expressed can be used.
  • the contact between RSKB and NF or HNF-4a and the test compound may be performed before the interaction between RSKB and HNF-4a, or may be performed by coexisting with the interaction.
  • a signal is a signal that itself can be directly detected by its physical or chemical properties, and a marker is a signal that can be detected indirectly using its physical or biological properties as an indicator. Point to.
  • signals include enzymes such as luciferase and green fluorescent protein, and radioactive isotopes.
  • the marker include a reporter gene such as chloramphenicol acetyl transferase gene, or a detection epitope tag such as 6 X His-tag. Signals and markers are not limited to these exemplified substances, and any labeling substance generally used in compound identification methods can be used. Can be. These signals or markers may be used alone or in combination of two or more. Methods for detecting these signals or markers are well known to those skilled in the art.
  • Still another embodiment of the present invention relates to a method for identifying a compound that inhibits phosphate phosphate of HNF-4a by RSKB.
  • This identification method can be constructed using a pharmaceutical screening system known per se.
  • this identification method can be carried out using the phosphorylation system or phosphorylation method according to the present invention.
  • RSKB and Z or HNF-4a are brought into contact with the test compound under the condition, and RSKB is used.
  • HNF-4 by RSKB by detecting the presence or absence or change of this signal and Z or marker using a system that uses the signal and Z or marker to detect HNF-4a phosphate
  • a compound that inhibits the phosphate of a can be identified.
  • whether or not the test compound inhibits HNF-4a phosphate by RSKB is determined by RSKB in the presence of the test compound. It can be carried out by comparing the signal generated by ⁇ or the phosphate marker and the signal or phosphate marker generated by the phosphate in the absence of the test compound. The signal generated by the phosphorylation in the absence of the test compound or the marker of phosphorylation is reduced or the signal generated by the phosphorylation in the presence of the test compound or the phosphate marker is reduced or When it disappears, it can be determined that the test compound inhibits phosphorylation of HNF-4a by R SKB.
  • RSKB and HNF-4a may be in vitro or in vivo.
  • cells in which RSKB and HNF-4 ⁇ are co-expressed can be used.
  • RSKB and ⁇ ⁇ ⁇ or HNF-4a can be contacted with the test compound before the phosphorylation reaction of RSKB and HNF-4 ⁇ ! Also good.
  • Phosphorylation of HNF-4a by RSKB can be conveniently detected by measuring the presence or absence and the Z or change of the amount of phosphorylated HNF-4a.
  • Phosphorylated HNF— 4 a Quantification of the amount can be carried out using a well-known method for measuring phosphate protein. For example, the amount of phosphate-type HNF-4 ⁇ can be quantified by Western blotting using an antibody against phosphate-type HNF-4a.
  • the detection of phosphorylated HNF- 4 ⁇ is, Arufatauro labeled radioisotope Rinsani ⁇ reaction, for example using [ ⁇ - 32 ⁇ ] ⁇ , amino acid residues RESULTS HNF- 4 a of the reaction This can be done by measuring the radioactivity of [ ⁇ - 32 ⁇ ⁇ ] transferred to the group. Specifically, referring to Example 2 to be described later, detection of phosphate salt of HNF-4 Q; by RSKB can be performed.
  • the detection of phosphorylation of HNF-4 ⁇ by RSKB can also be performed by measuring the presence or absence of HNF-4 ⁇ activity and wrinkles or changes.
  • the phosphorylation of HNF-4a by RSKB can be detected by detecting the transcriptional activity of the PEPCK gene promoter.
  • Detection of the transcriptional activity of the PEPCK gene promoter can be carried out by reporter assembly using a reporter gene having a PEPCK gene promoter.
  • cells expressing HNF-4 ⁇ and RSKB are transfected with a plasmid containing a reporter gene incorporating the PEPCK gene promoter region upstream, and the expression level of the reporter gene is measured. By doing so, detection of HNF-4 ⁇ phosphate by RSKB can be performed.
  • the expression level of the reporter gene when the test compound is brought into contact with the cell is compared with the expression level of the reporter gene when the test compound is not brought into contact with the test compound.
  • the expression level when the test compound is brought into contact with the cells is reduced or eliminated, it can be determined that phosphorylation of HNF-4a by RSKB is inhibited by the test compound.
  • Another embodiment of the present invention relates to a method for identifying a compound that inhibits the binding between RSKB and HNF-4a.
  • This identification method can be constructed using a pharmaceutical screening system known per se. Further, this identification method can be carried out using the phosphoric acid system or the phosphoric acid method according to the present invention.
  • whether or not the test compound inhibits the binding between RSKB and HNF-4a is determined by the signal generated by the binding of RSKB and HNF-4a in the presence of the test compound or the This can be carried out by comparing the marker of binding with the signal generated by the binding in the absence of the test compound or the marker of binding.
  • the test compound is It can be determined that the binding between RSKB and HNF-4a is inhibited.
  • the conditions enabling the binding of RSKB and HNF-4a may be any of in vitro and in vivo conditions.
  • cells in which RSKB and HNF-4 ⁇ are co-expressed can be used.
  • the contact between RSKB and ⁇ or HNF-4a and the test compound may be performed before the binding reaction of RSKB and HNF-4a, or may be carried out by coexisting in the binding reaction.
  • the binding between RSKB and HNF-4 ⁇ can be performed using a protein detection method known per se.
  • the binding between RSKB and HNF-4 ⁇ can be detected by detecting a complex containing RSKB and HNF-4a by a known protein detection method.
  • the protein detection method include Westamplot method, immunoprecipitation method, pull-down method, two-hybrid method, and fluorescence resonance energy transfer method. These methods can be used alone, or a combination of these methods can be used to detect the desired protein.
  • a complex containing RSKB and HNF-4a can be easily detected.
  • Preferred examples of the labeling substance include tag peptides such as HA-tag and FLAG-tag.
  • test compound examples include compounds derived from chemical libraries and natural products, or compounds obtained by drug design based on the primary structure and three-dimensional structure of RSKB and HNF-4a.
  • test compound examples include compounds derived from chemical libraries and natural products, or compounds obtained by drug design based on the primary structure and three-dimensional structure of RSKB and HNF-4a.
  • compounds obtained by drug design based on the primary structure and three-dimensional structure of RSKB and HNF-4a Alternatively, based on the peptide structure of the binding site of HNF-4 ⁇ and RSKB and the phosphorylation site of HNF-4a by Z or RSKB, The compound obtained by in-situ is also suitable as the test compound.
  • the compound obtained by the identification method according to the present invention can be used as a phosphate inhibitor of HNF-4a by RSKB or a gene product production inhibitor of a gene on which HNF-4a acts.
  • the inhibitor of phosphorylation of HNF-4a by RSKB or the gene product production inhibitor of the gene that HNF-4a acts on is selected by considering the balance between biological usefulness and toxicity. It can be prepared as a composition. In the preparation of the pharmaceutical composition, these inhibitors can be used alone or in combination.
  • Yet another embodiment of the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of the phosphate inhibitor according to the present invention as an active ingredient.
  • the pharmaceutical composition according to the present invention can be used as a preventive and Z or therapeutic agent for diseases caused by phosphate knot of HNF-4a by RSKB. It can also be used for prevention and Z or treatment of the disease.
  • another embodiment of the present invention relates to a preventive and Z or therapeutic agent for a disease caused by phosphorylation of HNF-4a by RSKB, and a preventive and Z or therapeutic method for the disease.
  • the preventive and Z or therapeutic agent for the disease contains the phosphorylation inhibitor.
  • the prevention and Z or treatment method of the disease can be achieved by using the phosphorylation inhibitor or the phosphorylation inhibition method.
  • Examples of diseases caused by phosphorylation of HNF-4a by RSKB include diseases caused by an increase in the gene product of a gene on which HNF-4a acts.
  • HNF-4a phosphorylated by RSKB promotes binding to the transcription site upstream of the PEPCK gene and contributes to the gene product production of the gene.
  • the disease caused by the phosphorylation of HNF-4a by RSKB is caused by an increase in the gene product of a gene involved in gluconeogenesis, for example, an increase in the gene product of the PEPCK gene.
  • diseases caused by abnormal gluconeogenesis such as diabetes.
  • the pharmaceutical composition according to the present invention is usually preferably prepared as a pharmaceutical composition containing one or more pharmaceutical carriers in addition to the active ingredient.
  • the amount of the active ingredient contained in the pharmaceutical preparation according to the present invention is appropriately selected from a wide range. . Usually, it is appropriate that the amount is in the range of about 0.0001 to 70% by weight, preferably about 0.0001 to 5% by weight.
  • Examples of pharmaceutical carriers include fillers, fillers, binders, moisturizers, disintegrants, lubricants, diluents and excipients that are generally used depending on the form of use of the preparation. it can. These are appropriately selected and used depending on the dosage form of the preparation to be obtained.
  • water pharmaceutically acceptable organic solvent
  • collagen polyvinyl alcohol, polybutylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin Xanthan gum, gum arabic gum, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, ratatose and the like.
  • These may be used alone or in combination of two or more according to the dosage form of the pharmaceutical composition.
  • the stabilizer examples include human serum albumin, ordinary L amino acids, sugars, and cellulose derivatives. These can be used alone or in combination with a surfactant or the like. In particular, according to this combination, the stability of the active ingredient may be further improved.
  • the L-amino acid is not particularly limited, and may be any of glycine, cysteine, glutamic acid and the like.
  • Sugars are not particularly limited, for example, monosaccharides such as glucose, mannose, galactose, and fructose, sugar alcohols such as mannitol, inositol, and xylitol, disaccharides such as sucrose, maltose, and lactose, dextran, hydroxypropyl starch, chondroitin Any deviation from polysaccharides such as sulfuric acid and hyaluronic acid, and their derivatives.
  • Cellulose derivatives are not particularly limited, such as methylcellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropenoresenorelose, hydroxypropinoremethenoresenorelose, sodium canoleboximethylcellulose, etc. ,.
  • surfactant there are no particular limitations on the surfactant, and both an ionic surfactant and a nonionic surfactant can be used.
  • the surfactant include polyoxyethylene glycol solvent. Vitan alkyl ester, polyoxyethylene alkyl ether, sorbitan monoacyl ester, fatty acid glyceride and the like are included.
  • Buffering agents include boric acid, phosphoric acid, acetic acid, citrate, ⁇ -aminocaproic acid, glutamic acid and sputum or their corresponding salts (for example, sodium salts, potassium salts, calcium salts, magnesium salts thereof) Examples thereof include alkali metal salts and alkaline earth metal salts.
  • Examples of the isotonic agent include sodium chloride sodium, potassium salt potassium, sugars, and glycerin.
  • Examples of the chelating agent include sodium edetate and citrate.
  • the pharmaceutical and pharmaceutical composition according to the present invention can be used as a solution preparation, and after freezing, drying and storing it, it contains water, physiological saline and the like at the time of use. It can be used after being dissolved in a buffer solution or the like to prepare an appropriate concentration.
  • the dose range of the medicine and the pharmaceutical composition is not particularly limited, and the effectiveness of the contained ingredients, the administration form, the administration route, the type of the disease, the nature of the subject (weight, age, medical condition and use of other medicines Or the like) and the judgment of the doctor in charge.
  • a suitable dose is, for example, in the range of about 0.01 ⁇ g to 100 mg, preferably about 0.1: g to lmg, per kg of body weight of the subject.
  • these dosage changes can be made using general routine experimentation for optimization well known in the art.
  • the above dose can be administered once to several times a day, and may be administered intermittently at a rate of once every several days or weeks.
  • the pharmaceutical composition When administering the pharmaceutical composition according to the present invention, the pharmaceutical composition may be used alone or in combination with other compounds or medicines necessary for the prevention and Z or treatment of the target disease. May be. For example, you may mix
  • the administration route can be selected from systemic administration or local administration! In this case, an appropriate administration route is selected according to the disease, symptoms and the like.
  • parenteral routes include normal intravenous administration and intraarterial administration, as well as subcutaneous, intradermal and intramuscular administration. Or it can be administered by the oral route.
  • transmucosal administration or transdermal administration can be performed.
  • Various administration forms can be selected according to the purpose. Typical examples are solid dosage forms such as tablets, pills, powders, powders, fine granules, granules, capsules, and aqueous solutions. Solutions, ethanol solution preparations, suspensions, fat emulsions, ribosome preparations, inclusions such as cyclodextrins, and liquid dosage forms such as syrups and elixirs. Depending on the route of administration, these preparations can also be administered orally, parenterally (infusions, injections), nasal preparations, inhalants, vaginal preparations, suppositories, sublingual, eye drops, ear drops, ointments, creams. And can be prepared, molded and prepared according to ordinary methods.
  • Powders, pills, capsules, and tablets are excipients such as ratatoses, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc.
  • it can be produced using a binder such as polybutyl alcohol, hydroxypropyl cellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin.
  • solid pharmaceutical carriers are used for the production of tablets and capsules.
  • Suspending agents can be produced using water, sugars such as sucrose, sorbitol and fructose, dallicols such as polyethylene glycol, and oils.
  • Solutions for injection can be prepared using a carrier that also has a salt solution, a glucose solution, or a mixture of saline and glucose solution.
  • Ribosomeization is performed by, for example, adding a solution in which the above active ingredient is dissolved in a solvent (such as ethanol) to a solution in which phospholipid is dissolved in an organic solvent (such as chloroform), and then distilling off the solvent. This can be carried out by adding a phosphate buffer, shaking, sonicating and centrifuging, and then collecting the supernatant by filtration.
  • a solvent such as ethanol
  • an organic solvent such as chloroform
  • Fat emulsification is carried out, for example, by mixing and heating the above active ingredients, oil components (vegetable oils such as soybean oil, sesame oil and olive oil, MCT, etc.), and emulsifiers (phospholipids etc.) It can be carried out by adding the required amount of water and emulsifying and homogenizing using an emulsifier (homogenizer, such as a high-pressure jet type or ultrasonic type). It can also be lyophilized.
  • emulsification aids that may be added with emulsification aids when making fat emulsions include dariserine and saccharides (eg, glucose, sorbitol, fructose, etc.).
  • cyclodextrin clathrate is a precipitate formed by adding a solution in which cyclodextrin is heated and dissolved in water to a solution in which the above active ingredient is dissolved in a solvent (ethanol or the like), and then cooling to precipitate. Can be carried out by filtering and sterilizing and drying.
  • cyclodex used For the string cyclodextrins ( ⁇ , 13, ⁇ type) having different pore diameters may be appropriately selected according to the size of the active ingredient.
  • reagent kit comprising a polynucleotide encoding a and at least one of a vector containing the polynucleotide.
  • the reagent kit can be used, for example, in the identification method according to the present invention.
  • RSKB or HNF-4a-derived partial peptide for example, a peptide having an amino acid sequence ability at a site where RSKB and HNF-4a interact, or a reagent having a combined force obtained by the above identification method , And reagent kits containing these are also included within the scope of the present invention.
  • This reagent kit measures signals and Z or markers for detecting phosphorylation of HNF-4a by RSKB, their detection agents, reaction diluents, buffers, detergents, and reaction stop solutions. It may contain substances required for the implementation of In addition, the reagent kit can include stabilizers and substances such as Z or preservatives. For formulation, it is sufficient to introduce formulation means for each substance to be used. These reagents and reagent kits are useful, for example, for molecular studies on the involvement of HNF-4a and RSKB in diseases caused by HKB-4 ⁇ phosphate caused by RSKB, such as diabetes.
  • Non-patent Document 34 ((p- Bromocmnamyl) ammo no etnyl] — 5— isoquino lines ulfonamide) (Non-patent Document 34) Judge the degree. The blood glucose concentration is reduced to about half as described above.
  • RSKB activity can be measured by preparing an anti-activated RSKB antibody and performing immunoprecipitation or immunoprecipitation with an anti-RSKB antibody and measuring in vitro kinase activity.
  • Prediction of the protein that interacts with RSKB was carried out as follows according to the in silico prediction method described in Patent Document 1: (i) The amino acid sequence of RSKB was decomposed into oligopeptides of a certain length, and (ii ) Search the database for the amino acid sequence of each oligopeptide or a protein having an amino acid sequence homologous to the amino acid sequence, (iii) perform local alignment between the obtained protein and RSKB, and (iv ) We predicted that proteins with high local alignment scores interacted with R SKB.
  • the local alignment score was set to 25.0 or higher, as in the method described in International Publication No. W001Z67299.
  • HNF is predicted to have a function of interacting with RSKB.
  • Human HNF-4a was phosphorylated by RSKB was examined in an in vitro phosphorylation test.
  • Human HNF-4 ⁇ and RSKB were each transiently expressed in cultured mammalian cells and then prepared from the cells. The activity of RSKB was confirmed using CREB1, and PKA known to phosphorylate HNF-4 was used as a positive control for phosphorylase. [0169] ⁇ Material>
  • RSKB expression plasmid (RSKB / pCMV-Tag2) was constructed as shown below.
  • Human RSKB cDNA was obtained by PCR from cDNA derived from HeLa cells (Human HeLa Quick-clone cDNA, manufactured by Clontech), and the sequence was confirmed by sequencing. Thereafter, the plasmid was incorporated into an animal cell expression plasmid pCMV-Tag2 (Stratagene), to which a FLAG tag was added at the N-terminus, to construct an RSKB expression plasmid (R SKB / pCMV-Tag2).
  • the amino acid sequence encoded by the cloned RSKB cDNA was identical to that disclosed in the NCBI protein database as accession number NP-003933 (protein name is RPS6KA4).
  • HNF-4a ZpcDNA3.1ZHis A human HNF-4 ⁇ expression plasmid (HNF-4a ZpcDNA3.1ZHis) was constructed as shown below.
  • Human HNF-4a cDNA was obtained from human brain polyA + RNA by RT-PCR.
  • an expression plasmid for animal cells to which a (6 X His) —Xpress tag is added at the N-terminus, incorporated into pcDNA3.1 / His (Invitrogen) HNF-4 ⁇ expression plasmid (HNF-4 a ZpcDNA 3.lZHis) was constructed.
  • the amino acid sequence encoded by the cloned HNF-4a cDNA was identical to that disclosed in the Swiss-Prot database as accession number P41235 (protein name HNF4A).
  • a human CREB1 expression plasmid (CREBlZpcDNA3.lZHis) was constructed as shown below.
  • Human CREB1 cDNA was obtained by PCR from cDNA derived from HeLa cells (Human HeLa Quick-clone cDNA, Clontech), and the sequence was confirmed by sequencing. Then, add (6 X His) -Xpress tag to the N-terminal to the expression plasmid for animal cells pcDNA3. L / His (Invitrogen) to construct a CREB1 expression plasmid (CR EBl / pcDNA3. L / His) did.
  • the amino acid sequence encoded by the cloned CREB1 cDNA was identical to that disclosed in the NCBI protein database as accession number NP-004370 (protein name CREB1).
  • RSKBZpCMV— Tag2 A 10 gZ Petri dish RSKB expression plasmid (RSKBZpCMV— Tag2) The cells were tranfected using 6 (Roche Diagnostics). After culturing for 2 days, sodium arsenite was added to the medium to a final concentration of 50 mM and treated at 37 ° C. for 30 minutes to recover the cells.
  • buffer A 50 mM Tris-HCl (pH 7.5) / lmM esteramine tetraacetic acid (EDTA) / lmM Ethylene glycol bis (2-aminoethyl ether) —N, N, ⁇ ', ,, tetraacetic acid (EGTA) ⁇ 10% glycerol ⁇ 500 ⁇ dithiothreitol (DTT) / 5 mM NaPPi / lmM Na VO / 50 mM NaF / 1% Triton—XI 00) 4ml
  • FLAG M2 affinity gel (manufactured by Sigma) was added in an amount of 100 1 and mixed by inversion at 4 ° C for 1 hour. After mixing, the gel is washed twice with 10 times the volume of buffer B, and 10 times the volume of buffer C (50 mM Tris—HCl (pH 7.5) / 0. ImM EGTA / 10% Glycerol ⁇ . 5 M DTT / 150 mM NaCl ) Washed once. The gel was then washed with 400 / zl of buffer D (50 mM Tris—HCl (pH 7.5) / 0.
  • cAMP-dependent protein kinase As the cAMP-dependent protein kinase (PKA), Ushi PKA (cAMP-dependent protein kinase, catalytic subunit, manufactured by Promega) was used.
  • the immunoprecipitation phosphate test was performed as shown below.
  • HEK293T cells with 7 x 10 5 cells were sputum cultured at 37 ° C in the presence of 5% CO (60 mm diameter petri dish), 5 g HNF-4a expression plasmid (HNF-4a / pcDNA3.l / His) or CREB1 expression plasmid (CREBlZpcDNA3.l / His) was transfected into cells using FuGENE6 (Roche's Diagnostatus) . After culturing for 2 days, the cells were washed with chilled PBS (—) and RIPA buffer (50 mM Tris—HCl (pH 8.0) / 150 mM).
  • adenosine triphosphate 0.5 1 ⁇ 32 ⁇ — ⁇ (5 Ci)
  • 8.5 1 distilled water totaled 29 1 and reacted at 30 ° C for 30 minutes I let you.
  • 10 HNF-4a / protein G-sepharose prepared as above, was added to 1.5 1 ⁇ , 3 1 10X kinase buffer, 2 1 ⁇ ⁇ ATP, 0. ⁇ 32 ⁇ —ATP (5 / zCi), 3.5 / zl IX kinase buffer, 8.5 1 distilled water totaled 29 ⁇ 1 and incubated at 30 ° C for 30 minutes .
  • EMSA The effect of phosphate on the ability of HNF-4a to bind to the AF1 sequence in the PEPCK gene promoter region was examined by EMSA. Phosphorylation of HNF-4a was performed using RSKB. In addition, in order to investigate the specificity of RSKB, EMSA was similarly performed using PKA, which is known to phosphorylate HNF-4a.
  • the AF1 probe used was the following two probes designed and synthesized by the AF1 equivalent sequence force, which is the HNF-4a binding site in the promoter region of the human PEPCK gene (Sigma Genosis Japan). ): AF1 / S / HNF4 (5′-GTG ACCTTTG ACTA-3 ′) (SEQ ID NO: 7) and AF1ZASZHNF4 (5′—AT AGTCAAAGGTCA-3 ′) (SEQ ID NO: 8) (FIG. 4).
  • the promoter region (SEQ ID NO: 15) of the human PEPCK gene (NCBI accession number U31519) is shown in FIG.
  • the AF1 equivalent sequence is shown in FIG. 4 by surrounding it with a bold frame.
  • HNF-4 ⁇ expression plasmid (HNF-4a ZpCMV-Tag2) was constructed from the HNF-4 ⁇ expression plasmid (HNF-4a ZpcDNA 3. lZHis) constructed in Example 1 at the Eco RI site.
  • the cDNA sequence of HNF-4a was recombined into an expression plasmid for animal cells (pCMV-Tag2, manufactured by Stratagene) with an N-terminal FLAG tag.
  • HNF-4a expression of HNF-4a in cells and preparation and purification of HNF-4a from the cells were performed as follows. Cell count 1.2 ⁇ 10 6 HEK293T cells at 37 ° C, 5% CO
  • the F-4a expression plasmid (HNF-4a ZpCMV-Tag2) was transfected into cells using FuGENE6 (Roche Diagnostics).
  • the cells were washed with chilled PBS (-), and 4 ml of buffer A (50 mM Tris-HCl (pH 7.5) / ImM EDTA / lmM EGTA / 10% glycerol Z500 M DTT / 5 mM NaPPi / lmM Na3VO4 / 50 mM NaF / 1% Triton—XI 00) was added and pipetted well, and the mixture was stirred at 4 ° C. for 30 minutes. Thereafter, NaCl was added to a final concentration of 150 mM, and the mixture was centrifuged at 10,000 g for 10 minutes at 4 ° C.
  • buffer A 50 mM Tris-HCl (pH 7.5) / ImM EDTA / lmM EGTA / 10% glycerol Z500 M DTT / 5 mM NaPPi / lmM Na3VO4 / 50 mM NaF / 1% Triton—X
  • Radiolabeling and purification of the AF1 probe was performed as follows.
  • the synthesized A F1 probes were dissolved in distilled water so that each of them was lOOpmolZ ⁇ 1, heated at 100 ° C for 2 minutes and at 38 ° C for 1 hour, and then naturally cooled to anneal the DNA. Dilute this 10-fold to obtain 0.5 ⁇ l, 1 ⁇ l of ⁇ 4 polynucleotide kinase (manufactured by Koeisha), 6 1 ⁇ ” 2 P—ATP (60 Ci), 2 1 10 X Protruding End Kinase Buffer (500mM Tris—HCl (pH8.0) / 100mM MgCl / lOOmM 2—Mercaptoethanol
  • Protruding End means ⁇ protruding end '' and Protruding End Kinase Buffer is a T4 polynucleotide. This buffer is used when the photokinase adds a phosphate group at the ⁇ position of ⁇ -ATP to the ⁇ -end of the protruding end of double-stranded DNA.
  • HNF-4 ⁇ ′ DNA complex a band indicating a complex of HNF-4a and AF1 probe (hereinafter referred to as HNF-4 ⁇ ′ DNA complex) was detected.
  • the addition of the anti-HNF-4 ⁇ antibody showed a supershift in which the mobility of the HNF-4 ⁇ 'DNA complex was reduced. From this result, the band detected in the non-anti-HNF-4 ⁇ antibody-supplemented band was HNF-4 It was confirmed that the a-DNA complex was shown.
  • the amount of HNF-4a ′ DNA complex was increased by the phosphorylation treatment with RSKB. On the other hand, phosphorylation treatment with sputum did not detect an increase in the amount of HNF-4 ⁇ .DNA complex.
  • the amount of HNF-4a 'DNA complex decreased as the amount of RSKB used for the phosphorylation of HNF-4a decreased. From this result, it can be considered that the binding ability of HNF-4 ⁇ to the AF1 sequence decreased as the amount of RSKB used for the phosphorylation treatment of HNF-4a decreased.
  • phosphoric acid treatment with PKA increases the amount of HNF-4a 'DN A complex! That is, no enhancement of the binding ability of HNF-4 ⁇ to the AF1 sequence was observed.
  • HNF-4a phosphorylated with RSKB was found to have enhanced DNA binding ability to the AF1 sequence compared to the untreated sample. It was also found that the enhancement of the DNA binding ability of HNF-4a phosphorylated with RSKB was dependent on the dose of RSKB used for phosphorylation. On the other hand, HNF-4 Q; that had been phosphated with PKA showed only the same DNA binding ability as the untreated sample. This indicates that the DNA-binding ability of HNF-4a by RSKB treatment is specific.
  • the effect of RSKB on the transcriptional activity of HNF-4a in the PEPCK gene promoter was determined using HeLa cells (human cervical cancer-derived cell line) and HepG2 cells (human liver cancer-derived cell line) using luciferase reporter assembly. investigated.
  • a reporter gene a gene (PE PCK AF1 promoter-dependent luciferase reporter) in which a firefly luciferase gene is fused downstream of a region containing AF 1 which is a cis element of HNF-4a in the human PEPCK gene promoter region was used. .
  • RSKB expression plasmid used was RSKB / pCMV-Tag2 prepared in Example 2 [0189] Dominant negative RSKB (S343A) expression plasmid (RSKB (S343A) / pCMV- Tag2) is RSKB / pCMV- Tag2 as a saddle, using QuikChange Site-Directed Mutagenesis kit (Stratagene) RSKB The amino acid substitution at position 343 was performed. RSKB requires phosphorylation of 196th serine, 343th serine and 568th threonine for its kinase activity, and it is reported that the kinase activity disappears when one of these amino acids is substituted with alanine.
  • the protein RSKB (S343 A) expressed by this expression plasmid is an inactive mutant RSKB in which the kinase activity disappears by substitution of the 343rd serine of RSKB with alanine.
  • HNF-4a expression plasmid used was HNF-4a / pCMV-Tag2 prepared in Example 3.
  • a MAPK11 expression plasmid (MAPK11 / pCMV-Tag4) was constructed as shown below.
  • Human MAPK11 (p38-j82) cDNA was obtained from human brain whole cDNA (Human brain whole Quick-clone cDNA, Clontech) by PCR, and the sequence was confirmed by sequencing. Thereafter, the plasmid was incorporated into an expression plasmid for animal cells pCMV-Tag4 (Stratagene) to which a FLAG tag was added at the C terminus, and a MAPK11 expression plasmid (p38-beta2 / pCMV-Tag4) was constructed.
  • the amino acid sequence encoded by the cloned MAPK11 cDNA is the same as the Swiss-Prot database accession number Q 15759 (protein name is MAPK11ZSAPK2B).
  • PEPCK AF1 promoter-dependent luciferase reporter plasmid PEPCK
  • AF1 promoter / pGL3 The construction of AF1 promoter / pGL3) was performed as shown below.
  • PEPCK A AF1 promoter-dependent luciferase reporter plasmid (PEPC
  • the KA AFl promoter ZpGL3) was constructed as shown below. From the cloned human PEPCK gene promoter region, a portion not containing AF1 (base sequence 934-1406 of U31519) was obtained by PCR. After confirming the sequence by sequence
  • PGL3 Base (manufactured by Promega) and inserted.
  • pMMGR that expresses a rat glucocorticoid receptor was used (Non-patent Document 36).
  • phRL null plasmid or pRL—expressing Renilla luciferase as a reporter gene
  • SV40 plasmid (Promega) was used.
  • SB203580 was purchased from Promega.
  • Reporter assembly in HeLa cells was performed as follows. 6 X 10 4 Zwell HeLa cells at 37 ° C in the presence of 5% CO
  • the cells were washed with chilled PBS (—), and the firefly luciferase activity and the renilla luciferase activity in the cell lysate were measured with Dual-Lusif erase Reporter Assay kit (Promega).
  • the firefly luciferase activity value is divided by the Renilla luciferase activity value. After, viewed in multiples relative to controls group (PEPCK AF1 promoter ZpGL3 only).
  • Reporter assembly in HepG2 cells was performed as follows. 1 x 10 4 Zwell HepG2 cells at 37 ° C in the presence of 5% CO After sputum culture (24 well plate (2.0 cm 2 Z well), 0.5 g PEPCK AF1 promoter ZpGL3, 0.5 ⁇ g (HNF-4 a / pCMV ⁇ Tag2, 0.25 g and 0 5 ⁇ g RSKBZpCMV— Tag2, 0.5 ⁇ g MAPKl lZpCMV— Tag4, 0.5 ⁇ g pMMGR and 0.05 ⁇ g internal control pRL—SV40 with a preset combination of FuGENE6 (Roche The cells were transferred using the “Diagnostics Co., Ltd.” using the empty vector pC MV-Tag2 (Stratagene) so that the total amount of DNA was constant in each experimental group.
  • SB203580 was added to the medium so that the final concentration was 10 ⁇ immediately before transfection with FuGENE 6.
  • Dexamethasone was added to the final concentration at 1 hour 20 hours after the transfection.
  • PBS was added to the culture medium and cultured for 24 hours. After washing, the firefly luciferase activity and renilla luciferase activity in the cell lysate were measured with the Dual-Lusiferase Reporter Assay kit (Promega) .
  • the transcriptional activity was the value of the luciferase activity value. After dividing by RS, it was expressed as a multiple of the RSKB non-expression group (HNF-4a 0.5).
  • HNF-4a phosphorylated by RSKB promotes the transcriptional activity of the promoter through binding of the PEPCK gene promoter to AF1.
  • HNF-4 ⁇ from the group that co-expressed MAPK11, RSKB, and HNF-4a (HNF-4 ⁇ 0.5 + RSKB 0.5 + MAPK11 0.5)
  • Transcriptional activity against PEPCK AF1 promoter increased by 3 times or more compared to the RSKB non-expression group (HNF-4 ⁇ 0.5) (FIG. 12).
  • HNF-4 ⁇ phosphorylated by RSKB also promotes the transcriptional activity of the PEPCK gene promoter including AF1 in HepG2 cells.
  • HepG2 cells (human liver cancer-derived cell line) are transiently expressed with wild type RSKB or inactive mutant RS KB and MAPK11, an upstream kinase of RSKB, and the PEPCK gene The expression of was measured.
  • RSKB / pCMV-Tag2 prepared in Example 2 was used as the wild-type RSKB expression plasmid.
  • SKBZpCMV—Tag2 causes N-terminal FLAG tag RSKB (sometimes referred to as FLAG—RSKB) force S to be expressed.
  • the inactive mutant RSKB expression plasmid (RSKB—S196AZS343AZT568AZP CMV-Tag2) is the wild type RSKB expression plasmid RSKBZpCMV— Tag2 is used as a saddle and the QuikChange Site-Directed Mutagenesis kit (Stratagene) is used. The three amino acid residues 196, 343 and 568 were replaced with alanine. RSKB requires phosphorylation of 196th serine, 343th serine and 568th threonine for its kinase activity, and it is reported that the kinase activity disappears when one of these amino acids is substituted with alanine. (Non-patent document 34).
  • RSKB expressed by RSKB—S196A / S343A / T568A / pCMV—Tag has its kinase activity lost by substituting its 196th and 343th serine and 568th threonine with alanine.
  • This inactive mutant RSKB has a FLAG tag attached to its N-terminus.
  • MAPK11 expression plasmid As the MAPK11 expression plasmid, MAPKlZpCMV-Tag4 prepared in Example 4 was used.
  • the anti-phosphorylated RSKB antibody was prepared according to a previous report (Non-patent Document 34) by SCRAM, which produced anti-phosphorylated RSKB antibody PS360 that recognizes phosphorylation of the 360th serine of RS KB.
  • Hep G2 cells were seeded on a 6-well plate at a cell number of 5 ⁇ 10 5 Zwell and cultured in a MEM medium containing 10% FBS at 37 ° C. under conditions of 5% CO 2/95% air. -After sputum culture, wild type
  • RSKB expression plasmid (RSKBZpCMV—Tag2) or inactive mutant RSKB expression plasmid (RSKB—S196A / S343A / T568A / pCMV—Tag2) 2 ⁇ g 11 Expression plasmids (MAPKlZpCMV—Tag4, C-terminal FLAG tag) were transfected into cells using 2 ⁇ g of Lipofectamine 2000 (invitrogen). The total DNA amount was corrected by empty vector (P CMV-Tag2) such that 4 mu g in each experimental group. After transfection, the cells were cultured for 2 days.
  • PEPCK gene PCR was detected by RT-PCR. Specifically, the cells were washed with PBS, collected using trypsin ZEDTA, and total RNA was extracted from the cells using RNeasy Mini Kit (manufactured by Quiagen). CDNA was synthesized from the collected total RNA using Omniscript RT kit (manufactured by Qiagen) using random primer 9mer and oligo go dt primer. PEPCK gene PCR was performed using the obtained cDNA as a template. In addition, GAPDH gene PCR was performed as an internal control. Primer sequences and PCR cycle conditions were as previously reported (Non-patent Documents 40 and 41). Under these conditions, the PEPCK gene produces a 575 bp PCR product and the GAPDH gene produces a 209 bp PCR product.
  • each gene transfected into the cells was detected by Western blotting. Specifically, after washing the cells with PBS, the cells were collected using trypsin ZEDTA, 1501 of a cell lysis buffer (manufactured by Cell Signaling) was added, and the cells were allowed to stand for 15 minutes on ice to lyse the cells. . Thereafter, centrifugation was performed at 15, OOOrpm at 4 ° C for 10 minutes, and the supernatant was recovered and used as a cell lysate. The cell lysate was supplemented with an equal volume of 2 X SDS sample buffer and heated at 100 ° C for 5 minutes, and used as an SDS-PAGE sample.
  • a cell lysis buffer manufactured by Cell Signaling
  • Proteins were separated by SDS-PAGE using a 5 to 20% gradient gels, after transfer to Immobiron- P SQ membrane (Millipore (Millipore) Co. Ltd.) was detected for each protein.
  • FLAG—RSKB and MAPK11—FLAG were detected with an anti-FLAG M2 antibody (manufactured by Sigma).
  • Phosphorylated RSKB was detected with an anti-PS360 antibody, and / 3-tubulin was detected with an anti-tubulin antibody (-235 (manufactured by Santa Cruz). Detection was carried out using a fluorescently labeled secondary antibody using an Odyssey imaging system (Aloka).
  • This anti-phosphorylated RSKB antibody is an antibody recognizing the phosphate of the 360th serine of RSKB.
  • the RSKB kinase activity is important for the 360th serine phosphate, and the presence or absence of phosphate at this amino acid site is consistent with the kinase activity. (Non-patent Document 34). From this result, it was clarified that the inactive mutant RSKB used in this example lost its kinase activity.
  • HepG2 cells co-expressing wild-type RSKB and MAPK11, and inactive mutant RSKB (RSKB (S 196AZS343AZT5 68A)) and wild-type RSKB and inactive mutant RSKB in HepG2 cells co-expressing MAPK11
  • RSKB S 196AZS343AZT5 68A
  • MAPK11 wild-type RSKB and inactive mutant RSKB in HepG2 cells co-expressing MAPK11
  • RSKB and HNF-4a were transiently co-expressed in HeLa cells and immunorecipitated with RSKB. The binding of HNF-4a was examined.
  • RSKB / pCMV-Tag 2 prepared in Example 2 was used as the RSKB expression plasmid.
  • HNF-4a ZpCMV-Tag2 prepared in Example 3 was used.
  • RSKBZpCMV—Tag2 expresses the N-terminal FLAG tag HNF-4a (sometimes referred to as FLAG-HNF-4a).
  • B expression plasmid (RSKBZpCMV—Tag2) 0.5 ⁇ g and HNF-4 ⁇ expression plasmid ( ⁇ NF-4a / pCMV-Tag2) 0.5g or empty vector (pCMV-Tag2, Stratagene) ) 0.5 ⁇ g was transferred into cells using FuGENE6 (Roche Diagnostics) (total DNA amount was 1 ⁇ g). After transfection, the cells were cultured for 2 days.
  • the binding between RSKB and HNF-4a in the cells was detected by immunoprecipitation. Specifically, the cells were washed with cold PBS, and cell lysis buffer (20 mM HEPES (pH 7.5) / 150 mM NaCl / lmM EDTA / 1% Triton X—100Z Protea Zein Hibita Kuttel (manufactured by Sigma) )) 500 ⁇ l was added and left on ice for 15 minutes to lyse the cells. Thereafter, the lysed cells were centrifuged at 15, OOOrpm at 4 ° C for 10 minutes, and the supernatant was collected and used as a cell lysate.
  • cell lysis buffer (20 mM HEPES (pH 7.5) / 150 mM NaCl / lmM EDTA / 1% Triton X—100Z Protea Zein Hibita Kuttel (manufactured by Sigma)
  • HNF-4a was precipitated with an anti-HNF-4a antibody, and as a result, co-precipitation of RSKB was detected depending on HNF-4a (Panel A in Fig. 15). From these results, it was found that HNF-4 binds to RSKB in cells.
  • the liver was lysed under ice cooling (cell lysis buffer (manufactured by Cell Signaling) (20 mM Tris—HCl (pH 7.5), 150 mM NaCl / lmM Na EDTA / lmM EGTA / 1% Triton / 2. 5 mM pyrroline)
  • Anti-HNF-4a antibody (manufactured by Santa Cruz Co., Ltd.) was added to the prepared cell lysate and mixed by inverting for 1 hour at 4 ° C. Add protein G sepharose 4 Fast Flow (Amersham 'Falmacia' manufactured by Biotech) blocked with Saline (TBS) (50 mM Tris-HCl / 150 mM N aCl)) (pH 8.0), and add 1 at 4 ° C. Mix by inversion for an hour.
  • wash protein G sepharose twice with cell lysis buffer add 2X SDS sample buffer, heat at 100 ° C for 5 minutes, and use as SDS-PAGE sample.
  • Liver cell lysates are prepared from diabetic animals (ZDF faZfa) and normal animals (ZDF-lean) in the same manner as in Experimental Example 1 above.
  • Anti-RSKB antibody and protein G seph arose are added thereto, and RSKB is recovered by the same immunoprecipitation method as in Experimental Example 1 above.
  • the kinase activity of the recovered RSKB was measured using the synthetic substrate talebitide (CREBtide (manufactured by Santa Cruz). ;)) Detected by in vitro kinase assay using (SEQ ID NO: 9) (Non-patent Document 34).
  • RSKB-binding resin was added to CREBtide (33 ⁇ M) and kinase buffer (Kinase buffer (50 mM Tris-HCl (pH 7.5) / 0. ImM EGTA / 140 mM KCl / 5 mM NaPPi / 10 mM MgCl)). Mix with ⁇ - 32 P- ATP and bring to 22 ° C
  • H-8 9 blood glucose concentration and insulin concentration (Levis Insulin Kit, manufactured by Shibayagi) are measured.
  • the HNF-4 ⁇ phosphate and RSKB activities are measured in the same manner as in Experimental Examples 1 and 2 above, and the expression level of PEPCK is detected by Western blotting.
  • Administration of H-89 decreases both RSKB activity, phosphorylation of HNF-4 ⁇ , and expression of PE PCK, and gluconeogenesis is suppressed, resulting in a decrease in blood glucose concentration.
  • Inhibition of production of a gene product of a gene on which HNF-4a acts by the phosphorylation inhibitor of HNF-4a and / or the method of inhibiting phosphorylation by RSKB provided in the present invention, such as the PEPCK gene Inhibition of the production of the gene product can be carried out. It also makes it possible to prevent and / or treat diseases caused by increased gene products of genes that act on HNF-4 ⁇ . More specifically, for example, diseases caused by an increase in PEPCK gene products, more specifically, diabetes and the like can be prevented and Z or treated. Thus, the present invention is very useful for the prevention and Z or treatment of diseases caused by excessive HNF-4a phosphate involved in gluconeogenesis-related gene expression.
  • SEQ ID NO: 1 RSKB cDNA.
  • Sequence number 2 RSKB.
  • SEQ ID NO: 3 HNF-4 ⁇ cDNA.
  • SEQ ID NO: 4 HNF—4 ⁇ .
  • SEQ ID NO: 5 CREB 1 cDNA 0
  • SEQ ID NO: 6 CREB1.
  • SEQ ID NO: 7 Probe oligonucleotide designed based on the sequence of AF1.
  • SEQ ID NO: 8 Probe oligonucleotide designed based on the sequence of AF1.
  • SEQ ID NO: 9 Synthetic substrate CREBtide.
  • SEQ ID NO: 10 Partial RSKB oligopeptide showing high score in local alignment between RSKB and HNF-4 ⁇ .
  • SEQ ID NO: 11 A partial oligopeptide of HNF-4a which showed a score of RSKB and HNF-4 ⁇ for local alignment!
  • SEQ ID NO: 12 Partial oligopeptide of HNF-4a showing a score of RSKB and HNF-4 ⁇ for local alignment! /, High!
  • SEQ ID NO: 13 RSKB partial oligopeptide showing a score of RSKB and HNF-4 ⁇ for local alignment! /, High!
  • SEQ ID NO: 14 Partial oligopeptide of HNF-4a which showed a score of RSKB and HNF-4 ⁇ for local alignment! /, High!
  • SEQ ID NO: 15 Promoter region of the PEPCK gene, which corresponds to the sequence represented by nucleotides 841 to 1440 of the gene published as accession number U31519 in the NCBI nucleotide database .

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Abstract

[PROBLEMS] To provide a method of inhibiting the phosphorylation of a transcriptional factor for a gluconeogenesis-associated gene and a phosphorylation inhibitor. [MEANS FOR SOLVING PROBLEMS] By finding out that a transcriptional factor HNF-4α is phosphorylated by RSKB and the binding thereof to the promoter region of a gluconeogenesis-associated gene is promoted thereby, it is intended to provide: a method of phosphorylating HNF-4α by RSKB, a method of inhibiting the phosphorylation and a phosphorylation inhibitor; a method of producing a gene product of a gene on which HNF-4α acts as a transcriptional factor, a production inhibitor and a method of inhibiting the production; a drug for preventing and treating a disease caused by the phosphorylation of HNF-4α by RSKB and a method for preventing and treating the same; a method of identifying a compound inhibiting the interaction between RSKB and HNF-4α, the phosphorylation of HNF-4α by RSKB or the binding of RSKB to HNF-4α; a compound obtained by the identification method; a polynucleotide encoding RSKB, HNF-4α or RSKB or HNF-4α; and a reagent kit containing a vector having this polynucleotide.

Description

明 細 書  Specification
糖新生関連遺伝子の転写因子のリン酸ィヒ阻害方法およびリン酸化阻害 剤  Method for inhibiting phosphorylation of transcription factor of gluconeogenesis-related gene and phosphorylation inhibitor
技術分野  Technical field
[0001] 本発明は、へパトサイトヌクレア一ファクター 4 a (hepatocyte nuclear factor 4 a、以下 HNF— 4 aと略称する）のリン酸化方法、リン酸化剤、リン酸化阻害方法 およびリン酸ィ匕阻害剤に関する。より具体的にはリボソーム S6キナーゼ B (ribosoma 1 S6 kinase B、以下 RSKBと略称する）を用いることを特徴とする HNF— 4 αのリ ン酸化方法およびリン酸化剤に関する。また、 RSKBによる HNF— 4 Q;のリン酸ィ匕阻 害方法およびリン酸化阻害剤に関する。さらに、 HNF— 4 αが転写因子として作用 する遺伝子の遺伝子産物の産生促進方法に関する。また、 HNF— 4 aが転写因子 として作用する遺伝子の遺伝子産物の産生阻害方法および産生阻害剤に関する。 さらに、 HNF— 4ひのリン酸ィ匕に起因する疾患、例えば糖尿病の防止および Zまた は治療方法並びに防止および Zまたは治療剤に関する。また、 RSKBと HNF— 4 α の相互作用、 RSKBと HNF— 4 aの結合、または HNF— 4 aの RSKBによるリン酸 化を阻害する化合物の同定方法および該同定方法により同定された化合物に関す る。さらに、 RSKB、 HNF -4 aをコードするポリヌクレオチド、該ポリヌクレオチドを含 有するベクターを含んでなる試薬キットに関する。  [0001] The present invention relates to a method for phosphorylating hepatocyte nuclear factor 4a (hereinafter referred to as HNF-4a), a phosphorylating agent, a method for inhibiting phosphorylation, and phosphoric acid Relates to inhibitors. More specifically, the present invention relates to a method for phosphorylating HNF-4α and a phosphorylating agent characterized by using ribosome 1 S6 kinase B (abbreviated as RSKB hereinafter). The present invention also relates to a method for inhibiting HNF-4 Q; Furthermore, the present invention relates to a method for promoting the production of a gene product of a gene in which HNF-4α acts as a transcription factor. The present invention also relates to a method for inhibiting the production of a gene product of a gene for which HNF-4a acts as a transcription factor and a production inhibitor. Further, the present invention relates to prevention and Z or treatment methods and prevention and Z or treatment agents for diseases caused by phosphate lees from HNF-4. Further, the present invention relates to a method for identifying a compound that inhibits the interaction between RSKB and HNF-4α, the binding of RSKB and HNF-4a, or phosphorylation of HNF-4a by RSKB, and the compound identified by the identification method. The Furthermore, the present invention relates to a polynucleotide encoding RSKB, HNF-4a, and a reagent kit comprising a vector containing the polynucleotide.
背景技術  Background art
[0002] 肝臓はグルコースの恒常性維持にとって重要な臓器であり、グルコースを産生する 糖新生（gluconeogenesis)とグルコースからグリコーゲンを生成するグリコーゲン合 成（glycogenesis)によって生体内のグルコース量のバランスを保っている。糖尿病 患者では、肝臓での過剰なグルコース産生が起こっており、それが高血糖症の原因 の一つとされて ヽる（非特許文献 1)。  [0002] The liver is an important organ for maintaining glucose homeostasis, and maintains the balance of glucose in the living body by gluconeogenesis that produces glucose and glycogen that produces glycogen from glucose. Yes. In diabetic patients, excessive glucose production occurs in the liver, which is considered to be one of the causes of hyperglycemia (Non-patent Document 1).
[0003] 糖新生はピルビン酸力もグルコースを合成する経路であり、そのほとんどが肝臓で 行われる。糖新生を進める一連の酵素群の中で、ホスホェノールピルビン酸カルボキ シキナーゼ (phosphoenol pyruvate carboxy kinase、以下 PEPCKと略称す る）は糖新生の律速酵素と考えられている（非特許文献 2〜7)。肝臓中の PEPCKの 活性は PEPCK遺伝子からの転写量により調節されて 、る。この転写はホルモンによ つて調節されており、ダルココルチコイドは PEPCK遺伝子の転写を促進し (非特許 文献 8)、インスリンは PEPCK遺伝子の転写を抑制する（非特許文献 4、 6、 8)。さら に PEPCK遺伝子の mRNAの半減期は 40分と短ぐその転写量は数時間単位で劇 的に増減する（非特許文献 3)。このように、 PEPCK遺伝子からの転写量により、 PE PCKの酵素活性、糖新生、さらには血糖値が調節される（非特許文献 3)。糖尿病病 態時の肝臓では、ダルココルチコイドにより PEPCK遺伝子の発現が増加しており、 その結果、糖新生が亢進している（非特許文献 8および 9)。 [0003] Glucogenesis is a pathway in which pyruvate also synthesizes glucose, most of which is performed in the liver. Among a series of enzymes that promote gluconeogenesis, phosphoenol pyruvate carboxy kinase (hereinafter abbreviated as PEPCK). Is considered to be the rate-limiting enzyme for gluconeogenesis (Non-Patent Documents 2 to 7). The activity of PEPCK in the liver is regulated by the amount of transcription from the PEPCK gene. This transcription is regulated by hormones, darcocorticoids promote the transcription of the PEPCK gene (Non-patent Document 8), and insulin represses the transcription of the PEPCK gene (Non-patent Documents 4, 6, and 8). In addition, the half-life of PEPCK gene mRNA is as short as 40 minutes, and the amount of transcription dramatically increases or decreases in units of several hours (Non-patent Document 3). Thus, PE PCK enzyme activity, gluconeogenesis, and blood glucose level are regulated by the amount of transcription from the PEPCK gene (Non-patent Document 3). In the liver in the diabetic state, PEPCK gene expression is increased by darcocorticoids, and as a result, gluconeogenesis is enhanced (Non-patent Documents 8 and 9).
[0004] 糖尿病は 1型糖尿病 (インスリン依存性糖尿病、 IDDM)と 2型糖尿病 (インスリン非 依存性糖尿病、 NIDDM)に分類される。 2型糖尿病はさらに 2種類に大別される。そ の一方はインスリン分泌低下が主体の糖尿病であり、他方はインスリンは分泌されて いるが標的細胞でのグルコースに対するインスリン感受性の低下が主体の糖尿病で ある。後者は特にインスリン抵抗性と言われている。インスリンの標的組織は肝臓、脂 肪、筋肉であり、肝臓における糖新生と糖放出の抑制により血糖値が下がる。高血糖 の持続により肝臓のインスリン感受性が低下し、それによりインスリン依存性の糖新生 抑制作用は期待できなくなる。しカゝしながら、高血糖持続による肝臓のインスリン感受 性低下メカニズムについては、インスリンレセプターのダウンレギュレーション等が想 定されている力 未だ明らかにされていない。  [0004] Diabetes is classified into type 1 diabetes (insulin-dependent diabetes mellitus, IDDM) and type 2 diabetes (non-insulin-dependent diabetes mellitus, NIDDM). Type 2 diabetes is further divided into two types. One is diabetes mainly due to a decrease in insulin secretion, and the other is diabetes where insulin is secreted but the insulin sensitivity to glucose in target cells is mainly reduced. The latter is particularly said to be insulin resistant. The target tissues of insulin are the liver, fat, and muscle, and blood glucose levels are lowered by suppressing gluconeogenesis and sugar release in the liver. Sustained hyperglycemia reduces the sensitivity of the liver to insulin, thereby making it impossible to expect an insulin-dependent inhibitory effect on gluconeogenesis. However, the mechanism that lowers insulin sensitivity in the liver due to persistent hyperglycemia has not been clarified yet, such as the potential for insulin receptor down-regulation.
[0005] HNF-4 aは核内レセプターの一つであり、肝臓、脾臓の β細胞、腎臓および小 腸で発現している。 HNF— 4 αは転写因子として作用することが知られており、コレ ステロール、脂肪酸およびグルコース等の代謝や肝臓の発生および分化に関与する 蛋白質をコードする種々の遺伝子の発現に関与している。糖新生関連では、 HNF —4 aは PEPCK遺伝子のプロモーターに存在する AF1部位に結合し、 PEPCK遺 伝子の発現に関与している（非特許文献 16)。転写因子はリン酸ィ匕により DNA結合 能が変化することが分かっている（非特許文献 17)。 HNF— 4 αは、 PKA (cAMP依 存性プロテインキナーゼ、 cAMP— dependent protein kinase)によりリン酸化さ れ、その結果、 L型ピルビン酸キナーゼのプロモーターに対する結合能が低下するこ とが報告されて 1、る (非特許文献 10)。 [0005] HNF-4a is one of the nuclear receptors and is expressed in β cells, kidney and small intestine of liver and spleen. HNF-4α is known to act as a transcription factor, and is involved in the expression of various genes encoding proteins involved in metabolism and liver development and differentiation, such as cholesterol, fatty acids and glucose. In gluconeogenesis, HNF-4a binds to the AF1 site present in the promoter of the PEPCK gene and is involved in the expression of the PEPCK gene (Non-patent Document 16). Transcription factors have been shown to change their DNA-binding ability by phosphorylation (Non-patent Document 17). HNF-4α is phosphorylated by PKA (cAMP-dependent protein kinase), and as a result, the ability of L-type pyruvate kinase to bind to the promoter decreases. 1 is reported (Non-patent Document 10).
[0006] RSKBは、ストレス応答型 MAPK (マイト一ジェン活性化キナーゼ、 mytogen act ivated kinase)である p38キナーゼファミリ一により活性ィ匕されるキナーゼで、核に 分布する（非特許文献 11)。 P38キナーゼは色々な病態において活性化される（非 特許文献 12)。例えば、糖尿病病態下、持続的に高血糖に曝されている細胞で、活 性酸素による酸化ストレス力も P38キナーゼが活性ィ匕される（非特許文献 13および 1 4)。具体的には、高血糖下（16. 5mM)で培養された血管平滑筋細胞 (非特許文献 15)や糖尿病モデル動物の obZobマウスの肝臓で p38キナーゼの活性ィ匕が報告さ れている（非特許文献 29)。  [0006] RSKB is a kinase activated by the p38 kinase family, which is a stress-responsive MAPK (mitogen activated kinase), and is distributed in the nucleus (Non-patent Document 11). P38 kinase is activated in various pathological conditions (Non-patent Document 12). For example, P38 kinase is also activated by oxidative stress caused by active oxygen in cells that are continuously exposed to hyperglycemia under diabetic conditions (Non-patent Documents 13 and 14). Specifically, p38 kinase activity has been reported in vascular smooth muscle cells cultured under hyperglycemia (16.5 mM) (Non-patent Document 15) and in obZob mouse livers of diabetes model animals ( Non-patent document 29).
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非特許文献 41：「： Biol. Pharm. Bull.」2005年，第 28卷，第 4号， p. 565— 568。 発明の開示  Non-Patent Document 41: “: Biol. Pharm. Bull.” 2005, Vol. 28, No. 4, p. 565-568. Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0008] 本発明の課題は、糖新生律速酵素であると考えられる PEPCKをコードする遺伝子 の転写に関与する HNF— 4 aをリン酸ィ匕することにより、 PEPCK遺伝子プロモータ 一領域へのその結合能を促進させる物質を見出し、 PEPCK遺伝子の転写量増加 により引起こされる疾患、例えば、糖尿病の防止および Zまたは治療手段を提供す ることである。 [0008] An object of the present invention is to phosphorylate HNF-4a involved in transcription of a gene encoding PEPCK, which is considered to be a gluconeogenic rate-determining enzyme, and to bind the PEPCK gene promoter to a region. To provide a means to prevent and / or treat diseases caused by increased transcription of the PEPCK gene, such as diabetes.
課題を解決するための手段  Means for solving the problem
[0009] 本発明者らは鋭意努力し、 HNF-4 aと RSKBが相互作用することをインシリコ（in silico)で予測した。 RSKBがキナーゼであることから、本発明者らは、 HNF-4 a 力 SKBとの相互作用によりリン酸ィ匕されると予測した。  [0009] The present inventors diligently predicted that in silico that HNF-4a interacts with RSKB. Since RSKB is a kinase, we predicted that it would be phosphorylated by interaction with HNF-4 a force SKB.
[0010] そして、 HNF— 4 aと RSKBが結合すること、 RSKBによって HNF— 4 aカ^ン酸 ィ匕されること、 HNF— 4 aは RSKBによりリン酸化されることで PEPCK遺伝子プロモ 一ター領域内の AF1 (アクセサリー因子結合部位 1、 accessory factor binding sitel)への結合能が促進されること、および力かる促進により PEPCK遺伝子の転写 活性が亢進することを実証した。また、 RSKBによる HNF— 4 aのリン酸化には、 RS KBの活'性ィ匕が必要であることを明らかにした。  [0010] Then, HNF-4a and RSKB are combined, HKB-4a is cleaved by RSKB, HNF-4a is phosphorylated by RSKB, and the PEPCK gene promoter It was demonstrated that the ability to bind to AF1 (accessory factor binding site 1, accessory factor binding sitel) within the region is promoted, and that the transcriptional activity of the PEPCK gene is enhanced by vigorous promotion. It was also clarified that RSKB activity is required for phosphorylation of HNF-4a by RSKB.
[0011] PEPCK遺伝子は糖新生律速酵素であると考えられる PEPCKをコードする遺伝子 である。このことから、 RSKBによるリン酸化により HNF— 4 αの PEPCK遺伝子のプ 口モーター領域内の AF1への結合能が促進され、その結果、 PEPCK遺伝子の転 写活性が亢進することにより、該遺伝子の発現が促進され、さらには糖新生が亢進す ると考えることができる。  [0011] The PEPCK gene is a gene encoding PEPCK which is considered to be a gluconeogenic rate-determining enzyme. From this, phosphorylation by RSKB promotes the ability of HNF-4α to bind to PE1 in the motor region of PEPCK gene, and as a result, the transcriptional activity of PEPCK gene is enhanced, thereby It can be considered that expression is promoted and gluconeogenesis is enhanced.
[0012] RSKBは、 p38キナーゼにより活性化されることが知られて!/、る。 p38キナーゼは、 色々な病態下で活性化される。例えば、糖尿病病態下、持続的に高血糖に曝されて いる細胞で、活性酸素による酸化ストレスカゝら p38キナーゼが活性ィ匕される。したがつ て、糖尿病病態下では、 p38キナーゼが活性ィ匕され、それにより RSKBが活性ィ匕さ れると考えることができる。 [0012] RSKB is known to be activated by p38 kinase! p38 kinase is It is activated under various pathological conditions. For example, p38 kinase, such as oxidative stress caused by active oxygen, is activated in cells that are continuously exposed to hyperglycemia under diabetic conditions. Therefore, it can be considered that p38 kinase is activated and thereby RSKB is activated under diabetic conditions.
[0013] 糖尿病病態時には、活性ィ匕された P38キナーゼにより RSKBが活性ィ匕され、さらに RSKBにより HNF— 4 aカ^ン酸化されると考えることができる。 RSKBによりリン酸 化された HNF— 4 Q;は、 PEPCK遺伝子の転写活性を促進することにより、糖新生を 亢進させ、高血糖を悪ィ匕させると考えることができる。 [0013] When patients suffering from diabetes, RSKB the active I spoon has been P 38 kinase is active I spoon, can be considered to be further HNF- 4 a mosquito ^ phosphorylated by RSKB. It can be considered that HNF-4Q; phosphorylated by RSKB enhances gluconeogenesis and worsens hyperglycemia by promoting the transcriptional activity of the PEPCK gene.
[0014] HNF-4 aは、転写因子として作用することが知られており、 PEPCK遺伝子以外 にも、種々の遺伝子、例えば、コレステロール、脂肪酸およびグルコース等の代謝や 肝臓の発生および分ィ匕に関与する蛋白質をコードする遺伝子の発現に関与している 。したがって、 PEPCK遺伝子以外の遺伝子であって、 HNF— 4 αが転写因子として 作用する遺伝子のプロモーター領域への HNF— 4 aの結合能が、 RSKBによる HN F— 4 αのリン酸により促進され、その結果、該遺伝子の発現が促進される可能性が あると考えることができる。  [0014] HNF-4a is known to act as a transcription factor. In addition to the PEPCK gene, HNF-4a is also involved in the metabolism of various genes, such as cholesterol, fatty acids and glucose, and the development and differentiation of the liver. It is involved in the expression of genes that code for the proteins involved. Therefore, the ability of HNF-4a to bind to the promoter region of a gene other than the PEPCK gene, where HNF-4α acts as a transcription factor, is promoted by the phosphate of HNF-4α by RSKB, As a result, it can be considered that expression of the gene may be promoted.
[0015] これらから、 RSKBと HNF— 4 aを相互作用させることにより HNF— 4 aをリン酸ィ匕 することができ、それにより、 HNF— 4 αが転写因子として作用する遺伝子のプロモ 一ター領域への HNF— 4 aの結合能を促進でき、さらには該遺伝子の遺伝子産物 の産生を促進できる。具体的には、 RSKBと HNF— 4ひを相互作用させることにより HNF— 4 αをリン酸ィ匕することができ、それにより、糖新生関連遺伝子のプロモータ 一領域、例えば PEPCK遺伝子のプロモーター領域内の AF1部位への HNF4 aの 結合能を促進でき、さらには該遺伝子の遺伝子産物の産生を促進できる。  [0015] From these, it is possible to phosphorylate HNF-4a by interacting RSKB with HNF-4a, thereby promoting a gene for which HNF-4α acts as a transcription factor. The ability to bind HNF-4a to the region can be promoted, and further the production of the gene product of the gene can be promoted. Specifically, HNF-4α can be phosphorylated by interacting with RSKB and HNF-4, thereby promoting a promoter region of a gluconeogenesis-related gene, such as the promoter region of the PEPCK gene. The ability to bind HNF4a to the AF1 site of can be promoted, and further the production of the gene product of the gene can be promoted.
[0016] また、 RSKBによる HNF— 4 aのリン酸化を阻害することにより、 HNF— 4 aが転 写因子として作用する遺伝子のプロモーター領域への HNF— 4 aの結合能を低下 させることができ、その結果、該遺伝子の遺伝子産物の産生を阻害できる。具体的に は、 RSKBによる HNF— 4 aのリン酸ィ匕を阻害することにより、糖新生関連遺伝子の プロモーター領域、例えば PEPCK遺伝子のプロモーター領域内の AF1への HNF 4 αの結合能を低下させることができ、それにより、該遺伝子の遺伝子産物の産生を 阻害でき、さら〖こは、糖新生の阻害を実施できる。 [0016] Furthermore, by inhibiting phosphorylation of HNF-4a by RSKB, the ability of HNF-4a to bind to the promoter region of a gene in which HNF-4a acts as a transcription factor can be reduced. As a result, production of the gene product of the gene can be inhibited. Specifically, by inhibiting the phosphorylation of HNF-4a by RSKB, the ability of HNF4α to bind to AF1 in the promoter region of a gluconeogenesis-related gene, such as the promoter region of the PEPCK gene, is reduced. Production of the gene product of the gene And can be inhibited, and can inhibit gluconeogenesis.
[0017] さらに、 HNF-4 aが転写因子として作用する遺伝子の遺伝子産物の産生を阻害 できることから、該遺伝子産物の増加に起因する疾患の防止および Zまたは治療を 実施できる。具体的には、糖新生関連遺伝子の遺伝子産物の増加に起因する疾患 、より具体的には PEPCK遺伝子の遺伝子産物の増加に起因する疾患、例えば糖尿 病の防止および Zまたは治療を実施できる。  [0017] Furthermore, since HNF-4a can inhibit the production of a gene product of a gene that acts as a transcription factor, prevention and Z or treatment of a disease caused by an increase in the gene product can be performed. Specifically, diseases caused by an increase in gene products of gluconeogenesis-related genes, more specifically diseases caused by an increase in gene products of PEPCK genes such as diabetes can be prevented and Z or treated.
[0018] 本発明は、上記知見により完成した。  [0018] The present invention has been completed based on the above findings.
[0019] すなわち、本発明は、リボソーム S6キナーゼ B (RSKB)とへノ トサイトヌクレアーフ アクター 4 a (HNF-4 a )の相互作用を可能にする条件下で共存させることを特徴と する、 RSKBによる HNF— 4 aのリン酸ィ匕方法に関する。  That is, the present invention is characterized in that ribosome S6 kinase B (RSKB) and hematocytonuclear factor 4 a (HNF-4 a) are allowed to coexist under conditions that allow interaction. It relates to the phosphorylation method of HNF-4a by RSKB.
[0020] また本発明は、リボソーム S6キナーゼ B (RSKB)の活性を阻害することを特徴とす る、 RKSBによるへパトサイトヌクレア一ファクター 4 a (HNF-4 a )のリン酸化阻害 方法に関する。 [0020] The present invention also relates to a method for inhibiting phosphorylation of hepatocyte nuclear factor 4a (HNF-4a) by RKSB, characterized by inhibiting the activity of ribosomal S6 kinase B (RSKB) .
[0021] さらに本発明は、リボソーム S6キナーゼ B (RSKB)とへノ トサイトヌクレア一ファクタ 一 4 a (HNF-4 a )の結合を阻害することを特徴とする、 RSKBによる HNF— 4 a のリン酸ィ匕阻害方法に関する。  [0021] Further, the present invention relates to HNF-4a by RSKB, characterized in that it inhibits the binding of ribosomal S6 kinase B (RSKB) to hetocytonuclea factor 1a (HNF-4a). The present invention relates to a method for inhibiting phosphorylation.
[0022] さらにまた本発明は、少なくともリボソーム S6キナーゼ B (RSKB)とへノ讣サイトヌク レアーファクター 4 a (HNF-4 a )とを発現している細胞を、 RSKB活性の阻害剤で 処理することを特徴とする RSKBによる HNF— 4 aのリン酸ィ匕阻害方法に関する。  [0022] Furthermore, the present invention provides treatment of a cell expressing at least ribosomal S6 kinase B (RSKB) and henocytonuclear factor 4a (HNF-4a) with an inhibitor of RSKB activity. It relates to a method for inhibiting HNF-4a phosphorylation by RSKB.
[0023] また本発明は、リボソーム S6キナーゼ B (RSKB)活性の阻害剤力 RSKBを認識 する抗体、へノ トサイトヌクレア一ファクター 4 α (HNF— 4ひ）を認識する抗体力も選 ばれる 1つまたは 2つ以上の抗体である前記 RSKBによる HNF— 4 aのリン酸化阻 害方法に関する。  [0023] The present invention also selects an antibody that recognizes RSKB, an inhibitory force of ribosomal S6 kinase B (RSKB) activity, and an antibody that recognizes hetocytonuclea factor 4 α (HNF-4) 1 The present invention relates to a method for inhibiting phosphorylation of HNF-4a by RSKB, which is one or more antibodies.
[0024] さらに本発明は、リボソーム S6キナーゼ B (RSKB)活性を阻害することを特徴とす る、 RSKBによるへパトサイトヌクレア一ファクター 4 a (HNF-4 a )のリン酸化阻害 剤に関する。  [0024] Furthermore, the present invention relates to a phosphorylation inhibitor of hepatocyte nuclear factor 4a (HNF-4a) by RSKB, characterized by inhibiting ribosomal S6 kinase B (RSKB) activity.
[0025] さらにまた本発明は、リボソーム S6キナーゼ B (RSKB)とへノ トサイトヌクレアーファ クタ一 4 a (HNF-4 a )の結合を阻害することを特徴とする、 RSKBによる HNF— 4 aのリン酸ィ匕阻害剤に関する。 [0025] Furthermore, the present invention relates to HNF-4 by RSKB, characterized by inhibiting the binding of ribosomal S6 kinase B (RSKB) to hetocytonuclea factor 4a (HNF-4a). It relates to a phosphate inhibitor of a.
[0026] また本発明は、リボソーム S6キナーゼ B (RSKB)活性の阻害剤を有効量含むこと を特徴とする RSKBによるへノ トサイトヌクレア一ファクター 4 a (HNF-4 a )のリン 酸ィ匕阻害剤に関する。 [0026] The present invention also includes an effective amount of an inhibitor of ribosomal S6 kinase B (RSKB) activity, and the phosphorylation of hepatocyte nuclear factor 4a (HNF-4a) by RSKB. It relates to an anther inhibitor.
[0027] さらに本発明は、リボソーム S6キナーゼ B (RSKB)活性の阻害剤力 RSKBを認 識する抗体、へノ トサイトヌクレア一ファクター 4 α (HNF— 4ひ）を認識する抗体から 選ばれる 1つまたは 2つ以上の抗体である前記 RSKBによる HNF— 4 aのリン酸ィ匕 阻害剤に関する。  [0027] Furthermore, the present invention is selected from an antibody that recognizes the inhibitory power RSKB of ribosomal S6 kinase B (RSKB) activity and an antibody that recognizes hetocytonuclea factor 4 α (HNF-4). The present invention relates to a phosphate inhibitor of HNF-4a by RSKB, which is one or more antibodies.
[0028] さらにまた本発明は、へパトサイトヌクレア一ファクター 4 aをリボソーム S6キナーゼ Bを用いてリン酸化することを特徴とする、糖新生関連遺伝子の遺伝子産物産生促 進方法に関する。  [0028] Furthermore, the present invention relates to a method for promoting gene product production of a gene involved in gluconeogenesis, which comprises phosphorylating hepatocyte nuclear factor 4a using ribosome S6 kinase B.
[0029] また本発明は、糖新生関連遺伝子がホスホェノールピルビン酸カルボキシキナー ゼ遺伝子である前記糖新生関連遺伝子の遺伝子産物産生促進方法に関する。  [0029] The present invention also relates to a method for promoting gene product production of the gluconeogenesis-related gene, wherein the gluconeogenesis-related gene is a phosphoenolpyruvate carboxykinase gene.
[0030] さらに本発明は、リボソーム S6キナーゼ Bによるへノ トサイトヌクレア一ファクター 4 aのリン酸化を阻害することを特徴とする、糖新生関連遺伝子の遺伝子産物産生阻 害方法に関する。  [0030] Furthermore, the present invention relates to a method for inhibiting the production of a gene product of a gluconeogenesis-related gene, which comprises inhibiting phosphorylation of hepatocyte nuclease factor 4a by ribosomal S6 kinase B.
[0031] さらにまた本発明は、リボソーム S6キナーゼ Bによるへノ讣サイトヌクレア一ファクタ 一 4 aのリン酸化を阻害することを特徴とする、ホスホェノールピルビン酸カルボキシ キナーゼ遺伝子の遺伝子産物産生阻害方法に関する。  [0031] Furthermore, the present invention relates to a method for inhibiting the production of a gene product of a phosphoenolpyruvate carboxykinase gene, which comprises inhibiting phosphorylation of henocytocytolase factor 14a by ribosome S6 kinase B. About.
[0032] また本発明は、前記いずれかのリン酸ィ匕阻害方法を用いることを特徴とする、糖新 生関連遺伝子の遺伝子産物産生阻害方法に関する。 [0032] The present invention also relates to a method for inhibiting the production of a glycoprotein-related gene product, characterized by using any one of the methods for inhibiting phosphate kneading.
[0033] さらに本発明は、前記いずれかのリン酸ィ匕阻害方法を用いることを特徴とする、へ ノ讣サイトヌクレア一ファクター 4 aが転写因子として作用する遺伝子の遺伝子産物 産生阻害方法に関する。 [0033] Furthermore, the present invention relates to a method for inhibiting the production of a gene product of a gene that acts as a transcription factor, which is characterized in that any one of the above-described methods for inhibiting phosphate kneading is used. .
[0034] さらにまた本発明は、前記いずれかのリン酸ィ匕阻害方法を用いることを特徴とする、 ホスホェノールピルビン酸カルボキシキナーゼ遺伝子の遺伝子産物産生阻害方法 に関する。 [0034] Furthermore, the present invention relates to a method for inhibiting the gene product production of a phosphoenolpyruvate carboxykinase gene, characterized in that any one of the above-described methods for inhibiting phosphate kine is used.
[0035] また本発明は、前記いずれかのリン酸ィ匕阻害方法を用いることを特徴とする、リボソ ーム S6キナーゼ Bによるへノ トサイトヌクレア一ファクター 4 aのリン酸化に起因する 疾患の防止および Zまたは治療方法に関する。 [0035] Further, the present invention provides a ribozyme characterized by using any one of the methods for inhibiting phosphorylation. The present invention relates to a method for preventing and / or treating a disease caused by phosphorylation of hetocytonuclea factor 4a by S6 kinase B.
[0036] さらに本発明は、前記いずれかのリン酸ィ匕阻害方法を用いることを特徴とする、糖 新生関連遺伝子の遺伝子産物の増加に起因する疾患の防止および Zまたは治療 方法に関する。 [0036] Furthermore, the present invention relates to a method for the prevention and Z or treatment of a disease caused by an increase in the gene product of a gluconeogenesis-related gene, characterized by using any one of the above-described methods for inhibiting phosphorylation.
[0037] さらにまた本発明は、前記いずれかのリン酸ィ匕阻害方法を用いることを特徴とする、 ホスホェノールピルビン酸カルボキシキナーゼ遺伝子の遺伝子産物の増加に起因 する疾患の防止および Zまたは治療方法に関する。  [0037] Furthermore, the present invention provides a method for preventing and / or treating a disease caused by an increase in the gene product of the phosphoenolpyruvate carboxykinase gene, characterized by using any one of the methods for inhibiting phosphate kneading. About.
[0038] また本発明は、前記いずれかのリボソーム S6キナーゼ Bによるへパトサイトヌクレア 一ファクター 4 aのリン酸ィ匕阻害方法を用いることを特徴とする、糖尿病の防止およ び Zまたは治療方法に関する。 [0038] The present invention also relates to prevention and / or treatment of diabetes, characterized by using the method for inhibiting hepatocyte nuclear factor 1a phosphorylation by any one of the above-mentioned ribosome S6 kinase B. Regarding the method.
[0039] さらに本発明は、前記いずれかのリボソーム S6キナーゼ Bによるへパトサイトヌクレ ァーファクター 4 aのリン酸ィ匕阻害剤および Zまたは RSKB活性の阻害剤を用いるこ とを特徴とする、糖尿病の防止および Zまたは治療方法に関する。 [0039] Further, the present invention uses a phosphate inhibitor of hepatocyte nuclear factor 4a and an inhibitor of Z or RSKB activity by any one of the above-mentioned ribosomal S6 kinase B. Prevention and Z or treatment methods.
[0040] さらにまた本発明は、前記いずれかのリボソーム S6キナーゼ Bによるへノ讣サイトヌ クレア一ファクター 4 aのリン酸ィ匕阻害剤および Zまたは RSKB活性の阻害剤を有効 量含んでなる、ホスホェノールピルビン酸カルボキシキナーゼ遺伝子の遺伝子産物 の産生阻害剤に関する。 [0040] Furthermore, the present invention provides a phosphoprotein comprising an effective amount of any one of the above-mentioned phosphosomal S6 kinase B phosphorylation inhibitors of henocytonuclear factor 4a and an inhibitor of Z or RSKB activity. The present invention relates to an inhibitor of production of the gene product of the enolpyruvate carboxykinase gene.
[0041] また本発明は、前記いずれかのリボソーム S6キナーゼ Bによるへパトサイトヌクレア 一ファクター 4 aのリン酸ィ匕阻害剤および Zまたは RSKB活性の阻害剤を有効量含 んでなる医薬組成物に関する。 [0041] Further, the present invention provides a pharmaceutical composition comprising an effective amount of a phosphorylation inhibitor of hepatocyte nuclear factor 1a by any one of the ribosome S6 kinase B and an inhibitor of Z or RSKB activity. About.
[0042] さらに本発明は、前記いずれかのリボソーム S6キナーゼ Bによるへパトサイトヌクレ ァーファクター 4 aのリン酸ィ匕阻害剤および Zまたは RSKB活性の阻害剤を有効量 含んでなる、ホスホェノールピルビン酸カルボキシキナーゼ遺伝子の遺伝子産物の 増加に起因する疾患の防止および Zまたは治療剤に関する。 [0042] Further, the present invention provides a phosphoenolpyruvate comprising an effective amount of any one of the aforementioned ribosomal S6 kinase B hepatocyte nuclear factor 4a phosphate inhibitors and inhibitors of Z or RSKB activity. The present invention relates to a preventive and Z or therapeutic agent for diseases caused by an increase in the gene product of a carboxykinase gene.
[0043] さらにまた本発明は、前記いずれかのリボソーム S6キナーゼ Bによるへノ讣サイトヌ クレア一ファクター 4 aのリン酸ィ匕阻害剤および Zまたは RSKB活性の阻害剤を有効 量含んでなる、糖尿病の防止および Zまたは治療剤に関する。 [0044] また本発明は、リボソーム S6キナーゼ B (RSKB)とへノ トサイトヌクレア一ファクター 4 a (HNF— 4 a )の相互作用を阻害する化合物の同定方法であって、 RSKBと HN F-4 aの相互作用を可能にする条件下、 RSKBおよび Zまたは HNF— 4 aと被検 化合物を接触させ、 RSKBと HNF— 4 aの結合を検出するシグナルおよび Zまたは マーカーを使用する系を用い、このシグナルおよび Zまたはマーカーの存在若しく は不存在および Zまたは変化を検出することにより、被検化合物が RSKBと HNF— 4 aの相互作用を阻害するか否かを決定することを含む同定方法に関する。 [0043] Furthermore, the present invention comprises a diabetic comprising an effective amount of any one of the above-mentioned phosphosomal S6 kinase B phosphoric acid phosphatase inhibitors of cytoplasmic factor 1a 4a and inhibitors of Z or RSKB activity. Prevention and Z or treatment. [0044] The present invention also relates to a method for identifying a compound that inhibits the interaction between ribosomal S6 kinase B (RSKB) and hetocytonuclea factor 4a (HNF-4a), wherein RSKB and HNF A system that uses a signal and Z or marker to detect the binding of RSKB and HNF-4a by contacting RSKB and Z or HNF-4a with the test compound under conditions that allow -4a interaction Use to determine whether the test compound inhibits the interaction between RSKB and HNF-4a by detecting the presence or absence of this signal and the Z or marker and the Z or change. The present invention relates to an identification method.
[0045] さらに本発明は、リボソーム S6キナーゼ B (RSKB)とへノ トサイトヌクレア一ファクタ 一 4 a (HNF— 4 a )の結合を阻害する化合物の同定方法であって、 RSKBと HNF 4 aの結合を可能にする条件下、 RSKBおよび Zまたは HNF— 4 aと被検化合物 を接触させ、 RSKBと HNF— 4 aの結合を検出するシグナルおよび Zまたはマーカ 一を使用する系を用い、このシグナルおよび Zまたはマーカーの存在若しくは不存 在および Zまたは変化を検出することにより、被検化合物力 ¾SKBと HNF— 4 aの 結合を阻害する力否かを決定することを含む同定方法に関する。  [0045] Furthermore, the present invention provides a method for identifying a compound that inhibits the binding of ribosomal S6 kinase B (RSKB) to hetocytonuclea factor 1a (HNF-4a), comprising RSKB and HNF4 Using a system using RSKB and Z or HNF-4a in contact with a test compound under conditions that allow binding of a, and using a signal that detects the binding of RSKB and HNF-4a and Z or marker The present invention relates to an identification method comprising determining whether or not a compound has a test compound strength ¾ SKB and a binding to HNF-4a by detecting the presence or absence of this signal and Z or a marker and Z or a change.
[0046] さらにまた本発明は、リボソーム S6キナーゼ B (RSKB)によるへパトサイトヌクレア 一ファクター 4 a (HNF— 4 α )のリン酸化を阻害する化合物の同定方法であって、 R SKBによる HNF— 4 aのリン酸化を可能にする条件下、 RSKBおよび Zまたは HN F-4 aと被検化合物を接触させ、 RSKBによる HNF— 4 aのリン酸化を検出するシ グナルおよび Zまたはマーカーを使用する系を用い、このシグナルおよび Zまたは マーカーの存在若しくは不存在および Zまたは変化を検出することにより、被検化合 物が RSKBによる HNF— 4 aのリン酸化を阻害するか否かを決定することを含む同 定方法に関する。  [0046] Furthermore, the present invention provides a method for identifying a compound that inhibits phosphorylation of hepatocyte nuclear factor 1a (HNF-4α) by ribosomal S6 kinase B (RSKB), comprising: — RSKB and Z or HNF-4a in contact with the test compound under conditions that allow phosphorylation of 4a, and HNF by RSKB — Uses signal and Z or marker to detect phosphorylation of 4a To determine whether the test compound inhibits phosphorylation of HNF-4a by RSKB by detecting the presence or absence and Z or change of this signal and Z or marker. It relates to identification methods including
[0047] また本発明は、リボソーム S6キナーゼ B (RSKB)、 RSKBをコードするポリヌクレオ チドおよび RSKBをコードするポリヌクレオチドを含有するベクターのうち少なくともい ずれか 1つと、へパトサイトヌクレア一ファクター 4 a (HNF— 4 a )、 HNF— 4 aをコ ードするポリヌクレオチドおよび該 HNF— 4 aをコードするポリヌクレオチドを含有す るベクターのうちの少なくともいずれか 1つを含んでなるキットに関する。  [0047] The present invention also relates to at least one of ribosomal S6 kinase B (RSKB), a polynucleotide encoding RSKB, and a vector containing a polynucleotide encoding RSKB, and hepatocyte nuclear factor 4 The present invention relates to a kit comprising at least one of a (HNF-4a), a polynucleotide encoding HNF-4a, and a vector containing the polynucleotide encoding HNF-4a.
発明の効果 [0048] 本発明により、 RSKBによる HNF— 4 aのリン酸化方法、リン酸化阻害方法および リン酸ィ匕阻害剤を提供できる。また、本発明により、 HNF-4 aが作用する遺伝子の 遺伝子産物の産性方法、産生阻害方法および産性阻害剤を提供できる。 HNF-4 aが作用する遺伝子の遺伝子産物の産生阻害方法は、 RSKBによる HNF— 4 aの リン酸ィ匕阻害剤および Zまたはリン酸ィ匕阻害方法を用いることにより実施できる。さら に、本発明により、 HNF-4 aが作用する遺伝子の遺伝子産物の増加に起因する 疾患の防止および Zまたは治療方法および防止および Zまたは治療剤を提供でき る。具体的には、糖新生関連遺伝子の遺伝子産物の増加に起因する疾患、より具体 的には PEPCK遺伝子糖尿病の遺伝子産物の増加に起因する疾患、例えば糖尿病 の防止および Zまたは治療方法および防止および Zまたは治療剤を提供できる。 The invention's effect [0048] According to the present invention, it is possible to provide a method for phosphorylating HNF-4a by RSKB, a method for inhibiting phosphorylation, and a phosphoric acid inhibitor. Further, according to the present invention, it is possible to provide a productivity method, a production inhibition method, and a productivity inhibitor of a gene product of a gene on which HNF-4a acts. A method for inhibiting the production of a gene product of a gene on which HNF-4a acts can be carried out by using a phosphoric acid inhibitor of HNF-4a and a Z or phosphoric acid inhibitor method by RSKB. Furthermore, according to the present invention, it is possible to provide a preventive and Z or therapeutic method and preventive and Z or therapeutic agent for diseases caused by an increase in the gene product of a gene on which HNF-4a acts. Specifically, diseases caused by an increase in gene products of gluconeogenesis-related genes, more specifically diseases caused by an increase in the gene product of PEPCK gene diabetes, such as diabetes prevention and Z or treatment methods and prevention and Z Alternatively, a therapeutic agent can be provided.
[0049] 本発明は、このように、糖新生関連遺伝子、例えば PEPCK遺伝子の発現に関与 する HNF— 4 aの過剰なリン酸ィ匕に起因する疾患の防止および Zまたは治療のた めに非常に有用である。このような疾患として、 PEPCK遺伝子の発現による糖新生 の亢進に起因する疾患、具体的には糖尿病を好ましく例示できる。  [0049] The present invention is thus very useful for the prevention and Z or treatment of diseases caused by excessive phosphorylation of HNF-4a involved in the expression of gluconeogenesis-related genes such as PEPCK gene. Useful for. Preferred examples of such diseases include diseases caused by increased gluconeogenesis due to expression of the PEPCK gene, specifically diabetes.
[0050] 正常の肝臓では、 PEPCK遺伝子の発現はインスリンにより抑制的に支配されてお り、必要に応じて数時間単位で遺伝子発現が起きる。しかし、何らかの理由により PE PCK遺伝子が持続的に過剰発現すると、肝臓での糖新生が亢進して高血糖となり、 インスリン耐性の糖尿病を引き起こす。また、脾臓の β細胞が崩壊し、血中インスリン 量が低下して 、る糖尿病の末期患者にぉ 、ては、インスリンによる PEPCK遺伝子の 抑制効果は減少して 、ると考えられる。  [0050] In normal liver, PEPCK gene expression is repressively controlled by insulin, and gene expression occurs in units of several hours as necessary. However, if the PE PCK gene is overexpressed continuously for some reason, gluconeogenesis in the liver is increased and hyperglycemia occurs, causing insulin-resistant diabetes. In addition, β-cells in the spleen have collapsed and the amount of insulin in the blood has decreased, so that it is considered that the inhibitory effect of insulin on the PEPCK gene is decreased in end-stage patients with diabetes.
[0051] 本発明に係る RSKBによる HNF— 4 aのリン酸ィ匕阻害剤および Zまたはリン酸ィ匕 阻害方法により、インスリン非依存性に PEPCK遺伝子の発現を阻害できるため、ィ ンスリン耐性の糖尿病にぉ 、ても、 PEPCK遺伝子が関与する糖新生を抑制すること ができると考えることができる。すなわち、本発明に係る RSKBによる HNF— 4 αのリ ン酸ィ匕阻害剤および Ζまたはリン酸ィ匕阻害方法により、インスリンの効果が期待でき な ヽ 2型糖尿病のインスリン抵抗性患者の肝臓における糖新生を抑制でき、その結 果、高血糖を抑制できると考えている。  [0051] Since the expression of the PEPCK gene can be inhibited in an insulin-independent manner by the phosphate inhibitor of HNF-4a and Z or phosphate inhibitory method by RSKB according to the present invention, insulin resistant diabetes However, it can be considered that gluconeogenesis involving the PEPCK gene can be suppressed. That is, in the liver of an insulin resistant patient with type 2 diabetes who can not be expected to have an insulin effect by the phosphate inhibitor of HNF-4α and the method of inhibiting phosphate or phosphate by RSKB according to the present invention We believe that gluconeogenesis can be suppressed, and as a result, hyperglycemia can be suppressed.
[0052] 近年、糖尿病治療薬としてインスリン抵抗性改善薬が開発されおり、その代表例と して、チアゾリジンジオン誘導体が臨床の場で用いられている。チアゾリジンジオン誘 導体は、脂肪組織におけるインスリン感受性の低 、大型脂肪細胞をインスリン感受性 の高い小型脂肪細胞に変化させる作用を有する。一方、本発明に係る RSKBによる HNF-4 aのリン酸ィ匕阻害剤は、肝臓における PEPCK遺伝子の発現を阻害する作 用を有し、その結果、糖新生を抑制することができる。このように、本発明に係る RSK Bによる HNF— 4 aのリン酸ィ匕阻害剤とチアゾリジンジオン誘導体とは、標的臓器も その作用機序も異なる。 [0052] In recent years, insulin sensitizers have been developed as antidiabetic drugs, Thus, thiazolidinedione derivatives are used in clinical settings. The thiazolidinedione derivative has an action of changing large adipocytes having low insulin sensitivity in adipose tissue to small adipocytes having high insulin sensitivity. On the other hand, the phosphate inhibitor of HNF-4a by RSKB according to the present invention has an action of inhibiting the expression of PEPCK gene in the liver, and as a result, can suppress gluconeogenesis. Thus, the phosphorylation inhibitor of HNF-4a by RSK B according to the present invention and the thiazolidinedione derivative differ in the target organ and the mechanism of action.
[0053] 本発明により RSKBによる HNF— 4 aのリン酸化を阻害し、 PEPCK遺伝子の遺伝 子産物の産生を抑制することは、インスリン非依存的に血糖降下作用を奏する可能 性があり非常に有用である。 [0053] According to the present invention, it is very useful to inhibit the phosphorylation of HNF-4a by RSKB and suppress the production of the gene product of PEPCK gene, which may exert a hypoglycemic effect independent of insulin. It is.
図面の簡単な説明  Brief Description of Drawings
[0054] [図 1]RSKBと HNF— 4 aの相互作用をインシリコで予測した結果を示す図である。  [0054] FIG. 1 is a diagram showing the results of in silico prediction of the interaction between RSKB and HNF-4a.
RSKBと HNF— 4 aの間でローカルァライメントを行い、高いスコアを示した領域を 図示した。上の配列および下の配列はそれぞれ、 RKSBに存在する配列および HN F— 4 αに存在する配列である。アミノ酸配列は 1文字表記した。図中の数字は、 RS KBまたは HNF— 4 aの各アミノ酸配列における、図示した各領域の N末端アミノ酸 の位置を意味する。（実施例 1)  Areas that showed high scores were shown by local alignment between RSKB and HNF-4a. The upper sequence and the lower sequence are the sequence present in RKSB and the sequence present in HNF-4α, respectively. The amino acid sequence is represented by one letter. The numbers in the figure mean the position of the N-terminal amino acid in each region shown in each amino acid sequence of RS KB or HNF-4a. (Example 1)
[図 2]N末端 FLAGタグ動物細胞用発現プラスミドにて HEK293T細胞に一過性発 現させ、 FLAG M2ァフィ二ティーゲルを用いて精製した活性型 FLAG— RSKBの クマシ一ブリリアントブルー（CBB)染色像を示す図である。 1、 2はそれぞれ FLAG による溶出画分 1、溶出画分 2を示す。実験には溶出画分 1を使用した。矢頭は精製 FLAG— RSKBを示す。図の左列に記載した数値は分子量マーカー（図中、 Mと表 示する）の分子量である。（実施例 2)  [Figure 2] Active FLAG-RSKB stained with Kumashi Brilliant Blue (CBB), transiently expressed in HEK293T cells using an N-terminal FLAG-tagged animal cell expression plasmid and purified using FLAG M2 affinity gel FIG. 1 and 2 show the elution fraction 1 and elution fraction 2 by FLAG, respectively. Elution fraction 1 was used for the experiment. Arrowheads indicate purified FLAG-RSKB. The numerical values shown in the left column of the figure are the molecular weights of the molecular weight markers (indicated as M in the figure). (Example 2)
[図 3]HNF— 4 aのインビトロ免疫沈降リン酸化試験の結果、 HNF— 4 aが RSKBに よりリン酸ィ匕されたことを示す図である。パネル Aは陽性対照の PKAによるリン酸ィ匕 試験の結果を、パネル Bは RSKBによるリン酸ィ匕試験の結果を示す。 CREB1 (サイク リック AMP応答配列結合蛋白質 1、 cAMP responsive element binding prot ein 1)の RSKBによるリン酸ィ匕は、精製された RSKBが活性を有することを示す。図 中の +および一はそれぞれの蛋白質の有無を示している。矢頭は HNF— 4 αを、ス ターマーク（* )は CREB1のリン酸ィ匕体を示す。図の左列に記載した数値は、分子 量マーカーの分子量である。（実施例 2) FIG. 3 shows that HNF-4a was phosphorylated by RSKB as a result of in vitro immunoprecipitation phosphorylation test of HNF-4a. Panel A shows the results of the phosphate control test with the positive control PKA, and Panel B shows the results of the phosphate test with RSKB. CREB1 (cyclic AMP responsive element binding protein 1, cAMP responsive element binding protein 1) phosphate by RSKB indicates that the purified RSKB has activity. Figure Inside + and one indicate the presence or absence of each protein. The arrowhead indicates HNF-4α, and the star (*) indicates CREB1 phosphate. The numbers listed in the left column of the figure are the molecular weights of the molecular weight markers. (Example 2)
[図 4]ヒト PEPCK遺伝子（NCBIァクセッション番号 U31519)のプロモーター領域の 塩基配列を示す図である。ゲルシフトアツセィで用いた AF1の配列を太字枠内に示 す。 PEPCK AF1プロモーター ZpGL3は図の矢印 1, 3で区切られた 882〜140 6番目の塩基配列（ボールド表示）を、 PEPCK Δ AF1プロモーター ZpGL3は矢 印 2, 3で区切られた 934〜 1406番目の塩基配列をそれぞれ使用した。左列の数字 は U31519における塩基番号を示す。（実施例 3)  FIG. 4 shows the nucleotide sequence of the promoter region of the human PEPCK gene (NCBI accession number U31519). The AF1 sequence used in the gel shift assembly is shown in bold. PEPCK AF1 promoter ZpGL3 is the 882 to 140 6th base sequence (bold display) separated by arrows 1 and 3, PEPCK Δ AF1 promoter ZpGL3 is the 934 to 1406 bases separated by arrows 2 and 3 Each sequence was used. The numbers in the left column indicate the base numbers in U31519. (Example 3)
[図 5]N末端 FLAGタグ動物細胞用発現プラスミドにて HEK293T細胞に一過性発 現させ、 FLAG M2ァフィ二ティーゲルを用いて精製した FLAG— HNF— 4 a (DC BB染色像を示す図である。 1、 2はそれぞれ FLAGによる溶出画分 1、溶出画分 2を 示す。実験には溶出画分 1を使用した。矢頭は精製 FLAG— HNF— 4 aを示す。図 の右列に記載した数値は分子量マーカー（図中、 Mと表示する）の分子量である。（ 実施例 3)  [Fig. 5] FLAG-HNF-4a (DC BB stained image, transiently expressed in HEK293T cells using an N-terminal FLAG-tagged animal cell expression plasmid and purified using FLAG M2 affinity gel. 1 and 2 indicate FLAG elution fraction 1 and elution fraction 2, respectively, and elution fraction 1 was used in the experiment, and arrowheads indicate purified FLAG-HNF-4a, as shown in the right column of the figure. The numerical value is the molecular weight of the molecular weight marker (shown as M in the figure) (Example 3)
[図 6]PEPCK遺伝子プロモーター領域内の AF1配列に対する HNF— 4 aの結合 能を EMSA (電気泳動移動度シフトアツセィ、 electrophoretic mobility shift a ssay)にて検討した結果を示す図である。パネル Aは、 HNF— 4 aが AF1配列に結 合したことを示す図である。抗 HNF— 4 α抗体の添カ卩により、 HNF-4 a ' DNA複 合体の移動度が小さくなるスーパーシフトが見られた。パネル Bは、 HNF— 4 αの A F 1配列への DNA結合能に対する RSKBによるリン酸化の影響を示す図面である。 RSKBによるリン酸ィ匕処理により、 HNF-4 aの AF1配列に対する結合能は亢進し た力 PKAによるリン酸ィ匕処理ではそのような亢進は見られな力つた。パネル Bにお ける HNF— 4 αの使用量はパネル Aにおける使用量の約 10分の 1である。図中の + および—は抗 HNF— 4 α抗体の添カ卩の有無を示している。 （実施例 3)  FIG. 6 is a diagram showing the results of examining the binding ability of HNF-4a to the AF1 sequence in the PEPCK gene promoter region using EMSA (electrophoretic mobility shift assay). Panel A shows that HNF-4a bound to the AF1 sequence. By adding anti-HNF-4α antibody, a supershift was observed in which the mobility of the HNF-4a 'DNA complex was reduced. Panel B shows the effect of phosphorylation by RSKB on the ability of HNF-4α to bind DNA to the A F 1 sequence. Phosphate treatment with RSKB enhanced the ability of HNF-4a to bind to the AF1 sequence. Phosphate treatment with PKA showed no such enhancement. The amount of HNF-4α used in Panel B is about one-tenth that of Panel A. In the figure, + and — indicate the presence or absence of anti-HNF-4α antibody supplements. (Example 3)
[図 7]RSKBによる HNF— 4 aのリン酸ィ匕により、 HNF— 4 aの AF1配列への結合 が用量依存的に亢進することを示す図である。 HNF-4 aのリン酸ィ匕処理に使う RS KB量を減少させるにつれ、 HNF— 4 αの AF1との結合活性が低下した。一方、 ΡΚ A処理（図中、 PKAと表示する）では、 HNF— 4 αの AF1との結合の亢進は見られ なかった。 I X、 1/2, 1Z4はそれぞれ原液、 2倍および 4倍希釈した RSKBによる リン酸化試料の EMS Αの結果を示す。スターマーク（ * )は、抗 HNF— 4 a抗体の 添加を示す。（実施例 3) FIG. 7 shows that the binding of HNF-4a to the AF1 sequence is enhanced in a dose-dependent manner by the phosphorylation of HNF-4a by RSKB. As the amount of RS KB used for HNF-4a phosphate treatment decreased, the binding activity of HNF-4α to AF1 decreased. Meanwhile, ΡΚ In the A treatment (indicated as PKA in the figure), no enhanced binding of HNF-4α to AF1 was observed. IX, 1/2, and 1Z4 show the EMS results of the phosphorylated samples with RSKB diluted 2 and 4 times, respectively. The star mark (*) indicates the addition of anti-HNF-4a antibody. (Example 3)
[図 8]PEPCK AF1プロモーターを用いたレポーターアツセィの結果を示す図であ る。 HNF-4 aの導入量に依存して転写活性が亢進した。この結果は、 HNF— 4 a 力 SPEPCK遺伝子を正に制御することを示す。 RSKBを発現させても HNF— 4 aの 転写活性には影響がな力つた。縦軸はコントロール群（PEPCK AF1プロモーター ZpGL3のみ導入)の転写活性を 1. 00とした時の倍数である。グラフの高さおよびグ ラフ上段の値は各群の n= 3の平均値を、エラーバーは標準偏差を示す。各群の蛋 白質の横の数字は導入 DNA量である（例： HNF— 4 a 0. 05は、 0. 05 μ gの ΗΝ F— 4 a発現プラスミドを導入した)。 * :コントロールに対して有意差あり（pく 0. 05) 、 # : HNF— 4 a 0. 05に対して有意差あり（pく 0. 05)、 $： HNF— 4 a 0. 1に 対して有意差あり（ρ< 0. 05)、 + : HNF-4 a 0. 2に対して有意差あり（p< 0. 05 ) , ¥ :RSKB 0. 25に対して有意差あり（pく 0. 05)。（実施例 4)  FIG. 8 shows the results of reporter assembly using PEPCK AF1 promoter. Transcriptional activity increased depending on the amount of HNF-4a introduced. This result indicates that HNF-4a force SPEPCK gene is positively controlled. Even when RSKB was expressed, the transcriptional activity of HNF-4a was not affected. The vertical axis is a multiple when the transcriptional activity of the control group (introducing only PEPCK AF1 promoter ZpGL3) is 1.00. The height of the graph and the value at the top of the graph indicate the average value of n = 3 for each group, and the error bar indicates the standard deviation. The number next to each group of proteins is the amount of DNA introduced (eg, HNF-4a 0.05 was introduced with 0.05 μg of F-4a expression plasmid). *: Significantly different from control (p <0.05), #: Significantly different from HNF— 4 a 0. 05 (p <0.05), $: HNF— 4 a 0.1 Significantly different (ρ <0. 05), +: Significantly different from HNF-4 a 0.2 (p <0. 05), ¥: Significantly different from RSKB 0.25 (p (0. 05). (Example 4)
[図 9]PEPCK AF1プロモーターを用いたレポーターアツセィの結果を示す図であ る。 HNF— 4 aによる転写活性力 MAPK11によって活性化された RSKBによって 著しく促進された。 RSKBを除いて MAPK11だけを発現させても HNF— 4 aの転 写活性に影響がなかったことから、この作用は RSKBによるものと判断できる。縦軸 はコントロール群（PEPCK AF1プロモーター ZpGL3のみ導入）の転写活性を 1. 00とした時の倍数である。グラフの高さおよびグラフ上段の値は各群の n= 3の平均 値を、エラーバーは標準偏差を示す。各群の蛋白質の横の数字は導入 DNA量であ る（例： HNF— 4 a 0. 05は、 0. gの HNF— 4 α発現プラスミドを導入した）。 *：コントロールに対して有意差あり（pく 0. 05)、 #： HNF— 4 a 0. 05に対して有 意差あり（Pく 0. 05)、 $： RSKB 0. 25 + MAPK11 0. 05に対して有意差あり（p < 0. 05) , + : HNF-4 a 0. 05 +RSKB 0. 25 + MAPK11 0. 05に対して有 意差あり（p< 0. 05)。（実施例 4) FIG. 9 shows the results of reporter assembly using PEPCK AF1 promoter. Transcriptional activity by HNF-4a Significantly enhanced by RSKB activated by MAPK11. Since only the expression of MAPK11 except RSKB had no effect on the transcriptional activity of HNF-4a, this action can be attributed to RSKB. The vertical axis is the multiple when the transcriptional activity of the control group (introducing only PEPCK AF1 promoter ZpGL3) is 1.00. The height of the graph and the value at the top of the graph indicate the average value of n = 3 for each group, and the error bar indicates the standard deviation. The number next to each group of proteins is the amount of DNA introduced (eg, HNF-4a 0.05 was introduced with 0. g of HNF-4α expression plasmid). *: Significantly different from control (p <0.05), #: Significantly different from HNF— 4 a 0. 05 (P <0.05), $: RSKB 0.25 + MAPK11 0 Significantly different from 05 (p <0. 05), +: Significantly different from HNF-4 a 0. 05 + RSKB 0. 25 + MAPK11 0. 05 (p <0. 05). (Example 4)
[図 10]PEPCK AF1プロモーターを用いたレポーターアツセィの結果を示す図であ る。 HNF— 4 αによる転写活性力 MAPK11によって活性化された RSKBによって 著しく促進された。一方、ドミナントネガティブ RSKB (S343A)では MAPK11を共 発現させても HNF— 4 aの転写活性は促進されな力つた。縦軸はコントロール群 (P EPCK AF1プロモーター ZpGL3のみ導入）の転写活性 1. 00とした時の倍数であ る。グラフの高さおよびグラフ上段の値は各群の n= 3の平均値を、エラーバーは標 準偏差を示す。各群の蛋白質の横の数字は導入 DNA量である（例: HNF— 4 a 0 . 05は、 0. 05 gの HNF— 4 α発現プラスミドを導入した）。 *：コントロールに対し て有意差あり（ρ< 0. 05)、 # : HNF— 4 α 0. 05に対して有意差あり（ρ< 0. 05)、 $： HNF-4 a 0. 05 +RSKB 0. 25 + MAPK11 0. 05に対して有意差あり（p < 0. 05)。（実施例 4) FIG. 10 is a diagram showing the results of reporter assembly using PEPCK AF1 promoter. The Transcriptional activity by HNF-4α was significantly enhanced by RSKB activated by MAPK11. On the other hand, dominant negative RSKB (S343A) did not promote the transcriptional activity of HNF-4a even when MAPK11 was co-expressed. The vertical axis is a multiple of the transcriptional activity of the control group (only PEPCK AF1 promoter ZpGL3 introduced) is assumed to be 1.00. The height of the graph and the value at the top of the graph indicate the average value of n = 3 for each group, and the error bar indicates the standard deviation. The number next to each group of proteins is the amount of DNA introduced (eg, HNF-4a 0.05 introduced 0.05 g of HNF-4α expression plasmid). *: Significantly different from control (ρ <0. 05), #: HNF—4 Significantly different from 4 α 0.05 (ρ <0.05), $: HNF-4 a 0. 05 + Significantly different from RSKB 0.25 + MAPK11 0. 05 (p <0. 05). (Example 4)
[図 11]PEPCK A AF1プロモーターを用いたレポーターアツセィの結果を示す図で ある。 AF1を欠失している PEPCK遺伝子プロモーターを用いた結果、 HNF— 4 a による転写活性の促進は、 HNF— 4 aを導入した細胞においても、 HNF— 4 a、 RS KBおよび MAPK11を共発現させた細胞にお!、ても認められなかった。この結果は 、 HNF— 4 αによる転写活性力 SPEPCK遺伝子プロモーター領域内の AF1に依存 することを示す。縦軸はコントロール群（PEPCK A AF1プロモーター ZpGL3のみ 導入)の転写活性を 1. 00とした時の倍数である。グラフの高さおよびグラフ上段の値 は各群の n= 3の平均値を、エラーバーは標準偏差を示す。各群の蛋白質の横の数 字は導入 DNA量である（例： HNF— 4 α 0. 05は、 0. 05 gの HNF— 4 α発現プ ラスミドを導入した)。 * :コントロールに対して有意差あり（ρく 0. 05)、 # : HNF— 4 a 0. 05に対して有意差あり（p< 0. 05)、 $： HNF— 4 α 0. 2に対して有意差あ り（ρ< 0. 05)。（実施例 4)  FIG. 11 shows the results of reporter assembly using PEPCK A AF1 promoter. As a result of using the PEPCK gene promoter lacking AF1, HNF-4a promotes transcriptional activity by co-expressing HNF-4a, RS KB and MAPK11 even in cells into which HNF-4a has been introduced. It was not recognized even in the cells! This result shows that HNF-4α is dependent on AF1 within the promoter region of the SPEPCK gene promoter. The vertical axis is a multiple when the transcriptional activity of the control group (in which only PEPCK A AF1 promoter ZpGL3 is introduced) is 1.00. The height of the graph and the value at the top of the graph indicate the average value of n = 3 for each group, and the error bar indicates the standard deviation. The number next to each group of proteins is the amount of DNA introduced (eg, HNF-4α 0.05 was introduced with 0.05 g of HNF-4α expression plasmid). *: Significantly different from the control (ρ 0. 05), #: Significantly different from HNF— 4 a 0. 05 (p <0. 05), $: HNF — 4 α 0.2 There is a significant difference (ρ <0.05). (Example 4)
[図 12]HepG2細胞における PEPCK AF1プロモーターを用いたレポーターアツセ ィの結果を示す図である。本結果は、 HepG2細胞に pMMGRをトランスフエクシヨン することによりダルココルチコイドレセプターを発現させた条件下で得た。 HNF-4 a による転写活性は、 MAPK11で活性化された RSKBによって促進した。 MAPK11 阻害剤 SB203580を添加すると、 MAPK11と RSKBの共発現による HNF— 4 aの 転写活性の促進が認められな力つた。この結果は、 RSKBの活性ィ匕が HNF— 4 α による転写活性に重要であることを示す。縦軸は HNF— 4ひ 0. 5処理群の転写活 性を 1. 00とした時の倍数である。グラフの高さおよびグラフ上段の値は各群の n= 3 の平均値を、エラーバーは標準偏差を示す。各群の蛋白質の横の数字は導入 DNA 量である（例： HNF— 4 a 0. 5は、 0. 5 gの HNF— 4 a発現プラスミドを導入した )。 * : HNF— 4 α 0. 5に対して有意差あり（ρく 0. 05)、 # : HNF— 4 α 0. 5 +R SKB 0. 25に対して有意差あり（pく 0. 05)、 $： HNF— 4 α 0. 5 + RSKB 0. 5 に対して有意差あり（p< 0. 05)、 + : RSKB 0. 5に対して有意差あり（p< 0. 05)、 ¥ : HNF-4 a 0. 5 +RSKB 0. 5 + MAPK11 0. 5に対して有意差あり（pく 0 . 05) , + + : HNF-4 a 0. 5 +RSKB 0. 5 + MAPK11 0. 5 + SB203580に 対して有意差あり（p< 0. Οδ)。（実施例4) FIG. 12 shows the results of reporter assembly using the PEPCK AF1 promoter in HepG2 cells. This result was obtained under the condition that the darcocorticoid receptor was expressed by transfecting pMMGR into HepG2 cells. Transcriptional activity by HNF-4a was promoted by RSKB activated with MAPK11. When the MAPK11 inhibitor SB203580 was added, co-expression of MAPK11 and RSKB did not promote the transcriptional activity of HNF-4a. This result shows that the activity of RSKB is HNF-4 α It is important for transcriptional activity. The vertical axis is a multiple of the transfer activity of the HNF-4 and 0.5 treatment groups as 1.00. The height of the graph and the value at the top of the graph indicate the average value of n = 3 for each group, and the error bar indicates the standard deviation. The number next to each group of proteins is the amount of DNA introduced (eg, HNF-4a 0.5 introduced 0.5 g of HNF-4a expression plasmid). *: Significantly different from HNF— 4 α 0.5 (ρ 0. 05), #: Significantly different from HNF— 4 α 0.5 + R SKB 0.25 (p 0. 05) ), $: HNF— 4 α 0.5 + RSKB 0.5 (p <0.05), +: RSKB 0.5 (p <0.05) ¥: HNF-4 a 0. 5 + RSKB 0.5 + MAPK11 There is a significant difference from 0.5 (p 0.05), + +: HNF-4 a 0. 5 + RSKB 0.5 + MAPK11 Significantly different from 0.5 + SB203580 (p <0. Οδ). (Example 4 )
[図 13]RSKBによりそのキナーゼ活性依存的に PEPCK遺伝子の発現が亢進するこ とを示す図面である。野生型 RSKB (図中、 RSKB (WT)と表示する）と MAPK11を 共発現させた細胞において、これらを発現させな力 た細胞と比較して、内因性 PE PCK遺伝子の発現量が増加した。これに対して、キナーゼ活性を示さない不活性型 変異体 RSKB (RSKB (S 196A/S343A/T568A) )と MAPK11を共発現細胞さ せた細胞では、内因性 PEPCK遺伝子の発現量の増加は認められな力つた。一方、 内因性コントロールである GAPDH遺伝子の発現量は、いずれの細胞でもほぼ同等 であった。各遺伝子の発現は、細胞カゝら調製したトータル RNAを用いて RT— PCR により検出した。 PEPCK遺伝子由来の PCR産物は 575bpであり、 GAPDH遺伝子 由来の PCR産物は 209bpである。図中左側のレーンに示した 50bp DNAラダー（1 adder)は、電気泳動時の DNAのサイズマーカーである。（実施例 5)  FIG. 13 is a drawing showing that expression of PEPCK gene is enhanced by RSKB depending on its kinase activity. In cells co-expressing wild-type RSKB (shown as RSKB (WT) in the figure) and MAPK11, the expression level of endogenous PE PCK gene increased compared to cells that did not express them. In contrast, an inactive mutant RSKB (RSKB (S 196A / S343A / T568A)) that does not exhibit kinase activity and MAPK11 co-expressing cells showed an increase in the expression level of endogenous PEPCK gene. I was helped. On the other hand, the expression level of the endogenous control GAPDH gene was almost the same in all cells. The expression of each gene was detected by RT-PCR using the total RNA prepared by the cells. The PCR product derived from the PEPCK gene is 575 bp, and the PCR product derived from the GAPDH gene is 209 bp. The 50 bp DNA ladder (1 adder) shown in the left lane in the figure is a DNA size marker during electrophoresis. (Example 5)
[図 14]野生型 RSKB (図中、 RSKB (WT)と表示する）と MAPK11を共発現させた HepG2細胞、不活性型変異体RSKB (RSKB (S196AZS343AZT568A) )とM APK11を共発現させた HepG2細胞、およびこれらを発現させなかった細胞におけ る、各蛋白質の発現量を示す図である。上パネルは、各細胞における FLAG— RSK Bおよび MAPKl l— FLAGの発現を抗 FLAG抗体を用いたィムノブロット（i. b. ) により検出した結果を示す。上パネル中で、抗 FLAG抗体を用いたィムノブロット (i. b. )により検出された N末端 FLAGタグ野生型 RSKBおよび N末端 FLAGタグ不活 性型変異体 RSKBを示すバンドは!、ずれも、パネルの右の FLAG— RSKBの表示 により示した。中パネルは、リン酸化 RSKBを抗リン酸化 RSKB抗体により検出した結 果を示す。本抗リン酸化 RSKB抗体は、 RSKBのキナーゼ活性に重要な 360番目の セリンのリン酸ィ匕を認識する抗体である。中パネルに示したように、野生型 RSKBと M APK11を共発現させた HepG2細胞でみられた 360番目のセリンのリン酸化は、不 活性型変異体 RSKBと MAPK11を共発現させた HepG2細胞では検出されなかつ たことから、不活性型変異体 RSKBはキナーゼ活性が消失していることが明らかにな つた。下パネルは、コントロールである j8チューブリンの発現が各細胞でほぼ同等で あったことを示す。（実施例 5) [FIG. 14] HepG2 cells co-expressed with wild-type RSKB (shown as RSKB (WT)) and MAPK11, inactive mutant RSKB (RSKB (S196AZS343AZT568A)) and HepG2 co-expressed with M APK11 FIG. 3 is a view showing the expression level of each protein in cells and cells in which these were not expressed. The upper panel shows the results of detecting the expression of FLAG-RSK B and MAPKll-FLAG in each cell by immunoblotting (ib) using an anti-FLAG antibody. In the upper panel, N-terminal FLAG tag wild type RSKB and N-terminal FLAG tag inactivation detected by immunoblotting (ib) using anti-FLAG antibody The band indicating the sexual variant RSKB! Is indicated by the FLAG-RSKB display on the right of the panel. The middle panel shows the results of detecting phosphorylated RSKB with anti-phosphorylated RSKB antibody. This anti-phosphorylated RSKB antibody recognizes the 360th serine phosphate that is important for the kinase activity of RSKB. As shown in the middle panel, phosphorylation of the 360th serine in HepG2 cells co-expressed with wild-type RSKB and M APK11 was observed in HepG2 cells co-expressed with the inactive mutant RSKB and MAPK11. From the fact that it was not detected, it became clear that the inactive mutant RSKB lost its kinase activity. The lower panel shows that the expression of the control j8 tubulin was similar in each cell. (Example 5)
[図 15]細胞内で HNF— 4 aと RSKBが結合することを示す図である。 FLAG— HNF -4 aと FLAG— RSKBを一過性共発現させた細胞力 調製した細胞ライセートに ぉ 、て、抗 HNF— 4 a抗体を用いた免疫沈降および抗 FLAG M2抗体を用いた 検出により、 FLAG-HNF-4 aを示すバンドおよび FLAG—RSKBを示すバンド がいずれも検出された (パネル A)。本結果は、 HNF— 4 α依存的に RSKBが共沈し たことを示す。パネル Aは、抗 HNF— 4 α抗体による免疫沈降試料 (i. p. )を抗 FLA G M2抗体でィムノブロット（i. b. )した結果を示す。パネル Bは、細胞ライセートを抗 FLAG M2抗体でィムノブロット（i. b. )した結果を示す。図中、 +および一はそれ ぞれ各発現プラスミドの有無を示す。図の右側の数字は分子量を示す。本実験は各 群 n= 2で実施した。（実施例 6)  FIG. 15 is a diagram showing that HNF-4a and RSKB bind in cells. Cell force in which FLAG—HNF-4a and FLAG—RSKB were transiently co-expressed Prepared cell lysate was prepared by immunoprecipitation using anti-HNF-4a antibody and detection using anti-FLAG M2 antibody. A band indicating FLAG-HNF-4a and a band indicating FLAG—RSKB were both detected (Panel A). This result indicates that RSKB co-precipitated in a HNF-4α-dependent manner. Panel A shows the results of immunoblotting (i. B.) Of an immunoprecipitation sample (i. P.) With anti-HNF-4α antibody with anti-FLA G M2 antibody. Panel B shows the results of immunoblotting (i.b.) the cell lysate with anti-FLAG M2 antibody. In the figure, + and 1 indicate the presence or absence of each expression plasmid, respectively. The numbers on the right side of the figure indicate molecular weight. This experiment was performed with n = 2 in each group. (Example 6)
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0055] 本明細書中で使用されている技術的および科学的用語は、別途定義されていない 限り、当業者により普通に理解されている意味を持つ。本明細書中では当業者に既 知の種々の方法が参照されている。以下、本発明について、発明の実施の態様をさ らに詳しく説明する。以下の詳細な説明は例示であり、説明のためのものに過ぎず、 本発明を何ら限定するものではない。  [0055] Technical and scientific terms used herein have meanings commonly understood by a person of ordinary skill in the art unless otherwise defined. Reference is made herein to various methods known to those skilled in the art. Hereinafter, embodiments of the present invention will be described in more detail. The following detailed description is exemplary and illustrative only and is not intended to limit the invention in any way.
[0056] 本明細書においては、アミノ酸を 1文字表記または 3文字表記することがある。また 、ペプチドとは、ペプチド結合または修飾されたペプチド結合により互いに結合して いる 2個またはそれ以上のアミノ酸を含む任意のペプチドを意味する。さらにペプチド は、オリゴマーと称する単離された若しくは合成の完全長オリゴペプチド等の短鎖ぺ プチド、並びに単離された若しくは合成の完全長ポリペプチドや単離された若しくは 合成の完全長蛋白質等の長鎖ペプチドをも意味する。 [0056] In the present specification, amino acids may be represented by one letter or three letters. Peptide also means any peptide comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. Peptide Short-chain peptides such as isolated or synthetic full-length oligopeptides, referred to as oligomers, and long chains such as isolated or synthetic full-length polypeptides and isolated or synthetic full-length proteins Also means peptide.
[0057] 本発明では、 HNF-4 aと RSKBが相互作用することをインシリコ（in silico)で予 測した。 RSKBがキナーゼであることから、本発明者らは、 HNF-4 aが RSKBとの 相互作用によりリン酸化されると予測した。  [0057] In the present invention, the interaction between HNF-4a and RSKB was predicted in silico. Since RSKB is a kinase, we predicted that HNF-4a would be phosphorylated by interaction with RSKB.
[0058] そして、 HNF— 4 aと RSKBが結合すること、 RSKBによって HNF— 4 aカ^ン酸 ィ匕されること、 HNF— 4 aは RSKBによりリン酸化されることで PEPCK遺伝子プロモ 一ター領域内の AF1への結合能が促進されること、および力かる促進により PEPCK 遺伝子の転写活性が亢進することを実証した。また、 RSKBによる HNF— 4 aのリン 酸化には、 RSKBの活性ィ匕が必要であることを明らかにした。  [0058] Then, HNF-4a and RSKB bind, HNF-4a cation is converted by RSKB, HNF-4a is phosphorylated by RSKB, and PEPCK gene promoter It was demonstrated that the ability to bind to AF1 within the region was promoted, and that the transcriptional activity of the PEPCK gene was enhanced by vigorous promotion. It was also clarified that the activity of RSKB is required for phosphorylation of HNF-4a by RSKB.
[0059] 本実証結果から、 RSKBによる HNF— 4 aのリン酸化力 PEPCK遺伝子プロモー ター領域内の AF1への HNF— 4 aの結合の促進を介した、 PEPCK遺伝子の転写 活性の正の調節に関与することが判明した。  [0059] Based on the results of this demonstration, the phosphorylation power of HNF-4a by RSKB can be used to positively regulate the transcriptional activity of the PEPCK gene through promoting the binding of HNF-4a to AF1 in the PEPCK gene promoter region. Turned out to be involved.
[0060] PEPCK遺伝子は糖新生律速酵素であると考えられる PEPCKをコードする遺伝子 である。このことから、 RSKBによるリン酸化により HNF— 4 αの PEPCK遺伝子のプ 口モーター領域内の AF1への結合能が促進され、その結果、 PEPCK遺伝子の転 写活性が亢進することにより、該遺伝子の発現が促進され、さらには糖新生が亢進す ると考えることができる。  [0060] The PEPCK gene is a gene encoding PEPCK which is considered to be a gluconeogenic rate-determining enzyme. From this, phosphorylation by RSKB promotes the ability of HNF-4α to bind to PE1 in the motor region of PEPCK gene, and as a result, the transcriptional activity of PEPCK gene is enhanced, thereby It can be considered that expression is promoted and gluconeogenesis is enhanced.
[0061] PEPCK遺伝子の転写はホルモンによって制御されており、ダルココルチコイドによ り促進され、インスリンにより抑制される（非特許文献 8および 27)。インスリンは PEPC K遺伝子プロモーターにおいて転写調節因子である LIP (肝特異的転写抑制蛋白質 、 liver enriched transcriptional inhibitory protein)を介して PEPCK遺 子の転写を抑制することで肝臓での糖新生を調節している (非特許文献 28)。また、 インスリン刺激が PEPCK遺伝子プロモーターに直接作用して転写活性を抑制すると いう報告もある（非特許文献 6および 37)。  [0061] Transcription of the PEPCK gene is regulated by hormones, promoted by darcocorticoids, and repressed by insulin (Non-patent Documents 8 and 27). Insulin regulates gluconeogenesis in the liver by suppressing transcription of PEPCK gene through LIP (liver-enriched transcriptional inhibitory protein), a transcriptional regulator in PEPC K gene promoter. (Non-patent document 28). There are also reports that insulin stimulation acts directly on the PEPCK gene promoter to suppress transcriptional activity (Non-patent Documents 6 and 37).
[0062] したがって、正常の肝臓では PEPCK遺伝子の発現はインスリンによって抑制的に 支配されている力 何らかの理由で PEPCK遺伝子の過剰発現が持続すると、肝臓 での糖新生が亢進して高血糖となり、さらにインスリン耐性になると糖尿病を発症する と考えられる。実際、インスリン非依存性糖尿病患者の PEPCKによる糖新生能は、 正常人と比較して約 3倍亢進しており、この糖新生能は患者の空腹時血漿中ダルコ ース濃度と相関して 、ることが報告されて 、る (非特許文献 26)。 [0062] Therefore, in normal liver, the expression of PEPCK gene is repressively controlled by insulin. For some reason, if overexpression of PEPCK gene persists, It is thought that diabetes develops when gluconeogenesis is increased and hyperglycemia occurs and insulin resistance is further developed. In fact, gluconeogenesis by PEPCK in non-insulin-dependent diabetic patients is about 3 times higher than that in normal individuals, and this gluconeogenesis is correlated with the fasting plasma glucose concentration in patients. (Non-patent Document 26).
[0063] PEPCK遺伝子の過剰発現には、 RSKBによるリン酸化により HNF— 4 aの PEPC K遺伝子プロモーター領域内の AF1への結合能が促進することと、それによる PEP CK遺伝子の転写活性の亢進が関与すると考えられる。  [0063] Overexpression of the PEPCK gene includes phosphorylation by RSKB, which promotes the ability of HNF-4a to bind to AF1 in the PEPC K gene promoter region and thereby increases the transcriptional activity of the PEP CK gene. It is thought to be involved.
[0064] PEPCK遺伝子プロモーターには、 GRU (ダルココルチコイド応答ユニット、 glucoc orticoid response unit)と呼ばれる領域が存在し、該領域内には AF1、 AF2 (ァ クセサリー因子結合部位 2、 accessory factor binding site 2)、 GR1, 2 (グル ココノレチコイドレセプター結合咅 |5位 1 , 2、 glucocorticoid receptor binding site 1, 2)、および CRE (サイクリック AMP応答配列、 cAMP responive element)が 存在する。 PEPCK遺伝子を効率よく発現させるために、これらの部位が必要と考え られている。  [0064] The PEPCK gene promoter has a region called GRU (Glucocorticoid Response Unit), and AF1, AF2 (accessory factor binding site 2, accessory factor binding site 2) in the region , GR1, 2 (glucocorticoreticoid receptor binding 咅 | positions 5 and 1, glucocorticoid receptor binding site 1, 2), and CRE (cyclic AMP response element, cAMP responive element). These sites are considered necessary for efficient expression of the PEPCK gene.
[0065] PEPCK遺伝子プロモーターに結合する転写因子のうち、 RSKBによりリン酸化さ れて該プロモーターに結合する転写因子として、 CREB (サイクリック AMP応答配列 結合蛋白賓、 cAMP responsive element binding protein)が知られている。 CREBは PEPCK遺伝子プロモーターの CREに結合する転写因子である（非特許 文献 19)。 CREは PEPCK遺伝子プロモーターに存在し、 PEPCK遺伝子発現を正 に調節している領域である（非特許文献 18)。 CREに結合して PEPCK遺伝子を正 に調節する遺伝子として、 CREBの他に、 CZEBP (CCAATZェンハンサー結合 蛋白質、 CCAATZenhancer binding protein)が報告されて!、る（非特許文献 19)。  [0065] Among transcription factors that bind to the PEPCK gene promoter, CREB (cyclic AMP response element binding protein, cAMP responsive element binding protein) is known as a transcription factor that is phosphorylated by RSKB and binds to the promoter. ing. CREB is a transcription factor that binds to CRE of PEPCK gene promoter (Non-patent Document 19). CRE is a region that exists in the PEPCK gene promoter and positively regulates PEPCK gene expression (Non-patent Document 18). In addition to CREB, CZEBP (CCAATZenhancer binding protein) has been reported as a gene that binds to CRE and positively regulates the PEPCK gene (Non-patent Document 19).
[0066] RSKBは CREBをリン酸化し、リン酸化された CREBは CREを有するプロモーター の転写活性を亢進する (非特許文献 11)。したがって、 RSKBが活性化された場合、 HNF-4 aを介することなく CREBがリン酸化され、 PEPCK遺伝子プロモーターの CREに CREBが結合し、 PEPCK遺伝子の発現が促進される可能性がある。  [0066] RSKB phosphorylates CREB, and phosphorylated CREB enhances the transcriptional activity of a promoter having CRE (Non-patent Document 11). Therefore, when RSKB is activated, CREB is phosphorylated without passing through HNF-4a, and CREB binds to CRE of PEPCK gene promoter, which may promote PEPCK gene expression.
[0067] し力しながら、生体内で実際に PEPCK遺伝子プロモーターの CREを調節している のは CREBではなく CZEBPであるという報告がある（非特許文献 20〜23)。 C/E BPが RSKBによりリン酸ィ匕されると 、う報告はな!/、。 [0067] However, it actually regulates the CRE of the PEPCK gene promoter in vivo. There is a report that CZEBP is not CREB (Non-patent Documents 20 to 23). When C / E BP is phosphorylated by RSKB, there is no report!
[0068] HNF-4 aが結合する AF1は CREと比較して、 PEPCK遺伝子の発現に、より大 きな影響を与える。具体的には、 AF1の置換や欠失により、 PEPCK遺伝子プロモー ターの転写活性は野生型 PEPCK遺伝子プロモーターの約 4分の 1以下に低下する (非特許文献 24)。これに対し、 CREの置換や欠失によっては、 PEPCK遺伝子プロ モーターの転写活性は野生型 PEPCK遺伝子プロモーターの 2分の 1程度にまでし か低下しな!ヽ (非特許文献 25)。  [0068] AF1 to which HNF-4a binds has a greater effect on the expression of the PEPCK gene than CRE. Specifically, substitution or deletion of AF1 reduces the transcriptional activity of the PEPCK gene promoter to about one-fourth that of the wild-type PEPCK gene promoter (Non-patent Document 24). On the other hand, depending on CRE substitution and deletion, the transcriptional activity of the PEPCK gene promoter can be reduced to about half that of the wild-type PEPCK gene promoter! (Non-patent Document 25).
[0069] このように、 HNF-4 aが結合する AF1は、 CREと比較して、 PEPCK遺伝子発現 調節においてより重要な因子であると考えることができる。  [0069] Thus, AF1 to which HNF-4a binds can be considered to be a more important factor in the regulation of PEPCK gene expression compared to CRE.
[0070] また、 PEPCK遺伝子プロモーター領域内の転写に関わる部位のうち AF1を欠失 させると、 RSKBによる PEPCK遺伝子プロモーターの転写活性の促進が認められな いことを本発明において明らかにした。具体的には、細胞内で RSKB、 RSKBを活 性化する MAPK11、および PEPCK遺伝子プロモーター領域内の AF1を欠失して いる PEPCK A AF1プロモーターを有するレポーター遺伝子を発現させたところ、 該レポーター遺伝子の転写活性はほとんど促進されな力つた（実施例 4および図 11 参照)。  [0070] It has also been clarified in the present invention that, when AF1 is deleted from the transcription-related site within the PEPCK gene promoter region, RSKB does not promote the transcriptional activity of the PEPCK gene promoter. Specifically, when a reporter gene having RSKB, MAPK11 that activates RSKB, and PEPCK A AF1 promoter lacking AF1 in the PEPCK gene promoter region was expressed in cells, the reporter gene Transcriptional activity was hardly promoted (see Example 4 and FIG. 11).
[0071] PEPCK遺伝子プロモーター領域内の転写に関わる部位のうち AF1には、 RSKB によって HNF— 4 aカ^ン酸化されることにより HNF— 4 aが結合し、その結果、 PE PCK遺伝子プロモーターの転写活性が促進される。 AF1を欠失させると、 RSKBに よって HNF— 4 aがリン酸化されても、 HNF— 4 aは PEPCK遺伝子プロモーター に結合できない。そのため、 RSKBによる PEPCK遺伝子プロモーターの転写活性 が促進されな力つたと考えることができる。  [0071] AFNF of the PEPCK gene promoter region involved in transcription binds to HNF-4a by being converted to HNF-4a by RSKB, resulting in transcription of the PE PCK gene promoter. Activity is promoted. When AF1 is deleted, even if HNF-4a is phosphorylated by RSKB, HNF-4a cannot bind to the PEPCK gene promoter. Therefore, it can be considered that the transcription activity of the PEPCK gene promoter by RSKB was not promoted.
[0072] このように、 PEPCK遺伝子プロモーター領域内の転写に関わる部位のうち AF1の みを欠失させたことにより、 RSKBによる PEPCK遺伝子プロモーターの転写活性の 促進が認められなかったことから、生体内においては RSKBによる PEPCK遺伝子の 発現調節は、主に HNF— 4 aの AF1への結合を介していると考えることができる。  [0072] As described above, since deletion of only AF1 in the site involved in transcription in the PEPCK gene promoter region did not promote the transcriptional activity of the PEPCK gene promoter by RSKB, The regulation of PEPCK gene expression by RSKB can be thought to be mainly mediated by the binding of HNF-4a to AF1.
[0073] RSKBは上流のキナーゼである p38により活性ィ匕されキナーゼ活性を示す。活性 化された RSKBは CREBをリン酸化することが知られている。したがって、 RSKBの 活性ィ匕を、 P38の活性ィ匕および CREBのリン酸ィ匕を指標にして判定することができる 。糖尿病モデル動物の肝臓では、 p38のリン酸ィ匕型 (活性型 p38)は正常個体のそ れに比べ約 2. 5〜3倍増加しており、 CREBのリン酸化型も約 2倍増加していたとの 報告がある（非特許文献 29および 30)。 [0073] RSKB is activated by p38, an upstream kinase, and exhibits kinase activity. Activity RSKB is known to phosphorylate CREB. Therefore, the activity of RSKB can be determined using the activity of P38 and the phosphate of CREB as indicators. In the liver of diabetic model animals, the p38 phosphoric acid type (active p38) is about 2.5 to 3 times higher than that of normal individuals, and the phosphorylated form of CREB is also about 2 times higher. Have been reported (Non-Patent Documents 29 and 30).
[0074] これら報告から、本発明者らは糖尿病病態下の肝臓では、酸化ストレス等により P3 8キナーゼが活性ィ匕し、続いて RSKBが活性ィ匕すると考えた。その結果、 HNF-4 aがリン酸化されることにより HNF— 4 a力PEPCK遺伝子プロモーターの AF1に結 合し、 PEPCK遺伝子の発現が促進され、ひいては肝臓における糖新生が促進され る。 [0074] From these reports, the present inventors considered that in the liver under diabetic condition, P 38 kinase is activated by oxidative stress and the like, and then RSKB is activated. As a result, HNF-4a is phosphorylated and bound to AF1 of the PEPCK gene promoter, and the expression of the PEPCK gene is promoted, which in turn promotes gluconeogenesis in the liver.
[0075] PEPCKの活性は mRNAレベルで調節されており、また、 PEPCKの mRNA量、 酵素活性、糖新生能および血糖値上昇はそれぞれ良好な相関を示している。具体 的には、肝細胞を用いた実験において、 PEPCK遺伝子の mRNA量が 2倍増加する と酵素活性は 2. 8倍増加し、さらに糖新生能は 2. 1倍亢進することが報告されている (非特許文献 6)。マウスでは肝臓での糖新生能が約 2. 5倍亢進すると、血糖値は 2 倍上昇し (非特許文献 7)、ヒトでは血糖値が 2. 5倍高い糖尿病患者では PEPCKに よる糖新生能が正常人より 3倍亢進して 、る (非特許文献 26)。  [0075] PEPCK activity is regulated at the mRNA level, and PEPCK mRNA level, enzyme activity, gluconeogenic potential, and blood glucose level increase are well correlated. Specifically, in experiments using hepatocytes, it was reported that when the amount of PEPCK gene mRNA was increased 2-fold, enzyme activity increased 2.8-fold and gluconeogenesis increased 2.1-fold. (Non-patent document 6). In mice, when the gluconeogenic potential in the liver is increased by about 2.5 times, the blood glucose level rises by a factor of 2 (Non-patent Document 7), and in humans, the glucose gluconeogenic potential by PEPCK is 2.5 times higher in humans. Is 3 times higher than normal (Non-patent Document 26).
[0076] 本発明者らは、 RSKBの活性ィ匕による血糖値上昇の程度を次のように考えた。 RS KBによる HNF— 4 aのリン酸ィ匕が CREBのリン酸ィ匕と同程度であると仮定すると、糖 尿病病態時の RSKBによる肝臓中 HNF— 4 aのリン酸ィ匕は正常時よりも 2倍程度亢 進していると推定できる。なぜなら、上記のように、糖尿病モデル動物の肝臓では、 C REBのリン酸化型が約 2倍増加しており、 CREBのリン酸化には p38により活性化さ れた RSKBが関与しているからである（非特許文献 29および 30)。また、後述する実 施例に示すように、 RSKBにより HNF— 4 αがリン酸化されることで、 HNF— 4 α力 S 関与する PEPCK遺伝子プロモーター活性は約 2〜3倍亢進した (実施例 4並びに図 9、 10および 12)。 PEPCK遺伝子のプロモーター活性を mRNA量に置き換えると、 糖尿病病態時では RSKBの活性ィ匕により PEPCK遺伝子の mRNA量は約 2〜3倍 増加して 、ると推定できる。 PEPCK遺伝子の mRNA量の増加は上記で述べたよう にそのまま酵素活性に反映され、糖新生能亢進、血糖値の上昇を招く。このことによ り、糖尿病病態時において、 RSKBによる HNF—4ひのリン酸ィ匕を阻害すれば、肝 臓中の PEPCK遺伝子の mRNA量は 2分の 1〜3分の 1程度に抑制されると考えるこ とができる。これにより PEPCKの酵素活性は 3分の 1程度、糖新生能は 2分の 1程度 に減少すると推定され、その結果、血糖値は病態時の半分程度に減少する。従って 糖尿病病態時において RSKBによる HNF— 4 aのリン酸化を阻害し、 PEPCK遺伝 子の遺伝子産物の産生を調節することにより、糖尿病の治療が可能となると考えた。 [0076] The present inventors considered the degree of increase in blood glucose level due to RSKB activity as follows. Assuming that the RSNF HNF-4a phosphate is similar to the CREB phosphate, the liver KBN HNF-4a phosphate is normal in the pathogenesis of diabetes It can be estimated that it is about 2 times higher. This is because, as described above, the phosphorylated form of CREB is increased about 2-fold in the liver of diabetic model animals, and RSKB activated by p38 is involved in phosphorylation of CREB. (Non-Patent Documents 29 and 30). In addition, as shown in the examples described later, phosphorylation of HNF-4α by RSKB increased the PEPCK gene promoter activity involving HNF-4α force S by about 2-3 times (Example 4). And Figures 9, 10 and 12). If the promoter activity of the PEPCK gene is replaced with the amount of mRNA, it can be estimated that the amount of mRNA of the PEPCK gene increases by about 2 to 3 times due to the activity of RSKB in diabetic conditions. The increase in the amount of PEPCK gene mRNA is as described above. It is directly reflected in the enzyme activity, leading to increased gluconeogenic potential and increased blood glucose level. As a result, in the diabetic state, inhibition of HNF-4 phosphophosphate by RSKB suppresses the mRNA level of PEPCK gene in the liver to about one-half to one-third. Can be considered. As a result, the enzyme activity of PEPCK is estimated to be reduced to about one third and the gluconeogenic potential is reduced to about one half. Therefore, it was considered that diabetes can be treated by inhibiting the phosphorylation of HNF-4a by RSKB and regulating the production of the gene product of PEPCK gene during diabetic conditions.
[0077] これら知見により達成した本発明の一態様は、 RSKBを用いることを特徴とする HN F-4 aのリン酸ィ匕方法に関する。本発明に係る HNF— 4 aのリン酸ィ匕方法は、 RS KBと HNF— 4 aを、これらの相互作用を可能にする条件下で共存させることにより 実施できる。 [0077] One embodiment of the present invention achieved by these findings relates to a method for phosphorylating HNF-4a, characterized by using RSKB. The HNF-4a phosphate method according to the present invention can be carried out by allowing RS KB and HNF-4a to coexist under conditions that allow their interaction.
[0078] また、本発明にお 、て、 RSKBを有効量含む HNF— aのリン酸ィ匕阻害剤を提供で きる。  [0078] Further, in the present invention, an HNF-a phosphate inhibitor containing an effective amount of RSKB can be provided.
[0079] 「RSKBと HNF— 4 aの相互作用」とは、 RSKBと HNF— 4 aが直接的に関連する ことを意味する。 RSKBと HNF— 4 aの直接的な関連には、 RSKBと HNF— 4 aが 結合する反応、および、該結合の結果 RSKBにより HNF— 4 αがリン酸ィヒされる反 応が含まれる。「RSKBと HNF— 4 αの相互作用」においては、まず RSKBが HNF 4 aと結合する反応が起こり、その結果、 RSKBの作用により HNF— 4 aカ^ン酸 化される反応が起こる。すなわち、 RSKBと HNF— 4 aが結合する反応を RSKBと H NF— 4 αの相互作用ということができるし、また、該結合反応の結果 RSKBの作用に より HNF— 4 aカ^ン酸化される反応を RSKBと HNF— 4 aの相互作用ということが できる。  [0079] “RSKB and HNF-4a interaction” means that RSKB and HNF-4a are directly related. The direct association between RSKB and HNF-4a includes a reaction in which RSKB and HNF-4a bind, and a reaction in which HNF-4α is phosphorylated by RSKB as a result of the binding. In the “interaction between RSKB and HNF-4α”, the reaction of RSKB first binds to HNF4a, and as a result, the reaction of HNF-4a cation is caused by the action of RSKB. That is, the reaction of RSKB and HNF-4a binding can be called the interaction of RSKB and HNF-4α, and as a result of the binding reaction, the reaction of RSKB causes HNF-4a to be oxidized. This reaction can be called the interaction between RSKB and HNF-4a.
[0080] 「RSKBと HNF— 4 aの結合」とは、 RSKBと HNF— 4 a力 複合体を形成するよう に、水素結合、疎水結合または静電的相互作用等の非共有結合により近接すること を意味する。ここでの結合とは、 RSKBと HNF— 4 aがその一部分において結合す れば足りる。例えば、 RSKBまたはその一部と、 HNF— 4 αまたはその一部との結合 を意味する。また、 RSKBと HNF— 4 αにより形成される複合体には、これら蛋白質 とは別種の蛋白質が含まれて!/、てもよ!/、。 [0081] 「RSKBによる HNF— 4 aのリン酸化」とは、 RSKBのキナーゼ活性により、 HNF -4 aのセリン、スレオ-ンまたはチロシン残基のヒドロキシル基に ATPの γ —リン酸 基が転移され、その結果、リン酸基が結合した HNF— 4 α蛋白質が生成する反応を 意味する。「RSKBのキナーゼ活性」とは、 RSKB力 基質となる他の蛋白質 (以下、 基質蛋白質と称することがある）と結合し、該基質蛋白質中のアミノ酸残基のヒドロキ シル基にアデノシン三リン酸 (ATP)の γ —リン酸基を転移させる反応を触媒すること により、該基質蛋白質をリン酸化する機能を意味する。「基質」とは、酵素によって触 媒作用を受ける化合物または分子を意味する。 [0080] “The bond between RSKB and HNF-4a” is closer to non-covalent bonds such as hydrogen bonds, hydrophobic bonds, or electrostatic interactions to form RSKB and HNF-4a force complexes. Means that. In this case, the bond is sufficient if RSKB and HNF-4a are bonded in part. For example, it means a bond between RSKB or part thereof and HNF-4α or part thereof. In addition, the complex formed by RSKB and HNF-4α contains a different type of protein! /, Or even! /. [0081] “phosphorylation of HNF-4a by RSKB” refers to the transfer of the γ-phosphate group of ATP to the hydroxyl group of the serine, threonine or tyrosine residue of HNF-4a by the kinase activity of RSKB. As a result, it means a reaction in which an HNF-4α protein having a phosphate group bound thereto is produced. “RSKB kinase activity” refers to an RSKB force that binds to another protein (hereinafter sometimes referred to as a substrate protein) and binds to the hydroxyl group of an amino acid residue in the substrate protein with adenosine triphosphate ( It means a function of phosphorylating the substrate protein by catalyzing a reaction of transferring the γ-phosphate group of (ATP). “Substrate” means a compound or molecule that is catalyzed by an enzyme.
[0082] RSKBと HNF— 4 aの相互作用を可能にする条件は、インビトロおよびインビボの いずれの条件であってもよい。すなわち、本発明に係る HNF— 4 αのリン酸ィ匕方法 は、インビトロおよびインビボの
Figure imgf000024_0001
、ても実施できる。 [0082] The conditions allowing the interaction between RSKB and HNF-4a may be any of in vitro and in vivo conditions. That is, the HNF-4α phosphate method according to the present invention is used in vitro and in vivo.
Figure imgf000024_0001
However, it can also be implemented.
[0083] 本発明に係る HNF— 4 aのリン酸ィ匕方法は、好ましくは、生体外試料または非ヒト 哺乳動物において実施することができる。「生体外試料」とは、動物、例えば哺乳動 物から調製された細胞や組織、動物由来の培養細胞、並びに前記細胞、組織およ び培養細胞から調製された蛋白質や遺伝子を含む溶液等の試料を意味する。細胞 として、好ましくは肝臓細胞および肝臓細胞株を例示できる。「非ヒト哺乳動物」とは、 ヒト以外の哺乳動物、例えばマウス、ラット、ゥサギ、ィヌ、ャギ等の哺乳動物を意味す る。好ましくはマウスが使用される。  [0083] The HNF-4a phosphate method according to the present invention can be preferably performed in an in vitro sample or a non-human mammal. “In vitro sample” refers to cells and tissues prepared from animals such as mammals, cultured cells derived from animals, and solutions containing proteins and genes prepared from the cells, tissues and cultured cells. Means sample. Preferred examples of cells include liver cells and liver cell lines. “Non-human mammal” means mammals other than human, for example, mammals such as mouse, rat, rabbit, nu, goat and the like. Preferably a mouse is used.
[0084] 本発明に係る HNF— 4 aのリン酸化方法は、具体的には、 HNF— 4 aを発現して いる細胞に RSKBを発現させること、または細胞において RSKBと HNF— 4 aを共 発現させることにより実施できる。あるいは、 RSKBとHNF— 4 Q；を、例えば試験管や マルチウエルプレート内で共存させることにより、 HNF— 4 aのリン酸化方法を実施 できる。  [0084] The method for phosphorylating HNF-4a according to the present invention specifically includes expressing RSKB in a cell expressing HNF-4a, or coexisting RSKB and HNF-4a in the cell. It can be implemented by expressing. Alternatively, the phosphorylation method of HNF-4a can be performed by coexisting RSKB and HNF-4Q; for example, in a test tube or a multiwell plate.
[0085] 本発明に係る HNF— 4 aのリン酸化方法を利用することにより、 RSKBと HNF— 4 aを共存させることを特徴とする HNF— 4 aのリン酸ィ匕系を構築できる。本発明に係 る HNF— 4 aのリン酸化系は、インビトロおよびインビボのいずれの系であってもよい 。 RSKBによる HNF— 4ひのリン酸ィ匕系は、好ましくは、生体外試料または非ヒト哺 乳動物を利用した系であることができる。 [0086] 本発明に係る HNF— 4 aのリン酸化系は、具体的には、 HNF— 4 aを発現してい る細胞に RSKBを発現させること、または細胞において RSKBと HNF— 4 aを共発 現させることにより構築できる。あるいは、 RSKBとHNF— 4 Q；を、例えば試験管やマ ルチウエルプレート内で共存させてリン酸化させることにより、 HNF-4 aのリン酸ィ匕 系を構築できる。 [0085] By using the phosphorylation method of HNF-4a according to the present invention, it is possible to construct an HNF-4a phosphate system characterized by coexistence of RSKB and HNF-4a. The phosphorylation system of HNF-4a according to the present invention may be either in vitro or in vivo. The HNF-4 phosphate system by RSKB can preferably be a system utilizing in vitro samples or non-human mammals. [0086] Specifically, the phosphorylation system of HNF-4a according to the present invention specifically allows RSKB to be expressed in cells expressing HNF-4a, or allows RSKB and HNF-4a to be co-expressed in cells. It can be constructed by expressing it. Alternatively, the phosphorylation system of HNF-4a can be constructed by phosphorylating RSKB and HNF-4Q; for example, in a test tube or a multiwell plate.
[0087] RSKBの発現および HNF— 4 aの発現は、それぞれ RSKBをコードする遺伝子を 含む適当なベクターおよび HNF— 4 aをコードする遺伝子を含む適当なベクターを 用いて慣用の遺伝子工学的手法でこれらを細胞にトランスフエクシヨンすることにより 達成できる。 RSKBによる HNF— 4 aのリン酸化は、細胞内において RSKBと HNF -4 aが相互作用することにより達成される。  [0087] RSKB expression and HNF-4a expression can be performed by a conventional genetic engineering technique using an appropriate vector containing a gene encoding RSKB and an appropriate vector containing a gene encoding HNF-4a, respectively. These can be achieved by transfecting the cells. Phosphorylation of HNF-4a by RSKB is achieved by the interaction of RSKB and HNF-4a in the cell.
[0088] 発現に用いる細胞は、蛋白質発現に一般的に使用されている細胞が使用できる。  [0088] As a cell used for expression, a cell generally used for protein expression can be used.
好ましくは、真核細胞を用いる。真核細胞は、真核生物から調製した細胞、初代培養 細胞、および培養細胞株のいずれでもあり得る。好ましくは哺乳動物由来の培養細 胞株、より好ましくはヒト由来の培養細胞株を用いる。また、好ましくは、哺乳動物の 肝臓由来の培養細胞株、より好ましくはヒト肝臓由来の培養細胞株を用いる。ヒト由来 の培養細胞株として、 HeLa細胞 (子宮頸癌由来細胞株）および HepG2細胞 (ヒト肝 臓癌由来細胞株）を好ましく例示できる。  Preferably, eukaryotic cells are used. Eukaryotic cells can be any of cells prepared from eukaryotes, primary cultured cells, and cultured cell lines. Preferably, a mammalian cell line, more preferably a human cell line is used. Preferably, a cultured cell line derived from a mammalian liver, more preferably a cultured cell line derived from a human liver is used. Preferred examples of human-derived cultured cell lines include HeLa cells (cervical cancer-derived cell lines) and HepG2 cells (human liver cancer-derived cell lines).
[0089] RSKBによる HNF— 4 αのリン酸ィ匕の検出は、自体公知の蛋白質リン酸ィ匕測定方 法を用いて、 RSKBと HNF— 4 aを接触させてリン酸ィ匕反応を行った後にリン酸ィ匕 型 HNF— 4ひを測定することにより実施できる。リン酸ィ匕型 HNF— 4ひの検出は、例 えば、リン酸ィ匕型 HNF— 4ひに対する抗体を用いてウェスタンブロッテイングにより実 施できる。また、リン酸ィ匕型 HNF— 4ひの検出は、リン酸化反応に放射性同位体標 識した ATP、例えば [ y 32 P] ATPを用いて、該反応の結果 HNF— 4 aのアミノ酸 残基に転移された [ γ—32P]の放射活性を測定することにより実施できる。具体的に は、後述する実施例 2を参照して、 RSKBによる HNF— 4 aのリン酸ィ匕の検出を実施 できる。 [0089] Detection of HNF-4α phosphate by RSKB was carried out by contacting RSKB with HNF-4a using a well-known protein phosphate assay method. After that, it can be carried out by measuring phosphoric acid type HNF-4. The detection of phosphate-type HNF-4 can be performed, for example, by Western blotting using an antibody against phosphate-type HNF-4. In addition, detection of phosphoric acid-type HNF-4 was performed using ATP labeled with a radioisotope for phosphorylation, such as [y 32 P] ATP. As a result of the reaction, amino acid residues of HNF-4 a It can be carried out by measuring the radioactivity of [γ- 32 P] transferred to. Specifically, with reference to Example 2 described later, detection of HNF-4a phosphate by RSKB can be performed.
[0090] RSKBおよび HNF— 4 αはヒト由来の蛋白質であることが好ましいが、該ヒト由来の 蛋白質と同質の機能を有し、かつ配列相同性を有する哺乳動物由来の蛋白質、例 えばマウス、ゥマ、ヒッジ、ゥシ、ィヌ、サル、ネコ、ラットまたはゥサギ等に由来する蛋 白質であることができる。また、 RSKBおよび HNF— 4 aをそれぞれコードする遺伝 子は、ヒト由来の遺伝子であることが好ましいが、該ヒト由来の蛋白質と同質の機能を 有しかつ配列相同性を有する哺乳動物由来の蛋白質をコードする遺伝子であれば、 例えばマウス、ゥマ、ヒッジ、ゥシ、ィヌ、サル、ネコ、ラットまたはゥサギ等に由来する 遺伝子であることができる。 RSKBの機能として、例えば、キナーゼ活性を挙げること ができる。 HNF— 4 αの機能として、例えば、 AF1との結合機能および PEPCK遺伝 子プロモーターの転写活性を促進する機能を挙げることができる。 [0090] RSKB and HNF-4α are preferably human-derived proteins, but mammal-derived proteins having the same functions as the human-derived proteins and having sequence homology, eg, For example, it can be a protein derived from mouse, horse, hidge, ushi, nu, monkey, cat, rat or rabbit. Further, the genes encoding RSKB and HNF-4a are preferably human-derived genes, but mammal-derived proteins having the same function and sequence homology as the human-derived proteins. Can be a gene derived from, for example, mouse, horse, hidge, ushi, inu, monkey, cat, rat or rabbit. Examples of RSKB functions include kinase activity. Examples of functions of HNF-4α include a function of binding to AF1 and a function of promoting the transcriptional activity of the PEPCK gene promoter.
[0091] RSKBをコードする遺伝子および RSKBとして、それぞれ配列番号 1に記載の塩基 配列で表されるヒト由来の遺伝子および配列番号 2に記載のアミノ酸配列で表される ヒト由来の蛋白質を好ましく例示できる。 RSKBをコードする遺伝子は上記例示した 遺伝子に制限されず、該遺伝子と配列相同性を有し、かつ該遺伝子コードする蛋白 質と同様の機能を有する蛋白質をコードする遺伝子である限りにおいていずれの遺 伝子も包含される。また、 RSKBは、上記例示した蛋白質に制限されず、該蛋白質と 配列相同性を有し、かつ該蛋白質と同様の機能を有する蛋白質である限りにおいて いずれの蛋白質も包含される。配列相同性は、通常、アミノ酸配列または塩基配列 の全体で 50%以上、好ましくは少なくとも 70%であることが適当である。より好ましく は 70%以上、さらに好ましくは 80%以上、さらにより好ましくは 90%以上、またさらに より好ましくは 95%以上であることが適当である。 RSKBの機能として、例えば、キナ ーゼ活性を挙げることができる。  [0091] Preferred examples of the RSKB-encoding gene and RSKB include the human-derived gene represented by the nucleotide sequence set forth in SEQ ID NO: 1 and the human-derived protein represented by the amino acid sequence set forth in SEQ ID NO: 2, respectively. . The gene encoding RSKB is not limited to the above-exemplified gene, and any gene may be used as long as it is a gene encoding a protein having sequence homology with the gene and having the same function as the protein encoding the gene. Denko is also included. RSKB is not limited to the above-exemplified proteins, and any protein is included as long as it has a sequence homology with the protein and has the same function as the protein. It is appropriate that the sequence homology is usually 50% or more, preferably at least 70% of the entire amino acid sequence or base sequence. More preferably, it is 70% or more, more preferably 80% or more, still more preferably 90% or more, and even more preferably 95% or more. Examples of RSKB functions include kinase activity.
[0092] HNF -4 aをコードする遺伝子および HNF— 4 aとして、それぞれ配列番号 3に 記載の塩基配列で表されるヒト由来の遺伝子および配列番号 4に記載のアミノ酸配 列で表されるヒト由来の蛋白質を好ましく例示できる。 HNF -4 aをコードする遺伝 子は上記例示した遺伝子に制限されず、該遺伝子と配列相同性を有し、かつ該遺伝 子コードする蛋白質と同様の機能を有する蛋白質をコードする遺伝子である限りにお いていずれの遺伝子も包含される。また、 HNF— 4 Q;は、上記例示した蛋白質に制 限されず、該蛋白質と配列相同性を有し、かつ該蛋白質と同様の機能を有する蛋白 質である限りにおいていずれの蛋白質も包含される。配列相同性は、通常、アミノ酸 配列または塩基配列の全体で 50%以上、好ましくは少なくとも 70%であることが適 当である。より好ましくは 70%以上、さらに好ましくは 80%以上、さらにより好ましくは 90%以上、またさらにより好ましくは 95%以上であることが適当である。 HNF-4 a の機能として、例えば、 AF1との結合機能および PEPCK遺伝子プロモーターの転 写活性を促進する機能を挙げることができる。 [0092] As a gene encoding HNF-4a and HNF-4a, a human-derived gene represented by the nucleotide sequence set forth in SEQ ID NO: 3 and the human represented by the amino acid sequence set forth in SEQ ID NO: 4, respectively Preferred examples are derived proteins. The gene encoding HNF-4a is not limited to the gene exemplified above, as long as it is a gene encoding a protein having sequence homology with the gene and having the same function as the protein encoded by the gene. In this regard, any gene is included. HNF-4Q; is not limited to the above-exemplified proteins, and any protein is included as long as it is a protein having sequence homology with the protein and having the same function as the protein. The Sequence homology is usually amino acids It is appropriate that the total sequence or base sequence is 50% or more, preferably at least 70%. More preferably, it is 70% or more, more preferably 80% or more, still more preferably 90% or more, and even more preferably 95% or more. Examples of the function of HNF-4a include a function of binding to AF1 and a function of promoting the transcriptional activity of the PEPCK gene promoter.
[0093] RSKBおよび HNF— 4 aは、これらをそれぞれ遺伝子工学的手法で発現させた細 胞ゃ生体試料力 調製したもの、無細胞系合成産物または化学合成産物であってよ く、あるいはこれらからさらに精製されたものであってもよい。また、 RSKBおよび HN F -4 aのうち少なくとも 1を遺伝子工学的手法で発現させた細胞を使用することもで きる。 [0093] RSKB and HNF-4a may each be a cell sample prepared by genetic engineering techniques, or may be a cell-free synthetic product or a chemical synthesis product. It may be further purified. In addition, cells in which at least one of RSKB and HNF-4a is expressed by a genetic engineering technique can also be used.
[0094] RSKBおよび HNF— 4 aは、その性質や機能に影響がない限りにお 、て、 N末端 側や C末端側に別種の蛋白質やポリペプチド等の標識物質を、直接的にまたはリン カーペプチド等を介して間接的に、遺伝子工学的手法等を用いて付加することがで きる。標識物質自体またはその機能を測定することにより、 RSKBと HNF— 4ひの結 合の検出や RSKBによる HNF— 4 aのリン酸ィ匕の検出を容易に実施できる。  [0094] RSKB and HNF-4a can be directly or phosphorylated with other types of labeling substances such as proteins and polypeptides on the N-terminal side and C-terminal side, as long as their properties and functions are not affected. It can be added indirectly via a carpeptide or the like using genetic engineering techniques. By measuring the labeling substance itself or its function, it is possible to easily detect the binding of RSKB and HNF-4 and the detection of phosphate of HNF-4a by RSKB.
[0095] 標識物質として、酵素類 (ダルタチオン S—トランスフェラーゼ、ホースラディッシュ パーォキシダーゼ、ァノレカリホスファターゼまたは 13ガラクトシダーゼ等）、タグべプチ ド類（His— tag、 Myc— tag、 HA -tag, FLAG— tagまたは Xpress— tag等）、蛍 光蛋白質類 (グリーン蛍光蛋白質、フルォレセインイソチオシァネート（fluoresceinis othiocyanate)またはフィコエリスリン（phycoerythrin)等）、マルトース結合蛋白質 、免疫グロブリンの Fc断片、またはピオチンを例示できるが、これらに限定されない。 あるいは放射性同位元素により標識ィ匕することができる。標識ィ匕するとき、 1種類の標 識物質を付加してもよ 、し、複数の標識物質を組合わせて付加することもできる。  [0095] As labeling substances, enzymes (Dartathione S-transferase, horseradish peroxidase, anolekali phosphatase or 13 galactosidase), tag peptides (His-tag, Myc-tag, HA-tag, FLAG) — Tag or Xpress— tag), fluorescent proteins (green fluorescent protein, fluoresceinis othiocyanate or phycoerythrin, etc.), maltose binding protein, immunoglobulin Fc fragment, Alternatively, piotin can be exemplified, but not limited thereto. Alternatively, it can be labeled with a radioisotope. When labeling is performed, one type of labeling substance may be added, or a plurality of labeling substances may be added in combination.
[0096] RSKBおよび HNF— 4 aをそれぞれコードする遺伝子は、例えば、各遺伝子の発 現が認められる適当な起源 (例えば、肝組織および肝組織由来の細胞）、あるいは、 ヒト cDNAライブラリーから、自体公知のクローユング方法等を用いて容易に取得で きる。これら遺伝子によりコードされる蛋白質は、例えば、それぞれの遺伝子を用いた 自体公知の遺伝子工学的手法により取得できる。具体的には、それぞれの遺伝子を 適当な発現ベクター DNA、例えば細菌プラスミド由来のベクター等に自体公知の遺 伝子工学的手法で導入し、該遺伝子を含有するベクターを得て、該ベクターを適当 な宿主細胞に導入にすることにより該遺伝子を発現する細胞を取得し、該細胞から 該遺伝子がコードする蛋白質を得ることができる。例えば、 RSKBは、 RSKBをコード する遺伝子を発現させた細胞のセルライセートから抗 RSKB抗体等を用いて得られ る免疫沈降物として調製できる。 HNF-4 aは、 HNF— 4 aをコードする遺伝子と抗 HNF -4 α抗体を用 V、て同様に調製できる。 [0096] The genes encoding RSKB and HNF-4a are, for example, from an appropriate origin (for example, liver tissue and cells derived from liver tissue) from which each gene is expressed, or from a human cDNA library. It can be easily obtained using a known clawing method or the like. Proteins encoded by these genes can be obtained by, for example, a known genetic engineering technique using each gene. Specifically, each gene An appropriate expression vector DNA, for example, a vector derived from a bacterial plasmid is introduced by a known genetic engineering technique to obtain a vector containing the gene, and the vector is introduced into an appropriate host cell. A cell expressing the gene can be obtained, and a protein encoded by the gene can be obtained from the cell. For example, RSKB can be prepared as an immunoprecipitate obtained from a cell lysate of a cell expressing a gene encoding RSKB using an anti-RSKB antibody or the like. HNF-4a can be similarly prepared using a gene encoding HNF-4a and an anti-HNF-4α antibody.
[0097] 本発明に係る HNF— 4 aのリン酸化剤、リン酸化方法、およびリン酸ィ匕系は、 HNF  [0097] The phosphorylating agent of HNF-4a, the phosphorylating method, and the phosphoric acid salt system according to the present invention include
4 aの機能解明や HNF— 4 aが関与する転写因子ネットワークについての研究、 および HNF— 4 aのリン酸ィ匕に起因する疾患、例えば糖尿病における HNF— 4 a や RSKBの関与についての分子レベルでの研究等に有用である。また、当該リン酸 化方法およびリン酸ィ匕系を用いて、 RSKBによる HNF— 4 aのリン酸化を阻害する 化合物の同定方法を構築することもできる。  Molecular level studies on the function of 4a, research on transcription factor networks involving HNF-4a, and the involvement of HNF-4a and RSKB in diseases caused by HNF-4a phosphate, such as diabetes Useful for research in Japan. In addition, a method for identifying a compound that inhibits phosphorylation of HNF-4a by RSKB can be constructed using the phosphorylation method and the phosphoric acid-containing system.
[0098] 本発明の別の一態様は、 RSKBによる HNF— 4 aのリン酸化阻害剤および阻害方 法に関する。本発明に係る HNF— 4 aのリン酸化阻害剤および阻害方法は、 RSKB 活性を阻害すること、あるいは、 RSKBと HNF— 4 αの相互作用を阻害すること、す なわち RSKBと HNF— 4 aの結合を阻害することまたは RSKBによる HNF— 4 aの リン酸ィ匕を阻害することにより実施できる。  [0098] Another embodiment of the present invention relates to a phosphorylation inhibitor and a method for inhibiting HNF-4a by RSKB. The phosphorylation inhibitor and inhibition method of HNF-4a according to the present invention inhibits RSKB activity or inhibits the interaction between RSKB and HNF-4α, that is, RSKB and HNF-4a. By inhibiting the binding of or by inhibiting the phosphate of HNF-4a by RSKB.
[0099] 本発明に係る阻害剤および阻害方法を適用する対象物として、少なくとも RSKBと HNF-4 aとを含む対象物、例えば少なくともこれらを含む生体外試料を例示できる 。また、少なくとも RSKBと HNF— 4 αを発現している細胞、例えば肝臓細胞、並び にかかる細胞を担持している非ヒト哺乳動物を例示できる。  [0099] Examples of the target to which the inhibitor and the inhibition method according to the present invention are applied include a target including at least RSKB and HNF-4a, for example, an in vitro sample including at least these. Examples thereof include cells expressing at least RSKB and HNF-4α, such as liver cells, and non-human mammals carrying such cells.
[0100] 「RSKB活性」とは、 RSKBが有する機能を意味し、他の蛋白質と結合する機能、 例えば HNF— 4 aと結合する機能、およびキナーゼとして他の蛋白質をリン酸ィ匕す る機能、例えば HNF— 4 aをリン酸化する機能が含まれる。  [0100] “RSKB activity” means a function of RSKB, a function to bind to other proteins, for example, a function to bind to HNF-4a, and a function to phosphorylate other proteins as kinases. For example, a function of phosphorylating HNF-4a is included.
[0101] 「RSKB活性を阻害すること」とは、 RSKBが有する機能を阻害することを意味し、 例えば HNF— 4 aと結合する機能を阻害すること、および HNF— 4 aをリン酸化す る機能、すなわち HNF4 aに対するキナーゼ活性を阻害することが含まれる。 [0102] 本発明に係る RSKBによる HNF— 4 aのリン酸ィ匕阻害剤は、その一態様において RSKB活性を阻害する効果を有する化合物 (RSKB活性阻害剤)の少なくとも 1つを 含有することを特徴とする。 [0101] “Inhibiting RSKB activity” means inhibiting the function of RSKB, for example, inhibiting the function of binding to HNF-4a and phosphorylating HNF-4a Inhibiting function, ie kinase activity against HNF4a. [0102] According to the present invention, the phosphate inhibitor of HNF-4a by RSKB according to the present invention contains at least one compound (RSKB activity inhibitor) having an effect of inhibiting RSKB activity in one aspect thereof. Features.
[0103] 「RSKB活性阻害剤」とは、 RSKB活性を阻害する機能を有する化合物を意味する 。すなわち、「RSKB活性阻害剤」には、 RSKBのキナーゼ活性阻害剤や、 RSKBH NF-4 aの結合阻害剤が含まれる。また、「RSKB活性阻害剤」は、 RSKB活性を 阻害する機能を有する化合物の 1種または 2種以上を含んでなる組成物であることが できる。  [0103] "RSKB activity inhibitor" means a compound having a function of inhibiting RSKB activity. That is, “RSKB activity inhibitors” include RSKB kinase activity inhibitors and RSKBH NF-4a binding inhibitors. The “RSKB activity inhibitor” may be a composition comprising one or more compounds having a function of inhibiting RSKB activity.
[0104] RSKB活性阻害剤として、拮抗阻害効果を有する抗体および低分子量ィ匕合物を例 示できる。抗体としては、 RSKBまたは HNF— 4 aを認識して結合する抗体であって 、 RSKBによる HNF— 4 αのリン酸化を阻害する抗体が挙げられる。抗体は RSKB または HNF— 4 a自体、これら由来の部分ペプチド、あるいはこれらが相互作用する 部位のアミノ酸配列力 なるペプチドを抗原として自体公知の抗体作成法により取得 できる。低分子量ィ匕合物とは、ペプチド、ペプチド様物質、ポリペプチド、ポリヌクレオ チド、有機化合物、および無機化合物が含まれ、その分子量が好ましくは 10000以 下、より好ましくは 5000以下、さらに好ましくは 1000以下、さらにより好ましくは 500 以下の化合物を意味する。低分子量ィ匕合物としては、 RSKBのキナーゼ活性を阻害 する化合物、好ましくは該キナーゼ活性を特異的に阻害する化合物が挙げられる。 カゝかる化合物は、例えば、本発明に係るリン酸化方法またはリン酸化系を利用して、 RSKBによる HNF— 4 aのリン酸ィ匕を阻害する力否かを決定することにより同定でき る。 RSKBのキナーゼ活性を特異的に阻害するとは、 RSKBのキナーゼ活性を強く 阻害するが、他の酵素の活性は阻害しないか、弱く阻害することを意味する。  [0104] Examples of RSKB activity inhibitors include antibodies having a competitive inhibitory effect and low molecular weight compounds. Examples of the antibody include an antibody that recognizes and binds to RSKB or HNF-4a and inhibits phosphorylation of HNF-4α by RSKB. The antibody can be obtained by an antibody production method known per se using RSKB or HNF-4a itself, a partial peptide derived therefrom, or a peptide having an amino acid sequence at the site where they interact as an antigen. Low molecular weight compounds include peptides, peptide-like substances, polypeptides, polynucleotides, organic compounds, and inorganic compounds, and the molecular weight is preferably 10,000 or less, more preferably 5000 or less, and even more preferably 1000. Hereinafter, even more preferably 500 or less compounds are meant. Low molecular weight compounds include compounds that inhibit the kinase activity of RSKB, preferably compounds that specifically inhibit the kinase activity. The compound to be obtained can be identified, for example, by determining whether it can inhibit the phosphorylation of HNF-4a by RSKB using the phosphorylation method or phosphorylation system according to the present invention. To specifically inhibit the kinase activity of RSKB means to strongly inhibit the kinase activity of RSKB, but not or weakly inhibit the activity of other enzymes.
[0105] RSKB活性を阻害する機能を有する低分子量ィ匕合物として、 RSKBと HNF— 4 a が相互作用する部位のアミノ酸配列力 なるペプチドを例示できる。かかるペプチドと して、 RSKBおよび HNF— 4 aのアミノ酸配列において、これら蛋白質が結合する部 位のアミノ酸配列を含むペプチドを例示できる。また、かかるペプチドとして、 HNF- 4 aのアミノ酸配列において RSKBによりリン酸ィ匕される部位のアミノ酸配列を含むぺ プチドを例示できる。かかるペプチドは、 RSKBによる HNF— 4 αのリン酸化を競合 的に阻害すると考えられる。これらペプチドは、 RSKBまたは HNF— 4 Q;のアミノ酸 配列から設計し、自体公知のペプチド合成方法により合成したものから、 RSKBによ る HNF— 4 aのリン酸化および Zまたは RSKBと HNF— 4 aの結合を阻害するもの を選択することにより取得できる。 [0105] As a low molecular weight compound having a function of inhibiting RSKB activity, a peptide having an amino acid sequence at the site where RSKB and HNF-4a interact can be exemplified. Examples of such peptides include peptides containing the amino acid sequences of the sites to which these proteins bind in the amino acid sequences of RSKB and HNF-4a. An example of such a peptide is a peptide containing the amino acid sequence of the site phosphorylated by RSKB in the amino acid sequence of HNF-4a. Such peptides compete for phosphorylation of HNF-4α by RSKB It is thought to inhibit. These peptides were designed from the amino acid sequence of RSKB or HNF-4Q; and synthesized by a peptide synthesis method known per se, phosphorylation of HNF-4a by RSKB and Z or RSKB and HNF-4a It can be obtained by selecting those that inhibit the binding of.
[0106] このように特定されたペプチドに 1個乃至数個のアミノ酸の欠失、置換、付加、また は挿入等の変異を導入したものも、 RSKBと HNF— 4 aの相互作用を阻害する限り において、本発明の範囲に包含される。  [0106] Mutations such as deletion, substitution, addition, or insertion of one to several amino acids introduced into the peptides thus identified also inhibit the interaction between RSKB and HNF-4a Insofar as included in the scope of the present invention.
[0107] 変異を有するペプチドは天然に存在するものであってよぐまた変異を導入したも のであってもよい。欠失、置換、付加または挿入等の変異を導入する手段は自体公 知であり、例えばウルマーの技術 (非特許文献 31)を利用できる。このような変異の導 入において、当該ペプチドの基本的な性質 (物性、機能または免疫学的活性等)を 変化させないという観点から、例えば、同族アミノ酸 (極性アミノ酸、非極性アミノ酸、 疎水性アミノ酸、親水性アミノ酸、陽性荷電アミノ酸、陰性荷電アミノ酸および芳香族 アミノ酸等）の間での相互の置換は容易に想定される。  [0107] The peptide having a mutation may be a naturally occurring peptide or a mutation introduced. Means for introducing mutation such as deletion, substitution, addition or insertion are known per se, and for example, Ulmer's technology (Non-patent Document 31) can be used. From the viewpoint of not changing the basic properties (physical properties, functions, immunological activity, etc.) of the peptide in introducing such mutations, for example, homologous amino acids (polar amino acids, nonpolar amino acids, hydrophobic amino acids, Mutual substitution between hydrophilic amino acids, positively charged amino acids, negatively charged amino acids and aromatic amino acids, etc.) is readily envisioned.
[0108] RSKBと HNF— 4 aとの相互作用を阻害するペプチドは、その構成アミノ基または カルボキシル基等を、例えばアミドィ匕修飾する等、機能の著しい変更を伴わない程 度に改変できる。特に、一般的にペプチドと他の蛋白質との相互作用を安定ィ匕し、ぺ プチドを解離し難くするために汎用される修飾、例えば C末端のアルデヒドィ匕または N末端のァセチル化等の修飾は、 RSKBと HNF— 4 aの相互作用を阻害するぺプ チドの有効性を高めるために有用である。  [0108] A peptide that inhibits the interaction between RSKB and HNF-4a can be altered to such an extent that it does not undergo a significant change in function, such as modification of its constituent amino group or carboxyl group, for example, by amido. In particular, modifications generally used to stabilize the interaction between peptides and other proteins and make it difficult to dissociate peptides, such as C-terminal aldehydes or N-terminal acetylation Is useful to increase the effectiveness of peptides that inhibit the interaction between RSKB and HNF-4a.
[0109] 上記ペプチドは、ペプチドィ匕学にぉ 、て知られる一般的な方法で製造できる。例え ば、公知文献に記載の方法 (非特許文献 32および 33)が挙げられるが、これらに限 らず公知の方法が広く利用できる。  [0109] The peptide can be produced by a general method known in peptide science. For example, methods (Non-Patent Documents 32 and 33) described in known literatures can be mentioned, but the known methods can be widely used without being limited thereto.
[0110] HNF-4 aはその機能の一つにおいて、肝臓における転写因子として糖新生関連 遺伝子発現に関与していることが知られている。「糖新生関連遺伝子」とは、生体に ぉ 、て糖新生を調節して 、る物質をコードする遺伝子のことであり、例えば PEPCK 遺伝子が好ましく挙げられる。糖尿病病態時における HNF— 4 aの活性化は糖新生 をさらに亢進し、高血糖を悪化させる原因になると考えられる。 [0111] 本発明のまた別の一態様は、糖新生関連遺伝子の遺伝子産物の産生調節方法に 関する。本発明に係る糖新生関連遺伝子の遺伝子産物の産生調節方法は、それら をコードする遺伝子の発現に係る HNF— 4 aの RSKBによるリン酸ィ匕を調節して、該 遺伝子の遺伝子産物の産生を調節することを特徴とする。 [0110] HNF-4a is known to be involved in gluconeogenesis-related gene expression as a transcription factor in the liver in one of its functions. The “gluconeogenesis-related gene” is a gene that encodes a substance that regulates gluconeogenesis in a living body, and preferably includes, for example, the PEPCK gene. Activation of HNF-4a during diabetic conditions is thought to further increase gluconeogenesis and cause exacerbation of hyperglycemia. [0111] Another aspect of the present invention relates to a method for regulating the production of a gene product of a gluconeogenesis-related gene. The method for regulating the production of gene products of gluconeogenesis-related genes according to the present invention regulates the phosphorylation by RSKB of HNF-4a related to the expression of genes encoding them, thereby regulating the production of the gene products of the genes. It is characterized by adjusting.
[0112] 糖新生関連遺伝子の遺伝子産物産生調節方法の一態様は、糖新生関連遺伝子 の遺伝子産物の産生促進方法であり、該遺伝子発現に係る HNF— 4 aを RSKBを 用いてリン酸化することを特徴とする。  [0112] One aspect of the method for regulating gene product production of a gluconeogenesis-related gene is a method for promoting the production of a gene product of a gluconeogenesis-related gene, and phosphorylating HNF-4a related to the gene expression using RSKB It is characterized by.
[0113] 糖新生関連遺伝子の遺伝子産物産生調節方法の別の一態様は、糖新生関連遺 伝子の遺伝子産物の産生阻害方法であり、該遺伝子発現に係る HNF— 4 aの RSK Bによるリン酸化を阻害することを特徴とする。当該産生阻害方法は具体的には、 RS KBによる HNF— 4 aのリン酸化阻害剤または RSKBによる HNF— 4 aのリン酸ィ匕 阻害方法を使用することにより達成できる。  [0113] Another aspect of the method for regulating gene product production of a gluconeogenesis-related gene is a method for inhibiting the production of a gene product of a gluconeogenesis-related gene, and the phosphorylation by RSK B of HNF-4a related to the gene expression. It is characterized by inhibiting oxidation. Specifically, the production inhibition method can be achieved by using a phosphorylation inhibitor of HNF-4a by RS KB or a phosphorylation inhibition method of HNF-4a by RSKB.
[0114] 糖新生関連遺伝子発現に係る HNF— 4 aが作用する遺伝子の遺伝子産物として 、例えば、 HNF— 4 αの結合部位をプロモーターまたはェンハンサー内に有する遺 伝子の遺伝子産物が挙げられる。具体的には、 HNF— 4 αが作用する遺伝子として 、 RSKBによりリン酸化された HNF— 4 aの結合部位である AF1をプロモーターまた はェンハンサー内に有する遺伝子、例えば PEPCK遺伝子が挙げられる。  [0114] Examples of a gene product of a gene on which HNF-4a acts on gluconeogenesis-related gene expression include a gene product of a gene having a binding site of HNF-4α in a promoter or enhancer. Specifically, a gene on which HNF-4α acts includes a gene having AF1 which is a binding site of HNF-4a phosphorylated by RSKB in a promoter or enhancer, for example, PEPCK gene.
[0115] 本発明のさらに別の一態様は、糖新生関連遺伝子の遺伝子産物の産生阻害剤に 関する。本遺伝子産物の産生阻害剤は、上記リン酸化阻害剤を有効成分としてその 有効量含んでなる。本遺伝子産物の産生阻害剤は、好ましくは、上記リン酸化阻害 剤を有効成分としてその有効量含んでなる PEPCK遺伝子産物の産生阻害剤である  [0115] Yet another embodiment of the present invention relates to a production inhibitor of a gene product of a gluconeogenesis-related gene. The gene product production inhibitor comprises the phosphorylation inhibitor as an active ingredient in an effective amount thereof. The production inhibitor of this gene product is preferably a PEPCK gene product production inhibitor comprising the above phosphorylation inhibitor as an active ingredient and an effective amount thereof.
[0116] 本発明のまた別の一態様は、 RSKBと HNF— 4 aの相互作用を阻害する化合物 の同定方法に関する。本同定方法は、自体公知の医薬品スクリーニングシステムを 利用して構築できる。また、本発明に係るリン酸化系またはリン酸化方法を利用して、 本同定方法を実施できる。 [0116] Another embodiment of the present invention relates to a method for identifying a compound that inhibits the interaction between RSKB and HNF-4a. This identification method can be constructed using a pharmaceutical screening system known per se. Moreover, this identification method can be implemented using the phosphorylation system or phosphorylation method according to the present invention.
[0117] 具体的には、 RSKBと HNF— 4 aの相互作用を可能にする条件を選択し、当該条 件下で RSKBおよび Zまたは HNF— 4 aとある化合物 (被検化合物）を接触させ、 R SKBと HNF— 4 aの相互作用を検出するシグナルおよび Zまたはマーカーを使用 する系を用いて、このシグナルおよび Zまたはマーカーの存在若しくは不存在または 変化を検出することにより、 RSKBと HNF— 4 aの相互作用を阻害する化合物を同 定する。 [0117] Specifically, a condition that enables interaction between RSKB and HNF-4a is selected, and a compound (test compound) with RSKB and Z or HNF-4a is brought into contact under these conditions. , R Using a system that uses a signal and Z or marker to detect the interaction between SKB and HNF-4a, by detecting the presence or absence or change of this signal and Z or marker, RSKB and HNF-4a Identify compounds that inhibit the interaction.
[0118] 本同定方法において、被検化合物が RSKBと HNF— 4 aの相互作用を阻害する か否かの判定は、被検化合物存在下における RSKBと HNF— 4 aの結合および Z または RSKBによる HNF— 4 aのリン酸化により生じるシグナルあるいは該結合およ び Zまたは該リン酸ィヒのマーカーと、被検化合物非存在下における該結合および Z または該リン酸ィ匕により生じるシグナルある ヽは該結合および Zまたは該リン酸化の マーカーを比較することにより実施できる。被検化合物非存在下における該結合およ び Zまたは該リン酸ィ匕により生じるシグナルある 、は該結合および Zまたは該リン酸 化のマーカーと比較し、被検化合物存在下における該結合および Zまたは該リン酸 化により生じるシグナルあるいは該結合および Zまたは該リン酸ィ匕のマーカーが低減 または消失する場合、該被検化合物は、 RSKBと HNF— 4 aの相互作用を阻害す ると判定できる。  [0118] In this identification method, whether a test compound inhibits the interaction between RSKB and HNF-4a is determined by the binding of RSKB and HNF-4a in the presence of the test compound and Z or RSKB. The signal generated by phosphorylation of HNF-4a or the binding and Z or phosphate marker and the signal generated by the binding and Z or phosphate in the absence of the test compound are This can be done by comparing the binding and Z or phosphorylation markers. In the absence of a test compound, there is a signal generated by the binding and Z or the phosphoric acid, and the binding and Z in the presence of the test compound are compared with the binding and Z or the phosphorylation marker. Alternatively, when the signal generated by the phosphorylation or the binding and Z or phosphate markers are reduced or eliminated, the test compound can be determined to inhibit the interaction between RSKB and HNF-4a. .
[0119] RSKBと HNF— 4 aの相互作用を可能にする条件は、インビトロおよびインビボの いずれの条件でもよい。例えば、 RSKBと HNF— 4 αを共発現させた細胞を用いる ことができる。 RSKBおよび Ζまたは HNF— 4 aと被検化合物の接触は、 RSKBと H NF-4 aの相互作用の前に行なってもよいし、相互作用に共存させることにより行な つてもよい。  [0119] The conditions allowing the interaction between RSKB and HNF-4a may be any of in vitro and in vivo conditions. For example, cells in which RSKB and HNF-4α are co-expressed can be used. The contact between RSKB and NF or HNF-4a and the test compound may be performed before the interaction between RSKB and HNF-4a, or may be performed by coexisting with the interaction.
[0120] シグナルとは、そのもの自体がその物理的または化学的性質により直接検出され得 るものを指し、マーカーとはそのものの物理的または生物学的性質を指標として間接 的に検出され得るものを指す。  [0120] A signal is a signal that itself can be directly detected by its physical or chemical properties, and a marker is a signal that can be detected indirectly using its physical or biological properties as an indicator. Point to.
[0121] シグナルとして、ルシフェラーゼゃグリーン蛍光蛋白質等の酵素類、および放射性 同位体を例示できる。マーカーとして、レポーター遺伝子、例えばクロラムフエニコー ルァセチルトランスフェラーゼ遺伝子等、または検出用のェピトープタグ、例えば 6 X His— tag等を例示できる。シグナルやマーカーは、これら例示した物質に限定され ず、一般的に化合物の同定方法に用いられている標識物質であれば、いずれも用 いることができる。これらシグナルまたはマーカーは、単独で使用してもよぐ 2つ以上 を組合せて用いてもよい。これらシグナルまたはマーカーの検出方法は当業者に周 知のものである。 [0121] Examples of signals include enzymes such as luciferase and green fluorescent protein, and radioactive isotopes. Examples of the marker include a reporter gene such as chloramphenicol acetyl transferase gene, or a detection epitope tag such as 6 X His-tag. Signals and markers are not limited to these exemplified substances, and any labeling substance generally used in compound identification methods can be used. Can be. These signals or markers may be used alone or in combination of two or more. Methods for detecting these signals or markers are well known to those skilled in the art.
[0122] 本発明のさらに別の一態様は、 RSKBによる HNF— 4 aのリン酸ィ匕を阻害するィ匕 合物の同定方法に関する。本同定方法は、自体公知の医薬品スクリーニングシステ ムを利用して構築できる。また、本発明に係るリン酸化系またはリン酸化方法を利用 して、本同定方法を実施できる。  [0122] Still another embodiment of the present invention relates to a method for identifying a compound that inhibits phosphate phosphate of HNF-4a by RSKB. This identification method can be constructed using a pharmaceutical screening system known per se. In addition, this identification method can be carried out using the phosphorylation system or phosphorylation method according to the present invention.
[0123] 具体的には、 RSKBによる HNF— 4 aのリン酸化を可能にする条件を選択し、当 該条件下で RSKBおよび Zまたは HNF—4 aと被検化合物を接触させ、 RSKBに よる HNF— 4 aのリン酸ィ匕を検出するシグナルおよび Zまたはマーカーを使用する 系を用いて、このシグナルおよび Zまたはマーカーの存在若しくは不存在または変 化を検出することにより、 RSKBによる HNF— 4 aのリン酸ィ匕を阻害する化合物を同 定できる。  [0123] Specifically, a condition that enables phosphorylation of HNF-4a by RSKB is selected, RSKB and Z or HNF-4a are brought into contact with the test compound under the condition, and RSKB is used. HNF-4 by RSKB by detecting the presence or absence or change of this signal and Z or marker using a system that uses the signal and Z or marker to detect HNF-4a phosphate A compound that inhibits the phosphate of a can be identified.
[0124] 本同定方法において、被検化合物が RSKBによる HNF— 4 aのリン酸ィ匕を阻害す る力否かの判定は、被検化合物存在下における RSKBによる HNF— 4 aのリン酸ィ匕 により生じるシグナルまたは該リン酸ィ匕のマーカーと、被検化合物非存在下における 該リン酸ィ匕により生じるシグナルまたは該リン酸ィ匕のマーカーを比較することにより実 施できる。被検化合物非存在下における該リン酸ィ匕により生じるシグナルまたは該リ ン酸化のマーカーと比較し、被検化合物存在下における該リン酸化により生じるシグ ナルまたは該リン酸ィ匕のマーカーが低減または消失する場合、該被検化合物は、 R SKBによる HNF— 4 aのリン酸ィ匕を阻害すると判定できる。  [0124] In this identification method, whether or not the test compound inhibits HNF-4a phosphate by RSKB is determined by RSKB in the presence of the test compound. It can be carried out by comparing the signal generated by 匕 or the phosphate marker and the signal or phosphate marker generated by the phosphate in the absence of the test compound. The signal generated by the phosphorylation in the absence of the test compound or the marker of phosphorylation is reduced or the signal generated by the phosphorylation in the presence of the test compound or the phosphate marker is reduced or When it disappears, it can be determined that the test compound inhibits phosphorylation of HNF-4a by R SKB.
[0125] RSKBと HNF— 4 aのリン酸化を可能にする条件は、インビト口およびインビボのい ずれの条件でもよい。例えば、 RSKBと HNF— 4 αを共発現させた細胞を用いること ができる。 RSKBおよび Ζまたは HNF— 4 aと被検化合物の接触は、 RSKBと HNF -4 αのリン酸ィ匕反応の前に行なってもよ!/、し、リン酸化反応に共存させることにより 行なってもよい。  [0125] The conditions that allow phosphorylation of RSKB and HNF-4a may be in vitro or in vivo. For example, cells in which RSKB and HNF-4α are co-expressed can be used. RSKB and お よ び or HNF-4a can be contacted with the test compound before the phosphorylation reaction of RSKB and HNF-4α! Also good.
[0126] RSKBによる HNF— 4 aのリン酸化は、簡便にはリン酸化型 HNF— 4 a量の存在 若しくは不存在および Zまたは変化の測定により検出できる。リン酸化型 HNF— 4 a 量の定量は、 自体公知のリン酸ィ匕蛋白質測定方法を用いて実施できる。例えば、リン 酸ィ匕型 HNF— 4 aに対する抗体を用いてウェスタンブロッテイングによりリン酸ィ匕型 HNF— 4 α量の定量を実施できる。また、リン酸化型 HNF— 4 αの検出は、リン酸ィ匕 反応に放射性同位体標識した ΑΤΡ、例えば [ γ— 32Ρ]ΑΤΡを用いて、該反応の結 果 HNF— 4 aのアミノ酸残基に転移された [ γ—32Ρ]の放射活性を測定することによ り実施できる。具体的には、後述する実施例 2を参照して、 RSKBによる HNF— 4 Q; のリン酸ィ匕の検出を実施できる。 [0126] Phosphorylation of HNF-4a by RSKB can be conveniently detected by measuring the presence or absence and the Z or change of the amount of phosphorylated HNF-4a. Phosphorylated HNF— 4 a Quantification of the amount can be carried out using a well-known method for measuring phosphate protein. For example, the amount of phosphate-type HNF-4α can be quantified by Western blotting using an antibody against phosphate-type HNF-4a. The detection of phosphorylated HNF- 4 α is, Arufatauro labeled radioisotope Rinsani匕reaction, for example using [γ- 32 Ρ] ΑΤΡ, amino acid residues RESULTS HNF- 4 a of the reaction This can be done by measuring the radioactivity of [γ- 32さ れ] transferred to the group. Specifically, referring to Example 2 to be described later, detection of phosphate salt of HNF-4 Q; by RSKB can be performed.
[0127] RSKBによる HNF— 4 αのリン酸化の検出は、その他、 HNF— 4 α活性の存在若 しくは不存在および Ζまたは変化の測定により行なうことができる。例えば、 HNF-4 aは RSKBによりリン酸化されると PEPCK遺伝子プロモーターの転写活性を促進す るため、 PEPCK遺伝子プロモーターの転写活性を検出することにより、 RSKBによる HNF-4 aのリン酸化を検出できる。 PEPCK遺伝子プロモーターの転写活性の検 出は、 PEPCK遺伝子プロモーターを有するレポーター遺伝子を用いたレポーター アツセィにより実施できる。具体的には、 HNF— 4 αと RSKBとを発現している細胞 に、 PEPCK遺伝子のプロモーター領域をその上流に組込んだレポーター遺伝子を 含むプラスミドをトランスフエクシヨンし、レポーター遺伝子の発現量を測定することに より、 RSKBによる HNF— 4 αのリン酸ィ匕の検出を実施できる。このような検出方法に おいて、被検化合物とこの細胞を接触させたときのレポーター遺伝子の発現量を、当 該被検化合物と接触させなかったときのレポーター遺伝子の発現量と比較し、被検 化合物とこの細胞を接触させたときの該発現量が低減または消失した場合、 RSKB による HNF— 4 aのリン酸化が当該被検化合物により阻害されたと判定できる。  [0127] The detection of phosphorylation of HNF-4α by RSKB can also be performed by measuring the presence or absence of HNF-4α activity and wrinkles or changes. For example, since phosphorylation of HNF-4a promotes the transcriptional activity of the PEPCK gene promoter when phosphorylated by RSKB, the phosphorylation of HNF-4a by RSKB can be detected by detecting the transcriptional activity of the PEPCK gene promoter. . Detection of the transcriptional activity of the PEPCK gene promoter can be carried out by reporter assembly using a reporter gene having a PEPCK gene promoter. Specifically, cells expressing HNF-4α and RSKB are transfected with a plasmid containing a reporter gene incorporating the PEPCK gene promoter region upstream, and the expression level of the reporter gene is measured. By doing so, detection of HNF-4α phosphate by RSKB can be performed. In such a detection method, the expression level of the reporter gene when the test compound is brought into contact with the cell is compared with the expression level of the reporter gene when the test compound is not brought into contact with the test compound. When the expression level when the test compound is brought into contact with the cells is reduced or eliminated, it can be determined that phosphorylation of HNF-4a by RSKB is inhibited by the test compound.
[0128] 本発明のまた別の一態様は、 RSKBと HNF— 4 aの結合を阻害する化合物の同 定方法に関する。本同定方法は、自体公知の医薬品スクリーニングシステムを利用し て構築できる。また、本発明に係るリン酸ィ匕系またはリン酸ィ匕方法を利用して、本同 定方法を実施できる。  [0128] Another embodiment of the present invention relates to a method for identifying a compound that inhibits the binding between RSKB and HNF-4a. This identification method can be constructed using a pharmaceutical screening system known per se. Further, this identification method can be carried out using the phosphoric acid system or the phosphoric acid method according to the present invention.
[0129] 具体的には、 RSKBと HNF— 4 aの結合を可能にする条件を選択し、当該条件下 で RSKBおよび Zまたは HNF— 4 aと被検化合物を接触させ、 RSKBと HNF— 4 aの結合を検出するシグナルおよび Zまたはマーカーを使用する系を用いて、この シグナルおよび Zまたはマーカーの存在若しくは不存在または変化を検出すること により、 RSKBと HNF— 4 aの結合を阻害する化合物を同定できる。 [0129] Specifically, conditions that enable the binding of RSKB and HNF-4a are selected, RSKB and Z or HNF-4a are contacted with the test compound under the conditions, and RSKB and HNF-4 are contacted. Using a signal that detects binding of a and a system that uses Z or a marker, By detecting the presence or absence or change of signal and Z or markers, compounds that inhibit the binding of RSKB to HNF-4a can be identified.
[0130] 本同定方法において、被検化合物が RSKBと HNF— 4 aの結合を阻害するか否 かの判定は、被検化合物存在下における RSKBと HNF— 4 aの結合により生じるシ グナルまたは該結合のマーカーと、被検化合物非存在下における該結合により生じ るシグナルまたは該結合のマーカーを比較することにより実施できる。被検化合物非 存在下における該結合により生じるシグナルまたは該結合のマーカーと比較し、被検 化合物存在下における該結合により生じるシグナルまたは該結合のマーカーが低減 または消失する場合、該被検化合物は、 RSKBと HNF— 4 aの結合を阻害すると判 定できる。 [0130] In this identification method, whether or not the test compound inhibits the binding between RSKB and HNF-4a is determined by the signal generated by the binding of RSKB and HNF-4a in the presence of the test compound or the This can be carried out by comparing the marker of binding with the signal generated by the binding in the absence of the test compound or the marker of binding. When the signal generated by the binding in the presence of the test compound or the marker of the binding is reduced or eliminated compared to the signal generated by the binding or the marker of the binding in the presence of the test compound, the test compound is It can be determined that the binding between RSKB and HNF-4a is inhibited.
[0131] RSKBと HNF— 4 aの結合を可能にする条件は、インビトロおよびインビボのいず れの条件でもよい。例えば、 RSKBと HNF— 4 αを共発現させた細胞を用いることが できる。 RSKBおよび Ζまたは HNF— 4 aと被検化合物の接触は、 RSKBと HNF— 4 aの結合反応の前に行なってもよいし、結合反応に共存させることにより行なっても よい。  [0131] The conditions enabling the binding of RSKB and HNF-4a may be any of in vitro and in vivo conditions. For example, cells in which RSKB and HNF-4α are co-expressed can be used. The contact between RSKB and Ζ or HNF-4a and the test compound may be performed before the binding reaction of RSKB and HNF-4a, or may be carried out by coexisting in the binding reaction.
[0132] RSKBと HNF— 4 αの結合は、自体公知の蛋白質検出法を用いて実施できる。例 えば、 RSKBと HNF— 4 aを含む複合体を、自体公知の蛋白質検出法で検出する ことにより、 RSKBと HNF— 4 αの結合を検出できる。蛋白質の検出方法として、ゥェ スタンプロット法、免疫沈降法、プルダウン法、ツーハイブリッド法および蛍光共鳴ェ ネルギー転移法を例示できる。これら方法を単独で用いて、またはこれら方法を組合 わせて用いて、所望の蛋白質の検出を実施できる。 RSKBおよび/または HNF— 4 aを予め標識しておき、その標識物質を検出することにより、 RSKBと HNF— 4 aを 含む複合体の検出を容易に実施できる。標識物質として HA - tagや FLAG - tag 等のタグペプチドを好ましく例示できる。  [0132] The binding between RSKB and HNF-4α can be performed using a protein detection method known per se. For example, the binding between RSKB and HNF-4α can be detected by detecting a complex containing RSKB and HNF-4a by a known protein detection method. Examples of the protein detection method include Westamplot method, immunoprecipitation method, pull-down method, two-hybrid method, and fluorescence resonance energy transfer method. These methods can be used alone, or a combination of these methods can be used to detect the desired protein. By labeling RSKB and / or HNF-4a in advance and detecting the labeling substance, a complex containing RSKB and HNF-4a can be easily detected. Preferred examples of the labeling substance include tag peptides such as HA-tag and FLAG-tag.
[0133] 被検化合物として、例えばィ匕学ライブラリーや天然物由来の化合物、または RSKB および HNF— 4 aの一次構造や立体構造に基づいてドラッグデザインして得られた 化合物等が挙げられる。あるいは、 HNF— 4 αと RSKBの結合部位および Zまたは RSKBによる HNF— 4 aのリン酸化部位のペプチドの構造に基づいてドラッグデザ インして得られたィ匕合物等も被検化合物として好適である。 [0133] Examples of the test compound include compounds derived from chemical libraries and natural products, or compounds obtained by drug design based on the primary structure and three-dimensional structure of RSKB and HNF-4a. Alternatively, based on the peptide structure of the binding site of HNF-4α and RSKB and the phosphorylation site of HNF-4a by Z or RSKB, The compound obtained by in-situ is also suitable as the test compound.
[0134] 本発明に係る同定方法で得られた化合物は、 RSKBによる HNF— 4 aのリン酸ィ匕 阻害剤あるいは HNF— 4 aが作用する遺伝子の遺伝子産物産生阻害剤として利用 できる。 RSKBによる HNF— 4 aのリン酸化阻害剤または HNF— 4 aが作用する遺 伝子の遺伝子産物産生阻害剤は、生物学的有用性と毒性のバランスを考慮して選 別することにより、医薬組成物として調製できる。医薬組成物の調製において、これら 阻害剤は、単独で使用することもできるし、複数を組合せて使用することもできる。  [0134] The compound obtained by the identification method according to the present invention can be used as a phosphate inhibitor of HNF-4a by RSKB or a gene product production inhibitor of a gene on which HNF-4a acts. The inhibitor of phosphorylation of HNF-4a by RSKB or the gene product production inhibitor of the gene that HNF-4a acts on is selected by considering the balance between biological usefulness and toxicity. It can be prepared as a composition. In the preparation of the pharmaceutical composition, these inhibitors can be used alone or in combination.
[0135] 本発明のさらに別の一態様は、本発明に係るリン酸ィ匕阻害剤を有効成分としてそ の有効量含んでなる医薬組成物に関する。  [0135] Yet another embodiment of the present invention relates to a pharmaceutical composition comprising an effective amount of the phosphate inhibitor according to the present invention as an active ingredient.
[0136] 本発明に係る医薬組成物は、 RSKBによる HNF— 4 aのリン酸ィ匕に起因する疾患 の防止および Zまたは治療剤として使用することができる。また、当該疾患の防止お よび Zまたは治療方法に使用することができる。  [0136] The pharmaceutical composition according to the present invention can be used as a preventive and Z or therapeutic agent for diseases caused by phosphate knot of HNF-4a by RSKB. It can also be used for prevention and Z or treatment of the disease.
[0137] すなわち、本発明のまた別の一態様は、 RSKBによる HNF— 4 aのリン酸化に起 因する疾患の防止および Zまたは治療剤、並びに当該疾患の防止および Zまたは 治療方法に関する。当該疾患の防止および Zまたは治療剤は、上記リン酸化阻害剤 を含んでいる。当該疾患の防止および Zまたは治療方法は、上記リン酸化阻害剤ま たは上記リン酸ィ匕阻害方法を使用することにより達成できる。  That is, another embodiment of the present invention relates to a preventive and Z or therapeutic agent for a disease caused by phosphorylation of HNF-4a by RSKB, and a preventive and Z or therapeutic method for the disease. The preventive and Z or therapeutic agent for the disease contains the phosphorylation inhibitor. The prevention and Z or treatment method of the disease can be achieved by using the phosphorylation inhibitor or the phosphorylation inhibition method.
[0138] RSKBによる HNF— 4 aのリン酸化に起因する疾患として、例えば HNF— 4 aが 作用する遺伝子の遺伝子産物の増加に起因する疾患が挙げられる。例えば RSKB によりリン酸ィ匕された HNF— 4 aは PEPCK遺伝子上流の転写部位への結合を促進 させ、該遺伝子の遺伝子産物産生に寄与している。  [0138] Examples of diseases caused by phosphorylation of HNF-4a by RSKB include diseases caused by an increase in the gene product of a gene on which HNF-4a acts. For example, HNF-4a phosphorylated by RSKB promotes binding to the transcription site upstream of the PEPCK gene and contributes to the gene product production of the gene.
[0139] RSKBによる HNF— 4 aのリン酸ィ匕に起因する疾患として具体的には、糖新生関 連遺伝子の遺伝子産物の増加に起因する疾患、例えば PEPCK遺伝子の遺伝子産 物の増加に起因する疾患を例示できる。より具体的には、糖新生異常による疾患、例 えば糖尿病等が挙げられる。  [0139] Specifically, the disease caused by the phosphorylation of HNF-4a by RSKB is caused by an increase in the gene product of a gene involved in gluconeogenesis, for example, an increase in the gene product of the PEPCK gene. Can be exemplified. More specifically, diseases caused by abnormal gluconeogenesis, such as diabetes.
[0140] 本発明に係る医薬組成物は、通常、有効成分に加えて 1種または 2種以上の医薬 用担体を含む医薬組成物として製造することが好ましい。  [0140] The pharmaceutical composition according to the present invention is usually preferably prepared as a pharmaceutical composition containing one or more pharmaceutical carriers in addition to the active ingredient.
[0141] 本発明に係る医薬製剤中に含まれる有効成分の量は、広範囲から適宜選択される 。通常、約 0. 00001〜70重量％、好ましくは 0. 0001〜5重量％程度の範囲とする のが適当である。 [0141] The amount of the active ingredient contained in the pharmaceutical preparation according to the present invention is appropriately selected from a wide range. . Usually, it is appropriate that the amount is in the range of about 0.0001 to 70% by weight, preferably about 0.0001 to 5% by weight.
[0142] 医薬用担体は、製剤の使用形態に応じて一般的に使用される、充填剤、増量剤、 結合剤、付湿剤、崩壊剤、滑沢剤、希釈剤および賦形剤を例示できる。これらは得ら れる製剤の投与形態に応じて適宜選択して使用される。  [0142] Examples of pharmaceutical carriers include fillers, fillers, binders, moisturizers, disintegrants, lubricants, diluents and excipients that are generally used depending on the form of use of the preparation. it can. These are appropriately selected and used depending on the dosage form of the preparation to be obtained.
[0143] より具体的には、水、医薬的に許容される有機溶剤、コラーゲン、ポリビニルアルコ ール、ポリビュルピロリドン、カルボキシビ-ルポリマー、アルギン酸ナトリウム、水溶性 デキストラン、カルボキシメチルスターチナトリウム、ぺクチン、キサンタンガム、ァラビ ァゴム、カゼイン、ゼラチン、寒天、グリセリン、プロピレングリコール、ポリエチレングリ コール、ワセリン、パラフィン、ステアリルアルコール、ステアリン酸、ヒト血清アルブミン 、マン-トール、ソルビトール、ラタトース等が挙げられる。これらは、本医薬組成物の 剤形に応じて適宜 1種類または 2種類以上を組合わせて使用される。  [0143] More specifically, water, pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polybutylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin Xanthan gum, gum arabic gum, casein, gelatin, agar, glycerin, propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, ratatose and the like. These may be used alone or in combination of two or more according to the dosage form of the pharmaceutical composition.
[0144] 所望により、通常の蛋白質製剤に使用され得る各種の成分、例えば安定化剤、殺 菌剤、緩衝剤、等張化剤、キレート剤、界面活性剤、および pH調整剤等を適宜使用 することちでさる。  [0144] Various components that can be used in normal protein preparations, for example, stabilizers, bactericides, buffers, isotonic agents, chelating agents, surfactants, pH adjusters, and the like are used as needed. I'll do it for you.
[0145] 安定化剤は、ヒト血清アルブミンや通常の L アミノ酸、糖類、セルロース誘導体を 例示できる。これらは単独でまたは界面活性剤等と組合わせて使用できる。特にこの 組合わせによれば、有効成分の安定性をより向上させ得る場合がある。 L—アミノ酸 は、特に限定はなぐ例えばグリシン、システィン、グルタミン酸等のいずれでもよい。 糖類も特に限定はなぐ例えばグルコース、マンノース、ガラクトース、果糖等の単糖 類、マン-トール、イノシトール、キシリトール等の糖アルコール、ショ糖、マルトース、 乳糖等の二糖類、デキストラン、ヒドロキシプロピルスターチ、コンドロイチン硫酸、ヒア ルロン酸等の多糖類等およびそれらの誘導体等の 、ずれでもよ 、。セルロース誘導 体も特に限定はなぐメチルセルロース、ェチルセルロース、ヒドロキシェチルセル口 ース、ヒドロキシプロピノレセノレロース、ヒドロキシプロピノレメチノレセノレロース、カノレボキ シメチルセルロースナトリウム等の!/、ずれでもよ!/、。  [0145] Examples of the stabilizer include human serum albumin, ordinary L amino acids, sugars, and cellulose derivatives. These can be used alone or in combination with a surfactant or the like. In particular, according to this combination, the stability of the active ingredient may be further improved. The L-amino acid is not particularly limited, and may be any of glycine, cysteine, glutamic acid and the like. Sugars are not particularly limited, for example, monosaccharides such as glucose, mannose, galactose, and fructose, sugar alcohols such as mannitol, inositol, and xylitol, disaccharides such as sucrose, maltose, and lactose, dextran, hydroxypropyl starch, chondroitin Any deviation from polysaccharides such as sulfuric acid and hyaluronic acid, and their derivatives. Cellulose derivatives are not particularly limited, such as methylcellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropenoresenorelose, hydroxypropinoremethenoresenorelose, sodium canoleboximethylcellulose, etc. ,.
[0146] 界面活性剤も特に限定はなぐイオン性界面活性剤および非イオン性界面活性剤 のいずれも使用できる。界面活性剤には、例えばポリオキシエチレングリコールソル ビタンアルキルエステル系、ポリオキシエチレンアルキルエーテル系、ソルビタンモノ ァシルエステル系、脂肪酸グリセリド系等が包含される。 [0146] There are no particular limitations on the surfactant, and both an ionic surfactant and a nonionic surfactant can be used. Examples of the surfactant include polyoxyethylene glycol solvent. Vitan alkyl ester, polyoxyethylene alkyl ether, sorbitan monoacyl ester, fatty acid glyceride and the like are included.
[0147] 緩衝剤は、ホウ酸、リン酸、酢酸、クェン酸、 ε アミノカプロン酸、グルタミン酸およ び Ζまたはそれらに対応する塩 (例えばそれらのナトリウム塩、カリウム塩、カルシウム 塩、マグネシウム塩等のアルカリ金属塩やアルカリ土類金属塩）を例示できる。  [0147] Buffering agents include boric acid, phosphoric acid, acetic acid, citrate, ε-aminocaproic acid, glutamic acid and sputum or their corresponding salts (for example, sodium salts, potassium salts, calcium salts, magnesium salts thereof) Examples thereof include alkali metal salts and alkaline earth metal salts.
[0148] 等張化剤は、塩ィ匕ナトリウム、塩ィ匕カリウム、糖類、グリセリンを例示できる。  [0148] Examples of the isotonic agent include sodium chloride sodium, potassium salt potassium, sugars, and glycerin.
[0149] キレート剤は、ェデト酸ナトリウム、クェン酸を例示できる。  [0149] Examples of the chelating agent include sodium edetate and citrate.
[0150] 本発明に係る医薬および医薬組成物は、溶液製剤として使用できる他に、これを凍 結乾燥ィ匕し保存し得る状態にした後、用時、水や生理的食塩水等を含む緩衝液等 で溶解して適当な濃度に調製した後に使用することができる。  [0150] The pharmaceutical and pharmaceutical composition according to the present invention can be used as a solution preparation, and after freezing, drying and storing it, it contains water, physiological saline and the like at the time of use. It can be used after being dissolved in a buffer solution or the like to prepare an appropriate concentration.
[0151] 医薬および医薬組成物の用量範囲は特に限定されず、含有される成分の有効性、 投与形態、投与経路、疾患の種類、対象の性質 (体重、年齢、病状および他の医薬 の使用の有無等）、および担当医師の判断等応じて適宜選択される。一般的には適 当な用量は、例えば対象の体重 lkgあたり約 0. 01 μ g〜100mg程度、好ましくは約 0.： g〜lmg程度の範囲であることが好ましい。し力しながら、当該分野において よく知られた最適化のための一般的な常套的実験を用いてこれらの用量の変更を行 うことができる。上記投与量は 1日 1回〜数回に分けて投与することができ、数日また は数週間に 1回の割合で間欠的に投与してもよい。  [0151] The dose range of the medicine and the pharmaceutical composition is not particularly limited, and the effectiveness of the contained ingredients, the administration form, the administration route, the type of the disease, the nature of the subject (weight, age, medical condition and use of other medicines Or the like) and the judgment of the doctor in charge. In general, a suitable dose is, for example, in the range of about 0.01 μg to 100 mg, preferably about 0.1: g to lmg, per kg of body weight of the subject. However, these dosage changes can be made using general routine experimentation for optimization well known in the art. The above dose can be administered once to several times a day, and may be administered intermittently at a rate of once every several days or weeks.
[0152] 本発明に係る医薬組成物を投与するときは、該医薬組成物を単独で使用してもよく 、あるいは目的の疾患の防止および Zまたは治療に必要な他の化合物または医薬と 共に使用してもよい。例えば、糖尿病治療薬の有効成分等を配合してもよい。  [0152] When administering the pharmaceutical composition according to the present invention, the pharmaceutical composition may be used alone or in combination with other compounds or medicines necessary for the prevention and Z or treatment of the target disease. May be. For example, you may mix | blend the active ingredient of a diabetes therapeutic agent, etc.
[0153] 投与経路は、全身投与または局所投与の!/、ずれも選択することができる。この場合 、疾患、症状等に応じた適当な投与経路を選択する。非経口経路として、通常の静 脈内投与、動脈内投与の他、皮下、皮内、筋肉内等への投与を例示できる。あるい は経口経路で投与することができる。さらに、経粘膜投与または経皮投与を実施する ことができる。  [0153] The administration route can be selected from systemic administration or local administration! In this case, an appropriate administration route is selected according to the disease, symptoms and the like. Examples of parenteral routes include normal intravenous administration and intraarterial administration, as well as subcutaneous, intradermal and intramuscular administration. Or it can be administered by the oral route. Furthermore, transmucosal administration or transdermal administration can be performed.
[0154] 投与形態は、各種の形態が目的に応じて選択できる。その代表的なものは、錠剤、 丸剤、散剤、粉末剤、細粒剤、顆粒剤、カプセル剤等の固体投与形態や、水溶液製 剤、エタノール溶液製剤、懸濁剤、脂肪乳剤、リボソーム製剤、シクロデキストリン等 の包接体、シロップ、エリキシル等の液剤投与形態が含まれる。これらはさらに投与 経路に応じて経口剤、非経口剤（点滴剤、注射剤）、経鼻剤、吸入剤、経膣剤、坐剤 、舌下剤、点眼剤、点耳剤、軟膏剤、クリーム剤、経皮吸収剤、経粘膜吸収剤等に分 類され、それぞれ通常の方法に従い、調合、成形、調製することができる。 [0154] Various administration forms can be selected according to the purpose. Typical examples are solid dosage forms such as tablets, pills, powders, powders, fine granules, granules, capsules, and aqueous solutions. Solutions, ethanol solution preparations, suspensions, fat emulsions, ribosome preparations, inclusions such as cyclodextrins, and liquid dosage forms such as syrups and elixirs. Depending on the route of administration, these preparations can also be administered orally, parenterally (infusions, injections), nasal preparations, inhalants, vaginal preparations, suppositories, sublingual, eye drops, ear drops, ointments, creams. And can be prepared, molded and prepared according to ordinary methods.
[0155] 散剤、丸剤、カプセル剤および錠剤は、ラタトース、グルコース、シユークロースおよ びマン-トール等の賦形剤、澱粉およびアルギン酸ソーダ等の崩壊剤、マグネシウム ステアレートおよびタルク等の滑沢剤、ポリビュルアルコール、ヒドロキシプロピルセル ロースおよびゼラチン等の結合剤、脂肪酸エステル等の界面活性剤、およびグリセリ ン等の可塑剤等を用いて製造できる。錠剤やカプセルを製造するには、固体の製薬 担体が用いられる。 [0155] Powders, pills, capsules, and tablets are excipients such as ratatoses, glucose, sucrose and mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc. In addition, it can be produced using a binder such as polybutyl alcohol, hydroxypropyl cellulose and gelatin, a surfactant such as fatty acid ester, and a plasticizer such as glycerin. For the production of tablets and capsules, solid pharmaceutical carriers are used.
[0156] 懸濁剤は、水、シユークロース、ソルビトールおよびフラクトース等の糖類、ポリェチ レンダリコール等のダリコール類、および油類を使用して製造できる。  [0156] Suspending agents can be produced using water, sugars such as sucrose, sorbitol and fructose, dallicols such as polyethylene glycol, and oils.
[0157] 注射用の溶液は、塩溶液、グルコース溶液、または塩水とグルコース溶液の混合物 力もなる担体を用いて調製できる。  [0157] Solutions for injection can be prepared using a carrier that also has a salt solution, a glucose solution, or a mixture of saline and glucose solution.
[0158] リボソーム化は、例えばリン脂質を有機溶媒 (クロ口ホルム等）に溶解した溶液に、上 記有効成分を溶媒 (エタノール等）に溶解した溶液を加えた後、溶媒を留去し、これ にリン酸緩衝液を加え、振とう、超音波処理および遠心処理した後、上清をろ過処理 して回収することにより実施できる。  [0158] Ribosomeization is performed by, for example, adding a solution in which the above active ingredient is dissolved in a solvent (such as ethanol) to a solution in which phospholipid is dissolved in an organic solvent (such as chloroform), and then distilling off the solvent. This can be carried out by adding a phosphate buffer, shaking, sonicating and centrifuging, and then collecting the supernatant by filtration.
[0159] 脂肪乳剤化は、例えば上記有効成分、油成分 (大豆油、ゴマ油およびォリーブ油 等の植物油、 MCT等)、および乳化剤 (リン脂質等)等を混合、加熱して溶液とした 後に、必要量の水を加え、乳化機 (ホモジナイザー、例えば高圧噴射型や超音波型 等)を用いて、乳化'均質化処理して実施できる。また、これを凍結乾燥ィ匕することも できる。なお、脂肪乳剤化するとき、乳化助剤を添加してもよぐ乳化助剤として、ダリ セリンや糖類 (例えばブドウ糖、ソルビトールおよび果糖等）を例示できる。  [0159] Fat emulsification is carried out, for example, by mixing and heating the above active ingredients, oil components (vegetable oils such as soybean oil, sesame oil and olive oil, MCT, etc.), and emulsifiers (phospholipids etc.) It can be carried out by adding the required amount of water and emulsifying and homogenizing using an emulsifier (homogenizer, such as a high-pressure jet type or ultrasonic type). It can also be lyophilized. Examples of emulsification aids that may be added with emulsification aids when making fat emulsions include dariserine and saccharides (eg, glucose, sorbitol, fructose, etc.).
[0160] シクロデキストリン包接ィ匕は、例えば上記有効成分を溶媒 (エタノール等）に溶解し た溶液に、シクロデキストリンを水等に加温溶解した溶液を加えた後、冷却して析出 した沈殿をろ過し、滅菌乾燥することにより実施できる。この際、使用されるシクロデキ ストリンは、上記有効成分の大きさに応じて、空隙直径の異なるシクロデキストリン（α 、 13、 γ型)を適宜選択すればよい。 [0160] For example, cyclodextrin clathrate is a precipitate formed by adding a solution in which cyclodextrin is heated and dissolved in water to a solution in which the above active ingredient is dissolved in a solvent (ethanol or the like), and then cooling to precipitate. Can be carried out by filtering and sterilizing and drying. At this time, the cyclodex used For the string, cyclodextrins (α, 13, γ type) having different pore diameters may be appropriately selected according to the size of the active ingredient.
[0161] 本発明のさらに別の一態様は、 RSKB、 RSKBをコードするポリヌクレオチド、 RSK Bをコードするポリヌクレオチドを含有するベクターのうちの少なくともいずれ力 1つと、 HNF-4 a、 HNF-4 aをコードするポリヌクレオチドおよび該ポリヌクレオチドを含 有するベクターのうちの少なくともいずれか 1つとを含んでなる試薬キットに関する。 当該試薬キットは、例えば本発明に係る同定方法に使用できる。 [0161] In another aspect of the present invention, at least one of RSKB, a polynucleotide encoding RSKB, a vector containing a polynucleotide encoding RSK B, and HNF-4a, HNF-4 The present invention relates to a reagent kit comprising a polynucleotide encoding a and at least one of a vector containing the polynucleotide. The reagent kit can be used, for example, in the identification method according to the present invention.
[0162] また、 RSKBまたは HNF— 4 a由来の部分ペプチド、例えば RSKBと HNF— 4 a が相互作用する部位のアミノ酸配列力 なるペプチド、あるいは上記同定方法で得ら れたィ匕合物力 なる試薬、およびこれらを含む試薬キットも本発明の範囲に包含され る。  [0162] Further, RSKB or HNF-4a-derived partial peptide, for example, a peptide having an amino acid sequence ability at a site where RSKB and HNF-4a interact, or a reagent having a combined force obtained by the above identification method , And reagent kits containing these are also included within the scope of the present invention.
[0163] 本試薬キットは、 RSKBによる HNF— 4 aのリン酸化を検出するためのシグナルお よび Zまたはマーカー、これらの検出剤、反応希釈液、緩衝液、洗浄剤および反応 停止液等、測定の実施に必要とされる物質を含むことができる。さらに、本試薬キット は、安定化剤および Zまたは防腐剤等の物質を含むことができる。製剤化にあたつ ては、使用する各物質それぞれに応じた製剤化手段を導入すればよい。これら試薬 および試薬キットは、例えば RSKBによる HNF— 4 αのリン酸ィ匕に起因する疾患、例 えば糖尿病における HNF— 4 aや RSKBの関与についての分子レベルでの研究等 に有用である。  [0163] This reagent kit measures signals and Z or markers for detecting phosphorylation of HNF-4a by RSKB, their detection agents, reaction diluents, buffers, detergents, and reaction stop solutions. It may contain substances required for the implementation of In addition, the reagent kit can include stabilizers and substances such as Z or preservatives. For formulation, it is sufficient to introduce formulation means for each substance to be used. These reagents and reagent kits are useful, for example, for molecular studies on the involvement of HNF-4a and RSKB in diseases caused by HKB-4α phosphate caused by RSKB, such as diabetes.
[0164] HNF-4 aのリン酸ィ匕を阻害することによる糖尿病の予防および Zまたは治療を 実施できることを確認するためには、以下のような実験を行えばよい。糖尿病モデル 動物の ZDFラットは、肝臓中の PEPCKの発現量が正常に比べ 2. 4倍増加している ことが知られている（非特許文献 38)。このモデル動物の RSKB活性ィ匕による HNF —4 aのリン酸ィ匕を、（1) 干臓中 HNF— 4 aのリン酸ィ匕量と（2)肝臓中 RSKBの活性 化量を測定することによりまず調べ、次に RSKB活性ィ匕を阻害するリン酸ィ匕阻害剤で ある H - 89 (N- [2- ( (p -ブロモシンナミル）ァミノ）ェチル] - 5—イソキノリンスル ホナマイト； N— [2— ( (p― Bromocmnamyl) ammoノ etnyl]— 5— isoquino lines ulfonamide) (非特許文献 34)を投与して、モデル動物個体群の血中グルコース濃 度を判定すればょ 、。血中グルコース濃度は前述したように約半分に減少する。 [0164] In order to confirm that prevention and / or treatment of diabetes by inhibiting HNF-4a phosphate can be performed, the following experiment may be performed. It is known that the expression level of PEPCK in the liver is increased 2.4-fold in the ZDF rat, a diabetic model animal (Non-patent Document 38). Measure HNF-4a phosphate by RSKB activity in this model animal. (1) Measure the amount of phosphate HNF-4a in the pig and (2) activate RSKB in the liver. H-89 (N- [2-((p-bromocinnamyl) amino) ethyl) -5-isoquinoline sulphonite; N—, a phosphate inhibitor that inhibits RSKB activity. [2— ((p- Bromocmnamyl) ammo no etnyl] — 5— isoquino lines ulfonamide) (Non-patent Document 34) Judge the degree. The blood glucose concentration is reduced to about half as described above.
[0165] 上記、肝臓中の（l) HNF— 4 αのリン酸化量の測定は、 HNF— 4 αを免疫沈降し て抗リン酸ィ匕抗体を用いてリン酸ィ匕の程度を検出することにより実施できる。また、 (2 ) RSKBの活性ィ匕量の測定は、抗活性化型 RSKB抗体を作成し免疫沈降するか、抗 RSKB抗体で免疫沈降し、インビトロのキナーゼ活性を測定することにより実施できる 実施例 1 [0165] The measurement of the amount of phosphorylation of (l) HNF-4α in the liver described above is performed by immunoprecipitation of HNF-4α and detecting the degree of phosphate using an anti-phosphate antibody. Can be implemented. In addition, (2) RSKB activity can be measured by preparing an anti-activated RSKB antibody and performing immunoprecipitation or immunoprecipitation with an anti-RSKB antibody and measuring in vitro kinase activity. 1
[0166] (RSKBと相互作用する蛋白質のインシリコでの探索）  [0166] (In silico search for proteins that interact with RSKB)
RSKBと相互作用する蛋白質の予測を、特許文献 1に記載のインシリコでの予測方 法に従って次のように実施した：（i) RSKBのアミノ酸配列をある長さのオリゴペプチド に分解し、（ii)各オリゴペプチドのアミノ酸配列あるいはそのアミノ酸配列と相同なアミ ノ酸配列を持った蛋白質をデータベース中で検索し、 (iii)得られた蛋白質と RSKB との間でローカルァライメントを行い、 (iv)ローカルァライメントのスコアの高いものを R SKBと相互作用する蛋白質であると予測した。ここではローカルァライメントのスコア を国際公開第 W001Z67299号公報に記載の方法と同様に、 25. 0以上とした。  Prediction of the protein that interacts with RSKB was carried out as follows according to the in silico prediction method described in Patent Document 1: (i) The amino acid sequence of RSKB was decomposed into oligopeptides of a certain length, and (ii ) Search the database for the amino acid sequence of each oligopeptide or a protein having an amino acid sequence homologous to the amino acid sequence, (iii) perform local alignment between the obtained protein and RSKB, and (iv ) We predicted that proteins with high local alignment scores interacted with R SKB. Here, the local alignment score was set to 25.0 or higher, as in the method described in International Publication No. W001Z67299.
[0167] 解析の結果、 RSKBと相互作用する機能を有すると予測される蛋白質として HNF  [0167] As a result of analysis, HNF is predicted to have a function of interacting with RSKB.
-4 aを見出した。 RSKBの部分アミノ酸配列力 なるオリゴペプチド（CRRCRQ (配 列番号 10)および VSRRILK (配列番号 13) )と相同なオリゴペプチド（CRYCRL ( 配列番号 11)、 CRFSRQ (配列番号 12)および VSIRILD (配列番号 14) )が HNF 4 αのアミノ酸配列中に認められた。図 1に、 RSKB (図中、上の配列）と HNF— 4 a (図中、下の配列）とのローカルァライメントの結果を示した。  -4 A was found. Oligopeptides (CRYCRL (SEQ ID NO: 11), CRFSRQ (SEQ ID NO: 12) and VSIRILD (SEQ ID NO: 14) that are homologous to the oligopeptides (CRRCRQ (SEQ ID NO: 10) and VSRRILK (SEQ ID NO: 13)) that have partial amino acid sequence capabilities of RSKB )) Was found in the amino acid sequence of HNF 4 α. Figure 1 shows the results of local alignment between RSKB (upper sequence in the figure) and HNF-4a (lower sequence in the figure).
実施例 2  Example 2
[0168] (RSKBによる HNF— 4 aのリン酸化）  [0168] (RSKB phosphorylation of HNF-4a)
ヒト HNF— 4 aが RSKBによってリン酸化されるか否かを、インビトロのリン酸化試 験で検討した。ヒト HNF— 4 αおよび RSKBは、それぞれ哺乳類培養細胞にて一過 性発現させた後、該細胞から調製した。 RSKBの活性は CREB1を用いて確認した、 また、リン酸化酵素の陽性対照として、 HNF— 4ひをリン酸ィ匕することが知られている PKAを使用した。 [0169] <材料 > Whether or not human HNF-4a was phosphorylated by RSKB was examined in an in vitro phosphorylation test. Human HNF-4α and RSKB were each transiently expressed in cultured mammalian cells and then prepared from the cells. The activity of RSKB was confirmed using CREB1, and PKA known to phosphorylate HNF-4 was used as a positive control for phosphorylase. [0169] <Material>
ヒト RSKB発現プラスミド (RSKB/pCMV— Tag2)は以下に示すように構築した。 ヒト RSKB cDNAを HeLa細胞由来 cDNA(Human HeLa Quick -clone cD NA、クローンテック（Clontech)社製）より PCRにより獲得し、シーケンスにより配列 を確認した。その後、 N末端に FLAGタグを付加させる動物細胞用発現プラスミド pC MV— Tag2 (ストラタジーン（stratagene)社製）に組込み、 RSKB発現プラスミド（R SKB/pCMV— Tag2)を構築した。クローユングした RSKB cDNAによりコードさ れるアミノ酸配列は NCBI proteinデータベースにァクセシヨン番号 NP— 003933 ( 蛋白質名は RPS6KA4)として開示されたものと同一であった。  A human RSKB expression plasmid (RSKB / pCMV-Tag2) was constructed as shown below. Human RSKB cDNA was obtained by PCR from cDNA derived from HeLa cells (Human HeLa Quick-clone cDNA, manufactured by Clontech), and the sequence was confirmed by sequencing. Thereafter, the plasmid was incorporated into an animal cell expression plasmid pCMV-Tag2 (Stratagene), to which a FLAG tag was added at the N-terminus, to construct an RSKB expression plasmid (R SKB / pCMV-Tag2). The amino acid sequence encoded by the cloned RSKB cDNA was identical to that disclosed in the NCBI protein database as accession number NP-003933 (protein name is RPS6KA4).
[0170] ヒト HNF— 4 α発現プラスミド（HNF— 4 a ZpcDNA 3. lZHis)は以下に示す ように構築した。ヒト HNF— 4 a cDNAを、ヒト脳 polyA+ RNAから RT— PCRに より獲得した。その後、 N末端に（6 X His)— Xpressタグを付加させる動物細胞用発 現プラスミド、 pcDNA3. l/His (インビトロジェン（Invitrogen)社製）に組込み HN F— 4 α発現プラスミド（HNF— 4 a ZpcDNA 3. lZHis)を構築した。クローニン グした HNF— 4 a cDNAによりコードされるアミノ酸配列は、 Swiss— Protデータ ベースにァクセッション番号 P41235 (蛋白質名は HNF4A)として開示されたものと 同一であった。 [0170] A human HNF-4α expression plasmid (HNF-4a ZpcDNA3.1ZHis) was constructed as shown below. Human HNF-4a cDNA was obtained from human brain polyA + RNA by RT-PCR. Subsequently, an expression plasmid for animal cells to which a (6 X His) —Xpress tag is added at the N-terminus, incorporated into pcDNA3.1 / His (Invitrogen) HNF-4α expression plasmid (HNF-4 a ZpcDNA 3.lZHis) was constructed. The amino acid sequence encoded by the cloned HNF-4a cDNA was identical to that disclosed in the Swiss-Prot database as accession number P41235 (protein name HNF4A).
[0171] ヒト CREB1発現プラスミド（CREBlZpcDNA3. lZHis)は以下に示すように構 築した。ヒト CREB1 cDNAを HeLa細胞由来 cDNA(Human HeLa Quick— cl one cDNA、クローンテック社）より PCRにより獲得し、シーケンスにより配列を確認 した。その後、 N末端に（6 X His)—Xpressタグを付加させる動物細胞用発現プラス ミド pcDNA3. l/His (インビトロジェン社製）に組込み、 CREB1発現プラスミド（CR EBl/pcDNA3. l/His)を構築した。クローユングした CREB1 cDNAによりコ ードされるアミノ酸配列は NCBI proteinデータベースにァクセシヨン番号 NP— 00 4370 (蛋白質名は CREB1)として開示されたものと同一であった。  [0171] A human CREB1 expression plasmid (CREBlZpcDNA3.lZHis) was constructed as shown below. Human CREB1 cDNA was obtained by PCR from cDNA derived from HeLa cells (Human HeLa Quick-clone cDNA, Clontech), and the sequence was confirmed by sequencing. Then, add (6 X His) -Xpress tag to the N-terminal to the expression plasmid for animal cells pcDNA3. L / His (Invitrogen) to construct a CREB1 expression plasmid (CR EBl / pcDNA3. L / His) did. The amino acid sequence encoded by the cloned CREB1 cDNA was identical to that disclosed in the NCBI protein database as accession number NP-004370 (protein name CREB1).
[0172] RSKBの活性ィ匕と精製は以下に示すように行った。細胞数 1. 2 X 106の HEK293 T細胞を 37°Cにて 5%COの存在下でー晚培養した後（直径 100mmシャーレ、 4枚 [0172] The activity and purification of RSKB were performed as follows. After cell culture of 1.2 x 10 6 HEK293 T cells at 37 ° C in the presence of 5% CO (100 mm diameter, 4 plates)
2  2
)、 10 gZシャーレの RSKB発現プラスミド（RSKBZpCMV— Tag2)を FuGENE 6 (ロッシュ 'ダイァグノスティックス（Roche Diagnostics)社製）を用いて細胞にトラ ンスフエクシヨンした。 2日間培養した後、終濃度 50mMとなるようにメタ亜ヒ酸ナトリウ ム（sodium arsenite)を培地中に添カ卩し、 37°Cにて 30分間処理して細胞を回収し た。細胞を冷却したリン酸緩衝生理食塩水（―）（以下、 PBS (―)と称する）で洗浄後 、バッファー A(50mM Tris-HCl (pH7. 5) /lmM ェチェンジアミン四酢酸（E DTA) /lmM エチレングリコールビス（2—アミノエチルエーテル）—N, N, Ν' , Ν ，一四酢酸（EGTA) Ζ10% グリセロール Ζ500 μ Μ ジチオスレィトール（DTT) /5mM NaPPi/lmM Na VO /50mM NaF/1% Triton— XI 00)を 4ml ), A 10 gZ Petri dish RSKB expression plasmid (RSKBZpCMV— Tag2) The cells were tranfected using 6 (Roche Diagnostics). After culturing for 2 days, sodium arsenite was added to the medium to a final concentration of 50 mM and treated at 37 ° C. for 30 minutes to recover the cells. After washing the cells with chilled phosphate buffered saline (-) (hereinafter referred to as PBS (-)), buffer A (50 mM Tris-HCl (pH 7.5) / lmM esteramine tetraacetic acid (EDTA) / lmM Ethylene glycol bis (2-aminoethyl ether) —N, N, Ν ', ，, tetraacetic acid (EGTA) Ζ10% glycerol Ζ500 μΜ dithiothreitol (DTT) / 5 mM NaPPi / lmM Na VO / 50 mM NaF / 1% Triton—XI 00) 4ml
3 4  3 4
加えてよくピペッティングし、 4°Cにて 30分間攪拌した。その後、終濃度 150mMとな るよう NaClを加え、 10, 000gで 4°Cにて 10分間遠心処理した。上清を回収し、バッ ファー B (50mM Tris— HCl(pH7. 5) /lmM EDTA/lmM EGTA/10% グリセロール Z500 M DTT/5mM NaPPi/lmM Na VO /50mM Na  In addition, it was pipetted well and stirred at 4 ° C for 30 minutes. Thereafter, NaCl was added to a final concentration of 150 mM, and the mixture was centrifuged at 10,000 g for 10 minutes at 4 ° C. The supernatant was collected and buffer B (50 mM Tris—HCl (pH 7.5) / lmM EDTA / lmM EGTA / 10% Glycerol Z500 M DTT / 5 mM NaPPi / lmM Na VO / 50 mM Na
3 4  3 4
F/150mM NaCl)で洗浄した FLAG M2ァフィ二ティーゲル（シグマ（Sigma)社 製)を 100 1カ卩え、 4°Cにて 1時間転倒混和した。混和後、 10倍容量のバッファー B でゲルを 2回洗浄し、 10倍容量のバッファー C (50mM Tris— HCl(pH7. 5) /0. ImM EGTA/10% グリセローノレ Ζθ. 5 M DTT/150mM NaCl)で 1回 洗浄した。その後ゲルを 400 /z lのバッファー D (50mM Tris—HCl(pH7. 5) /0. ImM EGTA/10% グリセロール /0. 5 ^ M DTT/150mM NaCl/0. 1% ノ-デット p— 40 (NP-40) /300 μ g/ml FLAGペプチド）で 3回溶出した。溶 出した各画分はバッファー Cによる透析を行い、活性型 RSKBとして使用した。透析 後の試料を用いて CBB染色を実施し、活性型 RSKBの精製度を確認した。図 2に透 析後の試料を CBB染色した結果を示す。。透析後の試料中には、活性型 RSKBを 示すバンドが認められた（図 2)。活性型 RSKBは使用まで 80°Cにて保存した。  F / 150 mM NaCl) washed FLAG M2 affinity gel (manufactured by Sigma) was added in an amount of 100 1 and mixed by inversion at 4 ° C for 1 hour. After mixing, the gel is washed twice with 10 times the volume of buffer B, and 10 times the volume of buffer C (50 mM Tris—HCl (pH 7.5) / 0. ImM EGTA / 10% Glycerol Ζθ. 5 M DTT / 150 mM NaCl ) Washed once. The gel was then washed with 400 / zl of buffer D (50 mM Tris—HCl (pH 7.5) / 0. ImM EGTA / 10% glycerol / 0.5 ^ M DTT / 150 mM NaCl / 0.1% nodette p—40 ( NP-40) / 300 μg / ml FLAG peptide). Each eluted fraction was dialyzed against buffer C and used as active RSKB. CBB staining was performed on the dialyzed sample to confirm the purity of active RSKB. Fig. 2 shows the results of CBB staining of the sample after permeation. . A band indicating active RSKB was observed in the dialyzed sample (Fig. 2). The activated RSKB was stored at 80 ° C until use.
[0173] cAMP依存性プロテインキナーゼ（PKA)は、ゥシ PKA (cAMP依存性プロテイン キナーゼ、触媒サブユニット、プロメガ (Promega)社製)を使用した。  As the cAMP-dependent protein kinase (PKA), Ushi PKA (cAMP-dependent protein kinase, catalytic subunit, manufactured by Promega) was used.
[0174] <方法 >  [0174] <Method>
免疫沈降リン酸ィ匕試験は以下に示すように行った。細胞数 7 X 105の HEK293T 細胞を 37°Cにて 5%COの存在下でー晚培養した後（直径 60mmシャーレ）、 5 g の HNF— 4 a発現プラスミド（HNF— 4 a /pcDNA3. l/His)または CREB1発 現プラスミド（CREBlZpcDNA3. l/His)を FuGENE6 (ロッシュ 'ダイァグノステ イツタス社製)を用いて細胞にトランスフエクシヨンした。 2日間培養後、細胞を冷却し た PBS (—）で洗浄し、 RIPAバッファー（50mM Tris— HCl(pH8. 0)/150mMThe immunoprecipitation phosphate test was performed as shown below. HEK293T cells with 7 x 10 5 cells were sputum cultured at 37 ° C in the presence of 5% CO (60 mm diameter petri dish), 5 g HNF-4a expression plasmid (HNF-4a / pcDNA3.l / His) or CREB1 expression plasmid (CREBlZpcDNA3.l / His) was transfected into cells using FuGENE6 (Roche's Diagnostatus) . After culturing for 2 days, the cells were washed with chilled PBS (—) and RIPA buffer (50 mM Tris—HCl (pH 8.0) / 150 mM).
NaCl/1% NP-40/0. 5% デォキシコール酸ナトリウム塩（deoxycholate sodium salt)/0. 1% ドデシル硫酸ナトリウム（SDS))にプロテアーゼインヒビタ 一力クテル (シグマ社製）を添カ卩したものを 500 μ 1加え、ピペッティングにて細胞を懸 濁し、氷上で 20分間静置して細胞を溶解させた。その後、軽くボルテックスをして 14 , OOOrpmで 4°Cにて 10分間遠心処理を行い、上清を採取した。上清 480 1〖こマウ ス IgGァガロース（シグマ社製）を 20 μ 1加え、 4°Cにて 1時間転倒混和した (pre-cle an)0その後、 14, OOOrpmで 4°Cにて 1分間で遠心処理を行ない、採取した上清 45 0 1に抗 Xpress抗体 (インビトロジェン社製） 1 μ 1を加え、 4°Cにて 1時間転倒混和し た後、 0. 1% BSA/TBS(pH8. 0)でブロッキングした Protein G Sepharose 4 Fast Flow (アマシャム'ファノレマシア'ノィォテック (Amersham Pharmacia Biotech社製）を 10 1カ卩え、さらに 4°Cにて 1時間転倒混和を行なった。その後、 Pr otein G Sepharoseを RIPAで 3回、キナーゼバッファー（セル'シグナリング（CellNaCl / 1% NP-40 / 0.5% deoxycholate sodium salt / 0.1% sodium dodecyl sulfate (SDS) supplemented with protease inhibitor Kuttel (manufactured by Sigma) 500 µ1 was added, the cells were suspended by pipetting, and allowed to stand on ice for 20 minutes to lyse the cells. Thereafter, the mixture was vortexed lightly and centrifuged at 14, OOOrpm at 4 ° C for 10 minutes, and the supernatant was collected. Supernatant 480 1 μm Mouse IgG agarose (manufactured by Sigma) 20 μ 1 was added and mixed by inversion for 1 hour at 4 ° C (pre-cle an) 0 Then, 14 rpm at 4 ° C at 1 ° C Centrifugation for 1 minute, add 1 μ 1 of anti-Xpress antibody (Invitrogen) to the collected supernatant 45 0 1 and mix by inverting at 4 ° C for 1 hour, then 0.1% BSA / TBS ( Protein G Sepharose 4 Fast Flow (Amersham 'Fanoremacia' Neotech (Amersham Pharmacia Biotech)), which was blocked with pH 8.0, was added in an amount of 101, and further mixed by inverting at 4 ° C for 1 hour. otein G Sepharose 3 times with RIPA, kinase buffer (Cell 'signaling (Cell
Signaling)社製）で 2回洗浄した。 10 1の洗浄した Protein G Sepharoseに 5 1の活性型 RSKB、 3 1の 10Xキナーゼバッファー（200mM HEPES(pH8. 0) /12mM EDTA/600mM KCl/lOmM DTT/50mM MgCl ), 2^1© 1 Washed twice with Signaling). 10 1 washed Protein G Sepharose, 5 1 active RSKB, 3 1 10X kinase buffer (200 mM HEPES (pH 8.0) / 12 mM EDTA / 600 mM KCl / lOmM DTT / 50 mM MgCl 2), 2 ^ 1 © 1
2  2
00 M アデノシン三リン酸 (ATP)、 0. 5 1の γ 32Ρ— ΑΤΡ(5 Ci)、 8. 5 1の蒸 留水をカ卩ぇ合計 29 1とし、 30°Cにて 30分間反応させた。 PKAを用いた試験では、 上記と同様に調整した 10 ΐの HNF— 4 a /protein G— sepharoseに、 1. 5 1 の ΡΚΑ、 3 1の 10Xキナーゼバッファー、 2 1の ΙΟΟ Μ ATP, 0. δμΚΌγ 32Ρ —ATP(5/zCi)、 3. 5 /zlの IXキナーゼバッファー、 8. 5 1の蒸留水をカ卩ぇ合計 2 9 μ 1とし、 30°C〖こて 30分間反応させた。反応は 20 μ 1の 2 X SDSサンプルバッファ 一を加え停止させ、 100°Cにて 5分間加熱し SDS— PAGE試料として用いた。 10% ゲルにて蛋白質を分離し、ハイブリパックに挟み、 BAS2000(富士フィルム（Fuji F ilm)社製)の画像処理により、リン酸ィ匕の有無の検出を行った。 [0175] <結果 > 00 M adenosine triphosphate (ATP), 0.5 1 γ 32 Ρ— ΑΤΡ (5 Ci), 8.5 1 distilled water totaled 29 1 and reacted at 30 ° C for 30 minutes I let you. In a test using PKA, 10 HNF-4a / protein G-sepharose, prepared as above, was added to 1.5 1 ΡΚΑ, 3 1 10X kinase buffer, 2 1 ΙΟΟ Μ ATP, 0. δμΚΌγ 32 Ρ —ATP (5 / zCi), 3.5 / zl IX kinase buffer, 8.5 1 distilled water totaled 29 μ 1 and incubated at 30 ° C for 30 minutes . The reaction was stopped by adding 20 µl of 2X SDS sample buffer, heated at 100 ° C for 5 minutes, and used as an SDS-PAGE sample. Proteins were separated with a 10% gel, sandwiched between hybrid packs, and the presence or absence of phosphate wrinkles was detected by image processing with BAS2000 (Fuji Film). [0175] <Result>
図 3のパネル Bに示したように、精製された RSKBによる CREB1のリン酸化が認め られたことから、使用した RSKBが活性を有することが分力ゝつた。この活性型 RSKBを 用いて HNF— 4 aに対するリン酸化反応を検討した結果、 RSKB依存的なリン酸ィ匕 が認められた（図 3のパネル B)。また、陽性対照である PKAにより、 HNF— 4 αのリ ン酸化が認められた（図 3のパネル Α)。  As shown in panel B of Fig. 3, the phosphorylation of CREB1 by purified RSKB was observed, indicating that RSKB used was active. As a result of examining the phosphorylation reaction against HNF-4a using this activated RSKB, RSKB-dependent phosphorylation was observed (Panel B in Fig. 3). In addition, phosphorylation of HNF-4α was observed by the positive control PKA (panel 3 in Fig. 3).
[0176] 本結果から、 HNF-4 aは RSKBの基質であることが明らかになった。 [0176] These results revealed that HNF-4a is a substrate for RSKB.
実施例 3  Example 3
[0177] (HNF-4 aの AF1配列に対する結合能の解析）  [0177] (Analysis of the binding ability of HNF-4 a to the AF1 sequence)
PEPCK遺伝子プロモーター領域内の AF1配列への HNF— 4 aの DNA結合能 に対するリン酸ィ匕の影響を EMSAにより検討した。 HNF-4 aのリン酸化は、 RSKB を用いて行った。また、 RSKBの特異性を検討するため、 HNF— 4 aをリン酸化する ことが知られて 、る PKAを用いて同様に EMSAを実施した。  The effect of phosphate on the ability of HNF-4a to bind to the AF1 sequence in the PEPCK gene promoter region was examined by EMSA. Phosphorylation of HNF-4a was performed using RSKB. In addition, in order to investigate the specificity of RSKB, EMSA was similarly performed using PKA, which is known to phosphorylate HNF-4a.
[0178] <材料 >  [0178] <Material>
AF1プローブは、ヒト PEPCK遺伝子のプロモーター領域内の HNF— 4 a結合部 位である AF1相当配列力 設計し合成した次に示す二本のプローブを使用した (シ グマジエノシスジャパン（Sigma Genosis Japan)社製）： AF1/S/HNF4 (5' - GTG ACCTTTG ACTA - 3') (配列番号 7)および AF1ZASZHNF4 (5'— AT AGTCAAAGGTCA- 3') (配列番号 8) (図 4)。ヒト PEPCK遺伝子（NCBIァクセ ッシヨン番号 U31519)のプロモーター領域（配列番号 15)は図 4に示した。 AF1相 当配列は、図 4において太字枠で囲むことにより示した。  The AF1 probe used was the following two probes designed and synthesized by the AF1 equivalent sequence force, which is the HNF-4a binding site in the promoter region of the human PEPCK gene (Sigma Genosis Japan). ): AF1 / S / HNF4 (5′-GTG ACCTTTG ACTA-3 ′) (SEQ ID NO: 7) and AF1ZASZHNF4 (5′—AT AGTCAAAGGTCA-3 ′) (SEQ ID NO: 8) (FIG. 4). The promoter region (SEQ ID NO: 15) of the human PEPCK gene (NCBI accession number U31519) is shown in FIG. The AF1 equivalent sequence is shown in FIG. 4 by surrounding it with a bold frame.
[0179] HNF— 4 α発現プラスミド（HNF— 4 a ZpCMV— Tag2)の構築は、実施例 1で 構築した HNF— 4 α発現プラスミド（HNF— 4 a ZpcDNA 3. lZHis)から、 Eco RI部位で、 HNF— 4 aの cDNA配列を N末端 FLAGタグの付いた動物細胞用発現 プラスミド (pCMV—Tag2、ストラタジーン社製）に組換えを行うことにより実施した。  [0179] The HNF-4α expression plasmid (HNF-4a ZpCMV-Tag2) was constructed from the HNF-4α expression plasmid (HNF-4a ZpcDNA 3. lZHis) constructed in Example 1 at the Eco RI site. The cDNA sequence of HNF-4a was recombined into an expression plasmid for animal cells (pCMV-Tag2, manufactured by Stratagene) with an N-terminal FLAG tag.
[0180] HNF-4 aの細胞での発現並びに該細胞からの HNF— 4 aの調製および精製は 以下に示すように行った。細胞数 1. 2 X 106の HEK293T細胞を 37°Cにて 5%CO [0180] Expression of HNF-4a in cells and preparation and purification of HNF-4a from the cells were performed as follows. Cell count 1.2 × 10 6 HEK293T cells at 37 ° C, 5% CO
2 の存在下でー晚培養した後（直径 100mmシャーレ、 4枚）、 10 μ gZシャーレの ΗΝ F -4 a発現プラスミド（HNF— 4 a ZpCMV— Tag2)を FuGENE6 (ロッシュ 'ダイ ァグノスティックス社製)を用いて細胞にトランスフエクシヨンした。 2日間培養後、細胞 を冷却した PBS (―)で洗浄し、 4mlのバッファー A (50mM Tris— HCl (pH7. 5) /ImM EDTA/lmM EGTA/10% グリセロール Z500 M DTT/5mM NaPPi/lmM Na3VO4/50mM NaF/1 % Triton— XI 00)を加えてよく ピペッティングし、 4°Cにて 30分間攪拌した。その後、終濃度 150mMとなるよう NaCl を加え、 10, 000gで 4°Cにて 10分間遠心処理した。上清を回収し、ノ ッファー B (50 mM Tris -HCl (pH7. 5) /ImM EDTA/lmM EGTA/10% グリセロー /V/500 μ M DTT/5mM NaPPi/lmM Na3VO4/50mM NaF/150m M NaCl)で洗浄した 100 1の FLAG M2 Affinity Gel (シグマ社製）をカロえ、 4°Cにて 1時間転倒混和した。混和後、 10倍容量のバッファー Bでゲルを 2回、ノ ッフ ァー C (50mM Tris— HCl (pH7. 5) /0. ImM EGTA/10% グリセロール Z 0. 5 ^ Μ DTT/150mM NaCl)で 1回洗浄した。その後ゲルを 400 1のバッフ ァー D (50mM Tris -HCl (pH7. 5) /0. ImM EGTA/10% glycerol/0. 5 ^ Μ DTT/150mM NaCl/0. 1 % ΝΡ -40/300 μ g/ml FLAGぺプチ ド)で 2回溶出した。溶出した各画分はそれぞれ SDS— PAGEを行い、ノ ッファー C にて透析した。透析後の試料は、 CBB染色によりその精製度を確認した。図 5に透 析後の試料をを CBB染色した結果を示す。透析後の試料中には、 FLAG -HNF- 4 aを示すバンドが認められた（図 5)。 After culturing in the presence of 2 (100 mm diameter petri dish, 4 plates), 10 μgZ petri dish The F-4a expression plasmid (HNF-4a ZpCMV-Tag2) was transfected into cells using FuGENE6 (Roche Diagnostics). After culturing for 2 days, the cells were washed with chilled PBS (-), and 4 ml of buffer A (50 mM Tris-HCl (pH 7.5) / ImM EDTA / lmM EGTA / 10% glycerol Z500 M DTT / 5 mM NaPPi / lmM Na3VO4 / 50 mM NaF / 1% Triton—XI 00) was added and pipetted well, and the mixture was stirred at 4 ° C. for 30 minutes. Thereafter, NaCl was added to a final concentration of 150 mM, and the mixture was centrifuged at 10,000 g for 10 minutes at 4 ° C. The supernatant was collected and nofer B (50 mM Tris-HCl (pH 7.5) / ImM EDTA / lmM EGTA / 10% glycero / V / 500 μM DTT / 5 mM NaPPi / lmM Na3VO4 / 50 mM NaF / 150 mM NaCl 100 1 of FLAG M2 Affinity Gel (manufactured by Sigma) was washed and mixed by inversion at 4 ° C for 1 hour. After mixing, gel twice with 10x volume of buffer B, Knoffer C (50 mM Tris—HCl (pH 7.5) / 0. ImM EGTA / 10% Glycerol Z 0.5 ^ Μ DTT / 150 mM NaCl ) Washed once. Then the gel was buffered with 400 1 buffer D (50 mM Tris-HCl (pH 7.5) / 0. ImM EGTA / 10% glycerol / 0.5 ^ Μ DTT / 150 mM NaCl / 0.1% ΝΡ -40/300 μ G / ml FLAG peptide) was eluted twice. Each eluted fraction was subjected to SDS-PAGE and dialyzed against Notfer C. The purity of the dialyzed sample was confirmed by CBB staining. Fig. 5 shows the results of CBB staining of the sample after permeation. In the sample after dialysis, a band indicating FLAG-HNF-4a was observed (FIG. 5).
<方法 > <Method>
AF1プローブの放射性標識化および精製は以下に示すように行った。合成した A F1プローブを各々 lOOpmolZ μ 1になるように蒸留水に溶解し、 100°Cにて 2分間、 38°Cにて 1時間加熱後、自然冷却して DNAをァニールさせた。これを 10倍希釈し て 0. 5 μ 1を分取し、 1 μ 1の Τ4ポリヌクレオチドキナーゼ (ΤΟΥΟΒΟ社製）、 6 1の γ "2P— ATP (60 Ci)、 2 1の 10 X Protruding End Kinase Buffer (500m M Tris— HCl (pH8. 0) /100mM MgCl /lOOmM 2—メルカプトエタノール Radiolabeling and purification of the AF1 probe was performed as follows. The synthesized A F1 probes were dissolved in distilled water so that each of them was lOOpmolZ μ1, heated at 100 ° C for 2 minutes and at 38 ° C for 1 hour, and then naturally cooled to anneal the DNA. Dilute this 10-fold to obtain 0.5 μl, 1 μl of Τ4 polynucleotide kinase (manufactured by Koeisha), 6 1 γ ” 2 P—ATP (60 Ci), 2 1 10 X Protruding End Kinase Buffer (500mM Tris—HCl (pH8.0) / 100mM MgCl / lOOmM 2—Mercaptoethanol
2  2
)、 10. 5 1の蒸留水をカ卩ぇ 37°Cにて 1時間インキュベーションした。 Protruding E ndとは「突出末端」を意味し、 Protruding End Kinase Bufferは、 T4ポリヌクレ ォチドキナーゼが 2本鎖 DNAの突出末端の ^—末端に γ—ATPの γ位のリン酸 基を付加する際に用いるバッファーである。その後、 5 1の 0. 25Μ EDTA, 5 μ 1 の 20mg/ml イースト RNAを加え、蒸留水で 100 μ 1とし、フエノール'クロ口ホルム 処理、エタノール沈殿を行い、 50 1の STE (100mM NaCl/lOmM Tris— HC 1 (ρΗ8. 0) /lmM EDTA (pH8. 0) )に溶解した。その後、 CENTRI ' SPIN 20 (プリンストン 'セパレーシヨンズ（Princeton Separations社製）でゲルろ過を行い、 放射性標識 AF1プローブを得た。 ), 10. 51 1 distilled water was incubated at 37 ° C for 1 hour. Protruding End means `` protruding end '' and Protruding End Kinase Buffer is a T4 polynucleotide. This buffer is used when the photokinase adds a phosphate group at the γ position of γ-ATP to the ^ -end of the protruding end of double-stranded DNA. Then add 0.25Μ EDTA of 5 1, 20 mg / ml yeast RNA of 5 μ 1 to 100 μ 1 with distilled water, treat with phenol 'chloroform, precipitate with ethanol, and add 50 1 STE (100 mM NaCl / lOmM Tris—dissolved in HC 1 (ρΗ8.0) / lmM EDTA (pH 8.0)). Then, gel filtration was performed with CENTRI 'SPIN 20 (Princeton Separations (manufactured by Princeton Separations)) to obtain a radiolabeled AF1 probe.
[0182] RSKBおよび PKAによるリン酸化処理と EMSAは以下に示すように行った。 6. 25 μ 1の FLAG—HNF—4 α〖こ、 5 1の段階的（1 X、 1/2, 1/4)に希釈した活性型 RSKB、 2 1の 10 Xキナーゼバッファー（200mM HEPES (pH8. 0) /12mM EDTA/600mM KCl/lOmM DTT/50mM MgCl )、 4 1の ImM ATP [0182] Phosphorylation with RSKB and PKA and EMSA were performed as shown below. 6. 25 μ 1 FLAG—HNF—4 α, 5 1 graded (1 X, 1/2, 1/4) diluted active RSKB, 2 1 10 X kinase buffer (200 mM HEPES ( pH 8.0) / 12mM EDTA / 600mM KCl / lOmM DTT / 50mM MgCl), 4 1 ImM ATP
2  2
と 2. 75 1の蒸留水を力卩ぇ計 20 /z lとし、 30°Cにて 30分間反応させた。 PKAによる リン酸ィ匕では RSKBの代わりに 1. 5 1の PKAを使用した。反応後、 4 1の反応液を 分取し、 2 1の 10 Xバインディングバッファー（lOOmM Tris— HCl (pH7. 5) /1 mM EDTA/500mM NaCl/lmM DTT/50% グリセロール）、 1 1の lm g/ml poly dIdC、 11 μ 1の蒸留水を混合し計 18 μ 1とし、氷上で 10分間反応させ た。この時、一部のサンプルには抗 HNF— 4 α抗体（C— 19、サンタクルヅ（Santa Cruz)社製） 1 μ 1を添加した。その後、放射性標識 AF1プローブを 2 1加え、室温 で 20分間反応させた。 6%アクリルアミドゲルを 0. 25 X TBE (0. 25 X EDTA含有ト リスホウ酸バッファー（12. 5mM Tris-HCl/12. ImM ホウ酸））で 4°Cにて 1時 間プレランした後、各試料を 15 1ずつアプライし、電気泳動を 1時間 50分、 4°Cにて 行った。ゲルを 65°Cにて 1時間乾燥させた後、 BAS 2000 (富士フィルム社製）での 画像処理により、プローブへの HNF— 4 aの結合の有無の検出を行った。  And 2.75 1 of distilled water was vigorously adjusted to 20 / z l and reacted at 30 ° C for 30 minutes. Phosphoric acid solution by PKA used 1.51 PKA instead of RSKB. After the reaction, separate the reaction mixture of 41, 2 1 of 10 X binding buffer (lOOmM Tris—HCl (pH 7.5) / 1 mM EDTA / 500 mM NaCl / lmM DTT / 50% glycerol), 1 1 lm G / ml poly dIdC and 11 μ1 of distilled water were mixed to make a total of 18 μ1, and reacted on ice for 10 minutes. At this time, 1 μ 1 of anti-HNF-4α antibody (C-19, manufactured by Santa Cruz) was added to some samples. Thereafter, 21 1 of radiolabeled AF1 probe was added and reacted at room temperature for 20 minutes. A 6% acrylamide gel was pre-run at 0.25 X TBE (0.25 mM EDTA-containing trisborate buffer (12.5 mM Tris-HCl / 12. ImM borate)) at 4 ° C for 1 hour, Samples were applied one by one and electrophoresis was performed at 4 ° C for 1 hour and 50 minutes. After the gel was dried at 65 ° C for 1 hour, the presence or absence of HNF-4a binding to the probe was detected by image processing with BAS 2000 (Fuji Film).
[0183] <結果 > [0183] <Result>
図 6のパネル Aに示したように、 HNF-4 aと AF1プローブとの複合体（以下、 HN F— 4 α ' DNA複合体と称する）を示すバンドが検出された。抗 HNF— 4 α抗体の添 加により、 HNF— 4 α ' DNA複合体の移動度が小さくなるスーパーシフトが見られた 。この結果から、抗 HNF— 4 α抗体非添カ卩において検出されたバンドは、 HNF-4 a - DNA複合体を示すものであることが確認できた。また、図 6のパネル Bに示したよ うに、 RSKBによるリン酸化処理により、 HNF— 4 a ' DNA複合体の量が増加した。 一方、 ΡΚΑによるリン酸化処理では、 HNF— 4 α . DNA複合体の量の増加は検出 されなかった。 As shown in panel A of FIG. 6, a band indicating a complex of HNF-4a and AF1 probe (hereinafter referred to as HNF-4α ′ DNA complex) was detected. The addition of the anti-HNF-4α antibody showed a supershift in which the mobility of the HNF-4α 'DNA complex was reduced. From this result, the band detected in the non-anti-HNF-4α antibody-supplemented band was HNF-4 It was confirmed that the a-DNA complex was shown. In addition, as shown in FIG. 6 panel B, the amount of HNF-4a ′ DNA complex was increased by the phosphorylation treatment with RSKB. On the other hand, phosphorylation treatment with sputum did not detect an increase in the amount of HNF-4α.DNA complex.
[0184] 図 6に示した結果から、 HNF-4 aの AF1配列への結合能力 RSKBによるリン酸 化処理特異的に促進されることが判明した。  [0184] From the results shown in FIG. 6, it was found that the binding ability of HNF-4a to the AF1 sequence was specifically promoted by the phosphorylation treatment by RSKB.
[0185] 図 7に示したように、 HNF-4 a ' DNA複合体の量は、 HNF— 4 aのリン酸化処理 に用いた RSKB量を減少させるに伴って減少した。本結果から、 HNF— 4 αの AF1 配列への結合能力 HNF-4 aのリン酸ィ匕処理に用いた RSKB量を減少させるに 伴って低下したと考えることができる。一方、 PKAによるリン酸ィ匕処理では、 HNF— 4 a ' DN A複合体の量の増力！]、すなわち HNF— 4 αの AF1配列への結合能の亢進 は認められなかった。  [0185] As shown in Fig. 7, the amount of HNF-4a 'DNA complex decreased as the amount of RSKB used for the phosphorylation of HNF-4a decreased. From this result, it can be considered that the binding ability of HNF-4α to the AF1 sequence decreased as the amount of RSKB used for the phosphorylation treatment of HNF-4a decreased. On the other hand, phosphoric acid treatment with PKA increases the amount of HNF-4a 'DN A complex! That is, no enhancement of the binding ability of HNF-4α to the AF1 sequence was observed.
[0186] このように、 RSKBでリン酸化処理された HNF— 4 aは、非処理試料に比べ AF1 配列に対する DNA結合能が亢進することが判明した。また、 RSKBでリン酸化処理 された HNF— 4 aの DNA結合能の亢進は、リン酸化処理に用いた RSKBの用量依 存的であることが判明した。一方、 PKAにてリン酸ィ匕処理された HNF— 4 Q;は、非処 理試料と同程度の DNA結合能しか示さなかった。このことは、 RSKB処理による HN F-4 aの DNA結合能の亢進力 特異的なものであることを示す。  [0186] Thus, HNF-4a phosphorylated with RSKB was found to have enhanced DNA binding ability to the AF1 sequence compared to the untreated sample. It was also found that the enhancement of the DNA binding ability of HNF-4a phosphorylated with RSKB was dependent on the dose of RSKB used for phosphorylation. On the other hand, HNF-4 Q; that had been phosphated with PKA showed only the same DNA binding ability as the untreated sample. This indicates that the DNA-binding ability of HNF-4a by RSKB treatment is specific.
実施例 4  Example 4
[0187] (PEPCK遺伝子プロモーターを用いたレポーターアツセィ）  [0187] (Reporter assembly using PEPCK gene promoter)
PEPCK遺伝子プロモーターにおける HNF— 4 aの転写活性能に及ぼす RSKB の影響を、ルシフェラーゼレポーターアツセィにより、 HeLa細胞（ヒト子宮頸癌由来 細胞株）と HepG2細胞 (ヒト肝臓癌由来細胞株）を用いて検討した。レポーター遺伝 子として、ヒト PEPCK遺伝子プロモーター領域のうち HNF— 4 aのシスエレメントで ある AF 1を含む領域の下流にホタルルシフェラーゼ遺伝子を融合させた遺伝子 (PE PCK AF1プロモーター依存性ルシフェラーゼレポーター）を用いた。  The effect of RSKB on the transcriptional activity of HNF-4a in the PEPCK gene promoter was determined using HeLa cells (human cervical cancer-derived cell line) and HepG2 cells (human liver cancer-derived cell line) using luciferase reporter assembly. investigated. As a reporter gene, a gene (PE PCK AF1 promoter-dependent luciferase reporter) in which a firefly luciferase gene is fused downstream of a region containing AF 1 which is a cis element of HNF-4a in the human PEPCK gene promoter region was used. .
[0188] <材料 > [0188] <Material>
RSKB発現プラスミドは、実施例 2で作製した RSKB/pCMV— Tag2を使用した [0189] ドミナントネガティブ RSKB (S343A)発現プラスミド（RSKB (S343A) /pCMV- Tag2)は、 RSKB/pCMV— Tag2を铸型として、 QuikChange Site - Directed Mutagenesis kit (ストラタジーン社製）を用いて、 RSKBの第 343番目のアミノ酸 置換を実施して構築した。 RSKBはそのキナーゼ活性に 196番目のセリン、 343番 目のセリンおよび 568番目のスレオニンのリン酸化が必要でありこれらアミノ酸のいず れカ 1つをァラニンに置換するとキナーゼ活性が消失することが報告されて 、る（非 特許文献 34)。したがって、本発現プラスミドにより発現される蛋白質 RSKB (S343 A)は、 RSKBの第 343番目のセリンのァラニンへの置換によりそのキナーゼ活性が 消失して 、る不活性型変異体 RSKBである。 RSKB expression plasmid used was RSKB / pCMV-Tag2 prepared in Example 2 [0189] Dominant negative RSKB (S343A) expression plasmid (RSKB (S343A) / pCMV- Tag2) is RSKB / pCMV- Tag2 as a saddle, using QuikChange Site-Directed Mutagenesis kit (Stratagene) RSKB The amino acid substitution at position 343 was performed. RSKB requires phosphorylation of 196th serine, 343th serine and 568th threonine for its kinase activity, and it is reported that the kinase activity disappears when one of these amino acids is substituted with alanine. (Non-patent document 34). Therefore, the protein RSKB (S343 A) expressed by this expression plasmid is an inactive mutant RSKB in which the kinase activity disappears by substitution of the 343rd serine of RSKB with alanine.
[0190] HNF-4 a発現プラスミドは、実施例 3で作製した HNF— 4 a /pCMV— Tag2を 使用した。  [0190] The HNF-4a expression plasmid used was HNF-4a / pCMV-Tag2 prepared in Example 3.
[0191] MAPK11発現プラスミド（MAPK11 /pCMV - Tag4)の構築は以下に示すよう に行った。ヒト MAPK1 l (p38 - j8 2) cDNAはヒト脳由来 cDNA (Human brain whole Quick -clone cDNA、クローンテック社製）より PCRにより獲得し、シーケ ンスにて配列を確認した。その後、 C末端に FLAGタグを付加させる動物細胞用発 現プラスミド pCMV— Tag4 (ストラタジーン社製）に組込み、 MAPK11発現プラスミ ド（p38— beta2/pCMV— Tag4)を構築した。なお、クローユングした MAPK11 cDNAによりコードされるアミノ酸配列は Swiss— Protデータベースのァクセシヨン番 号 Q 15759 (蛋白質名は MAPK11ZSAPK2B)と同一である。  [0191] A MAPK11 expression plasmid (MAPK11 / pCMV-Tag4) was constructed as shown below. Human MAPK11 (p38-j82) cDNA was obtained from human brain whole cDNA (Human brain whole Quick-clone cDNA, Clontech) by PCR, and the sequence was confirmed by sequencing. Thereafter, the plasmid was incorporated into an expression plasmid for animal cells pCMV-Tag4 (Stratagene) to which a FLAG tag was added at the C terminus, and a MAPK11 expression plasmid (p38-beta2 / pCMV-Tag4) was constructed. The amino acid sequence encoded by the cloned MAPK11 cDNA is the same as the Swiss-Prot database accession number Q 15759 (protein name is MAPK11ZSAPK2B).
[0192] PEPCK AF1プロモーター依存性ルシフェラーゼレポータープラスミド（PEPCK  [0192] PEPCK AF1 promoter-dependent luciferase reporter plasmid (PEPCK
AF1プロモーター/ pGL3)の構築は、以下に示すように実施した。ヒト PEPCK (P CK1)プロモーター領域のうち HNF— 4 aのシスエレメントである AF1を含む領域（ NCBIデータベースのァクセッション番号 U31519の塩基配列 882〜1406番目）を ヒトゲノム DNA (クローンテック社製)より PCRにより獲得し、シーケンスにて配列を確 認した。この領域は PEPCK遺伝子のェキソン 1の 1番目の塩基位置を + 1とすると 469〜+ 63に相当する（非特許文献 35)。その後、この領域をレポーター遺伝子とし てホタルルシフェラーゼを発現させるレポータープラスミド pGL3— Basic (プロメガ社 製）に挿入し、 PEPCK AF1プロモーター ZpGL3を構築した。 The construction of AF1 promoter / pGL3) was performed as shown below. Human PEPCK (P CK1) promoter region containing AF1 which is HNF-4a cis element (base sequence 882 to 1406 of NCBI database accession number U31519) from human genomic DNA (Clontech) Obtained by PCR, the sequence was confirmed by sequencing. This region corresponds to 469 to +63, assuming that the first base position of exon 1 of PEPCK gene is +1 (Non-patent Document 35). Then, reporter plasmid pGL3-Basic (Promega Corp.) that expresses firefly luciferase using this region as a reporter gene. PEPCK AF1 promoter ZpGL3 was constructed.
[0193] PEPCK A AF1プロモーター依存性ルシフェラーゼレポータープラスミド（PEPC[0193] PEPCK A AF1 promoter-dependent luciferase reporter plasmid (PEPC
K A AFlプロモーター ZpGL3)の構築は以下に示すように行った。上記クロー- ングしたヒト PEPCK遺伝子プロモーター領域から、 AF 1を含まな 、部分（U31519 の塩基配列 934〜1406番目）を PCRにより獲得した。シーケンスにて配列を確認後The KA AFl promoter ZpGL3) was constructed as shown below. From the cloned human PEPCK gene promoter region, a portion not containing AF1 (base sequence 934-1406 of U31519) was obtained by PCR. After confirming the sequence by sequence
、 pGL3— Basic (プロメガ社製）に挿入して構築した。 PGL3—Basic (manufactured by Promega) and inserted.
[0194] ダルココルチコイドレセプター（Glucocorticoid receptor)発現プラスミドは、ラッ トグルココルチコイドレセプターを発現させる pMMGRを使用した（非特許文献 36)。 [0194] As a glucocorticoid receptor expression plasmid, pMMGR that expresses a rat glucocorticoid receptor was used (Non-patent Document 36).
[0195] 遺伝子導入効率補正プラスミド (インターナルコントロール）には、レポーター遺伝 子としてゥミシィタケルシフェラーゼを発現させる phRL— nullプラスミドまたは pRL—[0195] For gene transfer efficiency-corrected plasmids (internal control), phRL—null plasmid or pRL—expressing Renilla luciferase as a reporter gene
SV40プラスミド (プロメガ社製)を使用した。 SV40 plasmid (Promega) was used.
[0196] MAPKl l阻害剤には、 SB203580をプロメガ社より購入して用いた。 [0196] As the MAPKl l inhibitor, SB203580 was purchased from Promega.
[0197] <方法 > [0197] <Method>
HeLa細胞 (ヒト子宮頸癌由来細胞株）におけるレポーターアツセィは以下に示すよ うに行った。細胞数 6 X 104Zゥエルの HeLa細胞を 37°Cにて 5%COの存在下で Reporter assembly in HeLa cells (human cervical cancer-derived cell line) was performed as follows. 6 X 10 4 Zwell HeLa cells at 37 ° C in the presence of 5% CO
2 一 晚培養した後（24ゥエルプレート（2. 0cm2Zゥエル））、 0. 25 gの PEPCK AF1 プロモーター ZpGL3、 0. 05〜0. 2 μ g( HNF-4 a /pCMV~Tag2, 0. 25 μ gの RSKB/pCMV— Tag2、 0. 05 gの MAPKl l/pCMV— Tag4および 0. 0 25 μ gのインタナールコントロール phRL— nullを予め設定した組み合わせで FuGE NE6 (ロッシュ 'ダイァグノスティックス社製）を用いて細胞にトランスフエクシヨンした。 総 DNA量は各実験群で一定量となるように、空ベクター pCMV— Tag2 (ストラタジ ーン社製）にて補正した。 2日間培養後、細胞を冷却した PBS (—）で洗浄し、 Dual -Lusif erase Reporter Assay kit (プロメガ社製）にて細胞ライセート中のホタ ルルシフェラーゼ活性およびゥミシィタケルシフェラーゼ活性を測定した。転写活性 はホタルルシフェラーゼ活性値をゥミシィタケルシフェラーゼ活性値で除した後、コン トロール群（PEPCK AF1プロモーター ZpGL3のみ）に対する倍数で表示した。 2 After culturing (24 well plate (2.0 cm 2 Zwell)), 0.25 g of PEPCK AF1 promoter ZpGL3, 0.05-0.2 μg (HNF-4 a / pCMV ~ Tag2, 0.25 μg RSKB / pCMV— Tag2, 0.05 g MAPKl l / pCMV— Tag4 and 0.025 μg internal control phRL— FuGE NE6 (Roche Dia The cells were transfected with Gnostics Co., Ltd. The total DNA amount was corrected with an empty vector pCMV-Tag2 (Stratagene) so that the total amount of DNA was constant in each experimental group. After culturing, the cells were washed with chilled PBS (—), and the firefly luciferase activity and the renilla luciferase activity in the cell lysate were measured with Dual-Lusif erase Reporter Assay kit (Promega). The firefly luciferase activity value is divided by the Renilla luciferase activity value. After, viewed in multiples relative to controls group (PEPCK AF1 promoter ZpGL3 only).
[0198] HepG2細胞 (ヒト肝癌由来細胞株）におけるレポーターアツセィは以下に示すよう に行った。細胞数 1 X 104Zゥエルの HepG2細胞を 37°Cにて 5%COの存在下で一 晚培養した後（24ゥエルプレート（2. 0cm2Zゥエル）、 0. 5 gの PEPCK AF1プロ モーター ZpGL3、 0. 5 μ g( HNF-4 a /pCMV~Tag2, 0. 25 gおよび 0. 5 μ gの RSKBZpCMV— Tag2、 0. 5 ^ gの MAPKl lZpCMV— Tag4、 0. 5 ^ g の pMMGRおよび 0. 05 μ gのインターナルコントロール pRL—SV40を予め設定し た組み合わせで FuGENE6 (ロッシュ 'ダイァグノスティックス社製）を用いて細胞にト ランスフエクシヨンした。総 DNA量は各実験群で一定量となるように、空ベクター pC MV— Tag2 (ストラタジーン社製）にて補正した。 MAPK11阻害剤 SB203580を添 加した群は、 FuGENE6によるトランスフエクシヨン直前に終濃度 10 μ Μとなるように SB203580を培地に添加した。トランスフエクシヨン後 20時間にデキサメサゾンを終 濃度 1 Μとなるように培地に添加し、さらに 24時間培養後、細胞を冷却した PBS ( 一）で洗浄し、 Dual— Lusiferase Reporter Assay kit (プロメガ社製）にて細胞 ライセート中のホタルルシフェラーゼ活性およびゥミシィタケルシフェラーゼ活性を測 定した。転写活性はホタルルシフェラーゼ活性値をゥミシィタケルシフェラーゼ活性 値で除した後、 RSKB非発現群 (HNF -4 a 0. 5)に対する倍数で表示した。 [0198] Reporter assembly in HepG2 cells (human liver cancer-derived cell line) was performed as follows. 1 x 10 4 Zwell HepG2 cells at 37 ° C in the presence of 5% CO After sputum culture (24 well plate (2.0 cm 2 Z well), 0.5 g PEPCK AF1 promoter ZpGL3, 0.5 μg (HNF-4 a / pCMV ~ Tag2, 0.25 g and 0 5 μg RSKBZpCMV— Tag2, 0.5 ^ g MAPKl lZpCMV— Tag4, 0.5 ^ g pMMGR and 0.05 μg internal control pRL—SV40 with a preset combination of FuGENE6 (Roche The cells were transferred using the “Diagnostics Co., Ltd.” using the empty vector pC MV-Tag2 (Stratagene) so that the total amount of DNA was constant in each experimental group. In the group to which the MAPK11 inhibitor SB203580 was added, SB203580 was added to the medium so that the final concentration was 10 μΜ immediately before transfection with FuGENE 6. Dexamethasone was added to the final concentration at 1 hour 20 hours after the transfection. PBS was added to the culture medium and cultured for 24 hours. After washing, the firefly luciferase activity and renilla luciferase activity in the cell lysate were measured with the Dual-Lusiferase Reporter Assay kit (Promega) .The transcriptional activity was the value of the luciferase activity value. After dividing by RS, it was expressed as a multiple of the RSKB non-expression group (HNF-4a 0.5).
[0199] 統計解析は、バートレット検定によって分散を、多重比較検定によって平均値をそ れぞれ検定した。  [0199] For statistical analysis, variance was tested by Bartlett's test and mean was tested by multiple comparison test.
[0200] <結果 >  [0200] <Result>
HeLa細胞において、 PEPCK AF1プロモータールシフェラーゼレポーター遺伝 子と HNF— 4 a遺伝子を共発現させたときに、 HNF-4 aの用量依存的に転写活 性の増加が認められた（図 8)。この結果から、 HNF— 4 α力 PEPCK遺伝子の転写 活性を正に制御することが確認できた。  In HeLa cells, when the PEPCK AF1 promoter luciferase reporter gene and the HNF-4a gene were co-expressed, an increase in the transcriptional activity of HNF-4a was observed in a dose-dependent manner (Fig. 8). From this result, it was confirmed that the transcriptional activity of HNF-4α force PEPCK gene was positively controlled.
[0201] 一方、 RSKBを HNF— 4 aと共発現させ HNF— 4 aの転写活性に対する影響を 検討した力 RSKB非発現群と明瞭な差は認められな力つた（図 8)。これは RSKB 自身のキナーゼ活性が不十分であるためと考えられた。  [0201] On the other hand, RSKB was co-expressed with HNF-4a, and the effect on the transcriptional activity of HNF-4a was examined. There was no clear difference from the RSKB non-expression group (Fig. 8). This was thought to be due to insufficient kinase activity of RSKB itself.
[0202] RSKBを活性化させるために上流キナーゼである MAPK11 (p38 - β 2)を RSK Bと共発現させ、転写活性への影響を検討した。その結果、図 9に示したように、 HN F— 4 α発現群（HNF— 4 α 0. 05)に比べ、 HNF— 4 αと活性化 RSKBを共発現 させた群（HNF— 4 α 0. 05 +RSKB 0. 25 + MAPK11 0. 05)では転写活性 力 S3倍以上増加した。一方、 RSKBを除き、 HNF-4 aと MAPK11を共発現させた 群（HNF— 4 α 0. 05 + MAPK11 0. 05)では HNF— 4 αの転写活性の促進は 認められな力つた（図 9)。さらに、図 10に示したように、キナーゼ活性を持たないドミ ナントネガティブ RSKB (S343A)、 MAPK11および HNF— 4 aを共発現させた細 胞では、 HNF-4 aの転写活性の促進が見られなかった（HNF— 4 a 0. 05 +RS KB (S343A) 0. 25 + MAPK11 0. 05)。 [0202] In order to activate RSKB, MAPK11 (p38-β2), an upstream kinase, was co-expressed with RSK B, and the effect on transcriptional activity was examined. As a result, as shown in Fig. 9, compared to the HNF-4α expression group (HNF-4α 0.05), the group that co-expressed HNF-4α and activated RSKB (HNF-4α0). 05 + RSKB 0. 25 + MAPK11 0. 05) Power S3 more than increased. On the other hand, with the exception of RSKB, HNF-4 α and MAPK11 co-expressed group (HNF-4 α 0. 05 + MAPK11 0. 05) did not promote HNF-4 α transcriptional activity (Fig. 9). Furthermore, as shown in Fig. 10, the cells that co-expressed dominant negative RSKB (S343A), MAPK11, and HNF-4a that do not have kinase activity showed enhanced transcriptional activity of HNF-4a. (HNF— 4 a 0. 05 + RS KB (S343A) 0.25 + MAPK11 0. 05).
[0203] また、 AF1を除いた PEPCK A AF1プロモーターノレシフェラーゼレポーター遺伝 子を用いた場合は、 HNF— 4ひの転写活性が認められな力つた（図 11)。このことか ら、上記のように PEPCK AF1プロモーターノレシフェラーゼレポーター遺伝子を用 いたときに認められた HNF— 4 aの転写活性は、 HNF— 4 aの AF1への結合を介 して行われて 、ることが確認された。  [0203] Moreover, when the PEPCK A AF1 promoter noreciferase reporter gene excluding AF1 was used, the transcriptional activity of HNF-4 was not recognized (FIG. 11). From this, the transcriptional activity of HNF-4a observed when using the PEPCK AF1 promoter noreciferase reporter gene as described above is performed through the binding of HNF-4a to AF1. That was confirmed.
[0204] 上記結果から、 RSKBによってリン酸化された HNF— 4 aは PEPCK遺伝子プロモ 一ターの AF1への結合を介して該プロモーターの転写活性を促進することが実証さ れた。  [0204] From the above results, it was demonstrated that HNF-4a phosphorylated by RSKB promotes the transcriptional activity of the promoter through binding of the PEPCK gene promoter to AF1.
[0205] 肝癌細胞由来の HepG2細胞では、 MAPK11、 RSKBおよび HNF— 4 aを共発 現させた群（HNF— 4 α 0. 5 + RSKB 0. 5 + MAPK11 0. 5)の HNF— 4 αの PEPCK AF1プロモーターに対する転写活性が、 RSKB非発現群（HNF— 4 α 0 . 5)と比較して 3倍以上増加した（図 12)。さらに、これらを共発現させた細胞に MAP Kl 1阻害剤 SB203580を添加し、 MAPK11による RSKBの活性化を阻害すると（ HNF-4 a 0. 5 +RSKB 0. 5 + MAPK11 0. 5 + SB203580) , HNF-4 α の転写活性の増加が消失した（図 12)。  [0205] In HepG2 cells derived from hepatoma cells, HNF-4 α from the group that co-expressed MAPK11, RSKB, and HNF-4a (HNF-4 α 0.5 + RSKB 0.5 + MAPK11 0.5) Transcriptional activity against PEPCK AF1 promoter increased by 3 times or more compared to the RSKB non-expression group (HNF-4α0.5) (FIG. 12). Furthermore, when the MAP Kl 1 inhibitor SB203580 is added to cells co-expressed with these cells, and the activation of RSKB by MAPK11 is inhibited (HNF-4a 0.5 + RSKB 0.5 + MAPK11 0.5 + SB203580) Therefore, the increase in transcriptional activity of HNF-4α disappeared (Fig. 12).
[0206] 上記結果から、 HepG2細胞においても、 RSKBによってリン酸化された HNF— 4 αは AF1を含む PEPCK遺伝子プロモーターの転写活性を促進することが実証され た。  [0206] From the above results, it was demonstrated that HNF-4α phosphorylated by RSKB also promotes the transcriptional activity of the PEPCK gene promoter including AF1 in HepG2 cells.
実施例 5  Example 5
[0207] (PEPCK遺伝子の発現に対する RSKBの活性化の影響）  [0207] (Effect of RSKB activation on PEPCK gene expression)
HepG2細胞 (ヒト肝臓癌由来細胞株）に、野生型 RSKBまたは不活性型変異体 RS KBと、 RSKBの上流キナーゼである MAPK11を一過性発現させ、 PEPCK遺伝子 の発現を測定した。 HepG2 cells (human liver cancer-derived cell line) are transiently expressed with wild type RSKB or inactive mutant RS KB and MAPK11, an upstream kinase of RSKB, and the PEPCK gene The expression of was measured.
[0208] <材料 >  [0208] <Material>
野生型 RSKB発現プラスミドは、実施例 2で作製した RSKB/pCMV— Tag2を使 用した。 SKBZpCMV— Tag2により、 N末端 FLAGタグ RSKB (FLAG— RSKBと 称することがある）力 S発現される。  RSKB / pCMV-Tag2 prepared in Example 2 was used as the wild-type RSKB expression plasmid. SKBZpCMV—Tag2 causes N-terminal FLAG tag RSKB (sometimes referred to as FLAG—RSKB) force S to be expressed.
[0209] 不活性型変異体 RSKB発現プラスミド（RSKB— S196AZS343AZT568AZP CMV-Tag2)は、野生型 RSKB発現プラスミド RSKBZpCMV— Tag2を铸型とし て QuikChange Site -Directed Mutagenesis kit (ストラタジーン社製）を用い 、 RSKBの 196番目、 343番目および 568番目の 3個のアミノ酸残基のァラニンへの 置換を実施して構築した。 RSKBはそのキナーゼ活性に 196番目のセリン、 343番 目のセリンおよび 568番目のスレオニンのリン酸化が必要でありこれらアミノ酸のいず れカ 1つをァラニンに置換するとキナーゼ活性が消失することが報告されて 、る（非 特許文献 34)。したがって、 RSKB— S196A/S343A/T568A/pCMV— Tag により発現される RSKBは、その 196番目および 343番目のセリン並び〖こ 568番目の スレオニンがァラニンに置換されて 、ることによりキナーゼ活性が消失して 、る不活 性型変異体 RSKBである。本不活性型変異体 RSKBは、その N末端に FLAGタグ が付カ卩されている。 [0209] The inactive mutant RSKB expression plasmid (RSKB—S196AZS343AZT568AZP CMV-Tag2) is the wild type RSKB expression plasmid RSKBZpCMV— Tag2 is used as a saddle and the QuikChange Site-Directed Mutagenesis kit (Stratagene) is used. The three amino acid residues 196, 343 and 568 were replaced with alanine. RSKB requires phosphorylation of 196th serine, 343th serine and 568th threonine for its kinase activity, and it is reported that the kinase activity disappears when one of these amino acids is substituted with alanine. (Non-patent document 34). Therefore, RSKB expressed by RSKB—S196A / S343A / T568A / pCMV—Tag has its kinase activity lost by substituting its 196th and 343th serine and 568th threonine with alanine. This is an inactive mutant RSKB. This inactive mutant RSKB has a FLAG tag attached to its N-terminus.
[0210] MAPK11発現プラスミドは、実施例 4で作製した MAPKl lZpCMV—Tag4を使 用した。  [0210] As the MAPK11 expression plasmid, MAPKlZpCMV-Tag4 prepared in Example 4 was used.
[0211] 抗リン酸化 RSKB抗体の作成は、既報 (非特許文献 34)に従い、スクラム社にて RS KBの 360番目のセリンのリン酸化を認識する抗リン酸化 RSKB抗体 PS360を作成 した。  [0211] The anti-phosphorylated RSKB antibody was prepared according to a previous report (Non-patent Document 34) by SCRAM, which produced anti-phosphorylated RSKB antibody PS360 that recognizes phosphorylation of the 360th serine of RS KB.
[0212] <方法 >  [0212] <Method>
細胞培養および各遺伝子のトランスフエクシヨンは次のように実施した。まず、 Hep G2細胞を 6ゥエルプレートに細胞数 5 X 105Zwellで播種し、 10%FBS含有 MEM 培地で 37°Cにて 5%CO /95% エアの条件下で培養した。ー晚培養後、野生型 Cell culture and transfection of each gene were performed as follows. First, Hep G2 cells were seeded on a 6-well plate at a cell number of 5 × 10 5 Zwell and cultured in a MEM medium containing 10% FBS at 37 ° C. under conditions of 5% CO 2/95% air. -After sputum culture, wild type
2  2
RSKB発現プラスミド (RSKBZpCMV— Tag2)または不活性型変異体 RSKB発現 プラスミド（RSKB— S196A/S343A/T568A/pCMV—Tag2) 2 μ gを ΜΑΡΚ 11発現プラスミド（MAPKl lZpCMV— Tag4、 C末端 FLAGタグ） 2 μ gと共に Lip ofectamine 2000 (invitrogen)を用いて細胞にトランスフエクシヨンした。総 DNA 量は各実験群で 4 μ gとなるように空ベクター (PCMV-Tag2)で補正した。トランス フエクシヨン後、細胞を 2日間培養した。 RSKB expression plasmid (RSKBZpCMV—Tag2) or inactive mutant RSKB expression plasmid (RSKB—S196A / S343A / T568A / pCMV—Tag2) 2 μg 11 Expression plasmids (MAPKlZpCMV—Tag4, C-terminal FLAG tag) were transfected into cells using 2 μg of Lipofectamine 2000 (invitrogen). The total DNA amount was corrected by empty vector (P CMV-Tag2) such that 4 mu g in each experimental group. After transfection, the cells were cultured for 2 days.
[0213] PEPCK遺伝子の発現は RT— PCRにより検出した。具体的には、上記細胞を PB Sで洗浄後、トリプシン ZEDTAを用いて回収し、 RNeasy Mini Kit (キアジェン（ Quiagen)社製）にてトータル RNAを細胞から抽出した。採取したトータル RNAから ランダムプライマー 9merおよびォリゴー dtプライマーを用いて Omniscript RT k it (キアジェン社製）により cDNAを合成した。得られた cDNAをテンプレートとして P EPCK遺伝子の PCRを実施した。また、インターナルコントロールとして GAPDH遺 伝子の PCRを実施した。プライマー配列および PCRサイクル条件は既報に従った（ 非特許文献 40および 41)。本条件下で、 PEPCK遺伝子は 575bpの PCR産物を、 GAPDH遺伝子は 209bpの PCR産物を生成する。  [0213] The expression of PEPCK gene was detected by RT-PCR. Specifically, the cells were washed with PBS, collected using trypsin ZEDTA, and total RNA was extracted from the cells using RNeasy Mini Kit (manufactured by Quiagen). CDNA was synthesized from the collected total RNA using Omniscript RT kit (manufactured by Qiagen) using random primer 9mer and oligo go dt primer. PEPCK gene PCR was performed using the obtained cDNA as a template. In addition, GAPDH gene PCR was performed as an internal control. Primer sequences and PCR cycle conditions were as previously reported (Non-patent Documents 40 and 41). Under these conditions, the PEPCK gene produces a 575 bp PCR product and the GAPDH gene produces a 209 bp PCR product.
[0214] 上記細胞にトランスフエクシヨンした各遺伝子の発現をウェスタンブロッテイングによ り検出した。具体的には、上記細胞を PBSで洗浄後、トリプシン ZEDTAを用いて回 収し、細胞溶解バッファー（セルシグナリング社製)を 150 1添加し、氷上で 15分間 静置して細胞を溶解させた。その後、遠心処理を 15, OOOrpmで 4°Cにて 10分間行 い、その上清を回収して細胞ライセートとして用いた。この細胞ライセートに等量の 2 X SDS サンプルバッファーを添カロし、 100°Cにて 5分間加熱したものを SDS— PA GE試料として用いた。 5— 20% グラジェントゲルを用いた SDS— PAGEにて蛋白 質を分離し、 Immobiron— PSQ membrane (ミリポア（Millipore)社製）に転写後、 各蛋白質の検出を行った。 FLAG— RSKBおよび MAPK11— FLAGは抗 FLAG M2抗体 (シグマ社製）にて検出した。リン酸化 RSKBは抗 PS360抗体、 /3—チュ 一ブリンは抗 —チューブリン抗体 Η— 235 (サンタクルヅ社製)でそれぞれ検出し た。検出は蛍光標識された二次抗体を用い、 Odysseyイメージングシステム (ァロカ（ Aloka)社製)を用いて行なった。 [0214] The expression of each gene transfected into the cells was detected by Western blotting. Specifically, after washing the cells with PBS, the cells were collected using trypsin ZEDTA, 1501 of a cell lysis buffer (manufactured by Cell Signaling) was added, and the cells were allowed to stand for 15 minutes on ice to lyse the cells. . Thereafter, centrifugation was performed at 15, OOOrpm at 4 ° C for 10 minutes, and the supernatant was recovered and used as a cell lysate. The cell lysate was supplemented with an equal volume of 2 X SDS sample buffer and heated at 100 ° C for 5 minutes, and used as an SDS-PAGE sample. Proteins were separated by SDS-PAGE using a 5 to 20% gradient gels, after transfer to Immobiron- P SQ membrane (Millipore (Millipore) Co. Ltd.) was detected for each protein. FLAG—RSKB and MAPK11—FLAG were detected with an anti-FLAG M2 antibody (manufactured by Sigma). Phosphorylated RSKB was detected with an anti-PS360 antibody, and / 3-tubulin was detected with an anti-tubulin antibody (-235 (manufactured by Santa Cruz). Detection was carried out using a fluorescently labeled secondary antibody using an Odyssey imaging system (Aloka).
[0215] <結果 >  [0215] <Result>
RT— PCRの結果を図 13に示す。野生型 RSKBと MAPK11を共発現させた Hep G2細胞ではこれらを発現させなかった細胞に比べ、 PEPCKの RT— PCR産物が増 加しており、このことから内因性 PEPCK遺伝子の発現が亢進していることが示された 。一方、不活性型変異体 RSKB (S343AZS 196AZT568A)と MAPK11を共発 現させた HepG2細胞では PEPCKの RT—PCR産物の増加がみられなかった。本 結果から、野生型 RSKBと MAPK1 1を共発現させた HepG2細胞における PEPCK 遺伝子の発現の亢進は、 RSKBのキナーゼ活性に依存していることが判明した。 The results of RT-PCR are shown in FIG. Hep co-expressing wild-type RSKB and MAPK11 In G2 cells, the RT-PCR product of PEPCK increased compared to cells that did not express them, indicating that endogenous PEPCK gene expression was increased. On the other hand, no increase in PEPCK RT-PCR product was observed in HepG2 cells co-expressed with inactive mutant RSKB (S343AZS 196AZT568A) and MAPK11. From these results, it was found that the enhancement of PEPCK gene expression in HepG2 cells in which wild-type RSKB and MAPK11 were coexpressed was dependent on the kinase activity of RSKB.
[0216] 野生型 RSKBと MAPK11を共発現させた HepG2細胞、不活性型変異体 RSKB ( RSKB (S 196AZS343AZT568A) )と MAPK11を共発現させた HepG2細胞、 およびこれらを発現させなかった細胞における、同条件下での各蛋白質の発現量を 図 14に示す。抗リン酸化 RSKB抗体によるウェスタンブロッテイングの結果、野生型 RSKBと MAPK11を共発現させた細胞ではリン酸化 RSKBが検出された（中パネル ) oそれに対し、不活性変異体 RSKBと MAPK11を共発現させた細胞では、リン酸 化 RSKBが検出されなかった（中パネル）。本抗リン酸化 RSKB抗体は、 RSKBの 36 0番目のセリンのリン酸ィ匕を認識する抗体である。 RSKBのキナーゼ活性はその 196 番目、 343番目のセリンおよび 568番目のスレオニン以外に 360番目のセリンのリン 酸ィ匕が重要であり、本アミノ酸部位のリン酸ィ匕の有無はキナーゼ活性と一致している (非特許文献 34)。本結果から、本実施例で使用した不活性変異体 RSKBはキナー ゼ活性が消失していることが明らかになった。野生型 RSKBと MAPK11を共発現さ せた HepG2細胞、および不活性型変異体 RSKB (RSKB (S 196AZS343AZT5 68A) )と MAPK11を共発現させた HepG2細胞における野生型 RSKBおよび不活 性型変異体 RSKBの発現量に、大きな差はな力つた。また、これら細胞における MA PK11の発現量はほぼ同等であった（上パネル）。いずれの細胞においても、コント口 ールである 13チューブリンの発現はほぼ同等であった（下パネル)。  [0216] HepG2 cells co-expressed with wild-type RSKB and MAPK11, HepG2 cells co-expressed with inactive mutant RSKB (RSKB (S196AZS343AZT568A)) and MAPK11, and cells that did not express them Figure 14 shows the expression level of each protein under the conditions. As a result of Western blotting with anti-phosphorylated RSKB antibody, phosphorylated RSKB was detected in cells co-expressing wild-type RSKB and MAPK11 (middle panel) o In contrast, inactive mutants RSKB and MAPK11 were co-expressed In these cells, phosphorylated RSKB was not detected (middle panel). This anti-phosphorylated RSKB antibody is an antibody recognizing the phosphate of the 360th serine of RSKB. In addition to the 196th, 343th serine and 568th threonine, the RSKB kinase activity is important for the 360th serine phosphate, and the presence or absence of phosphate at this amino acid site is consistent with the kinase activity. (Non-patent Document 34). From this result, it was clarified that the inactive mutant RSKB used in this example lost its kinase activity. HepG2 cells co-expressing wild-type RSKB and MAPK11, and inactive mutant RSKB (RSKB (S 196AZS343AZT5 68A)) and wild-type RSKB and inactive mutant RSKB in HepG2 cells co-expressing MAPK11 There was no big difference in the expression level. In addition, the expression level of MA PK11 in these cells was almost the same (upper panel). The expression of 13 tubulin, the control console, was almost the same in all cells (lower panel).
[0217] 上記結果から、 RSKBはそのキナーゼ活性に依存して、肝細胞での PEPCK遺伝 子発現を増加させることが判明した。  [0217] From the above results, it was found that RSKB increases PEPCK gene expression in hepatocytes depending on its kinase activity.
実施例 6  Example 6
[0218] (RSKBと HNF— 4 aの細胞内における結合）  [0218] (Intracellular binding of RSKB and HNF-4a)
HeLa細胞に RSKBと HNF— 4 aを一過性共発現させ、免疫沈降により RSKBと HNF-4 aの結合を検討した。 RSKB and HNF-4a were transiently co-expressed in HeLa cells and immunorecipitated with RSKB. The binding of HNF-4a was examined.
[0219] <材料 > [0219] <Material>
RSKB発現プラスミドは、実施例 2で作製した RSKB/pCMV— Tag2を使用した 。 RSKB/pCMV— Tag2により、 N末端 FLAGタグ RSKB (FLAG— RSKBと称す ることがある）力 S発現される。 HNF— 4ひ発現プラスミドは、実施例 3で作製した HNF — 4 a ZpCMV— Tag2を使用した。 RSKBZpCMV— Tag2により、 N末端 FLAG タグ HNF— 4 a (FLAG -HNF -4 aと称することがある）が発現される。  RSKB / pCMV-Tag 2 prepared in Example 2 was used as the RSKB expression plasmid. RSKB / pCMV— Tag2 N-terminal FLAG tag RSKB (sometimes called FLAG—RSKB) force S is expressed. As the HNF-4 expression plasmid, HNF-4a ZpCMV-Tag2 prepared in Example 3 was used. RSKBZpCMV—Tag2 expresses the N-terminal FLAG tag HNF-4a (sometimes referred to as FLAG-HNF-4a).
[0220] <方法 > [0220] <Method>
細胞培養および各遺伝子のトランスフエクシヨンは次のように実施した。まず、 HeLa 細胞を 6ゥエルプレートに細胞数 3. 6 X 105Zwellで播種し、 10%FBS含有 DME M培地で 37°Cにて 5%CO /95% エアの条件下で培養した。ー晚培養後、 RSK Cell culture and transfection of each gene were performed as follows. First, HeLa cells were seeded on a 6-well plate at a cell number of 3.6 × 10 5 Zwell, and cultured in DME M medium containing 10% FBS at 37 ° C. under conditions of 5% CO 2/95% air. -After incubation, RSK
2  2
B発現プラスミド（RSKBZpCMV— Tag2) 0. 5 μ gと HNF— 4 α発現プラスミド（Η NF - 4 a /pCM V - Tag 2) 0. 5 gまたは空ベクター（pCMV— Tag2、ストラタジ ーン社製） 0. 5 μ gとを FuGENE6 (ロッシュ 'ダイァグノスティックス社製）を用いて細 胞にトランスフエクシヨンした（総 DNA量は 1 μ g)。トランスフエクシヨン後、細胞を 2日 間培養した。  B expression plasmid (RSKBZpCMV—Tag2) 0.5 μg and HNF-4α expression plasmid (ΗNF-4a / pCMV-Tag2) 0.5g or empty vector (pCMV-Tag2, Stratagene) ) 0.5 μg was transferred into cells using FuGENE6 (Roche Diagnostics) (total DNA amount was 1 μg). After transfection, the cells were cultured for 2 days.
[0221] 上記細胞における RSKBと HNF— 4 aの結合を、免疫沈降法により検出した。具 体的には、上記細胞を冷 PBSで洗浄し、細胞溶解バッファー（20mM HEPES (p H7. 5) /150mM NaCl/lmM EDTA/1% Triton X— 100Zプロテア一 ゼインヒビタ一力クテル (シグマ社製) ) 500 μ 1を添加し、氷上で 15分間静置して細胞 を溶解させた。その後、溶解した細胞を 15, OOOrpmで 4°Cにて 10分間遠心処理し て上清を採取し、これを細胞ライセートとして用いた。この細胞ライセートに、 protein G sepharose 4 FastFlow (アマシャムノィオケィエンス (Amersham Bioscie nce)社製）の 50%スラリーを 40 μ 1加え、 4°Cにて 1時間転倒混和した（pre - clean) 。その後、 10, OOOrpmにて 15秒間の遠心処理して protein G sepharoseを除き 、回収した上清に抗 HNF— 4 a抗体 C— 19 (サンタクルヅ社製） 2 μ 1を添加し、 4°C にて 1時間転倒混和した。次に新たに protein sepharose 4 FastFlow 50%ス ラリー 40 1をカ卩え、再度 4°Cにて 1時間転倒混和した。 Protein G sepharoseを 遠心処理により回収し、これを戦場バッファー（50mM Trsi-HCl (pH7. 5) /15 OmM NaCl/O. 2% NP— 40)で 2回洗浄した後、 40 /z lの 2 X SDS サンプル バッファーを加え、 100°Cにて 5分間加熱処理をしたものを SDS— PAGE試料として 用いた。 5 - 20% グラジェントゲルを用いた SDS— PAGEにて蛋白質を分離し、ゥ エスタンブロッテイングにより、抗 FLAG M2抗体（シグマ社製）で FLAG— RSKBお よび FLAG— HNF— 4 αを検出した。検出は蛍光標識された二次抗体を用い、 Od ysseyイメージングシステム (ァロカ社製）により行なった。 [0221] The binding between RSKB and HNF-4a in the cells was detected by immunoprecipitation. Specifically, the cells were washed with cold PBS, and cell lysis buffer (20 mM HEPES (pH 7.5) / 150 mM NaCl / lmM EDTA / 1% Triton X—100Z Protea Zein Hibita Kuttel (manufactured by Sigma) )) 500 μl was added and left on ice for 15 minutes to lyse the cells. Thereafter, the lysed cells were centrifuged at 15, OOOrpm at 4 ° C for 10 minutes, and the supernatant was collected and used as a cell lysate. To this cell lysate, 40 μl of a 50% slurry of protein G sepharose 4 FastFlow (Amersham Bioscience) was added and mixed by inverting at 4 ° C. for 1 hour (pre-clean). Thereafter, the protein G sepharose was removed by centrifugation at 10, OOOrpm for 15 seconds, and 2 μ 1 of anti-HNF-4a antibody C-19 (manufactured by Santa Cruz Co., Ltd.) was added to the collected supernatant. Inverted for 1 hour. Next, a new protein sepharose 4 FastFlow 50% slurry 40 1 was added and mixed again by inversion at 4 ° C for 1 hour. Protein G sepharose Collected by centrifugation, washed twice with battlefield buffer (50 mM Trsi-HCl (pH 7.5) / 15 OmM NaCl / O. 2% NP-40), and then washed with 40 / zl of 2 X SDS sample buffer. In addition, a sample heat-treated at 100 ° C for 5 minutes was used as an SDS-PAGE sample. Proteins were separated by SDS-PAGE using 5-20% gradient gel, and FLAG-RSKB and FLAG-HNF-4α were detected by anti-FLAG M2 antibody (manufactured by Sigma) by Western blotting did. Detection was carried out using an Odyssey Imaging System (Aloka) using a fluorescently labeled secondary antibody.
[0222] <結果 > [0222] <Result>
FLAG-HNF-4 aと FLAG—RSKBを一過性共発現させた細胞から調製した 細胞ライセートにおいて、抗 HNF— 4 α抗体を用いた免疫沈降および抗 FLAG M 2抗体を用いた検出により、 FLAG-HNF-4 aを示すバンドおよび FLAG—RSK Bを示すバンドがいずれも検出された（図 15のパネル A)。一方、 FLAG— RSKBの みを発現させた細胞カゝら調製した細胞ライセートでは、 FLAG— RSKBを示すバンド および FLAG— HNF— 4 aを示すバンドはいずれも検出されなかった（図 15のパネ ル A)。各細胞で発現された FLAG— RSKBおよび FLAG— HNF— 4 αを、抗 FLA G M2抗体で検出したところ、 FLAG— HNF— 4 aと FLAG— RSKBを一過性共 発現させた細胞でこれら蛋白質が！ヽずれも発現されて！ヽることが観察された（図 15の パネル: B)。また、 FLAG— RSKBのみを発現させた細胞においても、 FLAG—RSK Bの発現が観察された。 FLAG— RSKBの発現は、 FLAG— HNF— 4 aと FLAG RSKBを一過性共発現させた細胞および FLAG— RSKBのみを発現させた細胞 で同程度であった（図 15のパネル: B)。このことから、 FLAG— HNF— 4 αと FLAG RSKBを一過性共発現させた細胞力 調製した細胞ライセートにおいて検出され た FLAG— RSKBを示すバンドは、 FLAG-HNF-4 aと FLAG— RSKBの共沈 の結果を示すと考えることができる。  In cell lysates prepared from cells co-expressed transiently with FLAG-HNF-4a and FLAG-RSKB, immunoprecipitation using anti-HNF-4α antibody and detection using anti-FLAG M2 antibody A band indicating -HNF-4a and a band indicating FLAG-RSK B were both detected (panel A in FIG. 15). On the other hand, in the cell lysate prepared only from FLAG-RSKB, neither FLAG-RSKB band nor FLAG-HNF-4a band was detected (Fig. 15 panel). A). When FLAG-RSKB and FLAG-HNF-4α expressed in each cell were detected with anti-FLA G M2 antibody, these proteins were transiently co-expressed in FLAG-HNF-4a and FLAG-RSKB. But! Mistakes are also expressed! Speaking was observed (panel in Figure 15: B). In addition, expression of FLAG-RSK B was also observed in cells expressing only FLAG-RSKB. The expression of FLAG-RSKB was similar in cells transiently co-expressing FLAG-HNF-4a and FLAG RSKB and in cells expressing only FLAG-RSKB (FIG. 15, Panel: B). From this, it was shown that FLAG-HNF-4a and FLAG-RSKB bands were detected in the cell lysates prepared by transient co-expression of FLAG-HNF-4α and FLAG RSKB. It can be considered that the result of coprecipitation is shown.
[0223] このように、抗 HNF— 4 a抗体で HNF— 4 aを沈降させた結果、 HNF— 4 a依存 的に RSKBの共沈が検出された（図 15のパネル A)。本結果から、 HNF— 4が細胞 内で RSKBと結合することが判明した。 [0223] As described above, HNF-4a was precipitated with an anti-HNF-4a antibody, and as a result, co-precipitation of RSKB was detected depending on HNF-4a (Panel A in Fig. 15). From these results, it was found that HNF-4 binds to RSKB in cells.
実施例 7 [0224] 実験例 1.糖尿病モデル動物での肝臓中 HNF 4 aのリン酸ィ匕の検出 糖尿病を発症する ZDF faZfa雄性ラットおよび正常動物の ZDF— lean雄性ラッ トを日本チャールズリバ一社より 5週齢で購入し、飼育維持する。 6、 8、 19週齢に採 血した後、ペントバルビタール麻酔下で屠殺し、肝臓を採取する（各週齢 n= 6)。採 取した血液は血中ダルコース濃度とインスリン濃度をそれぞれ測定し、 ZDF fa/fa で糖尿病が発症していることを確認する。肝臓は氷冷下で細胞溶解バッファー（cell lysis buffer (セル ·シグナリング社製）（20mM Tris— HCl (pH7. 5)、 150mM NaCl/lmM Na EDTA/lmM EGTA/1% Triton/2. 5mM ピロリン Example 7 [0224] Experimental Example 1. Detection of HNF 4 a phosphate in liver in diabetic model animals ZDF faZfa male rats that develop diabetes and ZDF— lean male rats from normal animals from Charles River, Japan 5 Purchase and maintain at the age of weeks. Blood samples are collected at 6, 8, and 19 weeks of age, then sacrificed under pentobarbital anesthesia, and livers are collected (each n = 6). The collected blood is measured for blood levels of dalcose and insulin, and ZDF fa / fa is used to confirm that diabetes has developed. The liver was lysed under ice cooling (cell lysis buffer (manufactured by Cell Signaling) (20 mM Tris—HCl (pH 7.5), 150 mM NaCl / lmM Na EDTA / lmM EGTA / 1% Triton / 2. 5 mM pyrroline)
2  2
酸ナトリウム ZlmM βーグリセ口リン酸 ZlmM Na VO ) )中でホモジナイズし、  Sodium ZlmM β-Glycetophosphate ZlmM Na VO))
3 4  3 4
氷冷下で 30分間静置後、 15, OOOrpmで 10分間遠心処理した上清を細胞ライセー トとして用いる。作製した細胞ライセートに抗 HNF— 4 a抗体 (サンタクルヅ社製)を 添加し 4°Cにて 1時間転倒混和した後、 0. 1%牛血清アルブミン (BSA) ZTris緩衝 生理食塩水（Tris— Buffered Saline (TBS) (50mM Tris-HCl/150mM N aCl) ) (pH8. 0)でブロッキングした protein G sepharose 4 Fast Flow (アマ シャム'フアルマシア 'バイオテック社製)を加え、さらに 4°Cにて 1時間転倒混和する。 その後、 protein G sepharoseを細胞溶解バッファーで 2回洗浄し、 2 X SDSサン プルバッファーを添カ卩し、 100°Cにて 5分間加熱したものを SDS— PAGE試料して 用いる。 5— 20%ゲルで蛋白質を分離し、抗リン酸ィ匕セリン抗体（トランスダクシヨン' ラボラトリーズ (Transduction Laboratories)社製）および抗リン酸化スレオ-ン抗 体 (ザィメッド (Zymed)社製)を用いたウェスタンブロッテイングにより、リン酸化型 HN F-4 aを検出する。糖尿病発症動物（ZDF faZfa)が正常動物（ZDF— lean)よ り HNF— 4 aのセリン 'スレオニン残基のリン酸化が増加することにより、 HNF-4 a のリン酸ィ匕が糖尿病病態下で亢進することが証明できる。  Let stand for 30 minutes under ice-cooling, and use the supernatant after centrifugation at 15, OOOrpm for 10 minutes as the cell lysate. Anti-HNF-4a antibody (manufactured by Santa Cruz Co., Ltd.) was added to the prepared cell lysate and mixed by inverting for 1 hour at 4 ° C. Add protein G sepharose 4 Fast Flow (Amersham 'Falmacia' manufactured by Biotech) blocked with Saline (TBS) (50 mM Tris-HCl / 150 mM N aCl)) (pH 8.0), and add 1 at 4 ° C. Mix by inversion for an hour. Then, wash protein G sepharose twice with cell lysis buffer, add 2X SDS sample buffer, heat at 100 ° C for 5 minutes, and use as SDS-PAGE sample. 5—Separate proteins with 20% gel and use anti-phosphoserine antibody (Transduction Laboratories) and anti-phosphothreonine antibody (Zymed) Detect phosphorylated HNF-4a by Western blotting. Diabetes-causing animals (ZDF faZfa) have increased phosphorylation of serine 'threonine residues in HNF-4a than normal animals (ZDF-lean), so that It can be proved to be enhanced.
[0225] 実験例 2.糖尿病モデル動物での肝臓中 RSKBの活性ィ匕の検出 [0225] Experimental example 2. Detection of liver RSKB activity in diabetic model animals
糖尿病発症動物 (ZDF faZfa)および正常動物 (ZDF— lean)から上記実験例 1 .と同様に肝臓細胞ライセートを作製する。これに抗 RSKB抗体と protein G seph aroseを添加し、上記実験例 1.と同様の免疫沈降法により RSKBを回収する。回収 した RSKBのキナーゼ活性は、合成基質タレビタイド（CREBtide (サンタクルヅ社製 ；) ) (配列番号 9)を用いた in vitro kinase assayにより検出する（非特許文献 34) 。具体的には、 RSKB結合レジンをキナーゼバッファー（Kinase buffer (50mM Tris-HCl (pH7. 5) /0. ImM EGTA/140mM KCl/5mM NaPPi/10 mM MgCl ) )中で CREBtide (33 μ M)および γ— 32P— ATPと混和し、 22°Cに Liver cell lysates are prepared from diabetic animals (ZDF faZfa) and normal animals (ZDF-lean) in the same manner as in Experimental Example 1 above. Anti-RSKB antibody and protein G seph arose are added thereto, and RSKB is recovered by the same immunoprecipitation method as in Experimental Example 1 above. The kinase activity of the recovered RSKB was measured using the synthetic substrate talebitide (CREBtide (manufactured by Santa Cruz). ;)) Detected by in vitro kinase assay using (SEQ ID NO: 9) (Non-patent Document 34). Specifically, RSKB-binding resin was added to CREBtide (33 μM) and kinase buffer (Kinase buffer (50 mM Tris-HCl (pH 7.5) / 0. ImM EGTA / 140 mM KCl / 5 mM NaPPi / 10 mM MgCl)). Mix with γ- 32 P- ATP and bring to 22 ° C
2  2
て 30分間インキュベーションする。その後、反応液を chromatography filter pap er P81 (ワットマン (Whatman)社製）に吸着させ、洗浄後、液体シンチレーシヨン力 ゥンターにて放射能を検出する。糖尿病発症動物 (ZDF faZfa)が正常動物 (ZD F— lean)より RSKBキナーゼ活性が増加することにより、肝臓中 RSKB活性が糖尿 病病態下で亢進することが証明できる。  Incubate for 30 minutes. Thereafter, the reaction solution is adsorbed on chromatography filter paper P81 (Whatman), washed, and then radioactivity is detected with a liquid scintillation force counter. It can be demonstrated that RSKB kinase activity in diabetic animals (ZDF faZfa) is increased under normal diabetes (ZDF-lean), and RSKB activity in the liver is enhanced under diabetic conditions.
[0226] 実験例 3.糖尿病モデル動物における RSKB阻害剤の抗糖尿病効果 [0226] Experimental Example 3. Anti-diabetic effect of RSKB inhibitor in diabetes model animals
糖尿病を発症する ZDF faZfa雄性ラットを日本チャールズリバ一社より 5週齢で 購入し、飼育維持する。動物を 2群に分け (各群 n= 5)、 6週齢から RSKB阻害剤の H89 (N- (2— ( (p ブロモシンナミル）ァミノ）ェチル） 5 イソキノリンスルホナマ イト； N— (2— ( (p― Bromocmnamyl) ammo) ethyl)― 5— isoquinolinesulfona mide) (非特許文献 34)を 5mgZkgZ日で 14日間連続皮下投与する。 14日間投与 終了後に動物をペントバルビタール麻酔下で屠殺し、肝臓を採取する。また、 H-8 9投与開始前と投与終了後に血液を採取し、血中グルコース濃度とインスリン濃度（ レビスインスリンキット、シバヤギ（Shibayagi)社製)を測定する。採取した肝臓を用い て HNF— 4 αのリン酸ィ匕および RSKB活性を上記実験例 1.および 2.と同様に測定 するとともに、 PEPCKの発現量をウェスタンブロッテイングにより検出する。上記実験 例 1.と同様に作製した肝臓細胞ライセートに 2 X SDSサンプルバッファーを等容量 添カロし 100°Cにて 5分間加熱したものを SDS— PAGEの試料として用いる。 5— 20 %ゲルで蛋白質を分離し、抗 PEPCK抗体を用いたウェスタンブロッテイングにより P EPCKを検出する。 H— 89投与により RSKB活性、 HNF— 4 αのリン酸化および PE PCKの発現のいずれも低下し、糖新生が抑制されることにより血中グルコース濃度 の低下が起こる。  ZDF faZfa male rats that develop diabetes are purchased from the Japan Charles River Company at 5 weeks of age and maintained. The animals were divided into two groups (n = 5 for each group), and from 6 weeks of age, the RSKB inhibitor H89 (N- (2— ((p bromocinnamyl) amino) ethyl) 5 isoquinoline sulfonamite; N— (2— ( (p-Bromocmnamyl) ammo) ethyl) -5-isoquinolinesulfona mide) (Non-patent Document 34) is administered subcutaneously continuously for 5 days at 5 mgZkgZ day. After 14 days of administration, animals are sacrificed under pentobarbital anesthesia and livers are collected. In addition, blood is collected before and after the administration of H-8 9 and the blood glucose concentration and insulin concentration (Levis Insulin Kit, manufactured by Shibayagi) are measured. Using the collected liver, the HNF-4α phosphate and RSKB activities are measured in the same manner as in Experimental Examples 1 and 2 above, and the expression level of PEPCK is detected by Western blotting. Use a liver cell lysate prepared in the same way as in Experimental Example 1 above with an equal volume of 2 X SDS sample buffer and heated at 100 ° C for 5 minutes as a sample for SDS-PAGE. Separate proteins on 5-20% gel and detect PEPCK by Western blotting with anti-PEPCK antibody. Administration of H-89 decreases both RSKB activity, phosphorylation of HNF-4α, and expression of PE PCK, and gluconeogenesis is suppressed, resulting in a decrease in blood glucose concentration.
[0227] 上記実験例 1.および 2.により、糖尿病モデル動物の肝臓では RSKBの活性ィ匕と それに伴う HNF— 4ひのリン酸ィ匕が亢進していることが示される。実験例 3.により、 R SKBを阻害することで PEPCKの発現が低下し、肝臓の糖新生が抑制されることが 証明できる。なお、 H— 89は RSKB以外に PKAを阻害する作用がある。 PKAは CR EBをリン酸ィ匕し、リン酸ィ匕された CREBは CREを有するプロモーターの転写活性を 亢進する（非特許文献 11)。従って、 PKAの抑制は CREBを介する PEPCK遺伝子 発現を低下させる可能性がある。しかし、糖尿病モデル動物の肝臓では CREBのリ ン酸ィ匕が増加するどころか逆に低下しており、 PKAも活性ィ匕されておらず、 PKAシ グナルは糖尿病病態下の肝臓での PEPCK発現増加に関与して ヽな 、ことが示され ている（非特許文献 39)。また、 PKAはHNF—4 Q；をリン酸ィ匕するカ このリン酸ィ匕 によって HNF— 4 aの PEPCK遺伝子プロモーターへの結合能が亢進することはな い（実施例 2、図 5)。従って、 H— 89が PEPCK発現を抑制した場合、それは PKAで はなく RSKB阻害に起因すると考えることができる。上記実験例により、生体内にお いて RSKBを阻害すれば肝臓での HNF— 4 aのリン酸化が抑制され、 PEPCKを介 する糖新生能が低下し、抗糖尿病効果が期待できる。 [0227] From the above experimental examples 1 and 2, it is shown that the activity of RSKB and the accompanying phosphate of HNF-4 are increased in the liver of diabetes model animals. From Experiment 3, R It can be demonstrated that inhibition of SKB reduces PEPCK expression and suppresses liver gluconeogenesis. H-89 has an effect of inhibiting PKA in addition to RSKB. PKA phosphorylates CREB, and CREB phosphorylated enhances the transcriptional activity of a promoter having CRE (Non-patent Document 11). Therefore, suppression of PKA may reduce PEPCK gene expression via CREB. However, in the liver of diabetic model animals, CREB phosphate increased rather than decreased, PKA was not activated, and PKA signal increased PEPCK expression in the liver under diabetic conditions. It has been shown to be involved in PKA also phosphorylates HNF-4 Q; This phosphoric acid does not enhance the ability of HNF-4a to bind to the PEPCK gene promoter (Example 2, Fig. 5). Thus, if H-89 suppresses PEPCK expression, it can be attributed to RSKB inhibition rather than PKA. According to the above experimental example, if RSKB is inhibited in vivo, phosphorylation of HNF-4a in the liver is suppressed, the ability of gluconeogenesis via PEPCK is reduced, and an antidiabetic effect can be expected.
産業上の利用可能性  Industrial applicability
[0228] 本発明において提供する RSKBによる HNF— 4 aのリン酸化阻害剤および/また はリン酸ィ匕阻害方法により、 HNF-4 aが作用する遺伝子の遺伝子産物の産生阻 害、例えば PEPCK遺伝子の遺伝子産物の産生阻害を実施できる。また、 HNF— 4 αが作用する遺伝子の遺伝子産物の増加に起因する疾患の防止および Zまたは治 療が可能になる。具体的には、例えば PEPCK遺伝子産物の増加に起因する疾患、 より具体的には糖尿病等の防止および Zまたは治療が可能になる。このように本発 明は、糖新生関連遺伝子発現に関与する HNF— 4 aの過剰なリン酸ィ匕に起因する 疾患の防止および Zまたは治療のために非常に有用である。  [0228] Inhibition of production of a gene product of a gene on which HNF-4a acts by the phosphorylation inhibitor of HNF-4a and / or the method of inhibiting phosphorylation by RSKB provided in the present invention, such as the PEPCK gene Inhibition of the production of the gene product can be carried out. It also makes it possible to prevent and / or treat diseases caused by increased gene products of genes that act on HNF-4α. More specifically, for example, diseases caused by an increase in PEPCK gene products, more specifically, diabetes and the like can be prevented and Z or treated. Thus, the present invention is very useful for the prevention and Z or treatment of diseases caused by excessive HNF-4a phosphate involved in gluconeogenesis-related gene expression.
配列表フリーテキスト  Sequence listing free text
[0229] 配列番号 1 :RSKB cDNA。  [0229] SEQ ID NO: 1: RSKB cDNA.
配列番号 2 :RSKB。  Sequence number 2: RSKB.
配列番号 3 : HNF— 4 α cDNA。  SEQ ID NO: 3: HNF-4α cDNA.
配列番号 4 : HNF— 4 α。  SEQ ID NO: 4: HNF—4α.
配列番号 5 : CREB 1 cDNA0 配列番号 6 : CREB1。 SEQ ID NO: 5: CREB 1 cDNA 0 SEQ ID NO: 6: CREB1.
配列番号 7 :AF1の配列に基づ ヽて設計された、プローブ用オリゴヌクレオチド。 配列番号 8 :AF1の配列に基づ ヽて設計された、プローブ用オリゴヌクレオチド。 配列番号 9：合成基質 CREBtide。 SEQ ID NO: 7: Probe oligonucleotide designed based on the sequence of AF1. SEQ ID NO: 8: Probe oligonucleotide designed based on the sequence of AF1. SEQ ID NO: 9: Synthetic substrate CREBtide.
配列番号 10 :RSKBと HNF— 4 αとのローカルァライメントにおいて高いスコアを示 した RSKBの部分オリゴペプチド。 SEQ ID NO: 10: Partial RSKB oligopeptide showing high score in local alignment between RSKB and HNF-4α.
配列番号 11： RSKBと HNF— 4 αとのローカルァライメントにお!/、て高!、スコアを示 した HNF— 4 aの部分オリゴペプチド。 SEQ ID NO: 11: A partial oligopeptide of HNF-4a which showed a score of RSKB and HNF-4α for local alignment!
配列番号 12： RSKBと HNF— 4 αとのローカルァライメントにお!/、て高!、スコアを示 した HNF— 4 aの部分オリゴペプチド。 SEQ ID NO: 12: Partial oligopeptide of HNF-4a showing a score of RSKB and HNF-4α for local alignment! /, High!
配列番号 13： RSKBと HNF— 4 αとのローカルァライメントにお!/、て高!、スコアを示 した RSKBの部分オリゴペプチド。 SEQ ID NO: 13: RSKB partial oligopeptide showing a score of RSKB and HNF-4α for local alignment! /, High!
配列番号 14： RSKBと HNF— 4 αとのローカルァライメントにお!/、て高!、スコアを示 した HNF— 4 aの部分オリゴペプチド。 SEQ ID NO: 14: Partial oligopeptide of HNF-4a which showed a score of RSKB and HNF-4α for local alignment! /, High!
配列番号 15 : PEPCK遺伝子のプロモーター領域であり、本領域の配列は NCBIヌ クレオチドデータベースにァクセッション番号 U31519として公開された該遺伝子の 第 841から第 1440番目のヌクレオチドで表される配列に相当する。 SEQ ID NO: 15: Promoter region of the PEPCK gene, which corresponds to the sequence represented by nucleotides 841 to 1440 of the gene published as accession number U31519 in the NCBI nucleotide database .

Claims

請求の範囲 The scope of the claims
[1] リボソーム S6キナーゼ B (RSKB)とへパトサイトヌクレア一ファクター 4 α (HNF— 4 a )を相互作用を可能にする条件下で共存させることを特徴とする、 RSKBによる H [1] RSKB H, characterized by the coexistence of ribosomal S6 kinase B (RSKB) and hepatocyte nuclear factor 4 α (HNF-4a) under conditions that allow interaction
NF— 4 αのリン酸化方法。 NF-4 α phosphorylation method.
[2] リボソーム S6キナーゼ B (RSKB)の活性を阻害することを特徴とする、 RKSBによる へノ トサイトヌクレア一ファクター 4 a (HNF-4 a )のリン酸化阻害方法。 [2] A method for inhibiting phosphorylation of hetocytonuclease factor 4a (HNF-4a) by RKSB, which comprises inhibiting the activity of ribosomal S6 kinase B (RSKB).
[3] リボソーム S6キナーゼ B (RSKB)とへパトサイトヌクレア一ファクター 4 a (HNF— 4 a )の結合を阻害することを特徴とする、 RSKBによる HNF— 4 aのリン酸化阻害方 法。 [3] A method for inhibiting phosphorylation of HNF-4a by RSKB, characterized by inhibiting the binding of ribosomal S6 kinase B (RSKB) to hepatocyte nuclear factor 4a (HNF-4a).
[4] 少なくともリボソーム S6キナーゼ B (RSKB)とへノ トサイトヌクレア一ファクター 4 a (H NF-4 a )とを発現している細胞を、 RSKB活性の阻害剤で処理することを特徴とす る RSKBによる HNF— 4 aのリン酸化阻害方法。  [4] It is characterized in that cells expressing at least ribosomal S6 kinase B (RSKB) and hetocytonuclea factor 4a (HNF-4a) are treated with an inhibitor of RSKB activity. The method of inhibiting phosphorylation of HNF-4a by RSKB.
[5] リボソーム S6キナーゼ B (RSKB)活性の阻害剤が、 RSKBを認識する抗体、へパト サイトヌクレア一ファクター 4 a (HNF-4 a )を認識する抗体力も選ばれる 1つまたは 2つ以上の抗体である請求項 4に記載の RSKBによる HNF— 4 aのリン酸化阻害方 法。  [5] An inhibitor of ribosomal S6 kinase B (RSKB) activity is an antibody that recognizes RSKB, and an antibody that recognizes hepatocyte factor 4a (HNF-4a). The method for inhibiting phosphorylation of HNF-4a by RSKB according to claim 4, wherein the antibody is an antibody.
[6] リボソーム S6キナーゼ B (RSKB)活性を阻害することを特徴とする、 RSKBによるへ ノ トサイトヌクレア一ファクター 4 a (HNF-4 a )のリン酸化阻害剤。  [6] An inhibitor of phosphorylation of hepatocyte nuclear factor 4a (HNF-4a) by RSKB, characterized by inhibiting ribosomal S6 kinase B (RSKB) activity.
[7] リボソーム S6キナーゼ B (RSKB)とへパトサイトヌクレア一ファクター 4 a (HNF— 4 a )の結合を阻害することを特徴とする、 RSKBによる HNF— 4 aのリン酸ィ匕阻害剤  [7] Phosphate inhibitor of HNF-4a by RSKB, characterized by inhibiting the binding of ribosomal S6 kinase B (RSKB) to hepatocyte nuclear factor 4a (HNF-4a)
[8] リボソーム S6キナーゼ B (RSKB)活性の阻害剤を有効量含むことを特徴とする RSK Bによるへノ トサイトヌクレア一ファクター 4 a (HNF-4 a )のリン酸化阻害剤。 [8] An inhibitor of phosphorylation of hetocytonuclea factor 4a (HNF-4a) by RSK B, comprising an effective amount of an inhibitor of ribosomal S6 kinase B (RSKB) activity.
[9] リボソーム S6キナーゼ B (RSKB)活性の阻害剤が、 RSKBを認識する抗体、へパト サイトヌクレア一ファクター 4 a (HNF-4 a )を認識する抗体力も選ばれる 1つまたは 2つ以上の抗体である請求項 8に記載の RSKBによる HNF— 4 aのリン酸ィ匕阻害剤  [9] An inhibitor of ribosomal S6 kinase B (RSKB) activity is an antibody that recognizes RSKB and an antibody that recognizes hepatocyte nuclear factor 4a (HNF-4a). The HNF-4a phosphate inhibitor by RSKB according to claim 8 which is an antibody of
[10] へノ トサイトヌクレア一ファクター 4 aをリボソーム S6キナーゼ Bを用いてリン酸化する ことを特徴とする、糖新生関連遺伝子の遺伝子産物産生促進方法。 [10] Phosphorylation of hetocytonuclea factor 4 a using ribosome S6 kinase B A method for promoting the production of a gene product of a gluconeogenesis-related gene.
[11] 糖新生関連遺伝子がホスホェノールピルビン酸カルボキシキナーゼ遺伝子である請 求項 10に記載の糖新生関連遺伝子の遺伝子産物産生促進方法。  [11] The method for promoting gene product production of a gluconeogenesis-related gene according to claim 10, wherein the gluconeogenesis-related gene is a phosphoenolpyruvate carboxykinase gene.
[12] リボソーム S6キナーゼ Bによるへノ トサイトヌクレア一ファクター 4 aのリン酸化を阻害 することを特徴とする、糖新生関連遺伝子の遺伝子産物産生阻害方法。 [12] A method for inhibiting the production of a gene product of a gluconeogenesis-related gene, which comprises inhibiting phosphorylation of hepatocyte nuclear factor 1a by ribosome S6 kinase B.
[13] リボソーム S6キナーゼ Bによるへノ トサイトヌクレア一ファクター 4 aのリン酸化を阻害 することを特徴とする、ホスホェノールピルビン酸カルボキシキナーゼ遺伝子の遺伝 子産物産生阻害方法。 [13] A method for inhibiting the production of a gene product of the phosphoenolpyruvate carboxykinase gene, which comprises inhibiting phosphorylation of hetocytonuclea factor 4a by ribosome S6 kinase B.
[14] 請求項 2から 5のいずれか 1項に記載のリン酸ィ匕阻害方法を用いることを特徴とする、 糖新生関連遺伝子の遺伝子産物産生阻害方法。  [14] A method for inhibiting the production of a gene product of a gluconeogenesis-related gene, characterized in that the method for inhibiting phosphorylation according to any one of claims 2 to 5 is used.
[15] 請求項 2から 5のいずれか 1項に記載のリン酸ィ匕阻害方法を用いることを特徴とする、 へパトサイトヌクレア一ファクター 4 aが転写因子として作用する遺伝子の遺伝子産 物産生阻害方法。 [15] A gene product of a gene in which hepatocyte nuclear factor 4a acts as a transcription factor, characterized in that the method for inhibiting phosphorylation according to any one of claims 2 to 5 is used. Live inhibition method.
[16] 請求項 2から 5のいずれか 1項に記載のリン酸ィ匕阻害方法を用いることを特徴とする、 ホスホェノールピルビン酸カルボキシキナーゼ遺伝子の遺伝子産物産生阻害方法。  [16] A method for inhibiting the production of a gene product of a phosphoenolpyruvate carboxykinase gene, characterized in that the method for inhibiting phosphorylation according to any one of claims 2 to 5 is used.
[17] 請求項 2から 5のいずれか 1項に記載のリン酸ィ匕阻害方法を用いることを特徴とする、 リボソーム S6キナーゼ Bによるへノ トサイトヌクレア一ファクター 4 aのリン酸化に起因 する疾患の防止および Zまたは治療方法。  [17] According to the phosphorylation inhibition method according to any one of claims 2 to 5, characterized by phosphorylation of hetocytonuclea factor 4a by ribosome S6 kinase B Disease prevention and Z or treatment methods.
[18] 請求項 2から 5のいずれか 1項に記載のリン酸ィ匕阻害方法を用いることを特徴とする、 糖新生関連遺伝子の遺伝子産物の増加に起因する疾患の防止および Zまたは治 療方法。 [18] Prevention or Z or treatment of a disease caused by an increase in a gene product of a gluconeogenesis-related gene, characterized in that the method for inhibiting phosphorylation according to any one of claims 2 to 5 is used. Method.
[19] 請求項 2から 5のいずれか 1項に記載のリン酸ィ匕阻害方法を用いることを特徴とする、 ホスホェノールピルビン酸カルボキシキナーゼ遺伝子の遺伝子産物の増加に起因 する疾患の防止および Zまたは治療方法。  [19] Prevention of a disease caused by an increase in the gene product of the phosphoenolpyruvate carboxykinase gene and the use of the method for inhibiting phosphorylation according to any one of claims 2 to 5 and Z Or treatment method.
[20] 請求項 2から 5のいずれか 1項に記載のリボソーム S6キナーゼ Bによるへパトサイトヌ クレア一ファクター 4 aのリン酸ィ匕阻害方法を用いることを特徴とする、糖尿病の防止 および Zまたは治療方法。 [20] Diabetes prevention and Z or treatment, characterized by using the method for inhibiting hepatocyte nuclear factor 4a phosphorylation by ribosome S6 kinase B according to any one of claims 2 to 5 Method.
[21] 請求項 6から 9のいずれか 1項に記載のリボソーム S6キナーゼ Bによるへパトサイトヌ クレア一ファクター 4 aのリン酸ィ匕阻害剤および Zまたは RSKB活性の阻害剤を用い ることを特徴とする、糖尿病の防止および Zまたは治療方法。 [21] Hepatocyte by ribosome S6 kinase B according to any one of claims 6 to 9 A method for preventing and / or treating diabetes, characterized by using a phosphate factor inhibitor of Claire factor 4a and an inhibitor of Z or RSKB activity.
[22] 請求項 6から 9のいずれか 1項に記載のリボソーム S6キナーゼ Bによるへパトサイトヌ クレア一ファクター 4 aのリン酸ィ匕阻害剤および Zまたは RSKB活性の阻害剤を有効 量含んでなる、ホスホェノールピルビン酸カルボキシキナーゼ遺伝子の遺伝子産物 の産生阻害剤。 [22] An effective amount of the hepatocyte nuclear factor 4a phosphate inhibitor and the inhibitor of Z or RSKB activity by ribosomal S6 kinase B according to any one of claims 6 to 9, Inhibitor of gene product of phosphoenolpyruvate carboxykinase gene.
[23] 請求項 6から 9のいずれか 1項に記載のリボソーム S6キナーゼ Bによるへパトサイトヌ クレア一ファクター 4 aのリン酸ィ匕阻害剤および Zまたは RSKB活性の阻害剤を有効 量含んでなる医薬組成物。  [23] A medicament comprising an effective amount of a hepatocyte nuclear factor 4a phosphate inhibitor and an inhibitor of Z or RSKB activity by ribosome S6 kinase B according to any one of claims 6 to 9 Composition.
[24] 請求項 6から 9のいずれか 1項に記載のリボソーム S6キナーゼ Bによるへパトサイトヌ クレア一ファクター 4 aのリン酸ィ匕阻害剤および Zまたは RSKB活性の阻害剤を有効 量含んでなる、ホスホェノールピルビン酸カルボキシキナーゼ遺伝子の遺伝子産物 の増加に起因する疾患の防止および Zまたは治療剤。  [24] It comprises an effective amount of the hepatocyte nuclear factor 4a phosphate inhibitor and the inhibitor of Z or RSKB activity by ribosome S6 kinase B according to any one of claims 6 to 9. A preventive and Z or therapeutic agent for diseases caused by an increase in the gene product of the phosphoenolpyruvate carboxykinase gene.
[25] 請求項 6から 9のいずれか 1項に記載のリボソーム S6キナーゼ Bによるへパトサイトヌ クレア一ファクター 4 aのリン酸ィ匕阻害剤および Zまたは RSKB活性の阻害剤を有効 量含んでなる、糖尿病の防止および Zまたは治療剤。  [25] It comprises an effective amount of the hepatocyte nuclear factor 4a phosphate inhibitor and the inhibitor of Z or RSKB activity by ribosomal S6 kinase B according to any one of claims 6 to 9. Diabetes prevention and Z or treatment.
[26] リボソーム S6キナーゼ B (RSKB)とへパトサイトヌクレア一ファクター 4 a (HNF— 4 a )の相互作用を阻害する化合物の同定方法であって、 RSKBと HNF— 4 aの相互 作用を可能にする条件下、 RSKBおよび Zまたは HNF— 4 aと被検化合物を接触 させ、 RSKBと HNF— 4 aの相互作用を検出するシグナルおよび Zまたはマーカー を使用する系を用い、このシグナルおよび Zまたはマーカーの存在若しくは不存在 および Zまたは変化を検出することにより、被検化合物力 ¾SKBと HNF— 4 aの相 互作用を阻害するか否かを決定することを含む同定方法。  [26] A method for identifying a compound that inhibits the interaction between ribosomal S6 kinase B (RSKB) and hepatocyte nuclear factor 4 a (HNF—4 a), comprising the interaction between RSKB and HNF—4 a. Using a system that uses a signal and a Z or marker to detect the interaction between RSKB and HNF-4a, contact RSKB and Z or HNF-4a with a test compound under conditions that enable it, and this signal and Z Or an identification method comprising determining whether or not to inhibit the interaction between a test compound force SKB and HNF-4a by detecting the presence or absence of a marker and Z or a change.
[27] リボソーム S6キナーゼ B (RSKB)とへパトサイトヌクレア一ファクター 4 a (HNF— 4 a )の結合を阻害する化合物の同定方法であって、 RSKBと HNF— 4 aの結合を可 能にする条件下、 RSKBおよび Zまたは HNF— 4 aと被検化合物を接触させ、 RS KBと HNF— 4 aの結合を検出するシグナルおよび Zまたはマーカーを使用する系 を用い、このシグナルおよび Zまたはマーカーの存在若しくは不存在および Zまた は変化を検出することにより、被検化合物が RSKBと HNF— 4 aの結合を阻害する か否かを決定することを含む同定方法。 [27] A method for identifying a compound that inhibits the binding of ribosomal S6 kinase B (RSKB) to hepatocyte nuclear factor 4a (HNF-4a), which can bind RSKB to HNF-4a. RSKB and Z or HNF-4a are brought into contact with the test compound under the conditions to be used, and a signal and Z or marker are used to detect the binding of RSKB and HNF-4a. The presence or absence of a marker and Z or Is an identification method comprising determining whether a test compound inhibits the binding between RSKB and HNF-4a by detecting a change.
[28] リボソーム S6キナーゼ B (RSKB)によるへノ トサイトヌクレア一ファクター 4 a (HNF  [28] Hetocytonuclease factor 4a (HNF by ribosomal S6 kinase B (RSKB)
4 a )のリン酸化を阻害する化合物の同定方法であって、 RSKBによる HNF— 4 a のリン酸ィ匕を可能にする条件下、 RSKBおよび Zまたは HNF— 4 aと被検化合物を 接触させ、 RSKBによる HNF— 4 aのリン酸ィ匕を検出するシグナルおよび Zまたは マーカーを使用する系を用い、このシグナルおよび Zまたはマーカーの存在若しく は不存在および Zまたは変化を検出することにより、被検化合物が RSKBによる HN F-4 aのリン酸ィ匕を阻害するか否かを決定することを含む同定方法。  4a) is a method for identifying a compound that inhibits phosphorylation of RSF and contacting a test compound with RSKB and Z or HNF-4a under conditions that allow phosphorylation of HNF-4a by RSKB. By using a system that uses a signal and Z or marker to detect HNF-4a phosphate by RSKB and detecting the presence or absence of this signal and Z or marker and Z or change, An identification method comprising determining whether or not a test compound inhibits phosphorylation of HNF-4a by RSKB.
[29] リボソーム S6キナーゼ B (RSKB)、 RSKBをコードするポリヌクレオチドおよび RSKB をコードするポリヌクレオチドを含有するベクターのうち少なくともいずれ力 1つと、へ ノ トサイトヌクレア一ファクター 4 a (HNF— 4 a )、 HNF— 4 aをコードするポリヌクレ ォチドおよび該 HNF— 4 aをコードするポリヌクレオチドを含有するベクターのうちの 少なくともいずれ力 1つを含んでなるキット。  [29] Ribosome S6 kinase B (RSKB), at least one of a polynucleotide encoding RSKB and a vector containing a polynucleotide encoding RSKB, and a hetocytonuclear factor 4a (HNF—4 a), a kit comprising at least one of a polynucleotide encoding HNF-4a and a vector containing the polynucleotide encoding HNF-4a.
PCT/JP2005/019518 2004-10-22 2005-10-24 Method of inhibiting phosphorylation of transcriptional factor for gluconeogenesis-associated gene and phosphorylation inhibitor WO2006043701A1 (en)

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Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
HALL R.K. ET AL: "The orphan receptors COUP-TF and HNF-4 serve as accessory factors required for induction of phosphoenolpyruvate carboxykinase gene transcription by glucocorticoids", PROC. NATL. ACAD. SCI. USA, vol. 92, no. 2, 1995, pages 412 - 416, XP002994007 *
HUNTER T. ET AL: "The regulation of transcription by phosphorylation", CELL, vol. 70, no. 3, 1992, pages 375 - 387, XP002994008 *
PIERRAT B. ET AL: "RSK-B, a novel ribosomal S6 kinase family member, is a CREB kinase under dominant control of p38alpha mitogen-activated protein kinase (p38alphaMAPK)", J. BIOL. CHEM., vol. 273, no. 45, 1998, pages 29661 - 29671, XP002120478 *
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