JP2008017701A - Method for inhibiting phosphorylation of transcriptional factor for gluconogenesis-associated gene and phosphorylation inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for inhibiting phosphorylation of a transcriptional factor for a gluconogenesis-associated gene and to provide a phosphorylation inhibitor. <P>SOLUTION: It is found out that an HNF-4α (hepatocyte nuclear factor 4α) which is the transcriptional factor is phosphorylated with an RSKB (ribosome S6 kinase B) to thereby promote the binding thereof to the promoter region of the gluconogenesis-associated gene. As a result, there are provided the method for phosphorylating the HNF-4α with the RSKB, a method for inhibiting the phosphorylation, the phosphorylation inhibitor, a production inhibitor of a gene product of the gene on which the HNF-4α acts as the transcriptional factor and a method for inhibiting the production, a preventive and/or a therapeutic agent for diseases caused by the phosphorylation of the HNF-4α with the RSKB and a method for preventing and/or treating the diseases, a method for identifying a compound inhibiting the phosphorylation of the HNF-4α with the RSKB, the compound obtained by the method for identification and further a reagent kit comprising the RSKB, HNF-4α, a polynucleotide encoding the HNF-4α and a vector containing the polynucleotide. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a phosphorylation method, phosphorylation agent, phosphorylation inhibition method and phosphorylation inhibitor of hepatocyte nuclear factor 4α (hereinafter abbreviated as HNF-4α). More specifically, the present invention relates to a method for phosphorylating HNF-4α and a phosphorylating agent characterized by using ribosomal S6 kinase B (hereinafter abbreviated as RSKB). The present invention also relates to a method for inhibiting phosphorylation of HNF-4α by RSKB and a phosphorylation inhibitor. Furthermore, the present invention relates to a method for inhibiting the production of a gene product of a gene for which HNF-4α acts as a transcription factor and a production inhibitor. The present invention also relates to a method for preventing and / or treating a disease caused by phosphorylation of HNF-4α, for example, diabetes, and a preventive and / or therapeutic agent. Furthermore, the present invention relates to a method for identifying a compound that inhibits phosphorylation of HNF-4α by RSKB and a compound identified by the identification method. The present invention also relates to a reagent kit comprising a polynucleotide encoding RSKB and HNF-4α and a vector containing the polynucleotide.

  The liver is an important organ for maintaining glucose homeostasis, and maintains the balance of the amount of glucose in the living body by gluconeogenesis that produces glucose and glycogen synthesis that produces glycogen from glucose. 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 for synthesizing glucose from pyruvate, most of which is performed in the liver. Among a series of enzymes that promote gluconeogenesis, phosphoenol pyruvate carboxykinase (hereinafter abbreviated as PEPCK) is considered to be a rate-limiting enzyme for gluconeogenesis (Non-Patent Documents 2 to 7). The activity of PEPCK in the liver is controlled by the amount of transcription from the PEPCK gene. This transcription is regulated by hormones, and glucocorticoid promotes the transcription of PEPCK (Non-patent Document 8) and suppresses insulin (Non-patent Documents 4, 6, and 8). Furthermore, 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). Thereby, the enzyme activity, gluconeogenesis, and blood glucose level of PEPCK are regulated (Non-patent Document 3). In the liver in a diabetic state, the expression of PEPCK gene is increased by glucocorticoid, and gluconeogenesis is enhanced (Non-patent Documents 8 and 9).

  Diabetes is classified into type 1 diabetes (insulin-dependent diabetes, IDDM) and type 2 diabetes (non-insulin-dependent diabetes, NIDDM). Type 2 diabetes is further divided into diabetes mainly composed of a decrease in insulin secretion, and insulin is secreted, but the insulin sensitivity to glucose in the target cells is mainly composed of diabetes, and the latter is particularly said to be insulin resistance. The target tissues of insulin are the liver, fat, and muscle, and the blood glucose level is lowered by suppressing gluconeogenesis and release of the liver. Sustained hyperglycemia reduces the sensitivity of the liver to insulin and prevents insulin-dependent gluconeogenesis suppression. However, the mechanism for lowering insulin sensitivity in the liver due to persistent hyperglycemia has not yet been clarified, and downregulation of the insulin receptor is assumed.

  HNF-4α is one of the nuclear receptors and is expressed in the liver, pancreatic β cells, kidney and small intestine, and various proteins encoding proteins involved in metabolism, liver development and differentiation, such as cholesterol, fatty acids and glucose It is known to be a gene transcription factor. Regarding gluconeogenesis, HNF-4α binds to the AF-1 region present in the promoter of the PEPCK gene and is involved in the expression of the PEPCK gene (Non-patent Document 16). It is known that the transcription factor changes the DNA binding ability by phosphorylation (Non-patent Document 17), but HNF-4α is phosphorylated by PKA (cAMP-dependent protein kinase, cAMP-dependent protein kinase), and L There is a report that the binding ability of the type pyruvate kinase to the promoter decreases (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). p38 kinase is activated in various pathological conditions (Non-patent Document 12). In cells that are continuously exposed to hyperglycemia under diabetic conditions, p38 kinase is activated from oxidative stress caused by active oxygen (Non-patent Documents 13 and 14). For example, activation of p38 kinase has been reported in vascular smooth muscle cells cultured under hyperglycemia (16.5 mM) (Non-patent Document 15) and in the liver of ob / ob mice of diabetes model animals (Non-patent Document). 29).
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  The present invention finds a substance that promotes the ability to bind to the PEPCK promoter region by phosphorylating HNF-4α, which regulates the transcription of the PEPCK gene, and causes diseases caused by an increase in the transcription amount of the PEPCK gene, such as diabetes Providing means for preventing and / or treating

  The inventors of the present invention have made diligent efforts and found that HNF-4α is phosphorylated by RSKB. Furthermore, HNF-4α was phosphorylated by RSKB, thereby accelerating the ability to bind the PEPCK promoter region to the AF1 site, and the promotion demonstrated that the transcription activity of the PEPCK gene was enhanced, thereby completing the present invention.

That is, the present invention
1. A method for phosphorylating HNF-4α by RSKB, characterized in that ribosome S6 kinase B (RSKB) and hepatocyte nuclear factor 4α (HNF-4α) coexist under conditions that allow interaction;
2. A method for inhibiting phosphorylation of hepatocyte nuclear factor 4α (HNF-4α) by RKSB, which comprises inhibiting the activity of ribosomal S6 kinase B (RSKB);
3. A method for inhibiting phosphorylation of HNF-4α by RSKB, which comprises inhibiting the interaction between ribosomal S6 kinase B (RSKB) and hepatocyte nuclear factor 4α (HNF-4α),
4). A cell expressing at least ribosomal S6 kinase B (RSKB) and hepatocyte nuclear factor 4α (HNF-4α) is treated with an inhibitor of RSKB activity, and the HNF-4α phosphorylation by RSKB Oxidation inhibition method,
5. The above-mentioned item 4, wherein the inhibitor of ribosomal S6 kinase B (RSKB) activity is one or more antibodies selected from an antibody recognizing RSKB and an antibody recognizing hepatocyte nuclear factor 4α (HNF-4α). A method for inhibiting phosphorylation of HNF-4α by RSKB according to claim 1,
6). An inhibitor of phosphorylation of hepatocyte nuclear factor 4α (HNF-4α) by RSKB, comprising an effective amount of an inhibitor of ribosomal S6 kinase B (RSKB) activity,
7). An inhibitor of phosphorylation of HNF-4α by RSKB, characterized by inhibiting the interaction between ribosomal S6 kinase B (RSKB) and hepatocyte nuclear factor 4α (HNF-4α),
8). An inhibitor of phosphorylation of hepatocyte nuclear factor 4α (HNF-4α) by RSKB, characterized by inhibiting ribosomal S6 kinase B (RSKB) activity;
9. The aforementioned item 8, wherein the inhibitor of ribosomal S6 kinase B (RSKB) activity is one or more antibodies selected from an antibody recognizing RSKB and an antibody recognizing hepatocyte nuclear factor 4α (HNF-4α) An inhibitor of phosphorylation of HNF-4α by RSKB,
10. A method for promoting the production of a gene product of a gluconeogenesis-related gene, which comprises phosphorylating hepatocyte nuclear factor 4α using ribosomal S6 kinase B;
11. The method for promoting the production of a gene product of a gluconeogenesis-related gene according to 10 above, wherein the gluconeogenesis-related gene is a phosphoenolpyruvate carboxykinase gene,
12 A method for inhibiting gene product production of a gluconeogenesis-related gene, which comprises inhibiting phosphorylation of hepatocyte nuclear factor 4α by ribosome S6 kinase B;
13. A method for inhibiting gene product production of a phosphoenolpyruvate carboxykinase gene, which comprises inhibiting phosphorylation of hepatocyte nuclear factor 4α by ribosomal S6 kinase B;
14 A method for inhibiting the production of a gene product of a gluconeogenesis-related gene, comprising using the method for inhibiting phosphorylation according to any one of 2 to 5 above,
15. A method for inhibiting the production of a gene product of a gene in which hepatocyte nuclear factor 4α acts as a transcription factor, comprising using the phosphorylation inhibition method according to any one of 2 to 5 above.
16. A method for inhibiting the production of a gene product of a phosphoenolpyruvate carboxykinase gene, which comprises using the method for inhibiting phosphorylation according to any one of items 2 to 5;
17. 6. A method for preventing and / or treating a disease caused by phosphorylation of hepatocyte nuclear factor 4α by ribosomal S6 kinase B, wherein the method for inhibiting phosphorylation according to any one of 2 to 5 above is used. Method,
18. A method for preventing and / or treating a disease caused by an increase in a gene product of a gluconeogenesis-related gene, which comprises using the method for inhibiting phosphorylation according to any one of items 2 to 5;
19. A method for preventing and / or treating a disease caused by an increase in a gene product of a phosphoenolpyruvate carboxykinase gene, comprising using the method for inhibiting phosphorylation according to any one of items 2 to 5;
20. A method for preventing and / or treating diabetes, comprising using the method for inhibiting phosphorylation of hepatocyte nuclear factor 4α by ribosome S6 kinase B according to any one of items 2 to 5,
21. 10. A method for preventing diabetes, comprising using the phosphorylation inhibitor of hepatocyte nuclear factor 4α and / or the inhibitor of RSKB activity by ribosome S6 kinase B according to any one of 6 to 9 above and / Or treatment method,
22. 10. A phosphoenolpyruvate carboxykinase comprising an effective amount of a phosphorylation inhibitor of hepatocyte nuclear factor 4α and / or an inhibitor of RSKB activity by ribosomal S6 kinase B according to any one of 6 to 9 above Production inhibitors of gene products of genes,
23. 10. A pharmaceutical composition comprising an effective amount of an inhibitor of phosphorylation of hepatocyte nuclear factor 4α and / or an inhibitor of RSKB activity by ribosome S6 kinase B according to any one of items 6 to 9 above,
24. 10. A phosphoenolpyruvate carboxykinase comprising an effective amount of a phosphorylation inhibitor of hepatocyte nuclear factor 4α and / or an inhibitor of RSKB activity by ribosomal S6 kinase B according to any one of 6 to 9 above Preventive and / or therapeutic agent for diseases caused by an increase in gene products of genes,
25. 10. An antidiabetic agent comprising an effective amount of a phosphorylation inhibitor of hepatocyte nuclear factor 4α and / or an inhibitor of RSKB activity by ribosome S6 kinase B according to any one of items 6 to 9 above and / or Or therapeutic agent,
26. A method for identifying a compound that inhibits the interaction between ribosomal S6 kinase B (RSKB) and hepatocyte nuclear factor 4α (HNF-4α), under conditions that allow the interaction between RSKB and HNF-4α And / or a system using a signal and / or a marker that contacts HNF-4α with a test compound and detects the interaction between RSKB and HNF-4α, and the presence or absence of this signal and / or marker and / or Or an identification method for determining whether a test compound inhibits the interaction between RSKB and HNF-4α by detecting a change,
27. A method for identifying a compound that inhibits phosphorylation of hepatocyte nuclear factor 4α (HNF-4α) by ribosomal S6 kinase B (RSKB), under conditions that allow phosphorylation of HNF-4α by RSKB And / or a system using a signal and / or a marker that contacts HNF-4α with a test compound and detects phosphorylation of HNF-4α by RSKB, and this signal and / or the presence or absence of the marker and / or Or an identification method for determining whether a test compound inhibits phosphorylation of HNF-4α by RSKB by detecting a change,
28. At least one of ribosomal S6 kinase B (RSKB), a polynucleotide encoding RSKB, and a vector containing a polynucleotide encoding RSKB, hepatocyte nuclear factor 4α (HNF-4α), and HNF-4α. A kit comprising at least one of a polynucleotide encoding and a vector containing the polynucleotide encoding HNF-4α,
About.

  In the present invention, it was found that HNF-4α phosphorylated by RSKB binds to AF1 in the promoter region of PEPCK gene. Thus, a phosphorylation method, phosphorylation inhibition method and phosphorylation inhibitor of HNF-4α by RSKB are provided. In diabetic conditions, HNF-4α activated by RSKB promotes transcriptional activity of PEPCK gene, thereby promoting gluconeogenesis and exacerbating hyperglycemia. Therefore, the inhibition of HNF-4α phosphorylation inhibitor and / or phosphorylation inhibition method by RSKB provided by the present invention inhibits the production of the gene product of the gene on which HNF-4α acts, for example, the inhibition of the production of the gene product of the PEPCK gene. It becomes possible. In addition, it is possible to prevent and / or treat diseases caused by an increase in the gene product of the gene on which HNF-4α acts. Specifically, it is possible to prevent and / or treat diseases caused by an increase in PEPCK gene products, more specifically diabetes. Thus, the present invention is very useful for prevention and / or treatment of diseases caused by excessive phosphorylation of HNF-4α involved in gluconeogenesis-related gene expression.

  Furthermore, by the phosphorylation inhibitor and / or phosphorylation inhibition method of HNF-4α by RSKB according to the present invention, it is possible to suppress gluconeogenesis in the liver of insulin resistant patients with type 2 diabetes who cannot expect the effect of insulin, and to prevent hyperglycemia It is believed to have an inhibitory effect. In recent years, insulin resistance improving drugs have been developed as antidiabetic drugs, and thiazolidinedione derivatives are used in clinical settings as a representative example. The thiazolidinedione derivative has an action of changing large adipocytes having low insulin sensitivity in adipose tissue to small adipocytes having high insulin sensitivity. However, the target organ is different between the phosphorylation inhibitor of HNF-4α by the RSKB of the present invention and the thiazolidinedione derivative. Moreover, it is considered that the inhibitory effect of the PEPCK gene by insulin is reduced in end-stage patients with diabetes in which pancreatic β cells are disrupted and the amount of insulin in the blood is decreased. Therefore, it is very useful to inhibit the phosphorylation of HNF-4α by RSKB and suppress the production of the gene product of the PEPCK gene, since it may exert a hypoglycemic effect independent of insulin.

  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.

  In the present specification, amino acids may be represented by one letter or three letters. A peptide also refers to any peptide comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds. In addition, peptides can be short peptides such as isolated or synthetic full-length oligopeptides, referred to as oligomers, as well as lengths such as isolated or synthetic full-length polypeptides and isolated or synthetic full-length proteins. Also means a chain peptide.

  In the present invention, a protein that interacts with RSKB was predicted according to the method described in International Publication No. WO01 / 67299, and as a result, it was found that RSKB interacts with HNF-4α. It is also revealed for the first time that RSKB phosphorylates HNF-4α, and that HNF-4α phosphorylated by RSKB promotes its binding to the PE1 of the PEPCK gene promoter region, resulting in an increase in PEPCK gene transcription. did.

  The PEPCK promoter has a region called GRU (glucocorticoid response unit, glucocorticoid response unit), AF1 (accessory factor binding site 1, accessory factor binding site 1), GR1, 2 (glucocorticoid receptor binding site, glucocorticoid receptor binding site). ), AF2 (accessory factor binding site 2, accessory factor binding site 2) and CRE (cyclic AMP response element, cAMP reactive element). These regions are considered necessary in order to efficiently express the PEPCK gene.

  As a transcription factor that is phosphorylated by RSKB and binds to the PEPCK promoter, there is CREB (cyclic AMP response element binding protein, cAMP responding element binding protein). CREB is a transcription factor that binds to the CRE of the PEPCK promoter (Non-patent Document 19). CRE is a region that exists in the PEPCK promoter and positively regulates PEPCK gene expression (Non-patent Document 18). CREB and C / EBP (CCAAT / enhancer binding protein, CCAAT / enhancer binding protein) have been reported as genes that positively regulate the PEPCK gene by binding to CRE (Non-patent Document 19).

  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-4α, and CREB binds to the CRE of the PEPCK promoter, which may promote the expression of the PEPCK gene. However, it is C / EBP, not CREB, that actually regulates the CRE of the PEPCK promoter in vivo (Non-Patent Documents 20 to 23), and there is no report that C / EBP is phosphorylated by RSKB.

  In addition, in the PEPCK promoter region, the transcriptional activity of the PEPCK promoter is reduced to about one-fourth or less of that of the control (wild-type PEPCK promoter) by AF1 substitution deletion (Non-patent Document 24). On the other hand, depending on the substitution deletion of CRE, the transcriptional activity of the PEPCK promoter is reduced only to about half that of the control (Non-patent Document 25). Therefore, AF1 to which HNF-4α binds is a sequence that affects the expression of the PEPCK gene, and is an important factor for regulation of gene expression that affects more than CRE1, which is a sequence of other PEPCK promoter regions. Thought. Furthermore, as shown in FIG. 11 of the Examples of the present specification, intracellular RSKB was activated using PEPCKΔAF1 promoter that is not affected by transcriptional activity by HNF-4α (co-expression of MAPK11 and RSKB), but PEPCK The transcriptional activity of the gene was hardly promoted. Therefore, in vivo, it is considered that regulation of PEPCK gene expression by RSKB is mediated by HNF-4α.

  It has been reported that the gluconeogenic potential of PEPCK in non-insulin dependent diabetic patients is about 3 times higher than that of normal individuals, and this gluconeogenic potential correlates with the fasting plasma glucose concentration of the patient ( Non-patent document 26). The transcription of the PEPCK gene is regulated by hormones, is enhanced by glucocorticoid, and is suppressed by insulin (Non-patent Documents 8 and 27). Insulin controls gluconeogenesis in the liver by suppressing transcription of the PEPCK gene via LIP (liver-enhanced transcriptional inhibitory protein), a transcriptional regulator in the PEPCK promoter (non-patented) Reference 28). There is also a report that insulin stimulation directly acts on the PEPCK promoter to suppress transcriptional activity (Non-patent Documents 6 and 37). Therefore, in normal liver, the expression of PEPCK gene is repressively controlled by insulin. However, if PEPCK gene overexpression persists for some reason, gluconeogenesis in the liver is increased, resulting in hyperglycemia and insulin resistance. It is thought to be diabetic.

  RSKB is activated by p38, an upstream kinase. RSKB also phosphorylates CREB (cAMP responding element binding protein). Thus, RSKB activation can be indexed by p38 activation and CREB phosphorylation. In the liver of diabetic model animals, the phosphorylated form of p38 (active p38) is increased by about 2.5 to 3 times that of normal individuals, and the phosphorylated form of CREB is also reported to increase by about 2 times. (Non-patent documents 29 and 30). Based on these facts, the inventor considered that in the liver in a diabetic state, p38 kinase is activated by oxidative stress or the like, and then RSKB is activated.

  PEPCK activity control is regulated at the mRNA level. From experiments using hepatocytes, it has been reported that when the amount of mRNA of the PEPCK gene increases 2-fold, the enzyme activity increases 2.8-fold and the gluconeogenic potential increases 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 is increased by a factor of 2 (Non-patent Document 7). 3 times higher than normal (Non-patent Document 26). That is, the amount of PEPCK mRNA, enzyme activity, gluconeogenic potential, and increase in blood glucose level show good correlations.

  The inventor considered the degree of increase in blood glucose level due to activation of RSKB as follows. Assuming that the phosphorylation of HNF-4α by RSKB is similar to that of CREB, it was estimated that the phosphorylation of HNF-4α in the liver by RSKB during diabetic pathology was about two times that of normal. 9, 10 and 12 showing the results of the examples, the ability of HNF-4α to activate PEPCK gene promoter transcription is enhanced about 2-3 times by phosphorylation by RSKB. When the promoter activity of the PEPCK gene was replaced with the amount of mRNA, it was considered that the amount of mRNA of the PEPCK gene was increased by about 2-3 times due to RSKB activation in diabetic conditions. As described above, an increase in the amount of mRNA of the PEPCK gene is directly reflected in the enzyme activity, leading to increased gluconeogenic potential and increased blood glucose level. From this, it was considered that if the phosphorylation of HNF-4α by RSKB is inhibited in a diabetic state, the amount of PEPCK gene mRNA in the liver is suppressed to about one-half to one-third. As a result, it is estimated that the enzyme activity of PEPCK is reduced to about one third and the gluconeogenic potential is reduced to about one half, and as a result, the blood glucose level is reduced to about half of that in the pathological condition. Therefore, it was considered that diabetes can be treated by inhibiting the phosphorylation of HNF-4α by RSKB and regulating the production of the gene product of the PEPCK gene during diabetic conditions.

  One embodiment of the present invention achieved by these findings relates to a method for phosphorylating HNF-4α, characterized by using RSKB. Detection of phosphorylation by RSKB can be carried out by a known method such as Western blotting after contacting RSKB and HNF-4α.

  The method for phosphorylating HNF-4α by RSKB according to the present invention is characterized in that RSKB and HNF-4α coexist. In addition, it is possible to construct and utilize a phosphorylation system of HNF-4α by RSKB characterized by coexistence of RSKB and HNF-4α.

  The phosphorylation method and phosphorylation system of HNF-4α by RSKB may be in vitro or an in vivo system. Specifically, a phosphorylation method and a phosphorylation system in which RSKB and HNF-4α are phosphorylated in the presence of, for example, a test tube or a multiwell plate can be exemplified. Alternatively, phosphorylation methods and phosphorylation systems using cells in which RSKB and HNF-4α are co-expressed can be exemplified.

  Cells used for protein expression can be used for expression. Specific examples include HeLa cells and HepG2 cells. Expression of these proteins can be performed using a known genetic engineering technique.

  RKSB and HNF-4α used in the present invention are prepared from cells, cell-free synthetic products, chemical synthesis products, or cells or biological samples in which one or more of these are expressed by genetic engineering techniques. It may be that which is further purified from these. In addition, these proteins are added by adding another protein or peptide to the N-terminal side or C-terminal side thereof directly or indirectly through a linker peptide using a genetic engineering technique. It may be labeled. The labeling is preferably performed by a method that does not inhibit the interaction between RSKB and HNF-4α and the functions of these proteins, for example, contact between RSKB and HNF-4α, RSKB enzyme activity, and HNF-4α function.

  Examples of the labeling substance include enzymes (glutathione S-transferase, horseradish peroxidase, alkaline phosphatase, β-galactosidase, etc.), tag peptides (His-tag, Myc-tag, HA-tag, FLAG-tag, Xpress-tag or Xpress-tag). ), Fluorescent proteins (such as green fluorescent protein, fluorescein isothiocyanate or phycoerythrin), maltose binding protein, Fc fragment of immunoglobulin, or biotin, but is not limited thereto. Alternatively, labeling with a radioisotope is also possible. When labeling, one type of labeling substance may be added, or a plurality of labeling substances may be added in combination. By measuring these labeling substances themselves or their functions, it is possible to detect phosphorylation of HNF-4α by RSKB.

  Genes encoding RSKB and HNF-4α used for gene introduction can be prepared from a human cDNA library by a known genetic engineering technique. These genes can be introduced into an appropriate expression vector DNA, for example, a vector derived from a bacterial plasmid, by a known genetic engineering technique to obtain a vector containing the gene, which can be used for gene introduction. Gene transfer can be performed using a genetic engineering technique known per se.

  The phosphorylating agent, phosphorylation method, and phosphorylation system of HNF-4α by RSKB are due to elucidation of the function of HNF-4α, studies on transcription factor networks involving HNF-4α, and phosphorylation of HNF-4α It is useful for molecular studies on the involvement of HNF-4α and RSKB in diseases such as diabetes. It is also possible to construct a method for identifying a compound that inhibits phosphorylation of HNF-4α by RSKB using the phosphorylation method and phosphorylation system.

  Another aspect of the present invention relates to a phosphorylation inhibitor and method for inhibiting HNF-4α by RSKB. The inhibitor and the inhibition method are characterized by inhibiting RSKB activity, inhibiting phosphorylation of HNF-4α by RSKB, or inhibiting the interaction between RSKB and HNF-4α.

  Inhibition of RSKB activity or inhibition of phosphorylation of HNF-4α by RSKB can be carried out, for example, by inhibiting the enzyme activity of RSKB. The phosphorylation inhibitor of HNF-4α by RSKB according to the present invention is characterized in that it synthesizes at least one compound (RSKB activity inhibitor) having an effect of inhibiting RSKB activity in one embodiment. 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-4α, for example, an in vitro sample including at least these. The subject also includes cells expressing at least RSKB and HNF-4α, such as liver cells, and non-human mammals carrying such cells.

  Examples of the inhibitor of RSKB activity include peptides, antibodies, and low molecular compounds having a competitive inhibitory effect. Examples of the antibody include an antibody that recognizes and binds RSKB or HNF-4α and inhibits phosphorylation of HNF-4α by RSKB. An antibody can be obtained by an antibody production method known per se using RSKB or HNF-4α itself, a partial peptide derived therefrom, or a peptide comprising an amino acid sequence of a site where these interact with each other as an antigen. Examples of the low molecular weight compound include compounds that inhibit the enzyme activity of RSKB, preferably compounds that specifically inhibit the enzyme activity. Such a compound can be obtained, for example, by identifying whether it inhibits phosphorylation of HNF-4α by RSKB using the phosphorylation method or phosphorylation system according to the present invention. Specifically inhibiting the enzyme activity of RSKB means strongly inhibiting the enzyme activity of RSKB, but not inhibiting or weakly inhibiting the activity of other enzymes.

  Inhibition of phosphorylation of HNF-4α by RSKB can also be performed by inhibiting the interaction between RSKB and HNF-4α. The reason why HNF-4α is phosphorylated by RSKB is that it is considered to be an aspect in which RSKB and HNF-4α interact. The interaction refers to an interaction due to non-covalent bonding force between proteins, and examples thereof include temporary binding, reversible binding, and irreversible binding.

In one aspect, the inhibitor of phosphorylation of HNF-4α by RSKB according to the present invention comprises an effective amount of at least one compound that inhibits the interaction between RSKB and HNF-4α as an active ingredient.

  In one aspect, the method for inhibiting phosphorylation of HNF-4α by RSKB according to the present invention is characterized by using at least one compound that inhibits the interaction between RSKB and HNF-4α.

  Inhibition of the interaction between RSKB and HNF-4α can be carried out, for example, using a peptide comprising an amino acid sequence at the site where both proteins interact. 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-4α. Such peptides are thought to competitively inhibit the phosphorylation of HNF-4α by RSKB. Inhibition of the interaction between RSKB and HNF-4α can also be carried out using a peptide consisting of the amino acid sequence of the interaction site between RSKB and HNF-4α.

  The above peptide is designed from the amino acid sequence of RSKB or HNF-4α and is synthesized by a peptide synthesis method known per se, and inhibits phosphorylation of HNF-4α by RSKB and / or the interaction between RSKB and the transcription factor. It can be obtained by selecting one.

  As long as the peptide in which a mutation such as deletion, substitution, addition, or insertion of one to several amino acids is introduced into the peptide thus identified inhibits the interaction between RSKB and HNF-4α, the present invention It is included in the range. The peptide into which such a mutation is introduced is preferably one that inhibits phosphorylation of HNF-4α by RSKB.

  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 technique (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, hydrophilic amino acids, etc.) Sex amino acids, positively charged amino acids, negatively charged amino acids and aromatic amino acids) are readily envisioned.

  Peptides capable of inhibiting the interaction between RSKB and HNF-4α can be altered to the extent that no significant change in function is caused, such as by amidation modification of the constituent amino group or carboxyl group. In particular, modifications commonly used to stabilize the interaction of the peptide with other proteins and make it difficult to dissociate the peptide, such as modifications such as C-terminal aldehyde or N-terminal acetylation, are RSKB and HNF. It is useful for enhancing the effectiveness of peptides that inhibit the −4α interaction.

  The peptide can be produced by a general method known in peptide chemistry. For example, methods (Non-Patent Documents 32 and 33) described in publicly known documents are exemplified, but not limited to these methods, publicly known methods are widely available.

  In one aspect, the inhibitor of phosphorylation of HNF-4α by RSKB according to the present invention is a compound that inhibits the interaction between HNF-4α and RSKB, a phosphorylation inhibitor of RSF of HNF-4α, and an inhibitor of RSKB activity. An effective amount is contained as an active ingredient. In this case, the inhibitor of phosphorylation of HNF-4α is an active ingredient comprising at least one of an interaction inhibition compound of HNF-4α and RSKB, an phosphorylation inhibitor of HNF-4α by RSKB, and an activity inhibitor of RSKB. As an inhibitor containing an effective amount. It can also be an inhibitor containing a combination of a plurality of such inhibitors for HNF-4α. More specifically, for example, it contains an effective amount as an active ingredient at least one of a compound that inhibits the interaction between HNF-4α and RSKB, a phosphorylation inhibitor of RSF of HNF-4α, and an activity inhibitor of RSKB. Is preferred.

  In one aspect of the method for inhibiting phosphorylation of HNF-4α by RSKB according to the present invention, an inhibitor of interaction between HNF-4α and RSKB, a phosphorylation inhibitor of HNF-4α by RSKB, and an activity inhibitor of RSKB At least one of the above is used. At this time, in the method of inhibiting phosphorylation of HNF-4α by RSKB, at least one of an interaction inhibition compound between HNF-4α and RSKB, an inhibitor of phosphorylation of HNF-4α by RSKB, and an RSKB activity inhibitor is used. In addition, a plurality of such inhibitory compounds against each transcription factor can be used in combination. More specifically, for example, it is preferable to use at least one of a compound that inhibits the interaction between HNF-4α and RSKB, a phosphorylation inhibitor of HNF-4α by RSKB, and an activity inhibitor of RSKB.

  One of the functions of HNF-4α is known to be involved in gluconeogenesis-related gene expression as a transcription factor in the liver. The gluconeogenesis-related gene is a gene encoding a substance that regulates gluconeogenesis in a living body, and includes, for example, the PEPCK gene. Activation of HNF-4α during a diabetic condition is thought to further promote gluconeogenesis and worsen 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 a gene product of a gluconeogenesis-related gene according to the present invention regulates the production of the gene product of the gene by regulating the phosphorylation of HNF-4α related to the expression of the gene encoding them by RSKB. It is characterized by.

  One aspect of a 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, characterized by inhibiting phosphorylation of HNF-4α by RSKB related to the gene expression. . Specifically, the production inhibition method can be achieved by using an inhibitor of phosphorylation of HNF-4α by RSKB or a method of inhibiting phosphorylation of HNF-4α by RSKB. A gluconeogenesis-related gene product production promoter comprising the phosphorylation inhibitor is also included in the scope of the present invention.

  Examples of a gene product of a gene on which HNF-4α is involved in 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 in the promoter or enhancer, which is a binding site of HNF-4α phosphorylated by RSKB, such as the PEPCK gene.

  Still another embodiment of the present invention relates to a preventive and / or therapeutic agent for a disease caused by phosphorylation of HNF-4α by RSKB, and a method for preventing and / or treating the disease. The preventive and / or therapeutic agent for the disease contains the phosphorylation inhibitor. The prevention method and / or treatment method of the disease can be achieved by using the phosphorylation inhibitor or the phosphorylation inhibition method.

  Examples of the disease caused by phosphorylation of HNF-4α by RSKB include a disease caused by an increase in the gene product of a gene on which HNF-4α acts. For example, HNF-4α phosphorylated by RSKB promotes the binding to the transcription site upstream of the PEPCK gene and contributes to the gene product production of the gene.

  Specific examples of diseases caused by phosphorylation of HNF-4α by RSKB include diseases caused by an increase in gene products of gluconeogenesis-related genes, such as diseases caused by an increase in the gene products of PEPCK gene. More specifically, diseases caused by abnormal gluconeogenesis, such as diabetes.

  Another embodiment of the present invention relates to a method for identifying a compound that inhibits the interaction between RSKB and HNF-4α. The identification method can be constructed using a pharmaceutical screening system known per se. Further, the identification method can be carried out using the phosphorylation system or phosphorylation method according to the present invention.

  Examples of the test compound of the present invention 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-4α. Alternatively, a compound obtained by drug design based on the peptide structure at the interaction site of HNF-4α and RSKB and / or the phosphorylation site of HNF-4α by RSKB is also suitable as the test compound.

  Specifically, a condition that enables the interaction between RSKB and HNF-4α is selected, RSKB and / or HNF-4α is brought into contact with a test compound under the condition, and the interaction between RSKB and HNF-4α is determined. A system that uses the signal and / or marker to be detected can be used to identify compounds that inhibit the interaction of RSKB and HNF-4α by detecting the presence or absence or change of this signal and / or marker. Detection of the interaction between RSKB and HNF-4α can be carried out using a detection method known per se, such as Western blotting.

  The conditions allowing the interaction between RSKB and HNF-4α may be an in vitro system or an in vivo system. For example, cells in which RSKB and the transcription factor are co-expressed can be used. The contact between RSKB and / or HNF-4α and the test compound may be performed before the interaction reaction between RSKB and HNF-4α, or may be performed by coexisting with the interaction reaction.

  In the present invention, a signal refers to a signal that can be directly detected by its physical or chemical properties, and a marker refers to a signal that can be detected indirectly using the physical or biological properties of the signal as an indicator. .

  Signals include luciferase, green fluorescent protein, and radioisotopes, markers include reporter genes such as chloramphenicol acetyltransferase gene, or epitope tags for detection such as 6 × His-tag Any labeling substance used in the compound identification method can be used. 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.

  Another aspect of the present invention relates to a method for identifying a compound that inhibits phosphorylation of HNF-4α by RSKB, which is related to gluconeogenesis-related gene expression by RSKB. The identification method can be constructed using a pharmaceutical screening system known per se. Further, the identification method can be carried out using the phosphorylation system or phosphorylation method according to the present invention.

  Specifically, conditions that enable phosphorylation of HNF-4α related to gluconeogenesis-related gene expression by RSKB are selected, RSKB and / or HNF-4α are contacted with the test compound under the conditions, and RSKB is used. Using a system that uses a signal and / or marker that detects phosphorylation of HNF-4α, the presence or absence or change of this signal and / or marker can be used to inhibit phosphorylation of HNF-4α by RSKB. Inhibiting compounds can be identified.

  Conditions that allow phosphorylation of HNF-4α by RSKB may be in vitro or an in vivo system. For example, cells in which RSKB and the transcription factor are co-expressed can be used. The contact between RSKB and / or HNF-4α and the test compound may be performed before the phosphorylation reaction of HNF-4α by RSKB, or may be performed by coexisting with the phosphorylation reaction. The phosphorylation of HNF-4α by RSKB can be conveniently detected by measuring the presence or absence and / or change of the amount of phosphorylated HNF-4α.

  The amount of phosphorylated HNF-4α can be quantified using a protein or peptide detection method known per se, such as Western blotting. Alternatively, detection of phosphorylation of HNF-4α by RSKB can be performed by measuring the presence or absence and / or change of HNF-4α activity. Specifically, for example, when HNF-4α acts on the PEPCK gene, a plasmid containing a reporter gene in which the promoter region of PEPCK gene is incorporated upstream is transferred into cells expressing HNF-4α and RSKB. 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, thereby identifying the desired compound. Can be done.

  In addition, a signal that allows the interaction between RSKB and HNF-4α is selected, RSKB and / or HNF-4α is brought into contact with the test compound under the conditions, and a signal for detecting the interaction between RSKB and HNF-4α is detected. By detecting the presence or absence or change of this signal and / or marker using a system that uses markers and / or markers, compounds that inhibit phosphorylation of HNF-4α by RSKB can be identified. Detection of the interaction between RSKB and HNF-4α can be carried out using a detection method known per se, such as Western blotting.

  The compound obtained by such an identification method can be used as an inhibitor of phosphorylation of HNF-4α by RSKB or a gene product production inhibitor of a gene on which HNF-4α acts. An inhibitor of phosphorylation of HNF-4α by RSKB or a gene product production inhibitor of a gene on which HNF-4α acts can be prepared as a pharmaceutical composition by selecting in consideration of the balance between biological usefulness and toxicity It is. In the preparation of the pharmaceutical composition, these inhibitors can be used alone or in combination.

  The pharmaceutical composition according to the present invention is usually preferably produced using one or more pharmaceutical carriers. Examples of pharmaceutical carriers include diluents and excipients such as fillers, extenders, binders, moisturizers, disintegrants, lubricants and the like that are usually used according to the form of use of the formulation. These are appropriately selected and used depending on the administration form of the resulting preparation. For example, water, pharmaceutically acceptable organic solvent, collagen, polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, sodium alginate, water-soluble dextran, sodium carboxymethyl starch, pectin, xanthan gum, gum arabic, casein, gelatin, agar, glycerin , Propylene glycol, polyethylene glycol, petrolatum, paraffin, stearyl alcohol, stearic acid, human serum albumin, mannitol, sorbitol, lactose and the like. These may be used alone or in combination of two or more according to the dosage form according to the present invention. If necessary, various ingredients that can be used in normal protein preparations, for example, stabilizers, bactericides, buffers, isotonic agents, chelating agents, pH adjusters, surfactants, etc. are used as appropriate. You can also.

  The pharmaceutical composition according to the present invention can be used as an agent for preventing and / or treating a disease caused by phosphorylation of HNF-4α by RSKB. Moreover, it can use for the prevention method and / or treatment method of the said disease. As a specific example, it can be used in a method for preventing and / or treating diabetes, more preferably in a method for preventing and / or treating non-insulin-dependent diabetes.

  The dose range of the pharmaceutical composition is not particularly limited, and the effectiveness of the contained components, the dosage form, the administration route, the type of disease, the nature of the subject (weight, age, medical condition and use of other medicines, etc.), It is appropriately selected according to the judgment of the doctor in charge. In general, an appropriate dose is, for example, in the range of about 0.01 μg to 100 mg, preferably about 0.1 μg to 1 mg, per kg of the subject's body weight. However, dosages can be varied using general routine experimentation for optimization well known in the art. The above dose can be divided into 1 to several times a day, and may be administered intermittently at a rate of once every several days or weeks.

  When administering a pharmaceutical composition according to the present invention, the pharmaceutical composition may be used alone or in combination with other compounds or medicaments required for treatment.

  As the administration route, either systemic administration or local administration can be selected. In this case, an appropriate administration route is selected according to the disease, symptoms and the like. For example, the parenteral route includes normal intravenous administration and intraarterial administration, as well as subcutaneous, intradermal and intramuscular administration. Or oral administration is also possible. Furthermore, transmucosal administration or transdermal administration is also possible.

  As the dosage form, various forms can be selected according to the therapeutic purpose, and representative examples include solid dosage forms such as tablets, pills, powders, powders, fine granules, granules, capsules and the like. , Aqueous preparations, ethanol solution preparations, suspensions, fat emulsions, liposome preparations, inclusions such as cyclodextrins, liquid dosage forms such as syrups and elixirs. Depending on the route of administration, these can be further administered orally, parenterally (instillation, injection), nasal, inhalation, vaginal, suppository, 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 lactose, glucose, sucrose, mannitol, disintegrants such as starch and sodium alginate, lubricants such as magnesium stearate and talc, polyvinyl alcohol, hydroxypropyl It can be produced using a binder such as 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.

  Suspending agents can be produced using sugars such as water, sucrose, sorbitol and fructose, glycols such as polyethylene glycol (PEG) having various molecular weights, and oils.

  Injectable solutions can be prepared using a carrier consisting of a salt solution, a glucose solution, or a mixture of saline and glucose solution.

  Liposomeization involves, for example, adding a solution obtained by dissolving the substance in a solvent (such as ethanol) to a solution obtained by dissolving phospholipid in an organic solvent (such as chloroform), and then distilling off the solvent. Additionally, after shaking, sonication and centrifugation, the supernatant can be filtered and collected.

  For fat emulsification, for example, the substance, oil components (vegetable oil such as soybean oil, sesame oil, olive oil, MCT, etc.), emulsifier (phospholipid, etc.), etc. are mixed and heated to form a solution, and then the required amount of water is added. It can be carried out by emulsification and homogenization using an emulsifier (homogenizer such as a high-pressure jet type or ultrasonic type). It can also be lyophilized. When emulsifying a fat, an emulsification aid may be added. Examples of the emulsification aid include glycerin and saccharides (for example, glucose, sorbitol, fructose, etc.).

  For cyclodextrin inclusion, for example, a solution in which cyclodextrin is heated and dissolved in water or the like is added to a solution in which the substance is dissolved in a solvent (such as ethanol), and then cooled and the deposited precipitate is filtered and sterilized and dried. It can be done by doing. At this time, the cyclodextrin used may be appropriately selected from cyclodextrins (α, β, γ types) having different pore diameters according to the size of the substance.

  Still another embodiment of the present invention is a polynucleotide encoding at least one of RSKB, a polynucleotide encoding RSKB, a vector containing a polynucleotide encoding RSKB, and a polynucleotide encoding HNF-4α and HNF-4α And a reagent kit comprising at least one of the vectors containing the polynucleotide. The reagent kit can be used, for example, in the identification method according to the present invention.

  Moreover, the reagent which consists of the said peptide and the compound obtained by the said identification method, and the reagent kit containing these are also included by the scope of the present invention. Reagent kits are required for performing measurements such as signals and / or markers for detecting phosphorylation of HNF-4α by RSKB, these detection agents, reaction diluents, buffers, washing agents, and reaction stop solutions. Substances may be included. Furthermore, substances such as stabilizers and / or preservatives may be included. In formulating, formulation means corresponding to each substance to be used may be introduced. These reagents and reagent kits are useful, for example, for molecular studies regarding the involvement of HNF-4α and RSKB in diseases caused by phosphorylation of HNF-4α by RSKB, such as diabetes.

  In order to confirm that diabetes can be prevented and / or treated by inhibiting phosphorylation of HNF-4α, the following experiment may be performed. It is known that the expression level of PEPCK in the liver is increased 2.4 times in the ZDF rat, which is a model animal for diabetes (Non-Patent Document 38). In this model animal, phosphorylation inhibition was carried out by first examining the phosphorylation of HNF-4α by activation of RSKB, 1) the amount of phosphorylation of HNF-4α in the liver, and 2) the amount of activation of RSKB in the liver. H-89 (N- [2-((p-bromocinnamyl) amino) ethyl] -5-isoquinolinesulfonamide; N- [2-((p-Bromocinnamyl) amino) ethyl) -5-isoquinolinessulfonamide) (Non-patent Document 34) may be used to determine the blood glucose concentration of the model animal population. The blood glucose concentration is reduced to about half as described above.

  The amount of phosphorylation of 1) HNF-4α in the liver may be determined by immunoprecipitation of HNF-4α and detecting the degree of phosphorylation using an anti-phosphorylated antibody. 2) The amount of activation of RSKB is: An anti-activated RSKB antibody may be prepared and immunoprecipitated, or immunoprecipitated with an anti-RSKB antibody, and in vitro kinase activity may be measured.

Example 1
(In silico search for proteins that interact with RSKB)
A protein that interacts with RSKB was predicted in silico in accordance with the prediction method described in International Publication No. W001 / 67299. That is, the amino acid sequence of RKSB is decomposed into oligopeptides of a certain length, proteins having the amino acid sequence of each oligopeptide or an amino acid sequence homologous to the amino acid sequence are searched in the database, and the obtained protein and RSKB The local alignment was performed, and those with high local alignment scores were predicted to interact with RSKB. Here, the score of local alignment was set to 25.0 or more, similarly to the method described in International Publication No. W001 / 67299.
As a result, oligopeptides [CRYCRL (SEQ ID NO: 11), CRFSRQ (SEQ ID NO: 12) homologous to oligopeptides [CRRCRQ (SEQ ID NO: 10), VSRRILK (SEQ ID NO: 13)] consisting of amino acid residues derived from RSKB, VSIRILD (SEQ ID NO: 14)] was found to be present in the amino acid sequence of HNF-4α. FIG. 1 shows the result of local alignment between RSKB (upper sequence in the figure) and HNF-4α (lower sequence in the figure).

Example 2
(HNF-4α phosphorylation by RSKB)
Whether or not human HNF-4α is phosphorylated by RSKB was examined by an in vitro phosphorylation test using HNF-4α and RSKB protein that were transiently expressed in cultured mammalian cells. The activity of RSKB is confirmed by cyclic AMP response element binding protein 1 [cAMP reactive element binding protein 1 (CREB1)], and cyclic AMP response element-dependent protein known to phosphorylate HNF-4α. Kinase [cAMP-dependent protein kinase (PKA)] was used as a positive control for phosphorylase.

<Material>
An RSKB expression plasmid (RSKB / pCMV-Tag2) was constructed as follows. Human RSKB cDNA was obtained by PCR from HeLa cell-derived cDNA (Human HeLa Quick-clone cDNA, 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 (RSKB / pCMV-Tag2). The amino acid sequence of the cloned RSKB cDNA is the same as the accession number NP_003933 (registered gene name is RPS6KA4) in the NCBI protein database.

  An HNF-4α expression plasmid (HNF-4α / pcDNA 3.1 / His) was constructed as follows. Human HNF-4α cDNA was obtained by RT-PCR from human brain poly A + RNA. Thereafter, an HNF-4α expression plasmid (HNF-4α / pcDNA 3.1 / His) is incorporated into an expression plasmid for animal cells to which a (6 × His) -Xpress tag is added at the N-terminus, pcDNA3.1 / His (Invitrogen). It was constructed. The amino acid translation sequence of the cloned HNF-4α cDNA is the same as the accession number P41235 (registered gene name is HNF4A) in the Swiss-Prot database.

  A human CREB1 [Human cAMP-responsible element binding protein (CREB1)] expression plasmid was constructed as follows. Human CREB1 cDNA was obtained by PCR from a HeLa cell-derived cDNA (Human HeLa Quick-clone cDNA, Clontech), and the sequence was confirmed by sequencing. After that, it was incorporated into an animal cell expression plasmid pcDNA3.1 / His (Invitrogen) to which a (6 × His) -Xpress tag was added at the N-terminus to construct a CREB1 expression plasmid (CREB1 / pcDNA3.1 / His). The amino acid sequence of the cloned CREB1 cDNA is the same as the accession number NP_004370 (registered gene name is CREB1) in the NCBI protein database.

Activation and purification of RSKB were performed as follows. After culturing HEK293T cells having a cell number of 1.2 × 10 ⁶ at 37 ° C. in the presence of 5% CO ₂ overnight (100 mm diameter, 4 plates), 10 μg / plate RSKB expression plasmid (RSKB / pCMV− Tag2) was transfected into the cells using FuGENE6 (Roche Diagnostics). After culturing for 2 days, sodium meta arsenite was added to the medium to a final concentration of 50 mM, and the cells were collected by treatment at 37 ° C. for 30 minutes. Phosphate buffered saline (-) in which the cells are cooled [hereinafter referred to as PBS (-). After washing, Buffer A and (50mM Tris-HCl (pH7.5) / 1mM EDTA / 1mM EGTA / 10% glycerol _{/ 500μM DTT / 5mM NaPPi / 1mM} Na 3 VO 4 / 50mM NaF / 1% Triton- X100) 4ml In] 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, followed by centrifugation at 10,000 g × 10 minutes (4 ° C.). The supernatant was collected and washed with BufferB (50mM Tris-HCl (pH7.5 ) / 1mM EDTA / 1mM EGTA / 10% glycerol _{/ 500μM DTT / 5mM NaPPi / 1mM} Na 3 VO 4 / 50mM NaF / 150mM NaCl) FLAG 100 μl of M2 affinity gel (Sigma) was added and mixed by inverting at 4 ° C. for 1 hour. After mixing, the gel was washed twice with 10 volumes of Buffer B, and 10 volumes of Buffer C (50 mM Tris-HCl (pH 7.5) /0.1 mM EGTA / 10% glycerol / 0.5 μM DTT / 150 mM NaCl). Washed once. The gel was then washed with 400 μl Buffer D (50 mM Tris-HCl (pH 7.5) /0.1 mM EGTA / 10% glycerol / 0.5 μM DTT / 150 mM NaCl / 0.1% NP-40 / 300 μg / ml FLAG peptide). Eluted once. Each eluted fraction was subjected to CBB staining, checked for purity, dialyzed with Buffer C, and stored at −80 ° C. as active RSKB until use. FIG. 1 shows the result of CBB staining of the active RSKB protein.

  As a cAMP-dependent protein kinase (PKA) enzyme, bovine PKA (cAMP-dependent protein kinase, catalytic subunit, Promega) was used.

<Method>
The immunoprecipitation phosphorylation test was performed as follows. HEK293T cells having a cell number of 7 × 10 ⁵ were cultured overnight at 37 ° C. in the presence of 5% CO ₂ (diameter 60 mm petri dish), 5 μg of HNF-4α expression plasmid (HNF-4α / pcDNA3.1 / His) ) Or CREB1 expression plasmid (CREB1 / pcDNA3.1 / His) was transfected into cells using FuGENE6 (Roche Diagnostics). After culturing for 2 days, the cells were washed with chilled PBS (−) and RIPA (50 mM Tris-HCl (pH 8.0) / 150 mM NaCl / 1% NP-40 / 0.5% deoxycholate sodium salt (deoxycholate sodium). salt) /0.1% SDS) to which protease inhibitor cocktail (Sigma) was added, 500 μl 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 lightly vortexed and centrifuged at 14,000 rpm × 10 minutes (4 ° C.), and the supernatant was collected. 20 μl of mouse IgG agarose (Sigma) was added to 480 μl of the supernatant, and mixed by inverting at 4 ° C. for 1 hour (pre-clean). Thereafter, centrifugation was performed at 14,000 rpm × 1 minute (4 ° C.), 1 μl of anti-Xpress antibody (Invitrogen) was added to 450 μl of the collected supernatant, and the mixture was mixed by inverting at 4 ° C. for 1 hour, and then 0.1% BSA / 10 μl of Protein G Sepharose 4 Fast Flow (Amersham Pharmacia Biotech) blocked with TBS (pH 8.0) was added, and the mixture was further mixed by inversion at 4 ° C. for 1 hour. Thereafter, Protein G Sepharose was washed 3 times with RIPA and 2 times with Kinase Buffer (Cell Signaling). 10 μl of washed Protein G Sepharose, 5 μl of active RSKB protein, 3 μl of 10 × kinase buffer (200 mM HEPES (pH 8.0) / 12 mM EDTA / 600 mM KCl / 10 mM DTT / 50 mM MgCl ₂ ), 2 μl of 100 μM ATP 0.5 μl of γ ³² P-ATP (5 μCi) and 8.5 μl of distilled water were added to make a total of 29 μl, and the mixture was reacted at 30 ° C. for 30 minutes. In the test using PKA, 10 μl of HNF-4α / protein G-sepharose prepared as described above was added to 1.5 μl of PKA, 3 μl of 10 × Kinase Buffer, 2 μl of 100 μM ATP, 0.5 μl of γ ³² P. -ATP (5 μCi), 3.5 μl of 10 × kinase buffer, and 8.5 μl of distilled water were added to make a total of 29 μl, followed by reaction at 30 ° C. for 30 minutes. The reaction was stopped by adding 20 μl of 2 × SDS sample buffer and heated at 100 ° C. for 5 minutes to obtain an SDS-PAGE sample. Proteins were separated with 10% gel, sandwiched between hybrid packs, and the presence or absence of phosphorylation was confirmed by image processing with BAS2000 (Fuji Film).

<Result>
As shown in FIG. 3, the phosphorylation of CREB1 protein by purified RSKB was observed, and it was confirmed that the RSKB protein used had activity. As a result of examining the phosphorylation reaction with respect to HNF-4α using this active RSKB protein, RSKB-dependent phosphorylation was observed, and it was revealed that HNF-4α is a substrate of RSKB.

Example 3
(EMSA for AF1 sequence of HNF-4α)
The effect of phosphorylation on the DNA binding ability of HNF-4α to the AF1 sequence was examined by electrophoretic mobility shift assay (EMSA). Moreover, in order to investigate the specificity of RSKB, EMSA was similarly performed also about PKA known to phosphorylate HNF-4α.

<Material>
As shown in FIG. 4, the AF1 probe was synthesized from the AF1 / S / HNF4 (5′-) from the AF1-equivalent sequence that is the binding site of HNF-4α from the promoter region (NCBI accession number U31519) of the human PEPCK gene. Two probes of GTGACCTTTGACTA-3 ′) (SEQ ID NO: 7) and AF1 / AS / HNF4 (5′-ATAGTCAAAGGTCA-3 ′) (SEQ ID NO: 8) were synthesized (Sigma Genosys Japan).

  The HNF-4α expression plasmid (HNF-4α / pCMV-Tag2) is the HNF-4α expression plasmid HNF-4α / pcDNA 3.1 / His constructed previously, and the cDNA sequence of HNF-4α is converted to the N-terminal FLAG. The expression plasmid pCMV-Tag2 (Stratagene) for animal cells with a tag was recombined to construct an expression plasmid HNF-4α / pCMV-Tag2.

Purification of HNF-4α protein was performed as follows. HEK293T cells with 1.2 × 10 ⁶ cells were cultured overnight at 37 ° C. in the presence of 5% CO ₂ (100 mm diameter, 4 plates), 10 μg / Petri dish of HNF-4α expression plasmid (HNF- 4α / pCMV-Tag2) was transfected into the 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) / 1 mM EDTA / 1 mM EGTA / 10% glycerol / 500 μM DTT / 5 mM NaPPi / 1 mM Na3VO4 / 50 mM NaF / 1% Triton-X100) was added and pipetted well, followed by stirring at 4 ° C. for 30 minutes. Thereafter, NaCl was added to a final concentration of 150 mM, followed by centrifugation at 10,000 g × 10 minutes (4 ° C.). The supernatant was collected and washed with Buffer B (50 mM Tris-HCl (pH 7.5) / 1 mM EDTA / 1 mM EGTA / 10% glycerol / 500 μM DTT / 5 mM NaPPi / 1 mM Na3VO4 / 50 mM NaF / 150 mM NaCl) 100 μl FLAG M2 Affinity Gel (Sigma) was added and mixed by inversion at 4 ° C. for 1 hour. After mixing, the gel was washed twice with 10 volumes of Buffer B and once with Buffer C (50 mM Tris-HCl (pH 7.5) /0.1 mM EGTA / 10% glycerol / 0.5 μM DTT / 150 mM NaCl). The gel was then added with 400 μl Buffer D (50 mM Tris-HCl (pH 7.5) /0.1 mM EGTA / 10% glycerol / 0.5 μM DTT / 150 mM NaCl / 0.1% NP-40 / 300 μg / ml FLAG peptide). Eluted once. Each eluted fraction was subjected to SDS-PAGE, dialyzed with Buffer C, and then stained with CBB to confirm the degree of purification. FIG. 5 shows the results of CBB staining of FLAG-HNF-4α protein.

<Method>
Radiolabeling and purification of the probe were performed as follows. The synthesized probes were each dissolved in distilled water so as to be 100 pmol / μl, heated at 100 ° C. for 2 minutes and at 38 ° C. for 1 hour, and then naturally cooled to anneal the DNA. This was diluted 10-fold, 0.5 μl was taken, 1 μl of T4 polynucleotide kinase (TOYOBO), 6 μl of γ ³² P-ATP (60 μCi), 2 μl of 10 × Protruding End Kinase Buffer (500 mM Tris-HCl) _{(pH8.0) / 100mM MgCl 2 /} 100mM 2- mercaptoethanol) and incubated for 1 hour at 37 ° C. was added distilled water 10.5Myueru. Protruding End means “protruding end”, and Protruding End Kinase Buffer is used when T4 polynucleotide kinase adds a phosphate group at the γ-position of γATP to the 5′-end of the protruding end of double-stranded DNA. It is a buffer. Thereafter, 5 μl of 0.25M EDTA, 5 μl of 20 mg / ml yeast RNA was added, and the mixture was made up to 100 μl with distilled water, treated with phenol / chloroform and ethanol precipitated, and 50 μl of STE (100 mM NaCl / 10 mM Tris-HCl (pH 8.0) was added. ) / 1 mM EDTA (pH 8.0)). Thereafter, gel filtration was performed with CENTRI SPIN 20 (Princeton Separations) to obtain a radiolabeled AF1 probe.

Phosphorylation with RSKBP and PKA and EMSA were performed as follows. 6. 25 μl of FLAG-HNF-4α protein, 5 μl of activated RSKB protein diluted stepwise (1 ×, 1/2, 1/4), 2 μl of 10 × kinase buffer (200 mM HEPES (pH 8.0)) / 12 mM EDTA / 600 mM KCl / 10 mM DTT / 50 mM MgCl ₂ ), 4 μl of 1 mM ATP and 2.75 μl of distilled water were added to make a total of 20 μl and reacted at 30 ° C. for 30 minutes. For phosphorylation with PKA, 1.5 μl of PKA was used instead of RSKB protein. After the reaction, 4 μl of the reaction solution was collected, and 2 μl of 10 × binding buffer (100 mM Tris-HCl (pH 7.5) / 1 mM EDTA / 500 mM NaCl / 1 mM DTT / 50% glycerol), 1 μl of 1 mg / ml poly dIdC 11 μl of distilled water was mixed to make a total of 18 μl, and reacted on ice for 10 minutes. At this time, 1 μl of anti-HNF-4α antibody (C-19, Santa Cruz) was added to some samples. Thereafter, 2 μl of radiolabeled AF1 probe was added and reacted at room temperature for 20 minutes. After pre-running a 6% acrylamide gel with 0.25 × TBE [Tris borate buffer (12.5 mM Tris-HCl / 12.1 mM boric acid) containing 0.25 × EDTA] at 4 ° C., 15 μl of each sample After applying, electrophoresis was performed at 4 ° C. for 1 hour and 50 minutes. Thereafter, the gel was dried at 65 ° C. for 1 hour, and the presence or absence of binding of HNF-4α to the probe was confirmed by image processing with BAS2000 (Fuji Film).

<Result>
As shown in FIGS. 6 and 7, HNF-4α phosphorylated with RSKB has an increased DNA binding ability to the AF1 sequence as compared to the untreated sample, and dose dependence of RSKB was also confirmed. . On the other hand, since the sample phosphorylated with PKA showed only the same level of binding as the non-treated sample, the enhancement of the DNA binding ability of HNF-4α by RSKB treatment is specific. It has been shown.

Example 4
(Reporter assay using PEPCK promoter)
The effect of RSKB on the transcriptional activity of HNF-4α at the PEPCK promoter was examined in HeLa cells and HepG2 cells by luciferase reporter assay.

<Material>
As the RSKB expression plasmid, RSKB / pCMV-Tag2 of “phosphorylation of HNF-4α by RSKB” previously performed was used.

  Since the dominant negative RSKB (S343A) expression plasmid (RSKB (S343A) / pCMV-Tag2) loses the kinase activity of RSKB when the 343rd serine is substituted with alanine (Non-patent Document 34), RSKB / pCMV-Tag2 The 343th amino acid substitution was carried out using QuikChange Site-Directed Mutagenesis kit (Stratagene) using as a template.

  As the HNF-4α expression plasmid, HNF-4α / pCMV-Tag2 of “EMSA for AF1 sequence of HNF-4α” previously performed was used.

  Construction of the MAPK11 expression plasmid (MAPK11 / pCMV-Tag4) was performed as follows. Human MAPK11 (p38-beta2) cDNA was obtained by PCR from human brain derived Quick-clone cDNA (Clontech), and the sequence was confirmed by sequencing. Thereafter, the MAPK11 expression plasmid (p38-beta2 / pCMV-Tag4) was constructed by incorporating it into an animal cell expression plasmid pCMV-Tag4 (Stratagene) to which a FLAG tag was added at the C-terminus. The amino acid sequence of the cloned MAPK11 cDNA is identical to Swiss-Prot database accession number Q15759 (registered gene name is MAPK11 / SAPK2B).

  The construction of PEPCK AF1 promoter-dependent luciferase reporter plasmid (PEPCK AF1 promoter / pGL3) was performed as follows. Of the human PEPCK (PCK1) promoter region, a region containing AF1 which is a cis element of HNF-4α (base sequence 882 to 1406 at NCBI database accession number U31519) was obtained by PCR from human genomic DNA (Clontech). The sequence was confirmed by sequencing. This region corresponds to −469 to +63 when the first base position of exon 1 of the PEPCK gene is +1 (Non-patent Document 35). Thereafter, this region was inserted into a reporter plasmid pGL3-Basic (Promega) for expressing firefly luciferase using the reporter gene to construct PEPCK AF1 promoter / pGL3.

  The PEPCK ΔAF1 promoter-dependent luciferase reporter plasmid (PEPCK ΔAF1 promoter / pGL3) was obtained by PCR from the previously cloned human PEPCK promoter region (the base sequence from 934 to 1406 of U31519) by PCR. After confirming the sequence by sequencing, it was constructed by inserting it into pGL3-Basic (Promega).

  PMMGR that expresses rat glucocorticoid was used as a glucocorticoid receptor expression plasmid (Non-patent Document 36).

  A phRL-null plasmid or a pRL-SV40 plasmid (Promega) that expresses Renilla luciferase was used as a reporter gene as a gene transfer efficiency correction plasmid (internal control).

  As the MAPK11 inhibitor, SB203580 was purchased from Promega and used.

<Method>
The reporter assay in HeLa cells was performed as follows. After culturing 6 × 10 ⁴ cells / well of HeLa cells at 37 ° C. in the presence of 5% CO ₂ overnight (24-well plate (2.0 cm ² / well)), 0.25 μg of PEPCK AF1 promoter / PGL3, 0.05-0.2 μg HNF-4α / pCMV-Tag2, 0.25 μg RSKB / pCMV-Tag2, 0.05 μg MAPK11 / pCMV-Tag4 and 0.025 μg internal control phRL-null Cells were transfected with FuGENE6 (Roche Diagnostics) in a preset combination. The total amount of DNA was corrected with an empty vector pCMV-Tag 2 (Stratagene) so that the total amount was constant in each experimental group. After culturing for 2 days, the cells were washed with chilled PBS (−), and firefly luciferase activity and Renilla luciferase activity in the cell lysate were measured with Dual-Lusiferase Reporter Assay kit (Promega). Transcriptional activity was expressed as a multiple of the control group (PEPCK AF1 promoter / pGL3 only) after dividing the firefly luciferase activity value by the Renilla luciferase activity value.

The reporter assay in HepG2 cells was performed as follows. After culturing 1 × 10 ⁴ cells / well of HepG2 cells at 37 ° C. in the presence of 5% CO ₂ overnight (24-well plate (2.0 cm ² / well), 0.5 μg of PEPCK AF1 promoter / pGL3, 0.5 μg HNF-4α / pCMV-Tag2, 0.25 μg and 0.5 μg RSKB / pCMV-Tag2, 0.5 μg MAPK11 / pCMV-Tag4, 0.5 μg pMMGR and 0.05 μg internal Control pRL-SV40 was transfected into cells using FuGENE6 (Roche Diagnostics) in a preset combination, and the empty vector pCMV-Tag2 (so that the total amount of DNA was constant in each experimental group. MAPK11 inhibitor SB203 In the group to which 80 was added, SB203580 was added to the medium to a final concentration of 10 μM immediately before transfection with FuGENE 6. Dexamethasone was added to the medium to a final concentration of 1 μM 20 hours after transfection, and further for 24 hours. After culturing, the cells were washed with chilled PBS (−), and firefly luciferase activity and Renilla luciferase activity in the cell lysate were measured with Dual-Lusiferase Reporter Assay kit (Promega). After dividing the value by the Renilla luciferase activity value, it was expressed as a multiple of the RSKB non-expression group (HNF-4α0.5).

  In statistical analysis, variance was tested by Bartlett's test, and average value was tested by multiple comparison test.

<Result>
As shown in FIG. 8, in HeLa cells, when HNF-4α is expressed with respect to the PEPCK AF1 promoter, an increase in transcriptional activity is observed in a dose-dependent manner, and HNF-4α positively controls the transcriptional activity of the PEPCK gene. It was confirmed. Next, RSKB was co-expressed with HNF-4α, and the effect on the transcriptional activity of HNF-4α was examined. As shown in FIG. 8, no clear difference was observed from the RSKB non-expression group. Since this was considered to be due to insufficient kinase activity of RSKB itself, in order to activate RSKB, MAPK11 (p38-beta2), which is an upstream kinase, was co-expressed with RSKB, and the effect on transcriptional activity was examined. did. As a result, as shown in FIG. 9, the transcriptional activity in the activated RSKB expression group (HNF-4α0.05 + RSKB 0.25 + MAPK11 0.05) was tripled compared to the HNF-4α expression group (HNF-4α0.05). More than that. On the other hand, in the MAPK11 expression group (HNF-4α0.05 + MAPK110.05) excluding RSKB, the promotion of the transcriptional activity of HNF-4α was not observed. Furthermore, as shown in FIG. 10, the dominant negative RSKB (S343A) having no kinase activity did not promote the transcriptional activity of HNF-4α even when co-expressed with MAPK11 (FIG. 10, HNF-4α0). .05 + RSKB (S343A) 0.25 + MAPK11 0.05). In addition, as shown in FIG. 11, the PEPCKΔAF1 promoter excluding AF1 does not show HNF-4α transcriptional activity, and the current transcriptional activity of HNF-4α is performed through binding to the AF1 region. Was confirmed. From the above results, it was demonstrated that HNF-4α phosphorylated by RSKB promotes transcriptional activity through binding of PEPCK promoter to AF1.

  As shown in FIG. 12, when MAPK11 is coexpressed with RSKB (HNF-4α 0.5 + RSKB 0.5 + MAPK11 0.5) in HepG2 cells derived from hepatocytes, the transcriptional activity of HNF-4α against the PEPCK AF1 promoter is It increased 3 times or more than the RSKB non-expression group (HNF-4α0.5). Further, when MAPK11 inhibitor SB203580 was added to inhibit activation of RSKB by MAPK11, the increase in transcriptional activity of HNF-4α disappeared (HNF-4α0.5 + RSKB 0.5 + MAPK11 0.5 + SB203580). From the above results, it was demonstrated that HNF-4α phosphorylated by RSKB also promotes the transcriptional activity of the PEPCK promoter including AF1 in HepG2 cells.

Example 5
Experimental Example 1 Detection of phosphorylation of HNF-4α in liver in diabetes model animals ZDF-fa / fa male rats that develop diabetes and normal ZDF-lean male rats were purchased from Charles River Japan, Inc. at 5 weeks of age and maintained. To do. After blood collection at 6, 8, and 19 weeks of age, the mice are sacrificed under pentobarbital anesthesia, and the livers are collected (each week n = 6). The collected blood is measured for blood glucose concentration and insulin concentration, respectively, and it is confirmed by ZDF-fa / fa that diabetes has developed. The liver was subjected to cell lysis buffer [cell lysis buffer (cell signaling) (20 mM Tris-HCl (pH 7.5), 150 mM NaCl / 1 mM Na ₂ EDTA / 1 mM EGTA / 1% Triton / 2.5 mM Sodium pyrophosphate / 1 mM β-glycerophosphate / 1 mM Na ₃ VO ₄ )], allowed to stand for 30 minutes under ice-cooling, and then centrifuged at 15,000 rpm × 10 minutes to obtain a cell lysate. Anti-HNF-4α antibody (Santa Cruz) was added to the prepared cell lysate and mixed by inverting at 4 ° C. for 1 hour, and then 0.1% bovine serum albumin (BSA) / Tris buffered saline [Tris-Buffered Saline ( Protein G Sepharose 4 Fast Flow (Amersham Pharmacia Biotech) blocked with TBS) (50 mM Tris-HCl / 150 mM NaCl)] (pH 8.0) is added, and the mixture is further mixed by inversion at 4 ° C. for 1 hour. Thereafter, protein G sepharose is washed twice with a cell lysis buffer, 2 × SDS sample buffer is added, and the mixture heated at 100 ° C. for 5 minutes is used as an SDS-PAGE sample. Protein is degraded with 5-20% gel, and phosphorylated HNF-4α is detected by Western blotting using an anti-phosphorylated serine antibody (Transduction Laboratories) and an anti-phosphorylated threonine antibody (Zymed). Diabetes-causing animals (ZDF-fa / fa) increase phosphorylation of HNF-4α in serine and threonine residues of normal animals (ZDF-lean), thereby enhancing phosphorylation of HNF-4α under diabetic conditions. I can prove that.

Experimental Example 2. Detection of Liver RSKB Activation in Diabetes Model Animals Liver cell lysates are prepared from diabetic animals (ZDF-fa / fa) and normal animals (ZDF-lean) in the same manner as in the above experimental example. An anti-RSKB antibody and protein G sepharose are added thereto, and RSKB is recovered by the same immunoprecipitation method as in the above experimental example. The kinase activity of the recovered RSKB protein is detected by in vitro kinase assay using a synthetic substrate clebitide [CREBtide (Santa Cruz)] (SEQ ID NO: 9) (Non-patent Document 34). That is, RSKB-binding resin was added to CREBtide (33 μM) and γ- ³² P- in a kinase buffer [Kinase buffer (50 mM Tris-HCl (pH 7.5) /0.1 mM EGTA / 140 mM KCl / 5 mM NaPPi / 10 mM MgCl ₂ )]. Mix with ATP and incubate at 22 ° C. for 30 minutes. Thereafter, the reaction solution is adsorbed on a chromatography filter paper P81 (Whatman), and after washing, the radioactivity is detected with a liquid scintillation counter. It can be proved that the RSKB kinase activity in the liver is enhanced in diabetic pathology by increasing the RSKB kinase activity in the diabetic animal (ZDF-fa / fa) compared to the normal animal (ZDF-lean).

Experimental Example 3. Anti-diabetic effect of RSKB inhibitor in diabetic model animals ZDF-fa / fa male rats that develop diabetes are purchased from Charles River Japan 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-isoquinolinesulfonamide; N- [ 2-((p-Bromocinnamyl) amino) ethyl] -5-isoquinolinessulfonamide) (Non-patent Document 34) is administered subcutaneously continuously at 5 mg / kg / day for 14 days. After 14 days of administration, the animals are sacrificed under pentobarbital anesthesia and the liver is collected. In addition, blood is collected before and after the administration of H-89, and the blood glucose concentration and insulin concentration (Levis Insulin Kit, Shibayagi Co., Ltd.) are measured. Using the collected liver, phosphorylation of HNF-4α and RSKB activity are measured in the same manner as in Experimental Examples 1 and 2, and the expression level of PEPCK protein is detected by Western blotting. An SDS-PAGE sample is prepared by adding an equal volume of 2 × SDS sample buffer to a liver cell lysate prepared in the same manner as in Experimental Example 1 and heating at 100 ° C. for 5 minutes. Proteins are separated on a 5-20% gel and PEPCK is detected by Western blot using an anti-PEPCK antibody. Administration of H-89 decreases all of RSKB activity, phosphorylation of HNF-4α and PEPCK protein expression, and gluconeogenesis is suppressed, resulting in a decrease in blood glucose concentration.

  Experimental Examples 1 and 2 show that activation of RSKB and accompanying phosphorylation of HNF-4α are enhanced in the liver of diabetic model animals. Experimental Example 3 demonstrates that inhibition of RSKB reduces PEPCK expression and suppresses liver gluconeogenesis. H-89 has an action of inhibiting PKA (cAMP-dependent protein kinase, cAMP-dependent protein kinase) in addition to RSKB. PKA phosphorylates CREB, and phosphorylated CREB 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 phosphorylation is increased rather than decreased, PKA is not activated, and PKA signal is not involved in increased expression of PEPCK in liver under diabetic condition. (Non-Patent Document 39). PKA phosphorylates HNF-4α, but this phosphorylation does not enhance the ability of HNF-4α to bind to the PEPCK promoter (Example 2, FIG. 5). Thus, when 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-4α in the liver is suppressed, the ability of gluconeogenesis via PEPCK is reduced, and an antidiabetic effect can be expected.

  As described above, the inhibition of HNF-4α phosphorylation inhibitor and / or phosphorylation inhibition method by RSKB provided in the present invention inhibits the production of the gene product of the gene on which HNF-4α acts, for example, the gene product of PEPCK gene Production inhibition becomes possible. In addition, it is possible to prevent and / or treat diseases caused by an increase in the gene product of the gene on which HNF-4α acts. Specifically, for example, it is possible to prevent and / or treat diseases caused by an increase in the PEPCK gene product, more specifically diabetes. Thus, the present invention is very useful for prevention and / or treatment of diseases caused by excessive phosphorylation of HNF-4α involved in gluconeogenesis-related gene expression.

The result of having predicted the interaction between RSKB and HNF-4α in silico is shown. Local alignment of RSKB and HNF-4α was performed, and a region showing a high score was displayed. The upper sequence and the lower sequence are the sequence present in RKSB and the sequence present in HNF-4α, respectively. The CBB dyeing | staining image of the active-type FLAG-RSKB protein which was made to express in HEK293T cell transiently with the expression plasmid for N terminal FLAG tag animal cells, and was refine | purified using the FLAG M2 affinity gel is shown. 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 protein. The numerical value described in the left column of the figure is the molecular weight of the molecular weight marker (M). As a result of in vitro immunoprecipitation phosphorylation test of HNF-4α, it is shown that HNF-4α is phosphorylated by RSKB. A shows the result of phosphorylation test with PKA as a positive control, and B shows the result of phosphorylation test with RSKB. Phosphorylation of CREB1 by RSKB indicates that the purified RSKB protein has activity. In the figure, + and-indicate the presence or absence of each protein. The arrowhead indicates HNF-4α, and the star mark (*) indicates a phosphorylated form of CREB1. The numerical value described in the left column of the figure is the molecular weight of the molecular weight marker. The nucleotide sequence of the promoter region of the human phosphoenolpyruvate carboxykinase gene (NCBI accession number U31519) is shown. The AF1 sequence used in the gel shift assay is shown in bold boxes. PEPCK AF1 promoter / pGL3 is the nucleotide sequence 882 to 1406 (bold) delimited by arrows 1 and 3, and PEPCK ΔAF1 promoter / pGL3 is the nucleotide sequence 934 to 1406 delimited by arrows 2 and 3 Were used respectively. The numbers in the left column indicate the base sequence number of U31519. The CBB dyeing | staining image of the FLAG-HNF-4 (alpha) protein which was transiently expressed by HEK293T cell with the expression plasmid for N terminal FLAG tag animal cells, and refine | purified using the FLAG M2 affinity gel is shown. 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-HNF-4α protein. The numerical value described in the right column of the figure is the molecular weight of the molecular weight marker (M). The EMSA result with respect to AF1 sequence of the PEPCK promoter of HNF-4 (alpha) is shown. A shows that HNF-4α binds to the AF1 sequence. The addition of the anti-HNF-4α antibody shows a supershift in which the mobility of the HNF-4α · DNA complex is reduced. B is a drawing showing the influence of phosphorylation by RSKB on the DNA binding activity of the AF1 sequence. The phosphorylation treatment with RSKB enhanced the binding activity of HNF-4α to the AF1 sequence, but PKA did not show its action. The amount of HNF-4α used in B is about one-tenth of A. + And-in the figure indicate the presence or absence of addition of anti-HNF-4α antibody. The phosphorylation of HNF-4α by RSKB shows a dose dependency of the effect of enhancing the binding to the AF1 sequence. As the amount of RSKB used for the phosphorylation of HNF-4α is decreased, the AF1 binding activity of HNF-4α is decreased. On the other hand, in PKA treatment (PKA), enhancement of AF1 binding of HNF-4α is not observed. 1 ×, 1/2, and 1/4 indicate EMSA of phosphorylation data with RSKB diluted 2 times and 4 times, respectively, in the stock solution. A star mark (*) indicates the addition of anti-HNF-4α antibody. The result of the reporter assay using the PEPCK AF1 promoter in HeLa cells is shown. It is a drawing showing that transcriptional activity is enhanced depending on the amount of HNF-4α introduced, and that HNF-4α positively controls the PEPCK gene. It also shows that RSKB expression does not affect the transcriptional activity of HNF-4α. The vertical axis is a multiple when the transcriptional activity of the control group (only PEPCKAF1 promoter / pGL3 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 of each group, and the error bar indicates the standard deviation. The number next to each group of proteins is the amount of introduced DNA (eg, 0.05 μg of HNF-4α expression plasmid was introduced into HNF-4α0.05). *: Significant difference with respect to control (p <0.05), #: Significant difference with respect to HNF-4α0.05 (p <0.05), $: Significant difference with respect to HNF-4α0.1 Yes (p <0.05), +: Significantly different from HNF-4α0.2 (p <0.05), ¥: Significantly different from RSKB0.25 (p <0.05). The result of the reporter assay using the PEPCK AF1 promoter in HeLa cells is shown. It is drawing which shows that transcriptional activity by HNF-4α is promoted by RSKB activated by MAPK11. Even if only MAPK11 is expressed except for RSKB, the transcriptional activity of HNF-4α is not affected, so this action can be judged to be due to RSKB. The vertical axis is a multiple when the transcriptional activity of the control group (only PEPCKAF1 promoter / pGL3 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 of each group, and the error bar indicates the standard deviation. The number next to each group of proteins is the amount of introduced DNA (eg, 0.05 μg of HNF-4α expression plasmid was introduced into HNF-4α0.05). *: Significantly different from control (p <0.05), #: Significantly different from HNF-4α0.05 (p <0.05), $: RSKB 0.25 + MAPK11 0.05 Significantly different (p <0.05), +: Significantly different from HNF-4α 0.05 + RSKB 0.25 + MAPK11 0.05 (p <0.05). The result of the reporter assay using the PEPCK AF1 promoter in HeLa cells is shown. It is a figure which shows that the transcriptional activity by HNF-4 (alpha) is promoted by RSKB. Dominant negative RSKB (S343A) does not promote HNF-4α transcriptional activity even when MAPK11 is co-expressed. The vertical axis is a multiple when the transcriptional activity of the control group (only PEPCKAF1 promoter / pGL3 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 of each group, and the error bar indicates the standard deviation. The number next to each group of proteins is the amount of introduced DNA (eg, 0.05 μg of HNF-4α expression plasmid was introduced into HNF-4α0.05). *: Significantly different from control (p <0.05), #: Significantly different from HNF-4α0.05 (p <0.05), $: HNF-4α0.05 + RSKB0.25 + MAPK110.05 There is a significant difference (p <0.05). The result of the reporter assay using the PEPCK ΔAF1 promoter in HeLa cells is shown. It is a figure which shows that the transcriptional activity by HNF-4 (alpha) is dependent on AF1 of a PEPCK promoter region. Since there is no AF1, even if HNF-4α is co-expressed, the transcriptional activity is not promoted. The vertical axis is a multiple when the transcriptional activity of the control group (only PEPCKΔAF1 promoter / pGL3 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 of each group, and the error bar indicates the standard deviation. The number next to each group of proteins is the amount of introduced DNA (eg, 0.05 μg of HNF-4α expression plasmid was introduced into HNF-4α0.05). *: Significant difference with respect to control (p <0.05), #: Significant difference with respect to HNF-4α0.05 (p <0.05), $: Significant difference with respect to HNF-4α0.2 Yes (p <0.05). The result of the reporter assay using the PEPCK AF1 promoter in HepG2 cells is shown. It is a figure which shows that the transcriptional activity by HNF-4 (alpha) is accelerated | stimulated by RSKB activated by MAPK11. When the MAPK11 inhibitor SB203580 was added, the transcriptional activity of HNF-4α was not promoted even when MAPK11 and RSKB were co-expressed, indicating that activation of RSKB was important. The vertical axis is a multiple when the transcriptional activity of the HNF-4α0.5 treatment group is 1.00. The height of the graph and the value at the top of the graph indicate the average value of n = 3 of 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-4α0.5 was introduced with 0.5 μg of HNF-4α expression plasmid). *: Significantly different from HNF-4α0.5 (p <0.05), #: Significantly different from HNF-4α0.5 + RSKB0.25 (p <0.05), $: HNF-4α0. Significantly different from 5 + RSKB0.5 (p <0.05), +: Significantly different from RSKB0.5 (p <0.05), ¥: HNF-4α0.5 + RSKB0.5 + MAPK110.5 Significantly different (p <0.05), ++: Significantly different from HNF-4α0.5 + RSKB0.5 + MAPK110.5 + SB203580 (p <0.05).

SEQ ID NO: 1 Base sequence of RSKB cDNA.
SEQ ID NO: 2: Amino acid sequence of RSKB protein.
SEQ ID NO: 3: Base sequence of HNF-4α cDNA.
SEQ ID NO: 4: Amino acid sequence of HNF-4α protein.
SEQ ID NO: 5: amino acid sequence of CREB1 cDNA.
SEQ ID NO: 6: amino acid sequence of CREB1 protein.
Sequence number 7: The base sequence of oligonucleotide for AF1 probe preparation.
SEQ ID NO: 8: Base sequence of oligonucleotide for preparing AF1 probe.
SEQ ID NO: 9: amino acid sequence of CREBtide.
SEQ ID NO: 10: Partial RSKB oligopeptide showing a high score in local alignment between RSKB and HNF-4α.
SEQ ID NO: 11: Partial oligopeptide of HNF-4α showing high score in local alignment between RSKB and HNF-4α.
SEQ ID NO: 12: Partial oligopeptide of HNF-4α showing high score in local alignment between RSKB and HNF-4α.
SEQ ID NO: 13: Partial RSKB oligopeptide showing a high score in the local alignment between RSKB and HNF-4α.
SEQ ID NO: 14: Partial oligopeptide of HNF-4α showing a high score in the local alignment between RSKB and HNF-4α.

Claims (28)

A method of phosphorylating HNF-4α by RSKB, characterized in that ribosome S6 kinase B (RSKB) and hepatocyte nuclear factor 4α (HNF-4α) coexist under conditions that allow interaction. A method for inhibiting phosphorylation of hepatocyte nuclear factor 4α (HNF-4α) by RKSB, which comprises inhibiting the activity of ribosomal S6 kinase B (RSKB). A method for inhibiting phosphorylation of HNF-4α by RSKB, which comprises inhibiting the interaction between ribosomal S6 kinase B (RSKB) and hepatocyte nuclear factor 4α (HNF-4α). A cell expressing at least ribosomal S6 kinase B (RSKB) and hepatocyte nuclear factor 4α (HNF-4α) is treated with an inhibitor of RSKB activity. Oxidation inhibition method. The inhibitor of ribosomal S6 kinase B (RSKB) activity is one or more antibodies selected from an antibody recognizing RSKB and an antibody recognizing hepatocyte nuclear factor 4α (HNF-4α). 5. A method for inhibiting phosphorylation of HNF-4α by RSKB according to 4. An inhibitor of phosphorylation of hepatocyte nuclear factor 4α (HNF-4α) by RSKB, comprising an effective amount of an inhibitor of ribosomal S6 kinase B (RSKB) activity. An inhibitor of phosphorylation of HNF-4α by RSKB, which inhibits the interaction between ribosomal S6 kinase B (RSKB) and hepatocyte nuclear factor 4α (HNF-4α). An inhibitor of phosphorylation of hepatocyte nuclear factor 4α (HNF-4α) by RSKB, characterized by inhibiting ribosomal S6 kinase B (RSKB) activity. The inhibitor of ribosomal S6 kinase B (RSKB) activity is one or more antibodies selected from an antibody recognizing RSKB and an antibody recognizing hepatocyte nuclear factor 4α (HNF-4α). The phosphorylation inhibitor of HNF-4α by RSKB according to 8. A method for promoting the production of a gene product of a gluconeogenesis-related gene, which comprises phosphorylating hepatocyte nuclear factor 4α using ribosomal S6 kinase B. The method of promoting gene product production of a gluconeogenesis-related gene according to claim 10, wherein the gluconeogenesis-related gene is a phosphoenolpyruvate carboxykinase gene. A method for inhibiting gene product production of a gluconeogenesis-related gene, which comprises inhibiting phosphorylation of hepatocyte nuclear factor 4α by ribosomal S6 kinase B. A method for inhibiting gene product production of a phosphoenolpyruvate carboxykinase gene, which comprises inhibiting phosphorylation of hepatocyte nuclear factor 4α by ribosomal S6 kinase B. A method for inhibiting gene product production of a gluconeogenesis-related gene, comprising using the phosphorylation inhibition method according to any one of claims 2 to 5. A method for inhibiting the production of a gene product of a gene in which hepatocyte nuclear factor 4α acts as a transcription factor, wherein the method for inhibiting phosphorylation according to any one of claims 2 to 5 is used. 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. A method for preventing a disease caused by phosphorylation of hepatocyte nuclear factor 4α by ribosomal S6 kinase B and / or using the phosphorylation inhibition method according to any one of claims 2 to 5. Method of treatment. A method for preventing and / or treating a disease caused by an increase in a gene product of a gluconeogenesis-related gene, wherein the method for inhibiting phosphorylation according to any one of claims 2 to 5 is used. A method for preventing and / or treating a disease caused by an increase in 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. A method for preventing and / or treating diabetes, comprising using the method for inhibiting phosphorylation of hepatocyte nuclear factor 4α by ribosome S6 kinase B according to any one of claims 2 to 5. A method for preventing diabetes, comprising using the phosphorylation inhibitor of hepatocyte nuclear factor 4α and / or the inhibitor of RSKB activity by ribosome S6 kinase B according to any one of claims 6 to 9. And / or treatment methods. A phosphoenolpyruvate carboxy comprising an effective amount of a phosphorylation inhibitor of hepatocyte nuclear factor 4α and / or an inhibitor of RSKB activity by ribosomal S6 kinase B according to any one of claims 6 to 9. Production inhibitor of gene product of kinase gene. A pharmaceutical composition comprising an effective amount of an inhibitor of phosphorylation of hepatocyte nuclear factor 4α and / or an inhibitor of RSKB activity by ribosomal S6 kinase B according to any one of claims 6 to 9. A phosphoenolpyruvate carboxy comprising an effective amount of a phosphorylation inhibitor of hepatocyte nuclear factor 4α and / or an inhibitor of RSKB activity by ribosomal S6 kinase B according to any one of claims 6 to 9. An agent for preventing and / or treating a disease caused by an increase in a gene product of a kinase gene. A diabetes prevention agent comprising an effective amount of a phosphorylation inhibitor of hepatocyte nuclear factor 4α and / or an inhibitor of RSKB activity by ribosomal S6 kinase B according to any one of claims 6 to 9, and / Or therapeutic agent. A method for identifying a compound that inhibits the interaction between ribosomal S6 kinase B (RSKB) and hepatocyte nuclear factor 4α (HNF-4α), under conditions that allow the interaction between RSKB and HNF-4α And / or a system using a signal and / or a marker that contacts HNF-4α with a test compound and detects the interaction between RSKB and HNF-4α, and the presence or absence of this signal and / or marker and / or Or the identification method which determines whether a test compound inhibits the interaction of RSKB and HNF-4 (alpha) by detecting a change. A method for identifying a compound that inhibits phosphorylation of hepatocyte nuclear factor 4α (HNF-4α) by ribosomal S6 kinase B (RSKB), under conditions that allow phosphorylation of HNF-4α by RSKB And / or a system using a signal and / or a marker that contacts HNF-4α with a test compound and detects phosphorylation of HNF-4α by RSKB, and this signal and / or the presence or absence of the marker and / or Alternatively, an identification method for determining whether or not a test compound inhibits phosphorylation of HNF-4α by RSKB by detecting a change. At least one of ribosomal S6 kinase B (RSKB), a polynucleotide encoding RSKB, and a vector containing a polynucleotide encoding RSKB, hepatocyte nuclear factor 4α (HNF-4α), and HNF-4α. A kit comprising at least one of a polynucleotide encoding and a vector containing the polynucleotide encoding HNF-4α.