WO1999050395A1

WO1999050395A1 - Serine-threonine protein kinase expressed in kidney

Info

Publication number: WO1999050395A1
Application number: PCT/JP1999/001575
Authority: WO
Inventors: Masaaki Muramatsu; Hiroshi Tokumitsu; Teruhisa Noguchi
Original assignee: Helix Research Institute
Priority date: 1998-03-27
Filing date: 1999-03-26
Publication date: 1999-10-07

Abstract

A novel rat gene encoding a serine-threonine protein kinase protein and being expressed specifically in the kidney which is successfully isolated by effecting a polymerase chain reaction by using synthetic DNAs corresponding to the kinase conserved region as primers and a rat brain-derived cDNA as a template and screening a rat kidney cDNA library by using the thus obtained DNA fragment as a probe. Moreover, primers are designed on the basis of the base sequence of the above rat gene and employed in screening a mouse testis cDNA library. Thus, a mouse gene seemingly belonging to the same family as the above rat gene is successfully isolated. It is also found out that use of the proteins encoded by these genes makes it possible to screen candidates for drugs regulating the activities of these proteins.

Description

Description ^: Serine / Threonine protein kinase expressed in kidney

The present invention relates to a serine / threonine protein kinase that shows strong expression in the kidney. North

Mass technology

Protein kinases are enzymes that phosphorylate serine, threonine, or tyrosine residues of the protein protein, and a large number of families are known. In general, protein kinases are involved in the control of various biological phenomena by regulating the intracellular signal transduction system via protein phosphorylation (Hunter, T. A thousand and one protein kinases. Cell 50). , 823-9 (1987)). Therefore, the inability of protein kinases to function properly in vivo is thought to cause various diseases.

For example, protein kinases are myotonic atrophy (DM kinase) (Brook JD, McCurrach ME, Harley HG, Buckler AJ, Church D, Aburatani H, Hunter K, Stanton VP, Thirion JP, Hudson T, et al olecular basis of myo tonic dystrophy: expansion of a trinucleotide (CTG) repeat at the 3, end of a transcript encoding a protein kinase family member.Cell 6 8: 799-808 (1992), glycogen storage disease (Phosphorylase kinase) (Hug G , Sc hubert WK, Chuck G. Phosphorylase kinase of the liver: deficiency in a girl with increased hepatic glycogen. Science 153: 1534-1535 (1966)), multiple endocrine neoplasia type II and Hirschschbung disease (Ret kinase) (Mulligan LM, Kwok JB, Healey CS, Elsdon MJ, Eng C, Gardner E, Love DR, Mole SE, Moore JK, Papi L, et al. Germ-line mutations of the R ET proto-oncogene in multiple endocrine neoplasia type 2A.Nature 36 3: 458-460 (1993), Edery P, Lyonnet S, Mulligan LM, Pelet A, Dow E, Ab el L, Holder S, Nihoul-Fekete C, Ponder BA, Munnich A. Mutations of the RET proto-oncogene in Hirschsprung's disease.Nature 367: 378-380 (1994)), chronic bone marrow Leukemia (abl kinase) (Walker LC, Ganesan TS, Dhut

S, Gibbons B, Lister TA, Rothbard J, Young BD. Novel chimaeric protein expressed in Philadelphia positive acute lymphoblastic leukaemia • Nature 329: 851-853 (1987)), Bulton aglobulinemia (btk kinase)

(Raw lings DJ, Saffran DC, Tsukada S, Largaespada DA, Grimaldi JC, Choen L, Mohr RN, Bazan JF, Howard M, Copeland NG, et al Mutation of unique region of Bruton's tyrosine kinase in immunodef icient XID mi ce. Science 261: 358-361 (1993), Thomas JD, Sideras P, Smith CI, Vorec hovsky I, Chapman V, Paul WE. Colocalization of X-linked agammaglobu linemia and X-linked immunodeficiency genes. Science 261: 355- 358 (1993)), severe combined immunodeficiency (JAK3 kinase) (Macchi P, Villa A, Gillani S,

Sacco MG, Frattini A, Porta F, Ugazio AG, Johnston JA, Candotti F,

0 'Shea JJ, et al. Mutations of Jak-3 gene in patients with autosoma 1 severe combined immune deficiency (SCID) Nature 377: 65-68 (1995)), colorectal cancer (TGF-beta receptor Yuichi II) ( Markowitz S, Wang J, Myeroff L, Parsons R, Sun L, Lutterbaugh J, Fan RS, Zborowska E, Kinzler KW, Voge lstein B, et al.Inactivation of the type II TGF-beta receptor in co Ion cancer cells with microsatellite instability. Science 268: 1336-1 338 (1995), Peutz-Jeghers syndrome (LKBl) Hemminki A, Markie D, Tomlinson I, Avizienyte E, Roth S, Loukola A, et al. A serine / threonine kinase gene defective in Peutz-Jeghers syndrome.Nature 391: 184-187 (1998)) It is known to be related to human diseases such as.

By the way, among the organs in the human body, the kidney is an organ that controls the removal of waste products in the blood and homeostasis of body fluids, and due to abnormal function, kidney disease such as acute and chronic renal failure, various hereditary renal diseases, renal tumors, etc. It is known to cause Various protein kinases also play an important role in such renal functions, and it is possible that abnormalities in signal transduction are involved in the above diseases. However, little is known about serine / threonine kinase related to renal function. Disclosure of the invention

An object of the present invention is to provide a serine / threonine kinase gene exhibiting strong expression in the kidney, a similar gene thereof, and a protein encoded by these genes. Another object of the present invention is to provide a molecule for performing such amplification, detection, production, and the like. Another object of the present invention is to provide a method for screening a drug candidate compound using the serine / threonine kinase.

The present inventors have conducted intensive studies to solve the above-mentioned problems. As a result, the polymerase chain reaction (PCR) was performed using a synthetic DNA corresponding to the kinase-conserved region as a primer and a rat brain-derived cDNA as type II. By screening the rat kidney cDNA library using the obtained DNA fragment as a probe, a novel rat gene that encodes a serine / threonine protein kinase protein and is specifically expressed in the kidney can be obtained. The isolation was successful. In addition, the present inventors designed a primer based on the nucleotide sequence of the obtained rat gene, and screened a mouse testis cDNA library using the primer. We succeeded in isolating mouse genes that are considered to belong to the family. Furthermore, the present inventors have decided to utilize the proteins encoded by these genes. It has been found that it is possible to screen drug candidate compounds that control the activity of the protein.

The present invention relates to a serine / threonine protein kinase gene that is specifically expressed in the kidney, a gene similar thereto, a protein encoded by these genes, a molecule used for amplification, detection, production, and the like thereof, and the serine / threonine protein kinase. Regarding a method for screening drug candidate compounds using threonine protein kinase, more specifically,

(1) a protein consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence in the protein in which one or more amino acids are substituted, deleted, or added, and the serine / threonine protein is A protein having an kinase activity,

(2) a protein encoded by DM, which hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1, which has a serine / threonine protein kinase activity;

(3) a protein consisting of the amino acid sequence of SEQ ID NO: 4, or having an amino acid sequence in which one or more amino acids have been substituted, deleted, or added in the amino acid sequence in the protein, and comprising a serine / threonine protein; A protein having an kinase activity,

(4) SEQ ID NO: a protein encoded by a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 3, which has a serine / threonine protein kinase activity;

(5) DNA encoding the protein of (1) or (3),

(6) a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 1, which encodes a protein having serine / threonine protein kinase activity,

(7) SEQ ID NO: DN hybridizing with DNA consisting of the nucleotide sequence of 3 A, which encodes a protein having serine / threonine protein kinase activity,

(8) a vector comprising the DNA according to any one of (5) to (7),

(9) a transformant that retains the DNA of any one of (5) to (7) in an expressible manner;

(10) The method for producing a protein according to any one of (1) to (4), comprising a step of culturing the transformant according to (9),

(11) a DNA which specifically hybridizes with the DNA according to any one of (5) to (7) and has a chain length of at least 15 nucleotides,

(12) Antisense DNA to the DNA according to any of (5) to (7) or a part thereof

(13) A method for screening a compound that binds to protein according to any one of (1) to (4),

(a) contacting the test sample with the protein according to any one of (1) to (4),

(b) a step of selecting a compound that binds to the protein according to any of (1) to (4),

Including the method,

(14) A method for screening a compound that regulates the serine / sleonine protein kinase activity of the protein according to any one of (1) to (4),

(b) a step of detecting serine / threonine protein kinase activity of the protein according to any of (1) to (4),

(c) Serine / threonine in evening protein according to any one of (1) to (4) Selecting a compound that promotes or inhibits protein kinase activity.

(15) a compound that binds to the protein according to any one of (1) to (4), which is isolated by the method according to (13);

(16) a compound that regulates the serine / threonine protein kinase activity of the protein according to any one of (1) to (4), which can be isolated by the method according to (14);

(17) The compound according to (15) or (16), which is derived from nature,

(18) An antibody that binds to protein according to any one of (1) to (4).

The present invention relates to a protein kinase protein that shows strong expression in the kidney. The rat-derived protein “rat PK80” (SEQ ID NO: 2) included in the protein of the present invention can be obtained by screening rat kidney cDNA using the nucleotide sequence of the conserved region of protein kinase. It is a protein encoded by the obtained gene. The present inventors have results of studies on Protein kinase activity of the "rack Bok PK80", "rat ΡΚ80" actually, ^{3 2} .rho. § - Arufatauro, and autophosphorylation in the presence Maguneshiyuu Ion peptide The activity of incorporation of phosphorus into substrate substrate (Sigma) was demonstrated (Example 4). In addition, Northern blot analysis revealed that the “rat PK80” gene was particularly strongly expressed in the kidney (see Example 2, FIG. 1). These facts suggest that "rat-80" is a protein kinase that is particularly involved in kidney function. The mouse-derived protein “mouse ΡΚ80” (SEQ ID NO: 4) included in the protein of the present invention is obtained by screening mouse testis cDNA using the nucleotide sequence of the “rat ΡΚ80” gene. The protein encoded by the obtained gene. Comparison of the amino acid sequences of “mouse PK80” and “rat # 80” revealed that both proteins had a high homology of about 80% in the amino acid sequence. This fact, "Mouse Ρ The K80j gene and the “rat PK80” gene belong to the same family.

, Suggesting that they have similar functions. Protein kinases are involved in the regulation of various biological phenomena by regulating intracellular signal transduction through phosphorylation of proteins (Hunter, T. A thousand and one protein kinases. Cell 50, 823-9 (1987)), it is thought that various diseases are caused by dysfunction of protein kinase in vivo. Therefore, the protein of the present invention and the compound that regulates the activity of the protein may be used for preventing or treating diseases related to the protein of the present invention (for example, kidney disease) as described later.

The protein of the present invention can be prepared as a recombinant protein or as a natural protein. The recombinant protein is prepared, for example, by introducing a vector into which a DNA encoding the protein kinase protein of the present invention has been introduced into an appropriate host cell and purifying the protein expressed in the transformant, as described later. It is possible to On the other hand, a natural protein can be prepared, for example, by an affinity column using an antibody (Cur retnt Protocols in Molecular Biology edit. Ausubel et al. (1987) Publich. Jhon Wily & Sons Section 16.1 -16.19). Also, in vitro translation (for example, see “0n the fidelity of mRNA translation in the nucl ease-treated rabbit retculocyte lysate system. Dasso, MC, Jackson, RJ. (1989) NAR 17: 3129-3144)” It is also possible to use the method. The antibody used for affinity purification may be a polyclonal antibody or a monoclonal antibody. The polyclonal antibody can be prepared, for example, by synthesizing an oligopeptide corresponding to the partial amino acid sequence of the protein of the present invention and immunizing rabbits (Curretnt Protocols in Molecular Biology). edit. Ausubel et al. (1987) Publich. Jhon Wily & Sons Section

11.4-11.11). On the other hand, monoclonal antibodies, for example, It can be obtained from hybridoma cells obtained by immunizing mice with the recombinant protein and fusing spleen cells and myeloma cells. (Curretnt Protocols in Molecular Biology edit. Ausubel et al. (1987) Publich. Jhon Wily & Sons (Sectionll.4-ll.ll) o

Antibodies that bind to the protein of the present invention may be used for purposes other than purification of the protein of the present invention, for example, for treatment of diseases associated with the protein of the present invention. When an antibody is used for the purpose of treating a patient, a human antibody or a humanized antibody is preferred because of its low immunogenicity. Human antibodies can be obtained from mice in which the immune system has been replaced by humans (e.g., `` Functional transplant oi megabase human immunoglobulin loci recapitulates human antibody response in mice, Mendez, MJ et al. (1997) Nat.Genet. 15: 146-156 ”). Furthermore, a humanized antibody can be prepared by genetic recombination using the hypervariable region of a monoclonal antibody (Methods in Enzymology 203, 99-121 (1991)).

The present invention also relates to protein functionally equivalent to “rat PK80” or “mouse # 80”. Those skilled in the art can use known methods (for example, site-directed mutagenesis method (Curretnt Protocols in Molecular Biology edit.Ausubel et al. (1 987) Publich.Jhon Wily & Sons Section 8.1-8.5)). Amino acids of “Rat PK80” (SEQ ID NO: 2) or “Mouse # 80” (SEQ ID NO: 4) are modified to prepare modified proteins that are functionally equivalent to these proteins. Can be. Amino acid mutations in proteins can also occur in nature. Thus, a protein in which one or more amino acids have been mutated by substitution, deletion or addition in the amino acid sequence of “rat ΡΚ80” (SEQ ID NO: 2) or “mouse ΡΚ80” (SEQ ID NO: 4). Thus, proteins functionally equivalent to these proteins are also included in the protein of the present invention. Here, “functionally equivalent” means that the protein is “rat ΡΚ80” or “mouse ΡΚ80”. "And has serine / threonine kinase activity. The term “serine / threonine protein kinase activity” refers to a biochemical activity that phosphorylates a substrate protein and a cell biological activity induced by this activity. The biochemical activity of serine / threonine protein kinase is detected as the activity of autophosphorylation and phosphorylation of myelin basic proteins, histones, synthases, and proteins in the presence of magnesium ion and ATP. (Protein kinases and phosphatases by DG Hardie, translated by Hiroyoshi Hidaka, Medical Science International, plOl-pl20), while the biological activity is the DNA encoding serine / threonine protein kinase. or variants thereof by expressing in a cell a Rukoto force ^s friendly affirmative έ be detected as a cellular changes (Muramatsu, et al prote in Kinase and Signal tr ansduct ion:. studies with mutant prote in kinases (1993 ) pl85-192, The mechanism and new approach on drug resistance of cancer cel ls.The Elsevier Science Publ ishers) o The amino acid mutation that retains serine / threonine kinase activity is usually 50 amino acids relative to the amino acid sequence of “rat PK80” (SEQ ID NO: 2) or “mouseΡΚ80” (SEQ ID NO: 4). Within 30 amino acids, preferably within 30 amino acids, more preferably within 10 amino acids, and still more preferably within 3 amino acids. It is functionally preferable that the amino acid mutation region is other than the kinase-conserved region of “Rat-80” or “mouse-80”, but as long as it retains serine / threonine kinase activity, it may be in such region. Good.

Other methods well known to those skilled in the art for isolating proteins functionally equivalent to "rat # 80" or "mouse # 80" include a method utilizing hybridization technology. That is, those skilled in the art can use the hybridization technology (Curretnt Protocols in Molecular Biology edit. Ausubel et al. (1987) Publich. Jhon Wily & Sons Section 6.3-6.4) to DNA having high homology to the DNA sequence (SEQ ID NO: 1 or SEQ ID NO: 3) or a part thereof encoding the ヅ PK80 ”or“ mouse ΡΚ80 ”is isolated from these DNAs. Obtaining a protein that is functionally equivalent to that is usually what can be done. Thus, a protein encoded by a DNA that hybridizes with a DNA encoding “rat PK80” or “mouse 、 80” and a protein functionally equivalent to these proteins is also a protein of the present invention. include . Here, “functionally equivalent” means that the protein has a serine / threonine kinase activity, similar to “rat-80” or “mouse-80”, as described above. Other organisms that isolate functionally equivalent proteins include, but are not limited to, for example, human, egret, monkey, monkey, bush. The stringency of hybridization for isolating DNA encoding a functionally equivalent protein is usually about `` lxSSC, 0.1% SDS, 37 ° C '', preferably `` 0.5xSSC, 0. 1% SDS, about 42 ° C ”, more preferably about“ 0.2xSSC, 0.1 0SDS, 65 ° C ”. However, the combination of the above SSC, SDS and temperature conditions is merely an example, and those skilled in the art will be able to achieve the same as above by appropriately combining the above or other factors that determine the stringency of the hybridization. It is possible to realize the stringency of this. Proteins isolated by such hybridization techniques usually have high homology in amino acid sequence to "rat PK80" or "mouse # 80". High homology refers to sequence identity of at least 60% or more, preferably 80% or more, and more preferably 95% or more. Identification of homology can be determined using, for example, a search algorithm such as BLAST, FAST A, and Smith-Waterman. The protein of the present invention can be used for screening candidate therapeutic compounds for diseases associated with the protein of the present invention (for example, renal disease). One embodiment of such compound screening includes: (a) a step of bringing a test sample into contact with the protein of the present invention; and (b) selecting a compound that binds to the protein. Process. Examples of such a method include, for example, a method of applying a kidney cell extract or the like to an affinity column of the protein kinase of the present invention to purify the protein kinase, a method using a two-hybrid system, a West Western Proteinting method, Many known methods such as a method using high-throughput screening in a combinatorial chemistry technology can be used. The test sample used for screening is not particularly limited, and examples thereof include a cell extract, an expression product of a gene library, a synthetic low-molecular compound, a synthetic peptide, and a natural compound. The compound isolated in this way is a candidate for a compound (agonist, angiogonist) that promotes or inhibits the activity of the protein of the present invention. In addition, it is a candidate for a compound that inhibits the interaction between the protein of the present invention and another protein that binds thereto in vivo. These compounds are considered to be applicable as pharmaceuticals for the prevention and treatment of diseases associated with the protein of the present invention (eg, kidney disease). Another embodiment of the screening method of the present invention is a method of directly screening for a compound that modulates the activity of the protein of the present invention, comprising: (a) contacting a test sample with a protein of the present invention; (B) detecting the serine / threonine protein kinase activity of the protein of the present invention, and (c) selecting a compound that promotes or inhibits the serine / threonine protein kinase activity of the protein of the present invention. . Test samples used for screening include, but are not limited to, cell extracts, expression products of gene libraries, synthetic low-molecular compounds, synthetic peptides, natural compounds, and the like, as in the screening method described above. Further, a compound that binds to the protein of the present invention isolated by the above screening method can also be used as a test sample. The activity of serine / threonine protein kinase in this screening method may be, for example, as described above, in the presence of magnesium ion and ATP, autophosphorylation and myelin basic protein, histone, synthamide, tautampa Can be detected as phosphorylation activity of proteins (protein protein Naze and Phosphatase DG Hardie, translated by Hiroyoshi Hidaka, Medical Science International, plOl-pl20).

When the compound isolated by the screening method of the present invention is used as a pharmaceutical, in addition to directly administering the isolated compound itself to a patient, it is formulated and administered by a known pharmaceutical method. It is also possible. For example, it is conceivable to administer the composition by appropriately combining it with a pharmacologically acceptable carrier or vehicle, specifically, sterile water, physiological saline, vegetable oil, emulsifier, suspension or the like. Administration to a patient can be performed by methods known to those skilled in the art, such as intraarterial injection, intravenous injection, and subcutaneous injection. The dose varies depending on the weight and age of the patient, the administration method, and the like, but those skilled in the art can appropriately select an appropriate dose.

The present invention also relates to a DNA encoding the protein of the present invention. The form of the DNA of the present invention is not particularly limited as long as it can encode the protein of the present invention, and includes genomic DNA, chemically synthesized DNA, etc. in addition to cDNA. The DNA of the present invention can be obtained, for example, by a hybridization method using a DNA sequence encoding “rat PK80” or “mouse # 80” (SEQ ID NO: 1, SEQ ID NO: 3) or a part thereof as a probe. It can be isolated by a conventional method such as a PCR method using a primer synthesized based on the primer.

The DNA of the present invention can be used to prepare the protein of the present invention as recombinant protein. That is, the recombinant protein can be prepared by incorporating the DNA of the present invention into an appropriate expression vector, introducing the DNA into an appropriate host cell, and purifying the protein expressed in the transformant. The expression vector is not particularly limited as long as it can express the protein in a test tube, in Escherichia coli, in a cultured cell, in an organism, and the like.For example, in the case of expression in a test tube, a pBEST vector (Promega), pET vector for Escherichia coli (Invitrogen), SRalpha vector for cultured cells or living organisms-(Mol. Cell. Biol. 8: 466-472 (1988)) Is preferred. Vector book The DNA insertion of the invention can be carried out in a conventional manner, for example, by a ligase reaction using a restriction enzyme site (Curretnt Protocols m Molecular Biology edit. Au subel et al. (1987) Publich. Jhon Wily & Sons Section 11.4-11. 11). The host cell into which the vector is introduced is not particularly limited, and includes, for example, cells capable of highly expressing a protein represented by COS cells, CH0 cells, and the like. The introduction of the vector into the host can be carried out, for example, by the calcium phosphate precipitation method or the electro-nores excision method (Curretnt Protocols in Molecular Biology edit. Ausubel et al. (1987) Publich. Jhon Wily & Sons Section 9.1) -9. 9), Ribofect Yumin method (manufactured by GIBCO-BRL) This method can be performed by a method such as microinjection method.

The DNA of the present invention or its antisense MA is also considered to be applicable to gene therapy for diseases (particularly diseases related to renal function) caused by abnormalities (abnormal function or abnormal expression) of the protein of the present invention. When used for gene therapy, the DNA or antisense of the present invention may be used, for example, using a viral vector such as a retrovirus vector, an adenovirus vector, an adeno-associated virus vector, or a non-viral vector such as a ribosome. DNA is administered to patients by ex vivo or in vivo methods. The antisense DNA has a chain length of at least 15 bp or more, preferably 100 bp, more preferably 500 bp or more in order to cause an antisense effect, and usually has a strand length of 3000 bp or less, preferably 2000 bp or less. Have a length.

The present invention also relates to a DNA that specifically hybridizes with the DNA of the present invention and has a chain length of at least 15 nucleotides. "Specifically hybridize" with the DNA of the present invention means that it hybridizes with the DNA of the present invention under ordinary hybridization conditions, preferably under strict conditions, and does not hybridize with other DNA. Means Such DNA is used as a probe for detecting and isolating the DNA of the present invention, and as a primer for amplifying the DNA of the present invention. It is possible to When used as a primer, it usually has a chain length of 15 bp to 100 bp, preferably 15 bp to 35 bp. When used as a probe, a DNA having at least a part or all of the sequence of the DNA of the present invention and having a chain length of at least 15 bp is used. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an electrophoresis photograph showing the results of detecting the expression of the “rat PK80” gene by Northern plotting.

FIG. 2 is a diagram showing a comparison of the amino acid sequences of “rat # 80” and “mouse # 80”. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described specifically with reference to examples, but the present invention is not limited to the examples.

[Example 1] Screening of "Rat @ 80" gene

In order to obtain a novel serine / threonine protein kinase gene, a synthetic DNA (SEQ ID NO: 5 and SEQ ID NO: 6) having codons corresponding to the kinase conserved region Vlb and the kinase conserved region VIII was used as a primer. PCR was performed using a single-stranded cDNA prepared from rat brain mRNA with an oligo dT primer as type III. As a result, a 141 bp fragment was obtained. Next, based on the obtained fragments, screening of rat kidney library (Clontech) was performed. Since the obtained cDNA lacked the 5 'end, the synthetic DNA (SEQ ID NO: 7 and SEQ ID NO: 8) was used as a primer and the 5' RACE kit (GIBC0-BRL) was used as the primer. The ends were cloned. The nucleotide sequence of the resulting cDNA is shown in SEQ ID NO: 1, and the deduced amino acid sequence is shown in SEQ ID NO: 2. The isolated cDNA has 0RF encoding 733 amino acid, and amino acids 446 to 693 are the protein kinase catalytic region. . The present inventors have named the protein encoded by the isolated cDNA "rat PK80".

[Example 2] Analysis of tissue specificity of expression of "rat # 80" gene

Isolated "rat PK80" cDNA fragment of 3155Bp (SEQ ID NO: 1: corresponding to 1 from 3155 position of the DNA represented by) and ^{3 2} P-labeled using the Megaprime kit (Amersham) and, Northern hybridization was performed to detect the expression of "rat PK80" in various tissues. As a result, the rat ΡΚ80 gene showed strong expression in the kidney (Fig. 1). Some expression was observed in other tissues, such as in the brain, but the intensity was much lower than in the kidney.

[Example 3] Screening of "mouse $ 80" gene

When GenBank was searched based on the base sequence of “rat PK80” cDNA (SEQ ID NO: 1), EST (GenBank accession NO. AA197833) with the highest homology was found. DNA (SEQ ID NO: 9, SEQ ID NO: 10) synthesized based on this sequence was ligated and nested PCR was performed to clone 5 ′ half of the full-length cDNA from a mouse testis SPORT vector library. Furthermore, using synthetic DNA (SEQ ID NO: 11 and SEQ ID NO: 12) as primers, three and half of the cDNA was cloned from the mouse testis SPORT vector library by nested PCR, and a total of 2976 bp full-length cDNA was obtained. Obtained. The nucleotide sequence of the full-length cDNA is shown in SEQ ID NO: 3, and the deduced amino acid sequence is shown in SEQ ID NO: 4. The amino acid sequence from position 445 to position 697 is the protein kinase catalytic region. Figure 2 shows the homology of the isolated rat and mouse serine / threonine protein kinases. The present inventors named the protein encoded by the isolated cDNA “mouse PK80”.

[Example 4] Detection of serine / threonine protein kinase activity

Rat “ΡΚ80” A 3123 bp fragment containing the catalytic region (corresponding to positions 33 to 3155 of the base sequence described in SEQ ID NO: 1) was added with His-DNA and a pET vector (Strata ne) And the vector was used to transform E. coli BL21. Transformation After culturing the Escherichia coli thus obtained, a protein kinase catalytic domain protein to which a His-tag was added by IPTG was produced, and a purified sample was recovered using a nickel column.

The serine / threonine protein kinase activity of the His-tagged protein kinase catalytic domain protein was detected as follows. "50mM Hep es (. PH7 5) , 10mM Mg (0Ac) 2, ImM DTT, 0.4mM 3 2 P- § - ATP (100- 200Cpm / pmol) , in a solution of 0. 2mM Syntidej, a His- tag The added and purified rat “PK80” was added, and a phosphorylation reaction was performed at 30 ° C. The reaction was terminated over time by dropping (25〃1) on Whatman P81 paper. WhatmanP81 paper, washed with 75mM Seiri phosphate was measured liquid scintillation counter evening one ^{3 2} P uptake. As a result, the protein, ^{3 2} P- § -ATP, and autophosphorylation in the presence magnetic Shiyu um ions were phosphorus incorporated into the peptide substrate Shin Yui de (Sigma Co.). This revealed that the "rat PK80" protein (SEQ ID NO: 2) had protein kinase activity. Industrial applicability

According to the present invention, a novel serine / threonine protein kinase gene showing strong expression in the kidney, a similar gene thereof, and proteins encoded by these genes have been provided. The serine / threonine protein kinase of the present invention is considered to particularly regulate protein kinase activity in the kidney, and is expected to be used for, for example, evaluation of renal function, diagnosis and treatment of renal diseases and the like. Further, the serine / threonine protein kinase of the present invention can also be used for screening a candidate compound for a drug for preventing or treating a disease (for example, a sick disease) caused by a functional abnormality or the like.

Claims

The scope of the claims

1. a protein consisting of the amino acid sequence of SEQ ID NO: 2 or an amino acid sequence in the protein in which one or more amino acids are substituted, deleted or added, and A protein having threonine protein kinase activity.

2. SEQ ID NO: a protein encoded by DM that hybridizes with the DNA consisting of the nucleotide sequence of SEQ ID NO: 1, which has serine / threonine protein kinase activity.

3. SEQ ID NO: having a protein consisting of the amino acid sequence of SEQ ID NO: 4, or having an amino acid sequence in which one or more amino acids are substituted, deleted or added in the amino acid sequence in the protein, A protein having threonine protein kinase activity.

4. SEQ ID NO: a protein encoded by a DNA that hybridizes with a DNA consisting of the nucleotide sequence of SEQ ID NO: 3, which has a serine / threonine protein kinase activity.

5. A DNA encoding the protein of claim 1 or 3.

6. SEQ ID NO: A DNA that hybridizes with the DNA consisting of the nucleotide sequence of 1, and encodes a protein having serine / threonine protein kinase activity.

7. SEQ ID NO: A DNA that hybridizes with the DNA consisting of the nucleotide sequence of SEQ ID NO: 3, which encodes a protein having serine / threonine protein kinase activity.

8. A vector containing the DNA according to any one of claims 5 to 7.

9. A transformant capable of expressing the DNA according to any one of claims 5 to 7 in an expressible manner.

10. The method for producing a protein according to any one of claims 1 to 4, comprising a step of culturing the transformant according to claim 9.

11. A DNA which specifically hybridizes with the DNA according to any one of claims 5 to 7, and has a chain length of at least 15 nucleotides.

12. Antisense DNA for the DNA according to any one of claims 5 to 7 or a part thereof.

13. A method for screening a compound that binds to the protein according to any one of claims 1 to 4,

(a) contacting a test sample with the protein according to any one of claims 1 to 4,

(b) selecting a compound that binds to the protein according to any one of claims 1 to 4,

A method that includes

14. A method for screening a compound that modulates serine / threonine protein kinase activity of a protein according to any one of claims 1 to 4, comprising:

(b) a step of detecting serine / threonine protein kinase activity of the protein according to any one of claims 1 to 4,

(c) selecting a compound that promotes or inhibits the serine / threonine protein kinase activity of the protein according to any one of claims 1 to 4.

15. A compound that binds to the protein according to any one of claims 1 to 4, which can be isolated by the method according to claim 13.

16. Any of claims 1 to 4, which can be isolated by the method of claim 14. A compound that regulates serine / threonine protein kinase activity of a protein according to any one of the above.

17. The compound according to claim 15 or 16, which is of natural origin.

18. An antibody that binds to the protein according to any one of claims 1 to 4.