US20030129606A1

US20030129606A1 - Cytokine-, stress-, and oncoprotein-activated human protein kinase kinases

Info

Publication number: US20030129606A1
Application number: US10/137,953
Authority: US
Inventors: Roger Davis; Alan Whitmarsh; Cathy Tournier
Original assignee: University of Massachusetts UMass
Current assignee: University of Massachusetts UMass
Priority date: 1995-05-19
Filing date: 2002-05-03
Publication date: 2003-07-10
Also published as: JP4377050B2; DE69833946D1; DE69833946T2; US6136596A; WO1999002547A1; JP2001509370A; AU8477898A; EP1005480B1; US6610523B1; KR100566101B1; JP2009131261A; CA2295876A1; EP1005480A4; EP1005480A1; AU756143B2; KR20010021609A; CA2295876C

Abstract

Disclosed are human mitogen-activated (MAP) kinase kinase isoforms (MKKs). MKKs mediate unique signal transduction pathways that activate human MAP kinases p38 and JNK, which result in activation of other factors, including activating transcription factor-2 (ATF2) and c-Jun. The pathways are activated by a number of factors, including cytokines and environmental stress. Methods are provided for identifying reagents that modulate MKK function or activity and for the use of such reagents in the treatment of MKK-mediated disorders.

Description

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of U.S. patent Ser. No. 08/530,950, filed Sep. 19, 1995, which is a continuation-in-part of pending application Ser. No. 08/446,083, filed May 19, 1995, which applications are incorporated herein by reference in their entirety.[0001]

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0002] This invention was made in part with National Cancer Institute research grant CA 58396 and CA 65861. The Federal government has certain rights in the invention.

BACKGROUND OF THE INVENTION

This invention relates to protein kinases.

Mitogen-activated protein (MAP) kinases are important mediators of signal transduction from the cell surface to the nucleus. Multiple MAP kinases have been described in yeast including SMK1, HOG1, MPK1, FUS3, and KSS1. In mammals, the MAP kinases identified are extracellular signal-regulated MAP kinase (ERK), c-Jun amino-terminal kinase (JNK), and p38 kinase (Davis (1994) Trends Biochem. Sci. 19:470). These MAP kinase isoforms are activated by dual phosphorylation on threonine and tyrosine.

Activating Transcription Factor-2 (ATF2), ATFa, and cAMP Response Element Binding Protein (CRE-BPa) are related transcription factors that bind to similar sequences located in the promoters of many genes (Ziff (1990) Trends in Genet. 6:69). The binding of these transcription factors leads to increased transcriptional activity. ATF2 binds to several viral proteins, including the oncoprotein E1a (Liu and Green (1994) Nature 388:520), the hepatitis B virus X protein (Maguire et al. (1991) Science 252:842), and the human T cell leukemia virus 1 tax protein (Wagner and Green (1993) Science 262:395). ATF2 also interacts with the tumor suppressor gene product Rb (Kim et al. (1992) Nature 358:331), the high mobility group protein HMG(I)Y (Du et al. (1993) Cell 74:887), and the transcription factors nuclear NF-κB (Du et al. (1993) Cell 74:887) and c-Jun (Benbrook and Jones (1990) Oncogene 5:295).

SUMMARY OF THE INVENTION

The invention is based on the identification and isolation of a new group of human mitogen-activated protein kinase kinases (MKKs). The MKK isoforms described herein, MKK3, MKK6, MKK4 (including MKK4-α, -β, and -γ), MKK7 (including murine MKK7, human MKK7, MKK7b, MKK7c, MKK7d, and MKK7e) have serine, threonine, and tyrosine kinase activity. MKK3, MKK4, and MKK6 specifically phosphorylate the human MAP kinase p38 at Thr ¹⁸⁰and Tyr¹⁸². The MKK4 isoforms also phosphorylate the human MAP kinases JNK (including JNK1, JNK2, and JNK5) at Thr¹⁸³and Tyr¹⁸⁵. The MKK7 isoforms phosphorylate JNK at Thr¹⁸³and Tyr¹⁸⁵.

Accordingly, the invention features a substantially pure human MKK polypeptide having serine, threonine, and tyrosine kinase activity that specifically phosphorylates human p38 MAP kinase. MKK3 has the amino acid sequence of SEQ ID NO: 2. The invention further includes MKK6 having the amino acid sequence of SEQ ID NO: 4 and having serine, threonine, and tyrosine kinase activity that specifically phosphorylates human p38 MAP kinase.

The invention further features a substantially pure human MKK polypeptide having serine, threonine, and tyrosine kinase activity that specifically phosphorylates human p38 MAP kinase and JNK. MKK4 isoform MKK4-α has the amino acid sequence of SEQ ID NO: 6. MKK4 isoform MKK4-β has the amino acid sequence of SEQ ID NO: 8. MKK4 isoform MKK4-γ has the amino acid sequence of SEQ ID NO: 10.

The invention also features a substantially pure MKK polypeptide (MKK7) having serine, threonine, and tyrosine kinase activity that specifically phosphorylates mitogen-activated protein kinase JNK. MKK isoforms MKK7 (murine) and MKK7 (human) have the amino acid sequences of SEQ ID NOS: 18 and 26, respectively. The MKK7 isoforms MKK7b, MKK7c, MKK7d, and MKK7e have the amino acid sequences of SEQ ID NO: 20, SEQ ID NO: 28, SEQ ID NO: 30, and SEQ ID NO: 32, respectively.

As used herein, the term “mitogen-activating protein kinase kinase” or “MKK” means a protein kinase which possesses the characteristic activity of phosphorylating and activating a human mitogen-activating protein kinase. Examples of MKKs include MKK3 and MKK6, which specifically phosphorylate and activate p38 MAP kinase at Thr ¹⁸⁰and Tyr¹⁸², MKK4 isoforms which specifically phosphorylate and activate p38 MAP kinase at Thr¹⁸⁰and Tyr¹⁸², and JNK at Thr¹⁸³and Tyr¹⁸⁵, and MKK7 isoforms which specifically phosphorylate JNK at Thr¹⁸³and Tyr¹⁸⁵.

An “MKK7” is a mammalian isoform of mitogen-activated protein kinase kinase (MKK) polypeptide having serine, threonine, and tyrosine kinase activity, and phosphorylating mitogen-activated protein (MAP) kinase JNK but not p38.

The invention includes the specific p38 and JNK MKKs disclosed, as well as closely related MKKs which are identified and isolated by the use of probes or antibodies prepared from the polynucleotide and amino acid sequences disclosed for the MKKs of the invention. This can be done using standard techniques, e.g., by screening a genomic, cDNA, or combinatorial chemical library with a probe having all or a part of the nucleic acid sequences of the disclosed MKKs. The invention further includes synthetic polynucleotides having all or part of the amino acid sequence of the MKKs herein described.

The term “polypeptide” means any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation), and includes natural proteins as well as synthetic or recombinant polypeptides and peptides.

The term “substantially pure,” when referring to a polypeptide, means a polypeptide that is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. A substantially pure MKK polypeptide (e.g., human) is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, MKK polypeptide. A substantially pure MKK can be obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding a MKK polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

In one aspect, the invention features isolated polynucleotides which encode the MKKs of the invention. In one embodiment, the polynucleotide is the nucleotide sequence of SEQ ID NO: 1. In other embodiments, the polynucleotide is the nucleotide sequence of SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31, respectively.

As used herein, “polynucleotide” refers to a nucleic acid sequence or deoxyribonucleotides or ribonucleotides in the form of a separate fragment or a component of a larger construct. DNA encoding portions or all of the polypeptides of the invention can be assembled from cDNA fragments or from oligonucleotides that provide a synthetic gene which can be expressed in a recombinant transcriptional unit. Polynucleotide sequences of the invention include DNA, RNA, and cDNA sequences, and can be derived from natural sources or synthetic sequences synthesized by methods known to the art.

An “isolated” polynucleotide is a nucleic acid molecule that is separated in some way from sequences in the naturally occurring genome of an organism. Thus, the term “isolated polynucleotide” includes any nucleic acid molecules that are not naturally occuring. The term therefore includes, for example, a recombinant polynucleotide which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequences.

The isolated polynucleotide sequences of the invention also include polynucleotide sequences that hybridize under stringent conditions to the polynucleotide sequences specified herein. The term “stringent conditions” means hybridization conditions that guarantee specificity between hybridizing polynucleotide sequences, such as those described herein, or more stringent conditions. One skilled in the art can select posthybridization washing conditions, including temperature and salt concentrations, which reduce the number of nonspecific hybridizations such that only highly complementary sequences are identified (Sambrook et al. (1989) in Molecular Cloning, 2d ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

The isolated polynucleotide sequences of the invention also include sequences complementary to the polynucleotides encoding MKK (antisense sequences). Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub (1990) Scientific American 262:40). The invention includes all antisense polynucleotides that inhibit production of MKK polypeptides. In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and introduced into a target MKK-producing cell. The use of antisense methods to inhibit the translation of genes is known in the art, and is described, e.g., in Marcus-Sakura Anal. Biochem., 172:289 (1988).

In addition, ribozyme nucleotide sequences for MKK are included in the invention. Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences encoding these RNAs, molecules can be engineered to recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech (1988) J. Amer. Med. Assn. 260:3030). A major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.

There are two basic types of ribozymes, tetrahymena-type (Hasselhoff (1988) Nature 334:585) and “hammerhead”-type. Tetrahymena-type ribozymes recognize sequences which are four bases in length, while “hammerhead”-type ribozymes recognize base sequences 11-18 bases in length. The longer the sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA species, and 18-base recognition sequences are preferable to shorter recognition sequences.

The MKK polypeptides can also be used to produce antibodies that are immunoreactive or bind epitopes of the MKK polypeptides. Accordingly, one aspect of the invention features antibodies to the MKK polypeptides of the invention. The antibodies of the invention include polyclonal antibodies which include pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations. Monoclonal antibodies are made from antigen-containing fragments of the MKK polypeptide by methods known in the art (see, for example, Kohler et al. (1975) Nature 256:495).

The term “antibody” as used herein includes intact molecules as well as fragments thereof, such as Fa, F(ab′) ₂, and Fv, which are capable of binding an epitopic determinant. Antibodies that specifically bind MKK polypeptides can be prepared using intact polypeptides or fragments containing small peptides of interest as the immunizing antigen. The polypeptide or peptide used to immunize an animal can be derived from translated cDNA or chemically synthesized, and can be conjugated to a carrier protein, if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin and thyroglobulin. The coupled peptide is then used to immunize the animal (e.g., a mouse, a rat, or a rabbit).

A molecule (e.g., antibody) that “specifically binds” is one that binds to a particular polypeptide, e.g., MKK7, but that does not substantially recoginze or bind to other molecules in a sample, e.g., a biological sample which includes MKK7. References to constructs made of an antibody (or fragment thereof) coupled to a compound comprising a detectable marker include constructs made by any technique, including chemical means and recombinant techniques.

The invention also features methods of identifying subjects at risk for MKK-mediated disorders by measuring activation of the MKK signal transduction pathway. Activation of the MKK signal transduction pathway can be determined by measuring MKK synthesis; activation of MKK isoforms; activation of MKK substrates p38 or JNK isoforms; or activation of p38 and JNK substrates such as ATF2, ATFa, CRE-BPa, and c-Jun. The term “JNK” or “JNK isoforms” includes JNK1, JNK2, and JNK3. The term “MKK substrate” as used herein includes MKK substrates, as well as MKK substrate substrates, e.g., p38, JNK, ATF2, and c-Jun.

In one embodiment, activation of the MKK signal transduction pathway is determined by measuring activation of the appropriate MKK signal transduction pathway substrates (for example, selected from p38, JNK isoforms, ATF2, ATFa, CRE-BPa, or c-Jun). MKK activity is measured by the rate of substrate phosphorylation as determined by quantitation of the rate of labelled phosphorus (e.g., [ ³²]P or [³³]P) incorporation. This can also be measured using phosphorylation-specific reagents, such as antibodies. The specificity of MKK substrate phosphorylation can be tested by measuring p38 activation, JNK activation, or both, or by employing mutated p38 or JNK molecules that lack the sites for MKK phosphorylations. Altered phosphorylation of the substrate relative to control values indicates alteration of the MKK signal transduction pathway, and increased risk in a subject of an MKK-mediated disorder. MKK activation of p38 and JNK can be detected in a coupled assay with the MKK signal transduction substrate ATF2, or related compounds such as ATFa and CRE-BPa. Activation can also be detected with the substrate c-Jun. When ATF2 is included in the assay, it is present as an intact protein or as a fragment of the intact protein, e.g., the activation domain (residues 1-109, or a portion thereof). ATF2 is incubated with a test sample in which MKK activity is to be measured and [γ-³²P]ATP, under conditions sufficient to allow the phosphorylation of ATF2. ATF2 is then isolated and the amount of phosphorylation quantitated. In a specific embodiment, ATF2 is isolated by immunoprecipitation, resolved by SDS-PAGE, and detected by autoradiography.

In another embodiment, activation of the MKK signal transduction pathway is determined by measuring the level of MKK expression in a test sample. In a specific embodiment, the level of MKK expression is measured by Western blot analysis. The proteins present in a sample are fractionated by gel electrophoresis, transferred to a membrane, and probed with labeled antibodies to MKK. In another specific embodiment, the level of MKK expression is measured by Northern blot analysis. Total cellular or polyadenylated [poly(A) ⁺] mRNA is isolated from a test sample. The RNA is fractionated by electrophoresis and transferred to a membrane. The membrane is probed with labeled MKK cDNA. In another embodiment, MKK expression is measured by quantitative PCR applied to expressed mRNA.

The MKKs of the invention are useful for screening reagents that modulate MKK activity. MKKs are activated by phosphorylation. Accordingly, in one aspect, the invention features methods for identifying a reagent which modulates MKK activity, by incubating MKK with the test reagent and measuring the effect of the test reagent on MKK synthesis, phosphorylation, function, or activity. In one embodiment, the test reagent is incubated with MKK and [ ³²]P-ATP, and the rate of MKK phosphorylation determined, as described above. In another embodiment, the test reagent is incubated with a cell transfected with an MKK polynucleotide expression vector, and the effect of the test reagent on MKK transcription is measured by Northern blot analysis, as described above. In a further embodiment, the effect of the test reagent on MKK synthesis is measured by Western blot analysis using an antibody to MKK. In still another embodiment, the effect of a reagent on MKK activity is measured by incubating MKK with the test reagent, [³²]P-ATP, and a substrate in the MKK signal transduction pathway, including one or more of p38, JNK, and ATF2. The rate of substrate phosphorylation is determined as described above.

The term “modulation of MKK activity” includes inhibitory or stimulatory effects.

The invention is particularly useful for screening reagents that inhibit MKK activity. Such reagents are useful for the treatment or prevention of MKK-mediated disorders, for example, inflammation and oxidative damage.

The invention further features a method of treating a MKK-mediated disorder by administering to a subject in need thereof, an effective dose of a therapeutic reagent that inhibits the activity of MKK.

An “MKK-mediated disorder” is a pathological condition resulting, at least in part, from excessive activation of an MKK signal transduction pathway. The MKK signal transduction pathways are activated by several factors, including inflammation and stress. MKK-mediated disorders include, for example, ischemic heart disease, burns due to heat or radiation (UV, X-ray, γ, β, etc.), kidney failure, liver damage due to oxidative stress or alcohol, respiratory distress syndrome, septic shock, rheumatoid arthritis, autoimmune disorders, and other types of inflammatory diseases.

A “therapeutic reagent” any compound or molecule that achieves the desired effect on an MKK-mediated disorder when administered to a subject in need thereof.

MKK-mediated disorders further include proliferative disorders, particularly disorders that are stress-related. Examples of stress-related MKK-mediated proliferative disorders are psoriasis, acquired immune deficiency syndrome, malignancies of various tissues of the body, including malignancies of the skin, bone marrow, lung, liver, breast, gastrointestinal system, and genito-urinary tract. Preferably, therapeutic reagents inhibit the activity or expression of MKK inhibit cell growth or cause apoptosis.

A therapeutic reagent that “inhibits MKK activity” interferes with a MKK-mediated signal transduction pathway. For example, a therapeutic reagent can alter the protein kinase activity of MKK, decrease the level of MKK transcription or translation, e.g., an antisense polynucleotide able to bind MKK mRNA, or suppress MKK phosphorylation of p38, JNK, or ATF2, thus disrupting the MKK-mediated signal transduction pathway. Examples of such reagents include antibodies that bind specifically to MKK polypeptides, and fragments of MKK polypeptides that competitively inhibit MKK polypeptide activity.

A therapeutic reagent that “enhances MKK activity” supplements a MKK-mediated signal transduction pathway. Examples of such reagents include the MKK polypeptides themselves, which can be administered in instances where the MKK-mediated disorder is caused by under expression of the MKK polypeptide, or expression of a mutant MKK polypeptide. In addition, portions of DNA encoding an MKK polypeptide can be introduced into cells that under express an MKK polypeptide.

A “therapeutically effective amount” is an amount of a reagent sufficient to decrease or prevent the symptoms associated with the MKK-mediated disorder.

Therapeutic reagents for treatment of MKK-mediated disorders identified by the methods of the invention are administered to a subject in a number of ways known to the art, including parenterally by injection, infusion, sustained-release injection or implant, intravenously, intraperitoneally, intramuscularly, subcutaneously, or transdermally. Epidermal disorders and disorders of the epithelial tissues are treated by topical application of the reagent. The reagent is mixed with other compounds to improve stability and efficiency of delivery (e.g., liposomes, preservatives, or dimethyl sulfoxide (DMSO)). Polynucleotide sequences, including antisense sequences, can be therapeutically administered by techniques known to the art resulting in introduction into the cells of a subject suffering from the MKK-mediated disorder. These methods include the use of viral vectors (e.g., retrovirus, adenovirus, vaccinia virus, or herpes virus), colloid dispersions, and liposomes.

The materials of the invention are ideally suited for the preparation of a kit for the detection of the level or activity of MKK. Accordingly, the invention features a kit comprising an antibody that binds MKK, or a nucleic acid probe that hybridizes to a MKK polynucleotide, and suitable buffers. The probe or monoclonal antibody can be labeled to detect binding to a MKK polynucleotide or protein. In a preferred embodiment, the kit features a labeled antibody to MKK.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the detailed description, and from the claims.

DETAILED DESCRIPTION

The drawings will first be described. [0042]
Drawings [0043]
FIG. 1 is a comparison of the amino acid sequences of MKK3 (SEQ ID NO: 2), MKK4-α (SEQ ID NO: 6), the human MAP kinase kinases MEK1 (SEQ ID NO: 11) and MEK2 (SEQ ID NO: 12), and the yeast HOG1 MAP kinase kinase PBS2 (SEQ ID NO: 13). Sequences were compared using the PILE-UP program (version 7.2; Wisconsin Genetics Computer Group). The protein sequences are presented in single letter code (A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp, and Y, Tyr). The PBS2 sequence is truncated at both the NH[0044] ₂— (<) and COOH— (>) termini. Gaps introduced into the sequences to optimize the alignment are illustrated by a dash. Identical residues are indicated by a period. The sites of activating phosphorylation in MEK are indicated by asterisks.
FIG. 2A is a dendrogram showing the relationship between members of the human and yeast MAP kinase kinases. The dendrogram was created by the unweighted pair-group method with the use of arithmetic averages (PILE-UP program). The human (hu) MAP kinase kinases MEK1, MEK2, MKK3, and MKK4; the [0045] Saccharomyces cerevisiae (sc) MAP kinase kinases PBS2, MKK1, and STE7; and the Saccharomyces pombe (sp) MAP kinase kinases WIS1 and BYR1 are presented.
FIG. 2B is a dendrogram showing the relationship between MKKs. The dendrogram was created as described for FIG. 2A. [0046]
FIG. 3 is a schematic representation of the ERK, p38, and JNK signal transduction pathways. MEK1 and MEK2 are activators of the ERK subgroup of MAP kinase. MKK3 and MKK4 are activators of the p38 MAP kinase. MKK4 is identified as an activator of both the p38 and JNK subgroups of MAP kinase. [0047]
FIGS. [0048] 4A-4D are a representation of the nucleic acid (SEQ ID NO: 1) and amino acid sequences (SEQ ID NO: 2) for MKK3.
FIGS. [0049] 5A-5C are a representation of the nucleic acid (SEQ ID NO: 3) and amino acid sequences (SEQ ID NO: 4) for MKK6.
FIGS. [0050] 6A-6F are a representation of the nucleic acid (SEQ ID NO: 5) and amino acid sequences (SEQ ID NO: 6) for MKK4α.
FIGS. [0051] 7A-7F are a representation of the nucleic acid (SEQ ID NO: 7) and amino acid sequences (SEQ ID NO: 8) for MKK4β.
FIGS. [0052] 8A-8F are a representation of the nucleic acid (SEQ ID NO: 9) and amino acid sequences (SEQ ID NO: 10) for MKK4γ.
FIG. 9 is a representation of the deduced primary structure of MKK7 (SEQ ID NO: 18) compared with hep (SEQ ID NO: 21), the MAP kinase kinases MEK1 ([0053] MKK 1; SEQ ID NO: 11), MEK2 (MKK2; SEQ ID NO: 12), MKK3 (SEQ ID NO: 2), MKK4γ (SEQ ID NO: 10), MKK5 (SEQ ID NO: 22), and MKK6 (SEQ ID NO: 4) using the PILE-UP program ( version 7,2; Wisconsin Genetics Computer Group). Gaps introduced into the sequences to optimize the alignment are illustrated with a dash (-). Identity is indicated with a dot (.). The sites of activating phosphorylation of MAP kinase kinases (2, 27, 37, and 38) are indicated with asterisks (*)
FIGS. [0054] 10A-10D are a representation of the nucleic acid (SEQ ID NO: 17) and amino acid (SEQ ID NO: 18) sequences for MKK7.
FIGS. [0055] 11A-11D are a representation of the nucleic acid (SEQ ID NO: 19) and amino acid (SEQ ID NO: 20) sequences of MKK7b.
FIGS. [0056] 12A-12B are a representation of the nucleic acid (SEQ ID NO: 25) and amino acid (SEQ ID NO: 26) sequences of human MKK7.
FIGS. [0057] 13A-13D are a representation of the nucleic acid (SEQ ID NO: 27) and amino acid (SEQ ID NO: 28) sequences of murine MKK7c.
FIGS. [0058] 14A-14D are a representation of the nucleic acid (SEQ ID NO: 29) and amino acid (SEQ ID NO: 30) sequences of murine MKK7d.
FIGS. [0059] 15A-15D are a representation of the nucleic acid (SEQ ID NO: 31) and amino acid (SEQ ID NO: 32) sequences of murine MKK7e.
FIG. 16A is a graph of data from a transfection assay in which cells were co-transfected with AP-1 reporter plasmid pTRE-Luciferase with expression vectors for MKK4, MKK7, JNK1, JNK1(APF), or control vector. [0060]
FIG. 16B is a graph of a transfection assay in which cells were co-transfected with a GAL4-ATF2 fusion vector and an expression vector for MKK4, MKK7, JNK1, JNK1(APF), or control vector.[0061]

HUMAN MITOGEN-ACTIVATED PROTEIN KINASE KINASES

The human MAP kinase kinases MKK3 and MKK4 (MKK3/4), and MKK7, described herein mediate the transduction of specific signals from the cell surface to the nucleus along specific pathways. These signal transduction pathways are initiated by factors such as cytokines, UV radiation, osmotic shock, and oxidative stress. Activation of MKK3/4, MKK6, and MKK7 results in activation of the MAP kinases. p38 is activated by MKK3 and MKK4. JNK is activated by MKK4 and MKK7. p38 and JNK in turn activate a group of related transcription factors such as ATF2, ATFa, and CRE-BPa. These transcription factors in turn activate expression of specific genes. For example, ATF2 in known to activate expression of human T cell leukemia virus 1 (Wagner and Green (1993) Science 262:395), transforming growth factor-b2 (Kim et al. (1992) supra), interferon-β (Du et al. (1993) Cell 74:887), and E-selectin (DeLuca et al. (1994) J. Biol. Chem. 269:19193). In addition, ATF2 is implicated in the function of a T cell-specific enhancer (Georgopoulos et al. (1992) Mol. Cell. Biol. 12:747). [0062]
The JNK group of MAP kinases is activated by exposure of cells to environmental stress or by treatment of cells with pro-inflammatory cytokines (Gupta et al. (1994) EMBO J. 15:2760-2770; Dérijard et al. (1991) Cell 76:1025-1037; Kyriakis et al. (1994) Nature 369:156-160; Sluss et al. (1994) Mol. Cell. Biol. 14:8376-8384; Kallunki et al. (1994) Genes & Dev. 8:2996-3007). Targets of the JNK signal transduction pathway include the transcription factors ATF2 and c-jun (Whitmarsh & Davis (1996) J. Mol. Med. 74:589-607). These transcription factors are members of the bZIP group that bind as homo- and hetero-dimeric complexes to AP-1 and AP-1-like sites in the promoters of many genes (Curran & Franza (1988) Cell 55:395-397). JNK binds to an NH[0063] ₂-terminal region of ATF2 and c-Jun and phosphorylates two sites within the activation domain of each transcription factor (Dérijard et al. (1994) Cell 76:1025-1037; van Dam et al. (1995) EMBO J. 14:1798-1811; Livingstone et al. (1995) EMBO J. 14:1785-1797). This phosphorylation leads to increased transcriptional activity (Whitmarsh, supra). Together, these biochemical studies indicate that the JNK signal transduction pathway contributes to the regulation of AP-1 transcriptional activity in response to cytokines and environmental stress (Whitmarsh, supra). Strong support for this hypothesis is provided by genetic evidence indicating that the JNK signaling pathway is required for the normal regulation of AP-1 transcriptional activity (Yang et al. (1997) Proc. Natl. Acad. Sci. USA, 94:3004-3009).
JNK is activated by dual phosphorylation on Thr-183 and Tyr-185 (Dérijard, supra). MKK4 (also known as SEKI) was the first MAP kinase kinase identified as a component of the JNK signal transduction pathway (Dérijard et al. (1995) Science 267:682-685; Lin et al. (1995) Science 268:286-290; Sanchez et al. (1994) Nature 372:794-798). Biochemical studies demonstrate that MKK4 phosphorylates and activates JNK (Dérijard et al. (1995) Science 267:682-685; Lin et al. (1995) Science 268:286-290; Sanchez et al. (1994) Nature 372:794-798). However, the function of MKK4 may not be restricted to the JNK signal transduction pathway because MKK4 also phosphorylates and activates p38 MAP kinase (Dérijard et al. (1995) Science 267:682-685; Lin et al. (1995) Science 268:286-290). This specificity of MKK4 to activate both JNK and p38 MAP kinase provides a mechanism that may account for the co-ordinate activation of these MAP kinases in cells treated with cytokines or environmental stress (Davis (1994) Trends Biochem. Sci. 19:470-473). However, this co-ordinate activation is not always observed. For example, JNK activation in the liver correlates with decreased p38 MAP kinase activity (Mendelson et al. (1996) Proc. Natl. Acad. Sci. USA 93:12908-12913). These data suggest that the properties of MKK4 are insufficient to account for the regulation of JNK in vivo. [0064]
The isolation of human MKKs is described in Example 1, Example 22, Dérijard et al. ((1995) Science 267:682-685, hereby specifically incorporated by reference), and Raingeaud et al. ((1995) Mol. Cell. Biol. 16:1247-1255). Distinctive regions of the yeast PBS2 sequence were used to design polymerase chain reaction (PCR) primers. Amplification of human brain mRNA with these primers resulted in the formation of specific products which were cloned into a plasmid vector and sequenced. Two different complementary DNAs (cDNAs) that encoded human protein kinases were identified: one encoding a 36 kD protein (MKK3), and one encoding a 44 kD protein (MKK4). MKK4 includes 3 isoforms that vary slightly at the NH[0065] ₂-terminal, identified as α, β, and γ. The amino acid sequences of MKK3 (SEQ ID NO: 2), MKK4-α (SEQ ID NO: 6), MKK4-β (SEQ ID NO: 8), and MKK4-γ (SEQ ID NO: 10) are shown in FIG. 1. The nucleic acid and amino acid sequences of MKK3 (FIG. 4), MKK6 (FIG. 5), MKK4-α (FIG. 6), MKK4-β (FIG. 7), and MKK4-γ (FIG. 8) are also provided. MKK6 was isolated from a human skeletal muscle library by cross-hybridization with MKK3. Except for differences at the N-terminus, MKK6 is highly homologous to MKK3. Other human MKK3 and MKK4 isoforms that exist can be identified by the method described in Example 1.
The expression of these human MKK isoforms was examined by Northern (RNA) blot analysis of mRNA isolated from eight adult human tissues (Example 2). Both protein kinases were found to be widely expressed in human tissues, with the highest expression seen in skeletal muscle tissue. [0066]
The substrate specificity of MKK3 was investigated in an in vitro phosphorylation assay with recombinant epitope-tagged MAP kinases (JNK1, p38, and ERK2) as substrates (Example 3). MKK3 phosphorylated p38, but did not phosphorylate JNK1 or ERK2. Phosphoaminoacid analysis of p38 demonstrated the presence of a phosphothreonine and phosphotyrosine. Mutational analysis of p38 demonstrated that replacement of phosphorylation sites Thr[0067] ¹⁸⁰and Tyr¹⁸²with Ala and Phe, respectively, blocked p38 phosphorylation. These results establish that MKK3 functions in vitro as a p38 MAP kinase kinase.
Studies of the in vitro substrate specificity of MKK4 are described in Example 4. MKK4 incubated with [γ-[0068] ³²P] ATP, and JNK1, p38, or ERK2 was found to phosphorylate both p38 and JNK1. MKK4 activation of JNK and p38 was also studied by incubating MKK4 with wild-type or mutated JNK1 or p38. The p38 substrate ATF2 was included in each assay. MKK4 was found to exhibit less autophosphorylation than MKK3. MKK4 was also found to be a substrate for activated MAP kinase. Unlike MKK3, MKK4 was also found to activate JNK1. MKK4 incubated with wild-type JNK1, but not mutated JNK1, resulted in increased phosphorylation of ATF2. These results establish that MKK4 is a p38 MAP kinase kinase that also phosphorylates the JNK subgroup of MAP kinases.
In vivo activation of p38 by UV-stimulated MKK3 is described in Example 5. Cells expressing MKK3 were exposed in the presence or absence of UV radiation. MKK3 was isolated by immunoprecipitation and used for protein kinase assays with the substrates p38 or JNK. ATF2 was included in some assays as a substrate for p38 and JNK. MKK3 from non-activated cultured COS cells caused a small amount of phosphorylation of p38 MAP kinase, resulting from basal activity of MKK3. MKK3 from UV-irradiated cells caused increased phosphorylation of p38 MAP kinase, but not of JNK1. An increase in p38 activity was also detected in assays in which ATF2 was included as a substrate. These results establish that MKK3 is activated by UV radiation. [0069]
The effect of expression of MKK3 and MKK4 on p38 activity was examined in COS-1 cells (Example 6). Cells were transfected with a vector encoding p38 and a MEK1, MKK3, or MKK4. Some of the cells were also exposed to EGF or UV radiation. p38 was isolated by immunoprecipitation and assayed for activity with [γ-[0070] ³²P]ATP and ATF2. The expression of the ERK activator MEK1 did not alter p38 phosphorylation of ATF2. In contrast, expression of MKK3 or MKK4 caused increased activity of p38 MAP kinase. The activation of p38 caused by MKK3 and MKK4 was similar to that observed in UV-irradiated cells, and was much greater than that detected in EGF-treated cells. These in vitro results provide evidence that MKK3 and MKK4 activate p38 in vivo.
A series of experiments was conducted to examine the potential regulation of ATF2 by JNK1. These experiments are described in Gupta et al. (1995) Science 267:389-393, hereby specifically incorporated by reference. The effect of UV radiation on ATF2 phosphorylation was investigated in COS-1 cells transfected with and without epitope-tagged JNK1 (Example 7). Cells were exposed to UV radiation, and JNK1 and JNK2 visualized by in-gel protein kinase assay with the substrate ATF2. JNK1 and JNK2 were detected in transfected and non-transfected cells exposed to UV radiation; however, JNK1 levels were higher in the transfected cells. These results demonstrate that ATF2 is a substrate for the JNK1 and JNK2 protein kinases, and that these protein kinases are activated in cells exposed to UV light. [0071]
The site of JNK1 phosphorylation of ATF2 was examined by deletion analysis (Example 8). Progressive NH[0072] ₂-terminal domain deletion GST-ATF2 fusion proteins were generated, and phosphorylation by JNK1 isolated from UV-irradiated cells was examined. The results showed that JNK1 requires the presence of ATF2 residues 1-60 for phosphorylation of the NH₂-terminal domain of ATF2.
The ATF2 residues required for binding of JNK1 were similarly examined. JNK1 was incubated with immobilized ATF2, unbound JNK1 was removed by extensive washing, and bound JNK1 was detected by incubation with [γ-[0073] ³²P]ATP. Results indicate that residues 20 to 60 of ATF2 are required for binding and phosphorylation by JNK1. A similar binding interaction between ATF2 and the 55 kD JNK2 protein kinase has also been observed.
Phosphorylation by JNK1 was shown to reduce the electrophoretic mobility of ATF2 (Example 9). Phosphoamino acid analysis of the full-length ATF2 molecule (residues 1-505) demonstrated that JNK phosphorylated both Thr and Ser residues. The major sites of Thr and Ser phosphorylation were located in the NH[0074] ₂and COOH terminal domains, respectively. The NH₂-terminal sites of phosphorylation were identified as Thr⁶⁹and Thr⁷¹by phosphopeptide mapping and mutational analysis. These sites of Thr phosphorylation are located in a region of ATF2 that is distinct from the sub-domain required for JNK binding (residues 20 to 60).
The reduced electrophoretic mobility seen with phosphorylation of ATF2 was investigated further (Example 10). JNK1 was activated in CHO cells expressing JNK1 by treatment with UV radiation, pro-inflammatory cytokine interleukin-1 (IL-1), or serum. A decreased electrophoretic mobility of JNK1-activated ATF2 was observed in cells treated with UV radiation and IL-1. Smaller effects were seen after treatment of cells with serum. These results indicate that ATF2 is an in viva substrate for JNK1. [0075]
The effect of UV radiation on the properties of wild-type (Thr[0076] ^{69, 71}) and phosphorylation-defective (Ala^{69, 71}) ATF2 molecules was investigated (Example 11). Exposure to UV caused a decrease in the electrophoretic mobility of both endogenous and over-expressed wild-type ATF2. This change in electrophoretic mobility was associated with increased ATF2 phosphorylation. Both the electrophoretic mobility shift and increased phosphorylation were blocked by the replacement of Thr⁶⁹and Thr⁷¹with Ala in ATF2. This mutation also blocked the phosphorylation of ATF2 on Thr residues in vivo.
Transcriptional activities of fusion proteins consisting of the GAL4 DNA binding domain and wild-type or mutant ATF2 were examined (Example 12). Point mutations at Thr[0077] ⁶⁹and/or Thr⁷¹of ATF2 significantly decreased the transcriptional activity of ATF2 relative to the wild-type molecule, indicating the physiological relevance of phosphorylation at these sites for activity.
The binding of JNK1 to the NH[0078] ₂-terminal activation domain of ATF2 (described in Example 8) suggested that a catalytically inactive JNK1 molecule could function as a dominant inhibitor of the wild-type JNK1 molecule. This hypothesis was investigated by examining the effect of a catalytically inactive JNK1 molecule on ATF2 function (Example 13). A catalytically-inactive JNK1 mutant was constructed by replacing the sites of activating Thr¹⁸³and Tyr¹⁸⁵phosphorylation with Ala and Phe, respectively (Ala¹⁸³,Phe¹⁸⁵, termed “dominant-negative”). Expression of wild-type JNK1 caused a small increase in serum-stimulated ATF2 transcriptional activity. In contrast, dominant-negative JNK1 inhibited both control and serum-stimulated ATF2 activity. This inhibitory effect results from the non-productive binding of the JNK1 mutant to the ATF2 activation domain, effectively blocking ATF2 phosphorylation.
The tumor suppressor gene product Rb binds to ATF2 and increases ATF2-stimulated gene expression (Kim et al. (1992) Nature 358:331). Similarly, the adenovirus oncoprotein E1A associates with the DNA binding domain of ATF2 and increases ATF2-stimulated gene expression by a mechanism that requires the NH[0079] ₂-terminal activation domain of ATF2 (Liu and Green (1994) Nature 368:520). ATF2 transcriptional activity was investigated with the luciferase reporter gene system in control, Rb-treated, and E1A-treated cells expressing wild-type or mutant ATF2 molecules (Example 14). Rb and E1A were found to increase ATF2-stimulated gene expression of both wild-type and mutant ATF2. However, mutant ATF2 caused a lower level of reporter gene expression than did wild-type ATF2. Together, these results indicate a requirement for ATF2 phosphorylation (on Thr⁶⁹and Thr⁷¹) plus either Rb or E1A for maximal transcriptional activity. Thus, Rb and E1A act in concert with ATF2 phosphorylation to control transcriptional activity.
A series of experiments were conducted to examine the action of p38 activation and to establish the relationship of the p38 MAP kinase pathway to the ERK and JNK signal transduction pathways (Raingeaud et al. (1995) J. Biol. Chem. 270:7420, hereby specifically incorporated by reference). Initially, the substrate specificity of p38 was investigated by incubating p38 with proteins that have been demonstrated to be substrates for the ERK and/or JNK groups of MAP kinases (Example 15). We examined the phosphorylation of MBP (Erickson et al. (1990) J. Biol. Chem. 265:19728), EGF-R (Northwood et al. (1991) J. Biol. Chem. 266:15266), cytoplasmic phospholipase A[0080] ₂(cPLA₂) (Lin et al. (1993) Cell 72:269), c-Myc (Alvarez et al. (1991) J. Biol. Chem. 266:15277), IκB, c-Jun, and wild-type (Thr^{69, 71}) or mutated (Ala^{69, 71}) ATF2. p38 phosphorylated MBP and EGF-R, and to a lesser extent IκB, but not the other ERK substrates, demonstrating that the substrate specificity of p38 differs from both the ERK and JNK groups of MAP kinases. Wild-type ATF2, but not mutated ATF2 (Ala^{69, 71}), was found to be an excellent p38 substrate.
The phosphorylation of ATF2 by p38 was associated with an electrophoretic mobility shift of ATF2 during polyacrylamide gel electrophoresis. We tested the hypothesis that p38 phosphorylates ATF2 at the same sites as JNK1 by replacing Thr[0081] ⁶⁹and Thr⁷¹with Ala (Ala^{69, 71}). It was found that p38 did not phosphorylate mutated ATF2, which demonstrates that p38 phosphorylates ATF2 within the NH2-terminal activation domain on Thr⁶⁹and Thr⁷¹.
A comparison of the binding of JNK and p38 to ATF2 was conducted by incubating extracts of cells expressing JNK1 or p38 with epitope alone (GST) or GST-ATF2 (residues 1-109 containing the activation domain) (Example 16). Bound protein kinases were detected by Western blot analysis. The results demonstrate that both p38 and JNK bind to the ATF2 activation domain. [0082]
EGF and phorbol ester are potent activators of the ERK signal transduction pathway (Egan and Weinberg (1993) Nature 365:781), causing maximal activation of the ERK sub-group of MAP kinases. These treatments, however, cause only a small increase in JNK protein kinase activity (Dérijard et al. (1994) supra; Hibi et al. (1993) supra). The effects of EGF or phorbol esters, as well UV radiation, osmotic shock, interleukin-1, tumor necrosis factor, and LPS, on p38 activity were all tested (Example 17). Significantly, EGF and phorbol ester caused only a modest increase in p38 protein kinase activity, whereas environmental stress (UV radiation and osmotic shock) caused a marked increase in the activity of both p38 and JNK. Both p38 and JNK were activated in cells treated with pro-inflammatory cytokines (TNF and IL-1) or endotoxic LPS. Together, these results indicate that p38, like JNK, is activated by a stress-induced signal transduction pathway. [0083]
ERKs and JNKs are activated by dual phosphorylation within the motifs Thr-Glu-Tyr and Thr-Pro-Tyr, respectively. In contrast, p38 contains the related sequence Thr-Gly-Tyr. To test whether this motif is relevant to the activation of p38, the effect of the replacement of Thr-Gly-Tyr with Ala-Gly-Phe was examined (Example 18). The effect of UV radiation on cells expressing wild-type (Thr[0084] ¹⁸⁰,Tyr¹⁸²) or mutant p38 (Ala¹⁸⁰, Phe¹⁸²) was studied. Western blot analysis using an anti-phosphotyrosine antibody demonstrated that exposure to UV radiation caused an increase in the Tyr phosphorylation of p38. The increased Tyr phosphorylation was confirmed by phosphoamino acid analysis of p38 isolated from [γ-³²P] phosphate-labeled cells. This analysis also demonstrated that UV radiation caused increased Thr phosphorylation of p38. Significantly, the increased phosphorylation on Thr¹⁸⁰and Tyr¹⁸²was blocked by the Ala¹⁸⁰/Phe¹⁸²mutation. This result demonstrates that UV radiation causes increased activation of p38 by dual phosphorylation.
It has recently been demonstrated that ERK activity is regulated by the mitogen-induced dual specificity phosphatases MKP1 and PAC1 (Ward et al. (1994) Nature 367:651). The activation of p38 by dual phosphorylation (Example 18) raises the possibility that p38 may also be regulated by dual specificity phosphatases. We examined the effect of MKP1 and PAC1 on p38 MAP kinase activation (Example 19). Cells expressing human MKP1 and PAC1 were treated with and without UV radiation, and p38 activity measured. The expression of PAC1 or MKP1 was found to inhibit p38 activity. The inhibitory effect of MKP1 was greater than PAC1. In contrast, cells transfected with a catalytically inactive mutant phosphatase (mutant PAC1 Cys[0085] ²⁵⁷/Ser) did not inhibit p38 MAP kinase. These results demonstrate that p38 can be regulated by dual specificity phosphatases PAC1 and MKP1.
The sub-cellular distribution of p38 MAP kinase was examined by indirect immunofluorescence microscopy (Example 20). Epitope-tagged p38 MAP kinase was detected using the M2 monoclonal antibody. Specific staining of cells transfected with epitope-tagged p38 MAP kinase was observed at the cell surface, in the cytoplasm, and in the nucleus. Marked changes in cell surface and nuclear p38 MAP kinase were not observed following UV irradiation, but an increase in the localization of cytoplasmic p38 MAP kinase to the perinuclear region was detected. [0086]
A series of experiments were conducted to study the activation of JNK by hyper-osmotic media (Example 21). These experiments were reported by Galcheva-Gargova et al. (1994) Science 265:806, hereby specifically incorporated by reference. CHO cells expressing epitope-tagged JNK1 were incubated with 0-1000 mM sorbitol, and JNK1 activity measured in an immune complex kinase assay with the substrate c-Jun. Increased JNK1 activity was observed in cells incubated for 1 hour with 100 mM sorbitol. Increased JNK1 activity was observed within 5 minutes of exposure to 300 mM sorbitol. Maximal activity was observed 15 to 30 minutes after osmotic shock with a progressive decline in JNK1 activity at later times. The activation of JNK by osmotic shock was studied in cells expressing wild-type (Thr[0087] ¹⁸³, Tyr¹⁸⁵) or mutated (Ala¹⁸³, Phe¹⁸⁵) JNK1. JNK1 activity was measured after incubation for 15 minutes with or without 300 mM sorbitol. Cells expressing wild-type JNK1 showed increased JNK1 activity, while cells expressing mutated JNK1 did not. These results demonstrate that the JNK signal transduction pathway is activated in cultured mammalian cells exposed to hyper-osmotic media.
The results of the above-described experiments are illustrated in FIG. 3, which diagrams the ERK, p38, and JNK MAP kinase signal transduction pathways. ERKs are potently activated by treatment of cells with EGF or phorbol esters. In contrast, p38 is only slightly activated under these conditions (Example 15). However, UV radiation, osmotic stress, and inflammatory cytokines cause a marked increase in p38 activity. This difference in the pattern of activation of ERK and p38 suggests that these MAP kinases are regulated by different signal transduction pathways. The molecular basis for the separate identity of these signal transduction pathways is established by the demonstration that the MAP kinase kinases that activate ERK (MEK1 and MEK2) and p38 (MKK3, MKK4, and MKK6) are distinct. [0088]
The isolation of murine and human MKK7 is described in Example 22. Distinctive regions of the Drosophila MAP kinase kinase hep sequence were used to design polymerase chain reaction (PCR) primers. Amplification of murine testis mRNA with these primers resulted in the formation of specific products which were cloned into a plasmid vector and sequenced. One sequence related to hep was identified and used to screen a murine testis library. Five DNAs (cDNAs) that encoded protein kinases were identified: one encoding a MAP protein kinase kinase (MKK7). The others encoded various splice variants: MKK7b (a partial sequence appears in FIG. 11), MKK7c (FIG. 13), MKK7d (FIG. 14), MKK7e (FIG. 15). The deduced amino acid sequences of MKK7 (SEQ ID NO: 18) and hep (SEQ ID NO: 21) are shown in FIG. 9, and compared to the MAP kinase kinases MEK1 (SEQ ID NO: 11), MEK2 (SEQ ID NO: 12), MKK3 (SEQ ID NO: 2), MKK4 (SEQ ID NO: 10), MKK5 (SEQ ID NO: 22), and MKK6 (SEQ ID NO: 4). A human MKK7 was identified by screening a human cDNA library with a full-length (mouse) MKK7 cDNA probe. The identified partial sequence (lacking the 3′ end) is homologous to mouse MKK7c. [0089]
The expression of MKK7 and MKK4 isoforms was examined by Northern (RNA) blot analysis of poly A+ mRNA isolated from eight murine tissues (Example 23). Both protein kinases were found to be widely expressed. [0090]
The substrate specificity of MKK7 was investigated in an in vitro phosphorylation assay with recombinant, epitope-tagged MAP kinases (JNK1, p38, and ERK2) as substrates (Example 24). MKK7 phosphorylated JNK, but did not phosphorylate p38 or ERK2. MKK7 was phosphorylated by p38 and JNK1. [0091]
MKK7 was found to specifically activate JNK protein kinase in vivo (Example 25). CHO cells were co-transfected with an epitope-tagged MAP kinase (JNK1, p38, or ERK2) together with an empty expression vector or an expression vector encoding MKK1, MKK4, MKK6, or MKK7 and the product of the phosphorylation reaction analyzed. MKK7 activated only JNK1, and did so to a greater extent than did MKK4. [0092]
To test whether MKK7 could cause increased AP-1 transcriptional activity, a co-transfection assay was employed (Example 26). Co-expression of MKK7 with JNK caused an increase in AP-1 reporter gene expression that was greater than the increase seen with MKK4 and JNK. A similar result was seen when ATF2 was used as the reporter gene. In addition, MKK7 alone was able to increase expression of ATF2 (FIG. 16). [0093]
MKK isoforms are useful for screening reagents which modulate MKK activity. Described in the Use section following the Examples are methods for identifying reagents capable of inhibiting or activating MKK activity. [0094]
The discovery of human MKK isoforms and MKK-mediated signal transduction pathways is clinically significant for the treatment of MKK-mediated disorders. One use of the MKK isoforms is in a method for screening reagents able to inhibit or prevent the activation of the MKK-MAP kinase-ATF2 pathways. [0095]

EXAMPLES

The following examples are meant to illustrate, not limit, the invention. [0096]

Example 1

MKK Protein Kinases [0097]
The primary sequences of MKK3 and MKK4 were deduced from the sequence of cDNA clones isolated from a human fetal brain library. [0098]
The primers TTYTAYGGNGCNTTYTTYATHGA (SEQ ID NO: 14) and ATBCTYTCNGGNGCCATKTA (SEQ ID NO: 15) were designed based on the sequence of PBS2 (Brewster et al. (1993) Science 259:1760; Maeda et al. (1994) Nature 369:242). The primers were used in a PCR reaction with human brain mRNA as template. Two sequences that encoded fragments of PBS2-related protein kinases were identified. Full-length human cDNA clones were isolated by screening of a human fetal brain library (Dérijard et al. (1995) Science 267:682-685). The cDNA clones were examined by sequencing with an Applied Biosystems model 373A machine. The largest clones obtained for MKK3 (2030 base pairs (bp)) and MKK4 (3576 bp) contained the entire coding region of these protein kinases. [0099]
The primary structures of MKK3 (SEQ ID NO: 2) and MKK4-α (SEQ ID NO: 6) are shown in FIG. 1. An in-frame termination codon is located in the 5═ untranslated region of the MKK3 cDNA, but not in the 5′ region of the MKK4 cDNA. The MKK4 protein sequence presented starts at the second in-frame initiation codon. [0100]
These sequences were compared to those of the human MAP kinase kinases MEK1 (SEQ ID NO : 11) and MEK2 (SEQ ID NO: 12) (Zheng and Guan (1993) J. Biol. Chem 268:11435) and of the yeast MAP kinase kinase PBS2 (SEQ ID NO: 13) (Boguslawaski and Polazzi (1987) Proc. Natl. Acad. Sci. USA 84:5848) (FIG. 1). The identity and similarity of the kinases with human MKK3 (between subdomains I and XI) were calculated with the BESTFIT program (version 7.2; Wisconsin Genetics Computer Group) (percent of identity to percent of similarity): MEK1, 41%/63%; MEK2, 41%/62%; MKK4α, 52%/73%; and PBS2, 40%/59%). The identity and similarity of the kinases with human MKK4α were calculated to be as follows (percent of identity to percent of similarity): MEK1, 44%/63%; MEK2, 45%/61%; MKK3, 52%/73%; and PBS2, 44%/58%. [0101]
The cDNA sequences of MKK3 and MKK4γ have been deposited in GenBank with accession numbers L36719 and L36870, respectively. The MKK4γ cDNA sequence contains both the cDNA sequences of MKK4α and MKK4β, which are generated in vivo from alternate splicing sites. One of ordinary skill in the art can readily determine the amino acid sequences of MKK3 and MKK4 isoforms from the deposited cDNA sequences. [0102]

Example 2

Expression of MKK3 and MKK4 mRNA in Adult Human Tissue [0103]
Northern blot analysis was performed with polyadenylated [poly(A)*] mRNA (2 μg) isolated from human heart, brain, placenta, lung, liver, muscle, kidney, and pancreas tissues. The mRNA was fractionated by denaturing agarose gel electrophoresis and was transferred to a nylon membrane. The blot was probed with the MKK3 and MKK4 cDNA labeled by random priming with [α-[0104] ³²P]ATP (deoxyadenosine triphosphate) (Amersham International PLC). MKK3 and MKK4 were expressed in all tissues examined; the highest expression of MKK3 and MKK4 was found in skeletal muscle tissue.
The relation between members of the human and yeast MAP kinase kinase group is presented as a dendrogram (FIG. 2). MKK3/4 form a unique subgroup of human MAP kinase kinases. [0105]

Example 3

In Vitro Phosphorylation of p38 MAP kinase by MKK3 [0106]
GST-JNK1, and GST-ERK2 have been described (Dérijard et al. (1994) supra; Gupta et al. (1995) Science 267:389; Wartmann and Davis (1994) J. Biol. Chem. 269:6695, each herein specifically incorporated by reference). GST-p38 MAP kinase was prepared from the expression vector pGSTag (Dressier et al. (1992) Biotechniques 13:866) and a PCR fragment containing the coding region of the p38 MAP kinase cDNA. GST-MKK3 and MKK4 were prepared with pGEX3X (Pharmacia-LKB Biotechnology) and PCR fragments containing the coding region of the MKK3 and MKK4 cDNAs. The GST fusion proteins were purified by affinity chromatography with the use of GSH-agarose (Smith and Johnson (1988) Gene 67:31). The expression vectors pCMV-Flag-JNK1 and pCMV-MEK1 have been described (Dérijard et al. (1994) supra; Wartmann and Davis (1994) supra). The plasmid pCMV-Flag-p38 MAP kinase was prepared with the expression vector pCMV5 (Andersson et al. (1989) J. Biol. Chem. 264:8222) and the p38 MAP kinase cDNA. The expression vectors for MKK3 and MKK4 were prepared by subcloning of the cDNAs into the polylinker of pCDNA3 (Invitrogen). The Flag epitope (Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 16); Immunex, Seattle, Wash.) was inserted between [0107] codons 1 and 2 of the kinases by insertional overlapping PCR (Ho et al. (1989) Gene 77:51).
Protein kinase assays were performed in kinase buffer (25 mM 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid, pH 7.4, 25 mM β-glycerophosphate, 25 mM MgCl[0108] ₂, 2 mM dithiothreitol, and 0.1 mM orthovanadate). Recombinant GST-MKK3 was incubated with [γ-³²P]ATP and buffer, GST-JNK1, GST-p38 MAP kinase, or GST-ERK2. The assays were initiated by the addition of 1 μg of substrate proteins and 50 μM [γ-³²P]ATP (10 Ci/mmol) in a final volume of 25 μl. The reactions were terminated after 30 minutes at 25° C. by addition of Laemmli sample buffer. The phosphorylation of the substrate proteins was examined after SDS-polyacrylamide gel electrophoresis (SDS-PAGE) by autoradiography. Phosphoaminoacid analysis was performed by partial acid hydrolysis and thin-layer chromatography (Dérijard et al. (1994) supra; Alvarez et al. (1991) J. Biol. Chem. 266:15277). Autophosphorylation of MKK3 was observed in all groups. MKK3 phosphorylated p38 MAP kinase, but not JNK1 or ERK2.
A similar insertional overlapping PCR procedure was used to replace Thr[0109] ¹⁸⁰and Tyr¹⁸²of p38, with Ala and Phe, respectively. The sequence of all plasmids was confirmed by automated sequencing on an Applied Biosystems model 373A machine. GST-MKK3 was incubated with [γ-³²P]ATP and buffer, wild-type GST-p38 MAP kinase (TGY), or mutated GST-p38 MAP kinase (AGF). The phosphorylated proteins were resolved by SDS-PAGE and detected by autoradiography. Only phosphorylation of wild-type p38 was observed.

Example 4

In Vitro Phosphorylation and Activation of JNK and p38 MAP Kinase by MKK4 [0110]
Protein kinase assays were conducted as described in Example 3. Recombinant GST-MKK4 was incubated with [γ-[0111] ³²P] ATP and buffer, GST-JNK1, GST-p38 MAP kinase, or GST-ERK2. JNK1 and p38 were phosphorylated, as was MKK4 incubated with JNK1 and p38.
GST-MKK4 was incubated with [γ-[0112] ³²P]ATP and buffer, wild-type JNK1 (Thr¹⁸³, Tyr¹⁸⁵), or mutated GST-JNK1 (Ala¹⁸³, Phe¹⁸⁵). The JNK1 substrate ATF2 (Gupta et al. (1995) supra) was included in each incubation. ATF2 was phosphorylated in the presence of MKK4 and wild-type JNK1. The results establish that MKK4 phosphorylates and activates both p38 and JNK1.

Example 5

Phosphorylation and Activation of p38 MAP Kinase by UV-stimulated MKK3 [0113]
Epitope-tagged MKK3 was expressed in COS-1 cells maintained in Dulbecco's modified Eagle's medium supplemented with fetal bovine serum (5%)(Gibco-BRL). The cells were transfected with the lipofectamine reagent according to the manufacturer's recommendations (Gibco-BRL) and treated with UV radiation or EGF as described (Dérijard et al. (1994) supra). [0114]
The cells were exposed in the absence and presence of UV-C (40 J/m[0115] ²). The cells were solubilized with lysis buffer (20 mM tris, pH 7.4, 1% TRITON® X-100, 10% glycerol, 137 mM NaCl, 2 mM EDTA, 25 mM β-glycerophosphate, 1 mM Na orthovanadate, 1 mM phenylmethylsulfonyl fluoride, and leupeptin (10 μg/ml)) and centrifuged at 100,000×g for 15 minutes at 4° C. MKK3 was isolated by immunoprecipitation. The epitope-tagged protein kinases were incubated for 1 hour at 4° C. with the M2 antibody to the Flag epitope (IBI-Kodak) bound to protein G-Sepharose (Pharmacia-LKB Biotechnology). The immunoprecipitates were washed twice with lysis buffer and twice with kinase buffer.
Protein kinase assays were conducted with the substrate GST-p38 MAP kinase or JNK1. ATF2 was included in some assays. Basal levels of MKK3 phosphorylation of p38 MAP kinase were observed. UV-irradiation resulted in increased phosphorylation of p38 MAP kinase, but not of JNK1. The increased p38 MAP kinase activity resulted in increased phosphorylation of ATF2. [0116]

Example 6

Activation of p38 MAP Kinase in Cells Expressing MKK3 and MKK4 [0117]
COS-1 cells were transfected with epitope-tagged p38 MAP kinase, together with an empty expression vector or an expression vector encoding MEK1, MKK3, or MKK4α. Some of the cultures were exposed to UV radiation (40 J/m[0118] ²) or treated with 10 nM EGF. p38 MAP kinase was isolated by immunoprecipitation with M2 monoclonal antibody, and the protein kinase activity was measured in the immunecomplex with [γ-³²P] ATP and ATF2 as substrates. The product of the phosphorylation reaction was visualized after SDS-PAGE by autoradiography. ATF2 was not phosphorylated in the control MEK1, or EGF-treated groups, but was phosphorylated in the MKK3, MKK4, and UV-irradiated groups. MKK3 and MKK4 phosphorylation of ATF2 was similar to that seen with p38 MAP kinase isolated from UV-irradiated cells.

Example 7

Phosphorylation of ATF2 by JNK1 and JNK2 [0119]
COS-1 cells were maintained in Dulbecco's modified Eagle's medium supplemented with bovine serum albumin (5%) (Gibco-BRL). Metabolic labeling with [[0120] ³²]P was performed by incubation of cells for 3 hours in phosphate-free modified Eagle's medium (Flow Laboratories Inc.) supplemented with [³²p] orthophosphate (2 mCi/ml) (Dupont-NEN). COS-1 cells were transfected without (Mock) and with epitope-tagged JNK1 (JNK1). Plasmid expression vectors encoding the JNK1 cDNA have previously been described (Dérijard et al. (1994) Cell 76:1025, herein specifically incorporated by reference). Plasmid DNA was transfected into COS-1 cells by the lipofectamine method (Gibco-BRL). After 48 hours of incubation, some cultures were exposed to 40 J/m²UV radiation and incubated for 1 hour at 37° C.
Cells were lysed in 20 mM Tris, pH 7.5, 25 mM β-glycerophosphate, 10% glycerol, 1% Triton® X-100, 0.5% (w/v) deoxycholate, 0.1% (w/v) SDS, 0.137 M NaCl, 2 mM pyrophosphate, 1 mM orthovanadate, 2 mM EDTA, 10 μg/ml leupeptin, 1 mM PMSF. Soluble extracts were prepared by centrifugation in a microfuge for 20 minutes at 4° C. JNK1 immunoprecipitates were also prepared by reaction with a rabbit antiserum prepared with recombinant JNK1 as an antigen. [0121]
In-gel protein kinase assays were performed with cell lysates and JNK1 immunoprecipitates after SDS-PAGE by renaturation of protein kinases, polymerization of the substrate (GST-ATF2, residues 1-505) in the gel, and incubation with [γ-[0122] ³²P]ATP (Dérijard et al. (1994) supra) The incorporation of [³²P] phosphate was visualized by autoradiography and quantitated with a Phosphorimager and ImageQuant software (Molecular Dynamics Inc., Sunnyvale, Calif.). The cell lysates demonstrate the presence of 46 kD and 55 kD protein kinases that phosphorylate ATF2 in extracts prepared from UV-irradiated cells. The 46 kD and 55 kD protein kinases were identified as JNK1 and JNK2, respectively.

Example 8

Binding of JNK1 to ATF2 and Phosphorylation of the NH[0123] ₂-Terminal Activation Domain
The site of JNK1 phosphorylation of ATF2 was investigated by generation of progressive NH[0124] ₂-terminal domain deletions of ATF2. Plasmid expression vectors encoding ATF2 (pECE-ATF2) (Liu and Green (1994) and (1990)), have been described. Bacterial expression vectors for GST-ATF2 fusion proteins were constructed by sub-cloning ATF2 cDNA fragments from a polymerase chain reaction (PCR) into pGEX-3X (Pharmacia-LKB Biotechnology Inc.). The sequence of all constructed plasmids was confirmed by automated sequencing with an Applied Biosystems model 373A machine. The GST-ATF2 proteins were purified as described (Smith and Johnson (1988) Gene 67:31), resolved by SDS-PAGE and stained with Coomassie blue. GST-ATF2 fusion proteins contained residues 1-505, 1-349, 350-505, 1-109, 20-109, 40-109, and 60-109.
The phosphorylation of GST-ATF2 fusion proteins by JNK1 isolated from UV-irradiated cells was examined in an immunocomplex kinase assay. Immunecomplex kinase assays were performed with Flag epitope-tagged JNK1 and the monoclonal antibody M2 (IBI-Kodak) as described by Dérijard et al. (1994) supra). Immunecomplex protein kinase assays were also performed with a rabbit antiserum prepared with recombinant JNK1 as an antigen. The cells were solubilized with 20 mM Tris, pH 7.5, 10% glycerol, 1% Triton® X-100, 0.137 M NaCl, 25 mM β-glycerophosphate, 2 mM EDTA, 1 mM orthovanadate, 2 mM pyrophosphate, 10 μg/ml leupeptin, and 1 mM PMSF. JNK1 was immunoprecipitated with protein G-Sepharose bound to a rabbit polyclonal antibody to JNK or the M2 monoclonal antibody to the Flag epitope. The beads were washed three times with lysis buffer and once with kinase buffer (20 mM Hepes, pH 7.6, 20 mM MgCl[0125] ₂, 25 mM β-glycerophosphate, 100 μM Na orthovanadate, 2 mM dithiothreitol). The kinase assays were performed at 25° C. for 10 minutes with 1 μg of substrate, 20 μM adenosine triphosphate and 10 μCi of [γ-³²P]ATP in 30 μl of kinase buffer. The reactions were terminated with Laemmli sample buffer and the products were resolved by SDS-PAGE (10% gel). JNK1 phosphorylates GST-ATF2 fusion proteins containing residues 1-505, 1-349, 1-109, 20-109, and 40-109, but not 60-109. These results indicate that the presence of ATF2 residues 1-60 are required for phosphorylation by JNK.
The binding of immobilized GST-ATF2 fusion proteins was examined in a solid-phase kinase assay as described by Hibi et al. ((1993) Genes Dev. 7:2135, herein specifically incorporated by reference). JNK1 from UV-irradiated cells was incubated with GST-ATF2 fusion proteins bound to GSH-agarose. The agarose beads were washed extensively to remove the unbound JNK1. Phosphorylation of the GST-ATF2 fusion proteins by the bound JNK1 protein kinase was examined by addition of [γ-[0126] ³²P]ATP. JNK1 bound GST-ATF2 fusion proteins containing residues 1-505, 1-349, 1-109, 20-109, and 40-109, indicating that the presence of residues 20-60 were required for binding of JNK1 to ATF2.

Example 9

Phosphorylation of the NH[0127] ₂-Terminal Activation Domain of ATF2 on Thr⁶⁹and Thr⁷¹by JNK1
The effect of UV radiation on the properties of wild-type (Thr[0128] ^{69, 71}) and phosphorylation-defective (Ala^{69, 71}) ATF2 molecules was examined. Mock-transfected and JNK1-transfected COS cells were treated without and with 40 J/m²UV radiation. The epitope-tagged JNK1 was isolated by immunoprecipitation with the M2 monoclonal antibody. The phosphorylation of GST-ATF2 (residues 1 to 109) was examined in an immunocomplex kinase assay as described above. The GST-ATF2 was resolved from other proteins by SDS-PAGE and stained with Coomassie blue. The phosphorylation of GST-ATF2 was detected by autoradiography. JNK1-transfected cells, but not mock-transfected cells, phosphorylated ATF2. JNK1 phosphorylation of ATF2 was greater in cells exposed to UV radiation. Phosphorylation of ATF2 by JNK1 was associated with a decreased electrophoretic mobility.
In a separate experiment, GST fusion proteins containing full-length ATF2 ([0129] residues 1 to 505), an NH₂-terminal fragment (residues 1 to 109), and a COOH-terminal fragment (residues 95 to 505) were phosphorylated with JNK1 and the sites of phosphorylation analyzed by phosphoamino acid analysis. The methods used for phosphopeptide mapping and phosphoamino acid analysis have been described (Alvarez et al. (1991) J. Biol. Chem. 266:15277). The horizontal dimension of the peptide maps was electrophoresis and the vertical dimension was chromatography. The NH₂-terminal sites of phosphorylation were identified as Thr⁶⁹and Thr⁷¹by phosphopeptide mapping and mutational analysis. Site-directed mutagenesis was performed as described above, replacing Thr⁶⁹and Thr⁷¹with Ala. Phosphorylation of mutated ATF2 was not observed.

Example 10

Reduced Electrophoretic Mobility of JNK-Activated ATF2 [0130]
CHO cells were maintained in Ham's F12 medium supplemented with 5% bovine serum albumin (Gibco-BRL). Cells were labeled and transfected with JNK1 as described above. CHO cells were treated with UV-C (40 J/m[0131] ²), IL-1α (10 ng/ml) (Genzyme), or fetal bovine serum (20%) (Gibco-BRL). The cells were incubated for 30 minutes at 37° C. prior to harvesting. The electrophoretic mobility of ATF2 after SDS-PAGE was examined by protein immuno-blot analysis. A shift in ATF2 electrophoretic mobility was observed in cells treated with UV, IL-1, and serum. These results indicate that JNK1 activation is associated with an electrophoretic mobility shift of ATF2, further suggesting that ATF2 is an in vivo substrate for JNK1.

Example 11

Increased ATF2 Phosphorylation After Activation of JNK [0132]
COS-1 cells were transfected without (control) and with an ATF2 expression vector (ATF2), as described above (Hai et al. (1989) supra). The effect of exposure of the cells to 40 J/m[0133] ²UV-C was examined. After irradiation, the cells were incubated for 0 or 30 minutes (control) or 0, 15, 30, and 45 minutes (ATF2) at 37° C. and then collected. The electrophoretic mobility of ATF2 during SDS-PAGE was examined by protein immuno-blot analysis as described above. The two electrophoretic mobility forms of ATF2 were observed in ATF2-transfected cells, but not in control cells.
The phosphorylation state of wild-type (Thr[0134] ^{69, 71}) ATF2 and mutated (Ala^{69, 71}) ATF2 was examined in cells labeled with [³²]P, treated without and with 40 J/m²UV-C, and then incubated at 37° C. for 30 minutes (Hai et al. (1989) supra). The ATF2 proteins were isolated by immunoprecipitation and analyzed by SDS-PAGE and autoradiography. The phosphorylated ATF2 proteins were examined by phosphoamino acid analysis as described above. Both forms of ATF2 contained phosphoserine, but only wild-type ATF2 contained phosphothreonine.
Tryptic phosphopeptide mapping was used to compare ATF2 phosphorylated in vitro by JNK1 with ATF2 phosphorylated in COS-1 cells. A map was also prepared with a sample composed of equal amounts of in vivo and in vitro phosphorylated ATF2 (Mix). Mutation of ATF2 at Thr[0135] ⁶⁹and Thr⁷¹resulted in the loss of two tryptic phosphopeptides in maps of ATF2 isolated from UV-irradiated cells. These phosphopeptides correspond to mono- and bis-phosphorylated peptides containing Thr⁶⁹and Thr⁷¹. Both of these phosphopeptides were found in maps of ATF2 phosphorylated by JNK1 in vitro.

Example 12

Inhibition of ATF2-Stimulated Gene Expression by Mutation of the Phosphorylation Sites Thr[0136] ⁶⁹and Thr⁷¹

A fusion protein consisting of ATF2 and the GAL4 DNA binding domain was expressed in CHO cells as described above. The activity of the GAL4-ATF2 fusion protein was measured in co-transfection assays with the reporter plasmid pG5E1bLuc (Seth et al. (1992) J. Biol. Chem. 267:24796, hereby specifically incorporated by reference). The reporter plasmid contains five GAL4 sites cloned upstream of a minimal promoter element and the firefly luciferase gene. Transfection efficiency was monitored with a control plasmid that expresses β-galactosidase (pCH110; Pharmacia-LKB Biotechnology). The luciferase and β-galactosidase activity detected in cell extracts was measured as the mean activity ratio of three experiments (Gupta et al. (1993) Proc. Natl. Acad. Sci. USA 90:3216, hereby specifically incorporated by reference). The results, shown in Table 1, demonstrate the importance of phosphorylation at Thr ⁶⁹and Thr⁷¹for transcriptional activity.

TABLE 1


INHIBITION OF ATF-2 STIMULATED GENE EXPRESSION BY
MUTATION OF THE PHOSPHORYLATION SITES THR^69,71

		LUCIFERASE ACTIVITY
	PROTEIN	(Light Units/OD)

	GAL4	45
	GAL4-ATF2 (wild type)	320,000
	GAL4-ATF2 (Ala⁶⁹)	24,000
	GAL4-ATF2 (Ala⁷¹)	22,000
	GAL4-ATF2 (Ala^69,71)	29,000
	GAL4-ATF2 (Glu⁶⁹)	27,000

Example 13

Effect of Dominant-Negative JNK1 Mutant on ATF2 Function [0138]
The luciferase reporter plasmid system was used to determine the effect of point mutations at the ATF2 phosphorylation sites Thr[0139] ⁶⁹and Thr⁷¹in serum-treated CHO cells transfected with wild-type (Thr¹⁸³, Tyr¹⁸⁵) or mutant (Ala¹⁸³, Phe¹⁸⁵) JNK1. Control experiments were done with mock-transfected cells. The CHO cells were serum-starved for 18 hours and then incubated without or with serum for 4 hours. Expression of wild-type ATF2 caused a small increase in serum-stimulated ATF2 transcriptional activity. In contrast, mutant JNK1 inhibited both control and serum-stimulated ATF2 activity.

Example 14

Effect of Tumor Suppressor Gene Product Rb and Adenovirus Oncoprotein E1A on ATF2-Stimulated Gene Expression [0140]
The effect of expression of the Rb tumor suppressor gene product and adenovirus oncoprotein E1A on ATF2 transcriptional activity were investigated with a luciferase reporter plasmid and GAL4-ATF2 (residues 1-505), as described above. Cells were transfected with wild-type (Thr[0141] ^{69, 71}) or mutated (Ala^{69, 71}) ATF2. No effect of Rb or E1A on luciferase activity was detected in the absence of GAL4-ATF2. Rb and E1A were found to increase ATF2-stimulated gene expression of both wild-type and mutated ATF2. However, mutated ATF2 caused a lower level of reporter gene expression than did wild-type ATF2. These results indicate a requirement for ATF2 phosphorylation (on Thr⁶⁹and Thr⁷¹) plus either Rb or E1A for maximal transcriptional activity.

Example 15

Substrate Specificity of p38 MAP Kinase [0142]
Substrate phosphorylation by p38 MAP kinase was examined by incubation of bacterially-expressed p38 MAP kinase with IκB, cMyc, EGF-R, cytoplasmic phospholipase A[0143] ₂(cPLA₂), c-jun, and mutated ATF2 (Thr^{69, 71}) and ATP(γ-³²P] (Raingeaud et al. (1995) J. Biol. Chem 270:7420, herein specifically incorporated by reference). GST-IκB was provided by Dr D. Baltimore (Massachusetts Institute of Technology). GST-cMyc (Alvarez et al. (1991) J. Biol. Chem. 266:15277), GST-EGF-R (residues 647-688) (Koland et al. (1990) Biochem. Biophys. Res. Commun. 166:90), and GST-c-Jun (Dérijard et al. (1994) supra) have been described. The phosphorylation reaction was terminated after 30 minutes by addition of Laemmli sample buffer. The phosphorylated proteins were resolved by SDS-PAGE and detected by autoradiography. The rate phosphorylation of the substrate proteins was quantitated by PhosphorImager (Molecular Dynamics Inc.) analysis. The relative phosphorylation of ATF2, MBP, EGF-R, and IκB was 1.0, 0.23, 0.04, and 0.001, respectively.

Example 16

Binding of p38 MAP Kinase to ATF2 [0144]
Cell extracts expressing epitope-tagged JNK1 and p38 MAP kinase were incubated with a GST fusion protein containing the activation domain of ATF2 (residues 1-109) immobilized on GSH agarose. The supernatant was removed and the agarose was washed extensively. Western blot analysis of the supernatant and agarose-bound fractions was conducted as follows: proteins were fractionated by SDS-PAGE, electrophoretically transferred to an Immobilon-P membrane, and probed with monoclonal antibodies to phosphotyrosine (PY20) and the Flag epitope (M2). Immunocomplexes were detected using enhanced chemiluminescence (Amersham International PLC). Control experiments were performed using immobilized GST. [0145]

Example 17

p38 MAP Kinase and JNK1 Activation by Pro-Inflammatory Cytokines and Environmental Stress [0146]
The effect of phorbol ester, EGF, UV radiation, osmotic stress, IL-1, tumor necrosis factor (TNF), and LPS on p38 MAP kinase and JNK1 activity were measured in immunecomplex protein kinase assays using ATP [γ-[0147] ³²P] and ATF2 as substrates. TNFα and IL-1α were from Genzyme Corp. Lipolysaccharide (LPS) was isolated from lyophilized Salmonella minesota Re595 bacteria as described (Mathison et a. (1988) J. Clin. Invest. 81:1925). Phorbol myristate acetate was from Sigma. EGF was purified from mouse salivary glands (Davis (1988) J. Biol. Chem. 263:9462). Kinase assays were performed using immunoprecipitates of p38 and JNK. The immunocomplexes were washed twice with kinase buffer (described above), and the assays initiated by the addition of 1 μg of ATF2 and 50 μM [γ-³²P]ATP (10 Ci/mmol) in a final volume of 25 μl. The reactions were terminated after 30 minutes at 30° C. by addition of Laemmli sample buffer. The phosphorylation of ATF2 was examined after SDS-PAGE by autoradiography, and the rate of ATF2 phosphorylation quantitated by PhosphorImager analysis.

The results are shown in Table 2. Exposure of HeLa cells to 10 nM phorbol myristate acetate very weakly activated p38 and JNK1. Similarly, treatment with 10 nM EGF only weakly activated p38 and JNK1. By contrast, treatment with 40 J/m ²UV-C, 300 mM sorbitol, 10 ng/ml interleukin-1, and 10 ng/ml TNFα strongly activated p38 and JNK1 activity. The effect of LPS on the activity of p38 was examined using CHO cells that express human CD14. Exposure of CHO cells to 10 ng/ml LPS only slightly activated p38 and JNK1 activity.

TABLE 2


p38 AND JNK1 ACTIVATION BY PRO-INFLAMMATORY
CYTOKINES AND ENVIRONMENTAL STRESS.
Relative Protein Kinase Activity

	JNK	p38

Control	1.0	1.0
Epidermal Growth Factor (10 nM)	1.9	2.1
Phorbol Ester (10 nM)	2.3	2.9
Lipopolysaccharide (10 ng/ml)	3.6	3.7
Osmotic Shock (300 mM sorbitol)	18.1	4.2
Tumor Necrosis Factor (10 ng/ml)	19.3	10.3
Interleukin-1 (10 ng/ml)	8.9	6.2
UV (40 J/M²)	7.4	17.1

Example 18

p38 MAP Kinase Activation by Dual Phosphorylation on Tyr and Thr [0149]
COS-1 cells expressing wild-type (Thr[0150] ¹⁸⁰, Tyr¹⁸²) or mutated (Ala¹⁸⁰, Phe¹⁸²) p38 MAP kinase were treated without and with UV-C (40 J/m²). The cells were harvested 30 minutes following exposure with or without UV radiation. Control experiments were performed using mock-transfected cells. The level of expression of epitope-tagged p38 MAP kinase and the state of Tyr phosphorylation of p38 MAP kinase was examined by Western blot analysis using the M2 monoclonal antibody and the phosphotyrosine monoclonal antibody PY20. Immune complexes were detected by enhanced chemiluminescence.
Wild-type and mutant p38 were expressed at similar levels. Western blot analysis showed that UV radiation caused an increase in the Tyr phosphorylation of p38. The increased Tyr phosphorylation was confirmed by phosphoamino acid analysis of p38 isolated from ([0151] ³²P]phosphate-labeled cells. The results also showed that UV radiation increased Thr phosphorylation of p38. The increased phosphorylation on Tyr and Thr was blocked by mutated p38. Wild-type and mutated p38 were isolated from the COS-1 cells by immunoprecipitation. Protein kinase activity was measured in the immune complex using [γ-³²P]ATP and GST-ATF2 as substrates. The phosphorylated GST-ATF2 was detected after SDS-PAGE by autoradiography. UV radiation resulted in a marked increase in the activity of wild-type p38, while the mutant p38 was found to be catalytically inactive. These results show that p38 is activated by dual phosphorylation within the Thr-Gly-Tyr motif.

Example 19

MAP Kinase Phosphatase Inhibits p38 MAP Kinase Activation [0152]
The cells were treated without and with 40 J/m[0153] ²UV-C. Control experiments were performed using mock-transfected cells (control) and cells transfected with the catalytically inactive mutated phosphatase mPAC1 (Cys²⁵⁷/Ser) and human MKP1. The activity of p38 MAP kinase was measured with an immunecomplex protein kinase assay employing [γ-³²P]ATP and GST-ATF2 as substrates. The expression of PAC1 or MKP1 was found to inhibit p38 phosphorylation, demonstrating that p38 can be regulated by the dual specificity phosphatases PAC1 and MKP1.

Example 20

Subcellular Distribution of p38 MAP Kinase [0154]
Epitope-tagged p38 MAP kinase was expressed in COS cells. The cells were treated without or with 40 J/m[0155] ²UV radiation and then incubated for 60 minutes at 37° C. The p38 MAP kinase was detected by indirect immunofluorescence using the M2 monoclonal antibody. The images were acquired by digital imaging microscopy and processed for image restoration.
Immunocytochemistry [0156]
Coverslips (22mm×22mm No. 1; Gold Seal Cover Glass; Becton-Dickinson) were pre-treated by boiling in 0.1 N HCl for 10 minutes, rinsed in distilled water, autoclaved and coated with 0.01% poly-L-lysine (Sigma; St. Louis Mo.). The coverslips were placed at the bottom of 35 mm multiwell tissue culture plates (Becton Dickinson, UK). Transfected COS-1 cells were plated directly on the coverslips and allowed to adhere overnight in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum (Gibco-BRL). Twenty-four hours post-transfection, the cells were rinsed once and incubated at 37° C. for 30 minutes in 25 mM Hepes, pH 7.4, 137 mM NaCl, 6 mM KCl, 1 MM MgCl[0157] ₂, 1 mM CaCl₂, 10 mM glucose. The cells were rinsed once with phosphate-buffered saline and the coverslips removed from the tissue culture wells. Cells were fixed in fresh 4% paraformaldehyde in phosphate-buffered saline for 15 minutes at 22° C. The cells were permeabilized with 0.25% Triton® X-100 in phosphate-buffered saline for 5 minutes and washed three times in DWB solution (150 mM NaCl, 15 mM Na citrate, pH 7.0, 2% horse serum, 1% (w/v) bovine serum albumin, 0.05% Triton® X-100) for 5 minutes. The primary antibody (M2 anti-FLAG monoclonal antibody, Eastman-Kodak Co., New Haven, Conn.) was diluted 1:250 in DWB and applied to the cells in a humidified environment at 22° C. for 1 hour. The cells were again washed three times as above and fluorescein isothiocyanate-conjugated goat anti-mouse Ig secondary antibody (Kirkegaard & Perry Laboratories Inc. Gaithersburg, Md.) was applied at a 1:250 dilution for 1 hour at 22° C. in a humidified environment. The cells were then washed three times in DWB and then mounted onto slides with Gel-Mount (Biomeda Corp. Foster City, Calif.) for immunofluorescence analysis. Control experiments were performed to assess the specificity of the observed immunofluorescence. No fluorescence was detected when the transfected cells were stained in the absence of the primary M2 monoclonal antibody, or mock-transfected cells.
Digital Imaging Microscopy and Image Restoration [0158]
Digital images of the fluorescence distribution in single cells were obtained using a [0159] Nikon 60× Planapo objective (numerical aperture=1.4) on a Zeiss IM-35 microscope equipped for epifluorescence as previously described (Carrington et al. (1990) in: Non-invasive Techniques in Cell Biology, Fosbett & Grinstein, eds., Wiley-Liss, NY; pp. 53-72; Fay et al. (1989) J. Microsci. 153:133-149). Images of various focal planes were obtained with a computer controlled focus mechanism and a thermoelectrically cooled charged-coupled device camera (model 220; Photometrics Ltd., Tucson, Ariz.). The exposure of the sample to the excitation source was determined by a computer-controlled shutter and wavelength selector system (MVI, Avon, Mass.). The charge-coupled device camera and microscope functions were controlled by a microcomputer, and the data acquired from the camera were transferred to a Silicon Graphics model 4D/GTX workstation (Mountainview, Calif.) for image processing. Images were corrected for non-uniformities in sensitivity and for the dark current of the charge coupled device detector. The calibration of the microscopy blurring was determined by measuring the instrument's point spread function as a series of optical sections at 0.125 μm intervals of a 0.3 μm diameter fluorescently labeled latex bead (Molecular Probes Inc.). The image restoration algorithm used is based upon the theory of ill-posed problems and obtains quantitative dye density values within the cell that are substantially more accurate than those in an unprocessed image (Carrington et al. (1990) supra; Fay et al. (1989) supra). After image processing, individual optical sections of cells were inspected and analyzed using computer graphics software on a Silicon Graphics workstation. p38 MAP kinase was observed at the cell surface, in the cytoplasm, and in the nucleus. After irradiation, an increased localization of cytoplasmic p38 to the perinuclear region was detected.

Example 21

Activation of the MKK Signal Transduction Pathway by Osmotic Shock [0160]
CHO cells were co-transfected with the plasmid pCMV-Flag-Jnk1 and pRSV-Neo (Dérijard et al. (1994) supra). A stable cell line expressing epitope-tagged Jnk1 (Flag; Immunex Corp.) was isolated by selection with Geneticin (Gibco-BRL). The cells were incubated with 0, 100, 150, 300, 600, or 1000 mM sorbitol for 1 hour at 37° C. The cells were collected in lysis buffer (20 mM Tris, pH 7.4, 1% TRITON® X-100, 2 mM EDTA, 137 mM NaCl, 25 mM β-glycerophosphate, 1 mM orthovanadate, 2 mM pyrophosphate, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin) and a soluble extract was obtained by centrifugation at 100,000 g for 30 minutes at 4° C. The[0161] 0 epitope-tagged JNK1 was isolated by immunoprecipitation with the monoclonal antibody M2 (Immunex Corp.). The immunoprecipitates were washed extensively with lysis buffer. Immunecomplex kinase assays were done in 25 μl of 25 mM Hepes, pH 7.4, 25 mM MgCl₂, 25 mM β-glycerophosphate, 2 mM dithiothreitol, 100 μM orthovanadate, and 50 μM ATP [γ-³²P] (10 Ci/mmole) with 2.5 μg of bacterially expressed c-Jun (residues 1-79) fused to glutathione-S-transferase (GST) as a substrate. The phosphorylation of c-Jun was examined after SDS-PAGE by autoradiography and PhosphorImager (Molecular Dynamics Inc.) analysis. JNK1 activation was observed at all concentrations of sorbitol exposure.
The time course of JNK1 protein kinase activation was measured in cells incubated in medium supplemented with 300 mM sorbitol as described above. Increased JNK1 activity was observed within 5 minutes of exposure to sorbitol, with maximum activity occurring after 15-30 minutes. [0162]
Mutation of JNK1 at the phosphorylation sites Thr[0163] ¹⁸³and Tyr¹⁸⁵blocked the activation of JNK1 protein kinase activity by osmotic shock. CHO cells were transfected with vector, wild-type JNK1 (Thr¹⁸³, Tyr¹⁸⁵), and mutated JNK1 (Ala¹⁸³, Phe¹⁸⁵). The cells were incubated in medium supplemented without or with 300 mM sorbitol for 15 minutes before measurement of JNK1 protein kinase activity as described above. JNK1 activation was seen in the wild-type but not mutated JNK1.

Example 22

Molecular Cloning of MKK7 [0164]
RT-PCR was employed to identify a fragment of a novel mammalian MAP kinase kinase. The primers designed for the protocol, ATNGCNGTNAARCARATG (SEQ ID NO; 23) and ATNCKYTCNGGNGCCATRTA (SEQ ID NO: 24), were based on the sequence of the Drosophila MAP kinase kinase hep (Glise et al. (1995) Cell 83:451-461). Murine testis mRNA was used as the template. A single product (461 bp) was detected following RT-PCR amplification of murine testis mRNA. Sequence analysis identified this PCR product as a fragment of a novel mammalian MAP kinase kinase. Full-length murine cDNA clones were isolated by screening a murine testis library (Stratagene Inc.). The cDNA clones were examined by sequencing with an Applied Biosystems model 373A machine. A group of seven clones was identified by sequence analysis to contain a single long open reading frame that encoded a putative protein kinase (FIG. 9 and FIG. 10; SEQ ID NO: 17 and SEQ ID NO: 18). In-frame termination codons were detected in the 5′ and 3′ regions of these clones. This sequence includes protein kinase sub-domains I-XI and is related to the MAP kinase kinase group. The novel protein kinase was designated MKK7. The sites of activating phosphorylation of MAP kinase kinases located in sub-domain VIII are conserved in MKK7. Comparison of MKK7 with other members of the mammalian MAP kinase kinase group demonstrates that MKK7 is related to the JNK activator MKK4. [0165]
One additional cDNA clone isolated from the λ phage library differed from the other seven clones. This clone contained the same 3′ untranslated region and coding region of MKK7, but had a different 5′ region that lacked an in-frame termination codon. This clone represents an alternatively spliced form of MKK7 (MKK7b; FIG. 11; SEQ ID NO: 19). The MKK7b cDNA clone does not have an initiation codon in the alternative 5′ region; this cDNA therefore encodes the same MKK7 protein kinase as the other clones that were isolated. However, if the MKK7b cDNA clone is not full-length it is possible that additional 5′ sequence may include an in-frame initiation codon. If true, MKK7b is predicted to fuse the sequence M-[?]-SPAPAPSQRAALQLPLANDGGSRSPSSESSPQHPTPPTRPRH-(SEQ ID NO: 33) to the initiating methionine of MKK7 (FIG. 9). Although the Drosophila MAP kinase kinase hep shares substantial sequence similarity with MKK7, the sequence of the NH2-terminal extension of MKK7b is not conserved in the hep protein kinase. Three additional clones encoded MKK7 splice variants that differ in the 5′ and 3′ regions. These clones (MKK7c (FIG. 13), MKK7d (FIG. 14), and MKK7e (FIG. 15)) are full-length because of the presence of in-frame termination codons in the 5′ and 3′ regions. [0166]
A human cDNA library was screened with a full-length mouse MKK7 cDNA probe. A single clone was identified and squenced. A partial MKK7 sequence was identified (FIG. 12; SEQ ID NO: 25 and SEQ ID NO: 26) that is missing the 3′ end. The sequence is most homologous to mouse MKK7c. [0167]
The sequences of MKK7, MKK7b, hep, and human MKK7 cDNAs have been deposited in Genbank with accession numbers U93030, U93031, U93032, and AF00319 respectively. [0168]

Example 23

Expression of MKK7 [0169]
MKK7 expression was examined by Northern blot analysis of mRNA isolated from different tissues. The analysis was done with poly A+ mRNA (2 μg) isolated from different tissues and fractionated by denaturing agarose gel electrophoresis and transferred to a nylon membrane (Clontech). The blot was probed with MKK4 and MKK7 cDNAs labeled by random priming with [α-[0170] ³²P]dATP (Amersham International PLC).
MKK7 was found to be widely expressed in murine tissues. A single MKK7 transcript (approximately 4.0-kb) was detected in all of the tissues examined, except for testis where two MKK7 transcripts (4.0 kb and 1.6 kb) were detected. The highest levels of MKK7 expression were in testis. Significant expression of MKK7 was also observed in heart, brain, lung, liver, and kidney. This contrasts with MKK4 expression which was highest in brain although significant amounts of expression were observed in brain, liver, muscle, heart, and kidney. Although MKK4 and MKK7 are co-expressed, the relative abundance of each MAP kinase kinase is different in each of the tissues examined. [0171]

Example 24

Specific Activation of JNK by MKK7 in vitro [0172]
To examine the specificity of MKK7, in vitro protein kinase assays were performed. A bacterial MKK7 expression vector was prepared by sub-cloning an MKK7 cDNA (Eco RI and Pvu II fragment) into the Eco RI and Sma I sites of pGEX-5X1 (Pharmacia-LKB). The glutathione-S-transferase (GST) fusion protein was purified by affinity chromatography (Smith and Johnson (1988) Gene 67:31-40). The recombinant proteins GST-ATF2 (Gupta et al. (1995) Science 267:389-393), GST-cJun (Dérijard (1994) supra), GST-cMyc (Alvarez et al. (1991) J. Biol. Chem. 266:15277-15285), GST-ERK2 (Seth et al. (1992) J. Biol. Chem. 267:24796-24804), GST-p38, (Raingeaud et al. (1995) J. Biol. Chem. 270:7420-7426), and GST-JNK1 (Dérijard (1994) supra) have been described. [0173]
Protein kinase assays were performed in kinase buffer (25 mM 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid (pH 7.4), 25 mM β-glycerophosphate, 25 mM MgCl[0174] ₂, 2 mM dithiothreitol, 0.1 mM orthovanadate). The assays were initiated by the addition of 1 μg of substrate proteins and 50 μM [γ-32P]ATP (10 Ci/mmol) in a final volume of 25 μl. The reactions were terminated after 30 minutes at 25° C. by addition of Laemmli sample buffer. The phosphorylation of the substrate proteins was examined after SDS-polyacrylamide gel electrophoresis (PAGE) by autoradiography.
Recombinant MAP kinases were incubated with GST (control) or GST-MKK7 using the substrate ATP[γ-[0175] ³²P]. Recombinant MKK7 purified from bacteria was not observed to autophosphorylate. Incubation of the recombinant MKK7 with MAP kinases demonstrated that MKK7 phosphorylated JNK1, but not p38 or ERK2. MKK7 was phosphorylated by p38 and JNK1. The significance of the retrophosphorylation of the MAP kinase kinase by the MAP kinase is unclear, but similar retrophosphorylation has been detected in kinase assays using MKK4 (Dérijard (1995) supra) and the Drosophila JNK activator hep (Sluss (1996) supra).
To test whether the phosphorylation of JNK1 by MKK7 caused increased protein kinase activity, experiments using ATF2 as the JNK substrate were performed. GST-MKK7 was incubated in a protein kinase assay with recombinant JNK1. JNK activity was measured by including the JNK substrate ATF2 in each assay. ATF2 was not phosphorylated by MKK7, but was weakly phosphorylated by JNK1. Incubation of MKK7 with JNK1 caused phosphorylation of JNK1 and a large increase in ATF2 phosphorylation. These data indicate that MKK7 phosphorylates and activates JNK1. To confirm this conclusion, the effect of replacement of the JNK dual phosphorylation motif Thr-Pro-Tyr with Ala-Pro-Phe was examined. MKK7 did not phosphorylate the mutated JNK1 (APF) protein. Furthermore, MKK7 did not increase ATF2 phosphorylation by the mutated JNK1 protein kinase. Thus, MKK7 is a JNK activator in vitro. [0176]

Example 25

Specific Activation of JNK by MKK7 In Vivo [0177]
To examine the specificity of MKK7 in vivo, cotransfection assays were performed. CHO cells were maintained in Dulbecco's modified Eagle's medium supplemented with fetal calf serum (5%; Gibco-BRL). The cells were transfected with the lipofectamine reagent according to the manufacturer's recommendations (Gibco-BRL) (Dérijard (1994) supra). Cells were co-transfected with vectors encoding epitope-tagged JNK1 together with an empty expression vector (control) or an expression vector encoding MKK4 or MKK7. The epitope tag was derived from the hemagglutinin protein (HA) of the influenza virus. JNK1 was isolated by immunoprecipitation of cell lysates. The cells were solubilized with lysis buffer (20 mM Tris (pH 7.4), 1% TRITON X-100®, 10% glycerol, 137 mM NaCl, 2 mM EDTA, 25 mM β-glycerophosphate, 1 mM Na orthovanadate, 2 mM pyrophosphate, 1 mM PMSF, 10 μg/ml leupeptin) and centrifuged at 100,000×g for 15 minutes at 4° C. The epitope-tagged protein kinases were immunoprecipitated by incubation for 3 hours at 4° C. with an anti-HA monoclonal antibody bound to protein-G Sepharose (Pharmacia-LKB Biotechnology Inc.). The immunoprecipitates were washed three times with lysis buffer (Gupta et al. (1995) Science 267:389-393). Protein kinase activity was measured in the immunecomplex with [γ-[0178] ³²P]ATP and c-Jun as substrates. The product of the phosphorylation reaction was visualized after SDS-PAGE by autoradiography. The ERK2 and p38 MAP kinases were not activated by co-expressed MKK7. Control experiments demonstrated that the ERK2 and p38 MAP kinases were activated by their respective cognate MAP kinase kinases, MKK1 and MKK6. In contrast, MKK7 did activate JNK1. Interestingly, the activation of JNK1 by co-expressed MKK7 was greater than that caused by the previously described JNK activator MKK4. Together, these data establish that MKK7 can function as a specific activator of JNK in cultured cells.

Example 26

Activation of the JNK Signal Transduction Pathway by MKK7 [0179]
The JNK signaling pathway is known to regulate AP-1 transcriptional activity (Whitmarsh (1996) supra). To test the hypothesis that the expression of MKK7 would cause increased AP-1 transcriptional activity, a co-transfection assay was employed using a luciferase reporter gene that contains three AP-1 sites cloned upstream of a minimal promoter element (Rincon and Flavell (1994) EMBO J. 13:4370-4381). Luciferase reporter gene expression was measured in co-transfection assays using the 0.5 μg of the reporter plasmid pTRE-luciferase (Rincon (1994) supra) and 0.25 μg of the β-galactosidase expression vector pCH110 (Pharmacia-LKB). Experiments using GAL4 fusion proteins were performed using 0.25 μg of pGAL4-ATF2 (residues 1-109), 0.5 μg of the reporter plasmid pG5E1bLuc, and 0.25 μg of pCH110 (Gupta et al. (1995) supra). The effect of protein kinases was examined by co-transfection with 0.3 μg of an empty expression vector or a protein kinase expression vector. The ERK2, p38, JNK1, MKK1, MKK3, MKK4, and MKK6 expression vectors have been described. The cells were harvested 36 hours post-transfection. The β-galactosidase and luciferase activity in the cell lysates was measured as described (Gupta (1995) supra). Expression of MKK4, MKK7, or JNK1 did not cause marked changes in AP-1 reporter gene expression (FIG. 16A). In contrast, co-expression of MKK7 with JNK1 caused increased AP-1-dependent reporter gene expression. Consistent with the observation that MKK4 causes weaker activation of JNK than MKK7, co-expression of MKK4 with JNK caused a smaller increase in AP-1 reporter gene expression (FIG. 16A). Together, these data demonstrate that MKK7 can function as an activator of the JNK signal transduction pathway. [0180]
To further examine the effect of MKK7 on transcriptional activity, the effect of MKK7 on the transcription factor ATF2 was investigated. Previous studies have demonstrated that ATF2 is a target of the JNK signal transduction pathway (van Dam et al. (1995) supra; Gupta et al. (1995) supra; Livingstone et al (1995) supra). JNK phosphorylates two sites (Thr-69 and Thr-71) in the NH[0181] ₂-terminal activation domain of ATF2 and increases transcriptional activity. A GAL4 fusion protein strategy was employed to monitor the transcriptional activity of the activation domain of ATF2 (Gupta (1995) supra). Measurement of reporter gene expression demonstrated that the co-expression of MKK4 with JNK1 caused increased transcriptional activity (FIG. 16B). A similar level of reporter gene expression was caused by expression of MKK7 and a larger increase was detected when MKK7 was co-expressed with JNK1. The more potent effect of MKK7, compared with MKK4, on transcriptional activity is consistent with the relative effects of MKK7 and MKK4 on JNK activation. To confirm that the increased reporter gene expression was mediated by ATF2 phosphorylation, the effect of replacement of the sites of ATF2 phosphorylation (Thr-69 and Thr-71) with Ala was examined. The mutated ATF2 protein was not regulated by MKK4, MKK7, or JNK1 (FIG. 16B). Together, these data demonstrate that MKK7 can regulate a physiological target of the JNK signaling pathway.
Use [0182]
The MKK polypeptides and polynucleotides of the invention are useful for identifying reagents that modulate the MKK signal transduction pathways. Reagents that modulate an MKK signal transduction pathway can be identified by their effect on MKK synthesis, MKK phosphorylation, or MKK activity. For example, the effect of a reagent on MKK activity can be measured by the in vitro kinase assays described above. MKK is incubated without (control) and with a test reagent under conditions sufficient to allow the components to react, then the effect of the test reagent on kinase activity is subsequently measured. Reagents that inhibit an MKK signal transduction pathway can be used in the treatment of MKK-mediated disorders. Reagents that stimulate an MKK signal transduction pathway can be used in a number of ways, including induction of programmed cell death (apoptosis) in tissues. For example, the elimination of UV damaged cells can be used to prevent cancer. [0183]
Generally, for identification of a reagent that inhibits the MKK signal transduction pathway, a kinase assay (see, for example, Example 3) is used. A range of reagent concentrations (e.g., 1.0 nM to 100 mM) are added to a test system that includes an MKK substrate and a radioactive marker such as [γ-[0184] ³²P]ATP. Appropriate substrate molecules include p38, JNK1, JNK2, or ATF2. The incorporation of labelled phosphorus (e.g., [³²]p or [³³]P) into the substrate is determined, and the results obtained with the test reagent compared to control values. Of particular interest are reagents that result in inhibition of [³²]P incorporation of about 80% or more. Phosphorylation may also be examined using a reagent that is phosphorylation-dependent, for example, an antibody. Phosphorylation-dependent antibodies may be made using MKK7 phosphorylated on the activating sites, Ser¹⁹⁸and Thr²⁰². This may be accomplished by immunizing animals with a synthetic peptide (for example, approximately 15 amino acids in length) corresponding to the MKK7 sequence with phosphorylated Ser¹⁹⁸and Thr²⁰². Methods of producing such antibodies are known in the art. Such antibodies are useful for the detection of activated MKK7 in tissues and cell extracts (e.g. on Western blots) and may be used in a kit.
Assays that test the effect of a reagent on MKK synthesis can also be used to identify compounds that inhibit MKK signal transduction pathways. The effect of the test reagent on MKK expression is measured by, for example, Western blot analysis with an antibody specific for an MKK. Antibody binding is visualized by autoradiography or chemiluminescence, and is quantitated. The effect of the test reagent on MKK mRNA expression can be examined, for example, by Northern blot analysis using a polynucleotide probe or by polymerase chain reaction. [0185]
Reagents found to inhibit MKK signal transduction pathways can be used as therapeutic agents for the treatment of MKK-mediated disorders. Such reagents are also useful in drug design for elucidation of the specific molecular features needed to inhibit MKK signal transduction pathways. [0186]
In addition, the invention provides a method for the treatment of MKK-mediated stress-related and inflammatory disorders. The method includes administration of an effective amount of a therapeutic reagent that inhibits MKK function. Suitable reagents inhibit either MKK activity or expression. The concentration of the reagent to be administered is determined based on a number of factors, including the appropriate dosage, the route of administration, and the specific condition being treated. The appropriate dose of a reagent is determined by methods known to those skilled in the art including routine experimentation to optimize the dosage as necessary for the individual patient and specific MKK-mediated disorder being treated. Specific therapeutically effective amounts appropriate for administration are readily determined by one of ordinary skill in the art (see, for example, [0187] Remington's Pharmaceutical Sciences. 18th ed., Gennaro, ed., Mack Publishing Company, Easton, Pa., 1990). Dosages may range from about 0.1-10 mg/kilo/day.
The invention provides methods for both acute and prophylactic treatment of stress-related and inflammatory disorders. For example, it is envisioned that ischemic heart disease will be treated during episodes of ischemia and oxidative stress following reperfusion. In addition, a patient at risk for ischemia can be treated prior to ischemic episodes. [0188]
In another example, a therapeutic agent that inhibits MKK function or activity is administered to control inflammatory responses by inhibiting the secretion of inflammatory cytokines, including TNF and IL-1. [0189]
Stress-related proliferative disorders can also be treated by the method of the invention by administering a therapeutic reagent that inhibits MKK function or activity. Such therapeutic reagents can be used alone or in combination with other therapeutic reagents, for example, with chemotherapeutic agents in the treatment of malignancies. Indeed, the control of stress-activated MKK by the therapeutic reagents provided by this invention can modulate symptoms caused by other therapeutic strategies that induce stress. [0190]
The therapeutic reagents employed are compounds which inhibit MKK function or activity, including polynucleotides, polypeptides, and other molecules such as antisense oligonucleotides and ribozymes, which can be made according to the invention and techniques known to the art. Polyclonal or monoclonal antibodies (including fragments or derivatives thereof) that bind epitopes of MKK also can be employed as therapeutic reagents. Dominant-negative forms of MKK which effectively displace or compete with MKK for substrate binding and/or phosphorylation can be used to decrease protein kinase activity. Dominant-negative forms can be created by mutations within the catalytic domain of the protein kinases, using methods known in the art, and as described above (Example 13). The catalytic residues are conserved in all the MKK isoforms. For example, mutation of Lys[0191] ⁷⁶inhibits MKK7 activity. Similarly, mutation of the conserved sites of activating phosphorylation (Ser¹⁹⁸, Thr²⁰²) inhibits MKK7 activity. These kinase-inactive forms of MKK7 act as dominant-negative inhibitors.
In some cases, augmentation of MKK activity is desirable, e.g., induction of apoptosis. The methods of the invention can be used to identify reagents capable of increasing MKK function or activity. Alternatively, increased activity is achieved by over-expression of MKK. When an MKK-mediated disorder is associated with under-expression of MKK, or expression of a mutant MKK polypeptide, a sense polynucleotide sequence (the DNA coding strand) or MKK polypeptide can be introduced into the cell to enhance normal MKK activity. If necessary, these treatments are targeted to specific cells by their mode of administration. (e.g., by use of cell-type specific viral vectors), or by placing MKK7 nucleic acids in recombinant constructs with cell-type specific or inducible promoters by methods known in the art. For example, MKK7 nucleic acid-containing vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the MKK7 nucleic acid can be by any promoter known in the art to act in mammalian, preferably human cells. Such promoters can be inducible or constitutive. Such promoters include, but are not limited to: the SV40 early promoter region (Bernoist et al., [0192] Nature 290:304, 1981); the promoter contained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al., Cell 22:787-797, 1988); the herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441, 1981); or the regulatory sequences of the metallothionein gene (Brinster et al., Nature 296:39, 1988).
The antibodies of the invention can be administered parenterally by injection or by gradual infusion over time. The monoclonal antibodies of the invention can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally. [0193]
Preparations for parenteral administration of a polypeptide or an antibody of the invention include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose) and the like. Preservatives and other additives can also be present, such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases, and the like. [0194]
Polynucleotide sequences, including antisense sequences, can be therapeutically administered by various techniques known to those skilled in the art. Such therapy would achieve its therapeutic effect by introduction of the MKK polynucleotide into cells of mammals having a MKK-mediated disorder. Delivery of MKK polynucleotides can be achieved using free polynucleotide or a recombinant expression vector such as a chimeric virus or a colloidal dispersion system. Especially preferred for therapeutic delivery of nucleotide sequences is the use of targeted liposomes. [0195]
Targeting of the therapeutic reagent to specific tissues is desirable to increase the efficiency of delivery. The targeting can be achieved by passive mechanisms via the route of administration. Active targeting to specific tissues can also be employed. The use of liposomes, colloidal suspensions, and viral vectors allows targeting to specific tissues by changing the composition of the formulation containing the therapeutic reagent, for example, by including molecules that act as receptors for components of the target tissues. Examples include sugars, glycoplipids, polynucleotides, or proteins. These molecules can be included with the therapeutic reagent. Alternatively, these molecules can be included by indirect methods, for example, by inclusion of a polynucleotide that encodes the molecule, or by use of packaging systems that provide targeting molecules. Those skilled in the art will know, or will ascertain with the use of the teaching provided herein, which molecules and procedures will be useful for delivery of the therapeutic reagent to specific tissues. [0196]
Transgenic Animals [0197]
MKK polypeptides can also be expressed in transgenic animals. These animals represent a model system for the study of disorders that are caused by or exacerbated by overexpression or underexpression of MKK, and for the development of therapeutic agents that modulate the expression or activity of MKK. For example, dominant-negative and constitutively activated alleles could be expressed in mice to establish physiological function. [0198]
Transgenic animals can be farm animals (pigs, goats, sheep, cows, horses, rabbits, and the like) rodents (such as rats, guinea pigs, and mice), non-human primates (for example, baboons, monkeys, and chimpanzees), and domestic animals (for example, dogs and cats). Transgenic mice are especially preferred. [0199]
Any technique known in the art can be used to introduce a MKK transgene into animals to produce the founder lines of transgenic animals. Such techniques include, but are not limited to, pronuclear microinjection (U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al., [0200] Proc. Natl. Acad. Sci., USA 82:6148, 1985); gene targeting into embryonic stem cells (Thompson et al., Cell 56:313, 1989); and electroporation of embryos (Lo, Mol. Cell. Biol. 3:1803, 1983). Especially useful are the methods described in Yang et al. (Proc. Natl Acac. Sci. USA 94:3004-3009, 1997)
The present invention provides for transgenic animals that carry the MKK transgene in all their cells, as well as animals that carry the transgene in some, but not all of their cells. That is, the invention provides for mosaic animals. The transgene can be integrated as a single transgene or in concatamers, e.g., head-to-head tandems or head-to-tail tandems. The transgene can also be selectively introduced into and activated in a particular cell type (Lasko et al., [0201] Proc. Natl. Acad. Sci. USA 89:6232, 1992). The regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
When it is desired that the MKK transgene be integrated into the chromosomal site of the endogenous MKK gene, gene targeting is preferred. Briefly, when such a technique is to be used, vectors containing some nucleotide sequences homologous to an endogenous MKK gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene. The transgene also can be selectively introduced into a particular cell type, thus inactivating the endogenous MKK gene in only that cell type (Gu et al., [0202] Science 265:103, 1984). The regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. These techniques are useful for preparing “knock outs” having no functional MKK gene.
Once transgenic animals have been generated, the expression of the recombinant MKK gene can be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to determine whether integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and RT-PCR. Samples of MKK gene-expressing tissue can also be evaluated immunocytochemically using antibodies specific for the MKK transgene product. [0203]
For a review of techniques that can be used to generate and assess transgenic animals, skilled artisans can consult Gordon ([0204] Intl. Rev. Cytol. 115:171-229, 1989), and may obtain additional guidance from, for example: Hogan et al. Manipulating the Mouse Embryo, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1986);, Krimpenfort et al. (Bio/Technology 9:86, 1991), Palmiter et al. (Cell 41:343, 1985), Kraemer et al. (Genetic Manipulation of the Early Mammalian Embryo, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 1985), Hammer et al. (Nature 315:680, 1985), Purcel et al. (Science, 244:1281, 1986), Wagner et al. (U.S. Pat. No. 5,175,385), and Krimpenfort et al. (U.S. Pat. No. 5,175,384) (the latter two publications are hereby incorporated by reference).
Other Embodiments [0205]
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, that the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. [0206]
1 34 2030 base pairs nucleic acid double linear cDNA Coding Sequence 338...1291 1 TGGCTGGCAA TGGCCTTGCT GACCTCGAGC CGGGCCCACG TGGGGACCTT TGGAGCACAG 60 CCTACGATCC TGGTGCAAGG CCGGTGGATG CAGAGGCCAG TCCATATACC ACCCAGGCCT 120 GCGAGGAGCG TGGTCCCCAC CCATCCAGCC CATATGTGCA AGTGCCCTTG ACAGAGAGGC 180 TGGTCATATC CATGGTGACC ATTTATGGGC CACAACAGGT CCCCATCTGC GCAGTGAACC 240 CTGTGCTGAG CACCTTGCAG ACGTGATCTT GCTTCGTCCT GCAGCACTGT GCGGGGCAGG 300 AAAATCCAAG AGGAAGAAGG ATCTACGGAT ATCCTGC ATG TCC AAG CCA CCC GCA 355 Met Ser Lys Pro Pro Ala 1 5 CCC AAC CCC ACA CCC CCC CGG AAC CTG GAC TCC CGG ACC TTC ATC ACC 403 Pro Asn Pro Thr Pro Pro Arg Asn Leu Asp Ser Arg Thr Phe Ile Thr 10 15 20 ATT GGA GAC AGA AAC TTT GAG GTG GAG GCT GAT GAC TTG GTG ACC ATC 451 Ile Gly Asp Arg Asn Phe Glu Val Glu Ala Asp Asp Leu Val Thr Ile 25 30 35 TCA GAA CTG GGC CGT GGA GCC TAT GGG GTG GTA GAG AAG GTG CGG CAC 499 Ser Glu Leu Gly Arg Gly Ala Tyr Gly Val Val Glu Lys Val Arg His 40 45 50 GCC CAG AGC GGC ACC ATC ATG GCC GTG AAG CGG ATC CGG GCC ACC GTG 547 Ala Gln Ser Gly Thr Ile Met Ala Val Lys Arg Ile Arg Ala Thr Val 55 60 65 70 AAC TCA CAG GAG CAG AAG CGG CTG CTC ATG GAC CTG GAC ATC AAC ATG 595 Asn Ser Gln Glu Gln Lys Arg Leu Leu Met Asp Leu Asp Ile Asn Met 75 80 85 CGC ACG GTC GAC TGT TTC TAC ACT GTC ACC TTC TAC GGG GCA CTA TTC 643 Arg Thr Val Asp Cys Phe Tyr Thr Val Thr Phe Tyr Gly Ala Leu Phe 90 95 100 AGA GAG GGA GAC GTG TGG ATC TGC ATG GAG CTC ATG GAC ACA TCC TTG 691 Arg Glu Gly Asp Val Trp Ile Cys Met Glu Leu Met Asp Thr Ser Leu 105 110 115 GAC AAG TTC TAC CGG AAG GTG CTG GAT AAA AAC ATG ACA ATT CCA GAG 739 Asp Lys Phe Tyr Arg Lys Val Leu Asp Lys Asn Met Thr Ile Pro Glu 120 125 130 GAC ATC CTT GGG GAG ATT GCT GTG TCT ATC GTG CGG GCC CTG GAG CAT 787 Asp Ile Leu Gly Glu Ile Ala Val Ser Ile Val Arg Ala Leu Glu His 135 140 145 150 CTG CAC AGC AAG CTG TCG GTG ATC CAC AGA GAT GTG AAG CCC TCC AAT 835 Leu His Ser Lys Leu Ser Val Ile His Arg Asp Val Lys Pro Ser Asn 155 160 165 GTC CTT ATC AAC AAG GAG GGC CAT GTG AAG ATG TGT GAC TTT GGC ATC 883 Val Leu Ile Asn Lys Glu Gly His Val Lys Met Cys Asp Phe Gly Ile 170 175 180 AGT GGC TAC TTG GTG GAC TCT GTG GCC AAG ACG ATG GAT GCC GGC TGC 931 Ser Gly Tyr Leu Val Asp Ser Val Ala Lys Thr Met Asp Ala Gly Cys 185 190 195 AAG CCC TAC ATG GCC CCT GAG AGG ATC AAC CCA GAG CTG AAC CAG AAG 979 Lys Pro Tyr Met Ala Pro Glu Arg Ile Asn Pro Glu Leu Asn Gln Lys 200 205 210 GGC TAC AAT GTC AAG TCC GAC GTC TGG AGC CTG GGC ATC ACC ATG ATT 1027 Gly Tyr Asn Val Lys Ser Asp Val Trp Ser Leu Gly Ile Thr Met Ile 215 220 225 230 GAG ATG GCC ATC CTG CGG TTC CCT TAC GAG TCC TGG GGG ACC CCG TTC 1075 Glu Met Ala Ile Leu Arg Phe Pro Tyr Glu Ser Trp Gly Thr Pro Phe 235 240 245 CAG CAG CTG AAG CAG GTG GTG GAG GAG CCG TCC CCC CAG CTC CCA GCC 1123 Gln Gln Leu Lys Gln Val Val Glu Glu Pro Ser Pro Gln Leu Pro Ala 250 255 260 GAC CGT TTC TCC CCC GAG TTT GTG GAC TTC ACT GCT CAG TGC CTG AGG 1171 Asp Arg Phe Ser Pro Glu Phe Val Asp Phe Thr Ala Gln Cys Leu Arg 265 270 275 AAG AAC CCC GCA GAG CGT ATG AGC TAC CTG GAG CTG ATG GAG CAC CCC 1219 Lys Asn Pro Ala Glu Arg Met Ser Tyr Leu Glu Leu Met Glu His Pro 280 285 290 TTC TTC ACC TTG CAC AAA ACC AAG AAG ACG GAC ATT GCT GCC TTC GTG 1267 Phe Phe Thr Leu His Lys Thr Lys Lys Thr Asp Ile Ala Ala Phe Val 295 300 305 310 AAG AAG ATC CTG GGA GAA GAC TCA TAGGGGCTGG GCCTCGGACC CCACTCCGGC 1321 Lys Lys Ile Leu Gly Glu Asp Ser 315 CCTCCAGAGC CCCACAGCCC CATCTGCGGG GGCAGTGCTC ACCCACACCA TAAGCTACTG 1381 CCATCCTGGC CCAGGGCATC TGGGAGGAAC CGAGGGGGCT GCTCCCACCT GGCTCTGTGG 1441 CGAGCCATTT GTCCCAAGTG CCAAAGAAGC AGACCATTGG GGCTCCCAGC CAGGCCCTTG 1501 TCGGCCCCAC CAGTGCCTCT CCCTGCTGCT CCTAGGACCC GTCTCCAGCT GCTGAGATCC 1561 TGGACTGAGG GGGCCTGGAT GCCCCCTGTG GATGCTGCTG CCCCTGCACA GCAGGCTGCC 1621 AGTGCCTGGG TGGATGGGCC ACCGCCTTGC CCAGCCTGGA TGCCATCCAA GTTGTATATT 1681 TTTTTAATCT CTCGACTGAA TGGACTTTGC ACACTTTGGC CCAGGGTGGC CACACCTCTA 1741 TCCCGGCTTT GGTGCGGGGT ACACAAGAGG GGATGAGTTG TGTGAATACC CCAAGACTCC 1801 CATGAGGGAG ATGCCATGAG CCGCCCAAGG CCTTCCCCTG GCACTGGCAA ACAGGGCCTC 1861 TGCGGAGCAC ACTGGCTCAC CCAGTCCTGC CCGCCACCGT TATCGGTGTC ATTCACCTTT 1921 CGTGTTTTTT TTAATTTATC CTCTGTTGAT TTTTTCTTTT GCTTTATGGG TTTGGCTTGT 1981 TTTTCTTGCA TGGTTTGGAG CTGATCGCTT CTCCCCCACC CCCTAGGGG 2030 318 amino acids amino acid linear protein internal 2 Met Ser Lys Pro Pro Ala Pro Asn Pro Thr Pro Pro Arg Asn Leu Asp 1 5 10 15 Ser Arg Thr Phe Ile Thr Ile Gly Asp Arg Asn Phe Glu Val Glu Ala 20 25 30 Asp Asp Leu Val Thr Ile Ser Glu Leu Gly Arg Gly Ala Tyr Gly Val 35 40 45 Val Glu Lys Val Arg His Ala Gln Ser Gly Thr Ile Met Ala Val Lys 50 55 60 Arg Ile Arg Ala Thr Val Asn Ser Gln Glu Gln Lys Arg Leu Leu Met 65 70 75 80 Asp Leu Asp Ile Asn Met Arg Thr Val Asp Cys Phe Tyr Thr Val Thr 85 90 95 Phe Tyr Gly Ala Leu Phe Arg Glu Gly Asp Val Trp Ile Cys Met Glu 100 105 110 Leu Met Asp Thr Ser Leu Asp Lys Phe Tyr Arg Lys Val Leu Asp Lys 115 120 125 Asn Met Thr Ile Pro Glu Asp Ile Leu Gly Glu Ile Ala Val Ser Ile 130 135 140 Val Arg Ala Leu Glu His Leu His Ser Lys Leu Ser Val Ile His Arg 145 150 155 160 Asp Val Lys Pro Ser Asn Val Leu Ile Asn Lys Glu Gly His Val Lys 165 170 175 Met Cys Asp Phe Gly Ile Ser Gly Tyr Leu Val Asp Ser Val Ala Lys 180 185 190 Thr Met Asp Ala Gly Cys Lys Pro Tyr Met Ala Pro Glu Arg Ile Asn 195 200 205 Pro Glu Leu Asn Gln Lys Gly Tyr Asn Val Lys Ser Asp Val Trp Ser 210 215 220 Leu Gly Ile Thr Met Ile Glu Met Ala Ile Leu Arg Phe Pro Tyr Glu 225 230 235 240 Ser Trp Gly Thr Pro Phe Gln Gln Leu Lys Gln Val Val Glu Glu Pro 245 250 255 Ser Pro Gln Leu Pro Ala Asp Arg Phe Ser Pro Glu Phe Val Asp Phe 260 265 270 Thr Ala Gln Cys Leu Arg Lys Asn Pro Ala Glu Arg Met Ser Tyr Leu 275 280 285 Glu Leu Met Glu His Pro Phe Phe Thr Leu His Lys Thr Lys Lys Thr 290 295 300 Asp Ile Ala Ala Phe Val Lys Lys Ile Leu Gly Glu Asp Ser 305 310 315 1602 base pairs nucleic acid double linear cDNA Coding Sequence 244...1245 3 TAGCTGCAGC ACAGCCTTCC CTAACGTTGC AACTGGGGGA AAAATCACTT TCCAGTCTGT 60 TTTGCAAGGT GTGCATTTCC ATCTTGATTC CCTGAAAGTC CATCTGCTGC ATCGGTCAAG 120 AGAAACTCCA CTTGCATGAA GATTGCACGC CTGCAGCTTG CATCTTTGTT GCAAAACTAG 180 CTACAGAAGA GAAGCAAGGC AAAGTCTTTT GTGCTCCCCT CCCCCATCAA AGGAAAGGGG 240 AAA ATG TCT CAG TCG AAA GGC AAG AAG CGA AAC CCT GGC CTT AAA ATT 288 Met Ser Gln Ser Lys Gly Lys Lys Arg Asn Pro Gly Leu Lys Ile 1 5 10 15 CCA AAA GAA GCA TTT GAA CAA CCT CAG ACC AGT TCC ACA CCA CCT AGA 336 Pro Lys Glu Ala Phe Glu Gln Pro Gln Thr Ser Ser Thr Pro Pro Arg 20 25 30 GAT TTA GAC TCC AAG GCT TGC ATT TCT ATT GGA AAT CAG AAC TTT GAG 384 Asp Leu Asp Ser Lys Ala Cys Ile Ser Ile Gly Asn Gln Asn Phe Glu 35 40 45 GTG AAG GCA GAT GAC CTG GAG CCT ATA ATG GAA CTG GGA CGA GGT GCG 432 Val Lys Ala Asp Asp Leu Glu Pro Ile Met Glu Leu Gly Arg Gly Ala 50 55 60 TAC GGG GTG GTG GAG AAG ATG CGG CAC GTG CCC AGC GGG CAG ATC ATG 480 Tyr Gly Val Val Glu Lys Met Arg His Val Pro Ser Gly Gln Ile Met 65 70 75 GCA GTG AAG CGG ATC CGA GCC ACA GTA AAT AGC CAG GAA CAG AAA CGG 528 Ala Val Lys Arg Ile Arg Ala Thr Val Asn Ser Gln Glu Gln Lys Arg 80 85 90 95 CTA CTG ATG GAT TTG GAT ATT TCC ATG AGG ACG GTG GAC TGT CCA TTC 576 Leu Leu Met Asp Leu Asp Ile Ser Met Arg Thr Val Asp Cys Pro Phe 100 105 110 ACT GTC ACC TTT TAT GGC GCA CTG TTT CGG GAG GGT GAT GTG TGG ATC 624 Thr Val Thr Phe Tyr Gly Ala Leu Phe Arg Glu Gly Asp Val Trp Ile 115 120 125 TGC ATG GAG CTC ATG GAT ACA TCA CTA GAT AAA TTC TAC AAA CAA GTT 672 Cys Met Glu Leu Met Asp Thr Ser Leu Asp Lys Phe Tyr Lys Gln Val 130 135 140 ATT GAT AAA GGC CAG ACA ATT CCA GAG GAC ATC TTA GGG AAA ATA GCA 720 Ile Asp Lys Gly Gln Thr Ile Pro Glu Asp Ile Leu Gly Lys Ile Ala 145 150 155 GTT TCT ATT GTA AAA GCA TTA GAA CAT TTA CAT AGT AAG CTG TCT GTC 768 Val Ser Ile Val Lys Ala Leu Glu His Leu His Ser Lys Leu Ser Val 160 165 170 175 ATT CAC AGA GAC GTC AAG CCT TCT AAT GTA CTC ATC AAT GCT CTC GGT 816 Ile His Arg Asp Val Lys Pro Ser Asn Val Leu Ile Asn Ala Leu Gly 180 185 190 CAA GTG AAG ATG TGC GAT TTT GGA ATC AGT GGC TAC TTG GTG GAC TCT 864 Gln Val Lys Met Cys Asp Phe Gly Ile Ser Gly Tyr Leu Val Asp Ser 195 200 205 GTT GCT AAA ACA ATT GAT GCA GGT TGC AAA CCA TAC ATG GCC CCT GAA 912 Val Ala Lys Thr Ile Asp Ala Gly Cys Lys Pro Tyr Met Ala Pro Glu 210 215 220 AGA ATA AAC CCA GAG CTC AAC CAG AAG GGA TAC AGT GTG AAG TCT GAC 960 Arg Ile Asn Pro Glu Leu Asn Gln Lys Gly Tyr Ser Val Lys Ser Asp 225 230 235 ATT TGG AGT CTG GGC ATC ACG ATG ATT GAG TTG GCC ATC CTT CGA TTT 1008 Ile Trp Ser Leu Gly Ile Thr Met Ile Glu Leu Ala Ile Leu Arg Phe 240 245 250 255 CCC TAT GAT TCA TGG GGA ACT CCA TTT CAG CAG CTC AAA CAG GTG GTA 1056 Pro Tyr Asp Ser Trp Gly Thr Pro Phe Gln Gln Leu Lys Gln Val Val 260 265 270 GAG GAG CCA TCG CCA CAA CTC CCA GCA GAC AAG TTC TCT GCA GAG TTT 1104 Glu Glu Pro Ser Pro Gln Leu Pro Ala Asp Lys Phe Ser Ala Glu Phe 275 280 285 GTT GAC TTT ACC TCA CAG TGC TTA AAG AAG AAT TCC AAA GAA CGG CCT 1152 Val Asp Phe Thr Ser Gln Cys Leu Lys Lys Asn Ser Lys Glu Arg Pro 290 295 300 ACA TAC CCA GAG CTA ATG CAA CAT CCA TTT TTC ACC CTA CAT GAA TCC 1200 Thr Tyr Pro Glu Leu Met Gln His Pro Phe Phe Thr Leu His Glu Ser 305 310 315 AAA GGA ACA GAT GTG GCA TCT TTT GTA AAA CTG ATT CTT GGA GAC TAAAA 1250 Lys Gly Thr Asp Val Ala Ser Phe Val Lys Leu Ile Leu Gly Asp 320 325 330 AGCAGTGGAC TTAATCGGTT GACCCTACTG TGGATTGGTG GGTTTCGGGG TGAAGCAAGT 1310 TCACTACAGC ATCAATAGAA AGTCATCTTT GAGATAATTT AACCCTGCCT CTCAGAGGGT 1370 TTTCTCTCCC AATTTTCTTT TTACTCCCCC TCTTAAGGGG GCCTTGGAAT CTATAGTATA 1430 GAATGAACTG TCTAGATGGA TGAATTATGA TAAAGGCTTA GGACTTCAAA AGGTGATTAA 1490 ATATTTAATG ATGTGTCATA TGAGTCCTCA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 1550 AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA AA 1602 334 amino acids amino acid linear protein internal 4 Met Ser Gln Ser Lys Gly Lys Lys Arg Asn Pro Gly Leu Lys Ile Pro 1 5 10 15 Lys Glu Ala Phe Glu Gln Pro Gln Thr Ser Ser Thr Pro Pro Arg Asp 20 25 30 Leu Asp Ser Lys Ala Cys Ile Ser Ile Gly Asn Gln Asn Phe Glu Val 35 40 45 Lys Ala Asp Asp Leu Glu Pro Ile Met Glu Leu Gly Arg Gly Ala Tyr 50 55 60 Gly Val Val Glu Lys Met Arg His Val Pro Ser Gly Gln Ile Met Ala 65 70 75 80 Val Lys Arg Ile Arg Ala Thr Val Asn Ser Gln Glu Gln Lys Arg Leu 85 90 95 Leu Met Asp Leu Asp Ile Ser Met Arg Thr Val Asp Cys Pro Phe Thr 100 105 110 Val Thr Phe Tyr Gly Ala Leu Phe Arg Glu Gly Asp Val Trp Ile Cys 115 120 125 Met Glu Leu Met Asp Thr Ser Leu Asp Lys Phe Tyr Lys Gln Val Ile 130 135 140 Asp Lys Gly Gln Thr Ile Pro Glu Asp Ile Leu Gly Lys Ile Ala Val 145 150 155 160 Ser Ile Val Lys Ala Leu Glu His Leu His Ser Lys Leu Ser Val Ile 165 170 175 His Arg Asp Val Lys Pro Ser Asn Val Leu Ile Asn Ala Leu Gly Gln 180 185 190 Val Lys Met Cys Asp Phe Gly Ile Ser Gly Tyr Leu Val Asp Ser Val 195 200 205 Ala Lys Thr Ile Asp Ala Gly Cys Lys Pro Tyr Met Ala Pro Glu Arg 210 215 220 Ile Asn Pro Glu Leu Asn Gln Lys Gly Tyr Ser Val Lys Ser Asp Ile 225 230 235 240 Trp Ser Leu Gly Ile Thr Met Ile Glu Leu Ala Ile Leu Arg Phe Pro 245 250 255 Tyr Asp Ser Trp Gly Thr Pro Phe Gln Gln Leu Lys Gln Val Val Glu 260 265 270 Glu Pro Ser Pro Gln Leu Pro Ala Asp Lys Phe Ser Ala Glu Phe Val 275 280 285 Asp Phe Thr Ser Gln Cys Leu Lys Lys Asn Ser Lys Glu Arg Pro Thr 290 295 300 Tyr Pro Glu Leu Met Gln His Pro Phe Phe Thr Leu His Glu Ser Lys 305 310 315 320 Gly Thr Asp Val Ala Ser Phe Val Lys Leu Ile Leu Gly Asp 325 330 3498 base pairs nucleic acid double linear cDNA Coding Sequence 40...1128 5 CTAGGGTCCC CGGCGCCAGG CCACCCGGCC GTCAGCAGC ATG CAG GGT AAA CGC 54 Met Gln Gly Lys Arg 1 5 AAA GCA CTG AAG TTG AAT TTT GCA AAT CCA CCT TTC AAA TCT ACA GCA 102 Lys Ala Leu Lys Leu Asn Phe Ala Asn Pro Pro Phe Lys Ser Thr Ala 10 15 20 AGG TTT ACT CTG AAT CCC AAT CCT ACA GGA GTT CAA AAC CCA CAC ATA 150 Arg Phe Thr Leu Asn Pro Asn Pro Thr Gly Val Gln Asn Pro His Ile 25 30 35 GAG AGA CTG AGA ACA CAC AGC ATT GAG TCA TCA GGA AAA CTG AAG ATC 198 Glu Arg Leu Arg Thr His Ser Ile Glu Ser Ser Gly Lys Leu Lys Ile 40 45 50 TCC CCT GAA CAA CAC TGG GAT TTC ACT GCA GAG GAC TTG AAA GAC CTT 246 Ser Pro Glu Gln His Trp Asp Phe Thr Ala Glu Asp Leu Lys Asp Leu 55 60 65 GGA GAA ATT GGA CGA GGA GCT TAT GGT TCT GTC AAC AAA ATG GTC CAC 294 Gly Glu Ile Gly Arg Gly Ala Tyr Gly Ser Val Asn Lys Met Val His 70 75 80 85 AAA CCA AGT GGG CAA ATA ATG GCA GTT AAA AGA ATT CGG TCA ACA GTG 342 Lys Pro Ser Gly Gln Ile Met Ala Val Lys Arg Ile Arg Ser Thr Val 90 95 100 GAT GAA AAA GAA CAA AAA CAA CTT CTT ATG GAT TTG GAT GTA GTA ATG 390 Asp Glu Lys Glu Gln Lys Gln Leu Leu Met Asp Leu Asp Val Val Met 105 110 115 CGG AGT AGT GAT TGC CCA TAC ATT GTT CAG TTT TAT GGT GCA CTC TTC 438 Arg Ser Ser Asp Cys Pro Tyr Ile Val Gln Phe Tyr Gly Ala Leu Phe 120 125 130 AGA GAG GGT GAC TGT TGG ATC TGT ATG GAA CTC ATG TCT ACC TCG TTT 486 Arg Glu Gly Asp Cys Trp Ile Cys Met Glu Leu Met Ser Thr Ser Phe 135 140 145 GAT AAG TTT TAC AAA TAT GTA TAT AGT GTA TTA GAT GAT GTT ATT CCA 534 Asp Lys Phe Tyr Lys Tyr Val Tyr Ser Val Leu Asp Asp Val Ile Pro 150 155 160 165 GAA GAA ATT TTA GGC AAA ATC ACT TTA GCA ACT GTG AAA GCA CTA AAC 582 Glu Glu Ile Leu Gly Lys Ile Thr Leu Ala Thr Val Lys Ala Leu Asn 170 175 180 CAC TTA AAA GAA AAC TTG AAA ATT ATT CAC AGA GAT ATC AAA CCT TCC 630 His Leu Lys Glu Asn Leu Lys Ile Ile His Arg Asp Ile Lys Pro Ser 185 190 195 AAT ATT CTT CTG GAC AGA AGT GGA AAT ATT AAG CTC TGT GAC TTC GGC 678 Asn Ile Leu Leu Asp Arg Ser Gly Asn Ile Lys Leu Cys Asp Phe Gly 200 205 210 ATC AGT GGA CAG CTT GTG GAC TCT ATT GCC AAG ACA AGA GAT GCT GGC 726 Ile Ser Gly Gln Leu Val Asp Ser Ile Ala Lys Thr Arg Asp Ala Gly 215 220 225 TGT AGG CCA TAC ATG GCA CCT GAA AGA ATA GAC CCA AGC GCA TCA CGA 774 Cys Arg Pro Tyr Met Ala Pro Glu Arg Ile Asp Pro Ser Ala Ser Arg 230 235 240 245 CAA GGA TAT GAT GTC CGC TCT GAT GTC TGG AGT TTG GGG ATC ACA TTG 822 Gln Gly Tyr Asp Val Arg Ser Asp Val Trp Ser Leu Gly Ile Thr Leu 250 255 260 TAT GAG TTG GCC ACA GGC CGA TTT CCT TAT CCA AAG TGG AAT AGT GTA 870 Tyr Glu Leu Ala Thr Gly Arg Phe Pro Tyr Pro Lys Trp Asn Ser Val 265 270 275 TTT GAT CAA CTA ACA CAA GTC GTG AAA GGA GAT CCT CCG CAG CTG AGT 918 Phe Asp Gln Leu Thr Gln Val Val Lys Gly Asp Pro Pro Gln Leu Ser 280 285 290 AAT TCT GAG GAA AGG GAA TTC TCC CCG AGT TTC ATC AAC TTT GTC AAC 966 Asn Ser Glu Glu Arg Glu Phe Ser Pro Ser Phe Ile Asn Phe Val Asn 295 300 305 TTG TGC CTT ACG AAG GAT GAA TCC AAA AGG CCA AAG TAT AAA GAG CTT 1014 Leu Cys Leu Thr Lys Asp Glu Ser Lys Arg Pro Lys Tyr Lys Glu Leu 310 315 320 325 CTG AAA CAT CCC TTT ATT TTG ATG TAT GAA GAA CGT GCC GTT GAG GTC 1062 Leu Lys His Pro Phe Ile Leu Met Tyr Glu Glu Arg Ala Val Glu Val 330 335 340 GCA TGC TAT GTT TGT AAA ATC CTG GAT CAA ATG CCA GCT ACT CCC AGC 1110 Ala Cys Tyr Val Cys Lys Ile Leu Asp Gln Met Pro Ala Thr Pro Ser 345 350 355 TCT CCC ATG TAT GTC GAT TGATATCGYT GCTACATCAG ACTCTAGAAA AAAGGGCT 1166 Ser Pro Met Tyr Val Asp 360 GAGAGGAAGC AAGACGTAAA GAATTTTCAT CCCGTATCAC AGTGTTTTTA TTGCTCGCCC 1226 AGACACCATG TGCAATAAGA TTGGTGTTCG TTTCCATCAT GTCTGTATAC TCCTGTCACC 1286 TAGAACGTGC ATCCTTGTAA TACCTGATTG ATCACACAGT GTTAGTGCTG GTCAGAGAGA 1346 CCTCATCCTG CTCTTTTGTG ATGAACATAT TCATGAAATG TGGAAGTCAG TACGATCAAG 1406 TTGTTGACTG TGATTAGATC ACATCTTAAA TTCATTTCTA GACTCAAAAC CTGGAGATGC 1466 AGCTACTGGA ATGGTGTTTT GTCAGACTTC CAAATCCTGG AAGGACACAG TGATGAATGT 1526 ACTATATCTG AACATAGAAA CTCGGGCTTG AGTGAGAAGA GCTTGCACAG CCAACGAGAC 1586 ACATTGCCTT CTGGAGCTGG GAGACAAAGG AGGAATTTAC TTTCTTCACC AAGTGCAATA 1646 GATTACTGAT GTGATATTCT GTTGCTTTAC AGTTACAGTT GATGTTTGGG GATCGATGTG 1706 CTCAGCCAAA TTTCCTGTTT GAAATATCAT GTTAAATTAG AATGAATTTA TCTTTACCAA 1766 AAACCATGTT GCGTTCAAAG AGGTGAACAT TAAAATATAG AGACAGGACA GAATGTGTTC 1826 TTTTCTCCTC TACCAGTCCT ATTTTTCAAT GGGAAGACTC AGGAGTCTGC CACTTGTCAA 1886 AGAAGGTGCT GATCCTAAGA ATTTTTCATT CTCAGAATTC GGTGTGCTGC CAACTTGATG 1946 TTCCACCTGC CACAAACCAC CAGGACTGAA AGAAGAAAAC AGTACAGAAG GCAAAGTTTA 2006 CAGATGTTTT TAATTCTAGT ATTTTATCTG GAACAACTTG TAGCAGCTAT ATATTTCCCC 2066 TTGGTCCCAA GCCTGATACT TTAGCCATCA TAACTCACTA ACAGGGAGAA GTAGCTAGTA 2126 GCAATGTGCC TTGATTGATT AGATAAAGAT TTCTAGTAGG CAGCAAAAGA CCAAATCTCA 2186 GTTGTTTGCT TCTTGCCATC ACTGGTCCAG GTCTTCAGTT TCCGAATCTC TTTCCCTTCC 2246 CCTGTGGTCT ATTGTCGCTA TGTGACTTGC GCTTAATCCA ATATTTTGCC TTTTTTCTAT 2306 ATCAAAAAAC CTTTACAGTT AGCAGGGATG TTCCTTACCG AGGATTTTTA ACCCCCAATC 2366 TCTCATAATC GCTAGTGTTT AAAAGGCTAA GAATAGTGGG GCCCAACCGA TGTGGTAGGT 2426 GATAAAGAGG CATCTTTTCT AGAGACACAT TGGACCAGAT GAGGATCCGA AACGGCAGCC 2486 TTTACGTTCA TCACCTGCTA GAACCTCTCG TAGTCCATCA CCATTTCTTG GCATTGGAAT 2546 TCTACTGGAA AAAAATACAA AAAGCAAAAC AAAACCCTCA GCACTGTTAC AAGAGGCCAT 2606 TTAAGTATCT TGTGCTTCTT CACTTACCCA TTAGCCAGGT TCTCATTAGG TTTTGCTTGG 2666 GCCTCCCTGG CACTGAACCT TAGGCTTTGT ATGACAGTGA AGCAGCACTG TGAGTGGTTC 2726 AAGCACACTG GAATATAAAA CAGTCATGGC CTGAGATGCA GGTGATGCCA TTACAGAACC 2786 AAATCGTGGC ACGTATTGCT GTGTCTCCTC TCAGAGTGAC AGTCATAAAT ACTGTCAAAC 2846 AATAAAGGGA GAATGGTGCT GTTTAAAGTC ACATCCCTGT AAATTGCAGA ATTCAAAAGT 2906 GATTATCTCT TTGATCTACT TGCCTCATTT CCCTATCTTC TCCCCCACGG TATCCTAAAC 2966 TTTAGACTTC CCACTGTTCT GAAAGGAGAC ATTGCTCTAT GTCTGCCTTC GACCACAGCA 3026 AGCCATCATC CTCCATTGCT CCCGGGGACT CAAGAGGAAT CTGTTTCTCT GCTGTCAACT 3086 TCCCATCTGG CTCAGCATAG GGTCACTTTG CCATTATGCA AATGGAGATA AAAGCAATTC 3146 TGGCTGTCCA GGAGCTAATC TGACCGTTCT ATTGTGTGGA TGACCACATA AGAAGGCAAT 3206 TTTAGTGTAT TAATCATAGA TTATTATAAA CTATAAACTT AAGGGCAAGG AGTTTATTAC 3266 AATGTATCTT TATTAAAACA AAAGGGTGTA TAGTGTTCAC AAACTGTGAA AATAGTGTAA 3326 GAACTGTACA TTGTGAGCTC TGGTTATTTT TCTCTTGTAC CATAGAAAAA TGTATAAAAA 3386 TTATCAAAAA GCTAATGTGC AGGGATATTG CCTTATTTGT CTGTAAAAAA TGGAGCTCAG 3446 TAACATAACT GCTTCTTGGA GCTTTGGAAT ATTTTATCCT GTATTCTTGT TT 3498 363 amino acids amino acid linear protein internal 6 Met Gln Gly Lys Arg Lys Ala Leu Lys Leu Asn Phe Ala Asn Pro Pro 1 5 10 15 Phe Lys Ser Thr Ala Arg Phe Thr Leu Asn Pro Asn Pro Thr Gly Val 20 25 30 Gln Asn Pro His Ile Glu Arg Leu Arg Thr His Ser Ile Glu Ser Ser 35 40 45 Gly Lys Leu Lys Ile Ser Pro Glu Gln His Trp Asp Phe Thr Ala Glu 50 55 60 Asp Leu Lys Asp Leu Gly Glu Ile Gly Arg Gly Ala Tyr Gly Ser Val 65 70 75 80 Asn Lys Met Val His Lys Pro Ser Gly Gln Ile Met Ala Val Lys Arg 85 90 95 Ile Arg Ser Thr Val Asp Glu Lys Glu Gln Lys Gln Leu Leu Met Asp 100 105 110 Leu Asp Val Val Met Arg Ser Ser Asp Cys Pro Tyr Ile Val Gln Phe 115 120 125 Tyr Gly Ala Leu Phe Arg Glu Gly Asp Cys Trp Ile Cys Met Glu Leu 130 135 140 Met Ser Thr Ser Phe Asp Lys Phe Tyr Lys Tyr Val Tyr Ser Val Leu 145 150 155 160 Asp Asp Val Ile Pro Glu Glu Ile Leu Gly Lys Ile Thr Leu Ala Thr 165 170 175 Val Lys Ala Leu Asn His Leu Lys Glu Asn Leu Lys Ile Ile His Arg 180 185 190 Asp Ile Lys Pro Ser Asn Ile Leu Leu Asp Arg Ser Gly Asn Ile Lys 195 200 205 Leu Cys Asp Phe Gly Ile Ser Gly Gln Leu Val Asp Ser Ile Ala Lys 210 215 220 Thr Arg Asp Ala Gly Cys Arg Pro Tyr Met Ala Pro Glu Arg Ile Asp 225 230 235 240 Pro Ser Ala Ser Arg Gln Gly Tyr Asp Val Arg Ser Asp Val Trp Ser 245 250 255 Leu Gly Ile Thr Leu Tyr Glu Leu Ala Thr Gly Arg Phe Pro Tyr Pro 260 265 270 Lys Trp Asn Ser Val Phe Asp Gln Leu Thr Gln Val Val Lys Gly Asp 275 280 285 Pro Pro Gln Leu Ser Asn Ser Glu Glu Arg Glu Phe Ser Pro Ser Phe 290 295 300 Ile Asn Phe Val Asn Leu Cys Leu Thr Lys Asp Glu Ser Lys Arg Pro 305 310 315 320 Lys Tyr Lys Glu Leu Leu Lys His Pro Phe Ile Leu Met Tyr Glu Glu 325 330 335 Arg Ala Val Glu Val Ala Cys Tyr Val Cys Lys Ile Leu Asp Gln Met 340 345 350 Pro Ala Thr Pro Ser Ser Pro Met Tyr Val Asp 355 360 3554 base pairs nucleic acid double linear cDNA Coding Sequence 6...1184 7 CAACA ATG GCG GCT CCG AGC CCG AGC GGT GGC GGC GGC AGC GGC ACC CCC 50 Met Ala Ala Pro Ser Pro Ser Gly Gly Gly Gly Ser Gly Thr Pro 1 5 10 15 GGC CCC GTA GGG TCC CCG GCG CCA GGC CAC CCG GCC GTC AGC AGC ATG 98 Gly Pro Val Gly Ser Pro Ala Pro Gly His Pro Ala Val Ser Ser Met 20 25 30 CAG GGT AAA CGC AAA GCA CTG AAG TTG AAT TTT GCA AAT CCA CCT TTC 146 Gln Gly Lys Arg Lys Ala Leu Lys Leu Asn Phe Ala Asn Pro Pro Phe 35 40 45 AAA TCT ACA GCA AGG TTT ACT CTG AAT CCC AAT CCT ACA GGA GTT CAA 194 Lys Ser Thr Ala Arg Phe Thr Leu Asn Pro Asn Pro Thr Gly Val Gln 50 55 60 AAC CCA CAC ATA GAG AGA CTG AGA ACA CAC AGC ATT GAG TCA TCA GGA 242 Asn Pro His Ile Glu Arg Leu Arg Thr His Ser Ile Glu Ser Ser Gly 65 70 75 AAA CTG AAG ATC TCC CCT GAA CAA CAC TGG GAT TTC ACT GCA GAG GAC 290 Lys Leu Lys Ile Ser Pro Glu Gln His Trp Asp Phe Thr Ala Glu Asp 80 85 90 95 TTG AAA GAC CTT GGA GAA ATT GGA CGA GGA GCT TAT GGT TCT GTC AAC 338 Leu Lys Asp Leu Gly Glu Ile Gly Arg Gly Ala Tyr Gly Ser Val Asn 100 105 110 AAA ATG GTC CAC AAA CCA AGT GGG CAA ATA ATG GCA GTT AAA AGA ATT 386 Lys Met Val His Lys Pro Ser Gly Gln Ile Met Ala Val Lys Arg Ile 115 120 125 CGG TCA ACA GTG GAT GAA AAA GAA CAA AAA CAA CTT CTT ATG GAT TTG 434 Arg Ser Thr Val Asp Glu Lys Glu Gln Lys Gln Leu Leu Met Asp Leu 130 135 140 GAT GTA GTA ATG CGG AGT AGT GAT TGC CCA TAC ATT GTT CAG TTT TAT 482 Asp Val Val Met Arg Ser Ser Asp Cys Pro Tyr Ile Val Gln Phe Tyr 145 150 155 GGT GCA CTC TTC AGA GAG GGT GAC TGT TGG ATC TGT ATG GAA CTC ATG 530 Gly Ala Leu Phe Arg Glu Gly Asp Cys Trp Ile Cys Met Glu Leu Met 160 165 170 175 TCT ACC TCG TTT GAT AAG TTT TAC AAA TAT GTA TAT AGT GTA TTA GAT 578 Ser Thr Ser Phe Asp Lys Phe Tyr Lys Tyr Val Tyr Ser Val Leu Asp 180 185 190 GAT GTT ATT CCA GAA GAA ATT TTA GGC AAA ATC ACT TTA GCA ACT GTG 626 Asp Val Ile Pro Glu Glu Ile Leu Gly Lys Ile Thr Leu Ala Thr Val 195 200 205 AAA GCA CTA AAC CAC TTA AAA GAA AAC TTG AAA ATT ATT CAC AGA GAT 674 Lys Ala Leu Asn His Leu Lys Glu Asn Leu Lys Ile Ile His Arg Asp 210 215 220 ATC AAA CCT TCC AAT ATT CTT CTG GAC AGA AGT GGA AAT ATT AAG CTC 722 Ile Lys Pro Ser Asn Ile Leu Leu Asp Arg Ser Gly Asn Ile Lys Leu 225 230 235 TGT GAC TTC GGC ATC AGT GGA CAG CTT GTG GAC TCT ATT GCC AAG ACA 770 Cys Asp Phe Gly Ile Ser Gly Gln Leu Val Asp Ser Ile Ala Lys Thr 240 245 250 255 AGA GAT GCT GGC TGT AGG CCA TAC ATG GCA CCT GAA AGA ATA GAC CCA 818 Arg Asp Ala Gly Cys Arg Pro Tyr Met Ala Pro Glu Arg Ile Asp Pro 260 265 270 AGC GCA TCA CGA CAA GGA TAT GAT GTC CGC TCT GAT GTC TGG AGT TTG 866 Ser Ala Ser Arg Gln Gly Tyr Asp Val Arg Ser Asp Val Trp Ser Leu 275 280 285 GGG ATC ACA TTG TAT GAG TTG GCC ACA GGC CGA TTT CCT TAT CCA AAG 914 Gly Ile Thr Leu Tyr Glu Leu Ala Thr Gly Arg Phe Pro Tyr Pro Lys 290 295 300 TGG AAT AGT GTA TTT GAT CAA CTA ACA CAA GTC GTG AAA GGA GAT CCT 962 Trp Asn Ser Val Phe Asp Gln Leu Thr Gln Val Val Lys Gly Asp Pro 305 310 315 CCG CAG CTG AGT AAT TCT GAG GAA AGG GAA TTC TCC CCG AGT TTC ATC 1010 Pro Gln Leu Ser Asn Ser Glu Glu Arg Glu Phe Ser Pro Ser Phe Ile 320 325 330 335 AAC TTT GTC AAC TTG TGC CTT ACG AAG GAT GAA TCC AAA AGG CCA AAG 1058 Asn Phe Val Asn Leu Cys Leu Thr Lys Asp Glu Ser Lys Arg Pro Lys 340 345 350 TAT AAA GAG CTT CTG AAA CAT CCC TTT ATT TTG ATG TAT GAA GAA CGT 1106 Tyr Lys Glu Leu Leu Lys His Pro Phe Ile Leu Met Tyr Glu Glu Arg 355 360 365 GCC GTT GAG GTC GCA TGC TAT GTT TGT AAA ATC CTG GAT CAA ATG CCA 1154 Ala Val Glu Val Ala Cys Tyr Val Cys Lys Ile Leu Asp Gln Met Pro 370 375 380 GCT ACT CCC AGC TCT CCC ATG TAT GTC GAT TGATATCGYT GCTACATCAG ACT 1207 Ala Thr Pro Ser Ser Pro Met Tyr Val Asp 385 390 CTAGAAAAAA GGGCTGAGAG GAAGCAAGAC GTAAAGAATT TTCATCCCGT ATCACAGTGT 1267 TTTTATTGCT CGCCCAGACA CCATGTGCAA TAAGATTGGT GTTCGTTTCC ATCATGTCTG 1327 TATACTCCTG TCACCTAGAA CGTGCATCCT TGTAATACCT GATTGATCAC ACAGTGTTAG 1387 TGCTGGTCAG AGAGACCTCA TCCTGCTCTT TTGTGATGAA CATATTCATG AAATGTGGAA 1447 GTCAGTACGA TCAAGTTGTT GACTGTGATT AGATCACATC TTAAATTCAT TTCTAGACTC 1507 AAAACCTGGA GATGCAGCTA CTGGAATGGT GTTTTGTCAG ACTTCCAAAT CCTGGAAGGA 1567 CACAGTGATG AATGTACTAT ATCTGAACAT AGAAACTCGG GCTTGAGTGA GAAGAGCTTG 1627 CACAGCCAAC GAGACACATT GCCTTCTGGA GCTGGGAGAC AAAGGAGGAA TTTACTTTCT 1687 TCACCAAGTG CAATAGATTA CTGATGTGAT ATTCTGTTGC TTTACAGTTA CAGTTGATGT 1747 TTGGGGATCG ATGTGCTCAG CCAAATTTCC TGTTTGAAAT ATCATGTTAA ATTAGAATGA 1807 ATTTATCTTT ACCAAAAACC ATGTTGCGTT CAAAGAGGTG AACATTAAAA TATAGAGACA 1867 GGACAGAATG TGTTCTTTTC TCCTCTACCA GTCCTATTTT TCAATGGGAA GACTCAGGAG 1927 TCTGCCACTT GTCAAAGAAG GTGCTGATCC TAAGAATTTT TCATTCTCAG AATTCGGTGT 1987 GCTGCCAACT TGATGTTCCA CCTGCCACAA ACCACCAGGA CTGAAAGAAG AAAACAGTAC 2047 AGAAGGCAAA GTTTACAGAT GTTTTTAATT CTAGTATTTT ATCTGGAACA ACTTGTAGCA 2107 GCTATATATT TCCCCTTGGT CCCAAGCCTG ATACTTTAGC CATCATAACT CACTAACAGG 2167 GAGAAGTAGC TAGTAGCAAT GTGCCTTGAT TGATTAGATA AAGATTTCTA GTAGGCAGCA 2227 AAAGACCAAA TCTCAGTTGT TTGCTTCTTG CCATCACTGG TCCAGGTCTT CAGTTTCCGA 2287 ATCTCTTTCC CTTCCCCTGT GGTCTATTGT CGCTATGTGA CTTGCGCTTA ATCCAATATT 2347 TTGCCTTTTT TCTATATCAA AAAACCTTTA CAGTTAGCAG GGATGTTCCT TACCGAGGAT 2407 TTTTAACCCC CAATCTCTCA TAATCGCTAG TGTTTAAAAG GCTAAGAATA GTGGGGCCCA 2467 ACCGATGTGG TAGGTGATAA AGAGGCATCT TTTCTAGAGA CACATTGGAC CAGATGAGGA 2527 TCCGAAACGG CAGCCTTTAC GTTCATCACC TGCTAGAACC TCTCGTAGTC CATCACCATT 2587 TCTTGGCATT GGAATTCTAC TGGAAAAAAA TACAAAAAGC AAAACAAAAC CCTCAGCACT 2647 GTTACAAGAG GCCATTTAAG TATCTTGTGC TTCTTCACTT ACCCATTAGC CAGGTTCTCA 2707 TTAGGTTTTG CTTGGGCCTC CCTGGCACTG AACCTTAGGC TTTGTATGAC AGTGAAGCAG 2767 CACTGTGAGT GGTTCAAGCA CACTGGAATA TAAAACAGTC ATGGCCTGAG ATGCAGGTGA 2827 TGCCATTACA GAACCAAATC GTGGCACGTA TTGCTGTGTC TCCTCTCAGA GTGACAGTCA 2887 TAAATACTGT CAAACAATAA AGGGAGAATG GTGCTGTTTA AAGTCACATC CCTGTAAATT 2947 GCAGAATTCA AAAGTGATTA TCTCTTTGAT CTACTTGCCT CATTTCCCTA TCTTCTCCCC 3007 CACGGTATCC TAAACTTTAG ACTTCCCACT GTTCTGAAAG GAGACATTGC TCTATGTCTG 3067 CCTTCGACCA CAGCAAGCCA TCATCCTCCA TTGCTCCCGG GGACTCAAGA GGAATCTGTT 3127 TCTCTGCTGT CAACTTCCCA TCTGGCTCAG CATAGGGTCA CTTTGCCATT ATGCAAATGG 3187 AGATAAAAGC AATTCTGGCT GTCCAGGAGC TAATCTGACC GTTCTATTGT GTGGATGACC 3247 ACATAAGAAG GCAATTTTAG TGTATTAATC ATAGATTATT ATAAACTATA AACTTAAGGG 3307 CAAGGAGTTT ATTACAATGT ATCTTTATTA AAACAAAAGG GTGTATAGTG TTCACAAACT 3367 GTGAAAATAG TGTAAGAACT GTACATTGTG AGCTCTGGTT ATTTTTCTCT TGTACCATAG 3427 AAAAATGTAT AAAAATTATC AAAAAGCTAA TGTGCAGGGA TATTGCCTTA TTTGTCTGTA 3487 AAAAATGGAG CTCAGTAACA TAACTGCTTC TTGGAGCTTT GGAATATTTT ATCCTGTATT 3547 CTTGTTT 3554 393 amino acids amino acid linear protein internal 8 Met Ala Ala Pro Ser Pro Ser Gly Gly Gly Gly Ser Gly Thr Pro Gly 1 5 10 15 Pro Val Gly Ser Pro Ala Pro Gly His Pro Ala Val Ser Ser Met Gln 20 25 30 Gly Lys Arg Lys Ala Leu Lys Leu Asn Phe Ala Asn Pro Pro Phe Lys 35 40 45 Ser Thr Ala Arg Phe Thr Leu Asn Pro Asn Pro Thr Gly Val Gln Asn 50 55 60 Pro His Ile Glu Arg Leu Arg Thr His Ser Ile Glu Ser Ser Gly Lys 65 70 75 80 Leu Lys Ile Ser Pro Glu Gln His Trp Asp Phe Thr Ala Glu Asp Leu 85 90 95 Lys Asp Leu Gly Glu Ile Gly Arg Gly Ala Tyr Gly Ser Val Asn Lys 100 105 110 Met Val His Lys Pro Ser Gly Gln Ile Met Ala Val Lys Arg Ile Arg 115 120 125 Ser Thr Val Asp Glu Lys Glu Gln Lys Gln Leu Leu Met Asp Leu Asp 130 135 140 Val Val Met Arg Ser Ser Asp Cys Pro Tyr Ile Val Gln Phe Tyr Gly 145 150 155 160 Ala Leu Phe Arg Glu Gly Asp Cys Trp Ile Cys Met Glu Leu Met Ser 165 170 175 Thr Ser Phe Asp Lys Phe Tyr Lys Tyr Val Tyr Ser Val Leu Asp Asp 180 185 190 Val Ile Pro Glu Glu Ile Leu Gly Lys Ile Thr Leu Ala Thr Val Lys 195 200 205 Ala Leu Asn His Leu Lys Glu Asn Leu Lys Ile Ile His Arg Asp Ile 210 215 220 Lys Pro Ser Asn Ile Leu Leu Asp Arg Ser Gly Asn Ile Lys Leu Cys 225 230 235 240 Asp Phe Gly Ile Ser Gly Gln Leu Val Asp Ser Ile Ala Lys Thr Arg 245 250 255 Asp Ala Gly Cys Arg Pro Tyr Met Ala Pro Glu Arg Ile Asp Pro Ser 260 265 270 Ala Ser Arg Gln Gly Tyr Asp Val Arg Ser Asp Val Trp Ser Leu Gly 275 280 285 Ile Thr Leu Tyr Glu Leu Ala Thr Gly Arg Phe Pro Tyr Pro Lys Trp 290 295 300 Asn Ser Val Phe Asp Gln Leu Thr Gln Val Val Lys Gly Asp Pro Pro 305 310 315 320 Gln Leu Ser Asn Ser Glu Glu Arg Glu Phe Ser Pro Ser Phe Ile Asn 325 330 335 Phe Val Asn Leu Cys Leu Thr Lys Asp Glu Ser Lys Arg Pro Lys Tyr 340 345 350 Lys Glu Leu Leu Lys His Pro Phe Ile Leu Met Tyr Glu Glu Arg Ala 355 360 365 Val Glu Val Ala Cys Tyr Val Cys Lys Ile Leu Asp Gln Met Pro Ala 370 375 380 Thr Pro Ser Ser Pro Met Tyr Val Asp 385 390 3576 base pairs nucleic acid double linear cDNA Coding Sequence 10...1206 9 CTCCCAACA ATG GCG GCT CCG AGC CCG AGC GGC GGC GGC GGC TCC GGG GGC 51 Met Ala Ala Pro Ser Pro Ser Gly Gly Gly Gly Ser Gly Gly 1 5 10 GGC AGC GGC AGC GGC ACC CCC GGC CCC GTA GGG TCC CCG GCG CCA GGC 99 Gly Ser Gly Ser Gly Thr Pro Gly Pro Val Gly Ser Pro Ala Pro Gly 15 20 25 30 CAC CCG GCC GTC AGC AGC ATG CAG GGT AAA CGC AAA GCA CTG AAG TTG 147 His Pro Ala Val Ser Ser Met Gln Gly Lys Arg Lys Ala Leu Lys Leu 35 40 45 AAT TTT GCA AAT CCA CCT TTC AAA TCT ACA GCA AGG TTT ACT CTG AAT 195 Asn Phe Ala Asn Pro Pro Phe Lys Ser Thr Ala Arg Phe Thr Leu Asn 50 55 60 CCC AAT CCT ACA GGA GTT CAA AAC CCA CAC ATA GAG AGA CTG AGA ACA 243 Pro Asn Pro Thr Gly Val Gln Asn Pro His Ile Glu Arg Leu Arg Thr 65 70 75 CAC AGC ATT GAG TCA TCA GGA AAA CTG AAG ATC TCC CCT GAA CAA CAC 291 His Ser Ile Glu Ser Ser Gly Lys Leu Lys Ile Ser Pro Glu Gln His 80 85 90 TGG GAT TTC ACT GCA GAG GAC TTG AAA GAC CTT GGA GAA ATT GGA CGA 339 Trp Asp Phe Thr Ala Glu Asp Leu Lys Asp Leu Gly Glu Ile Gly Arg 95 100 105 110 GGA GCT TAT GGT TCT GTC AAC AAA ATG GTC CAC AAA CCA AGT GGG CAA 387 Gly Ala Tyr Gly Ser Val Asn Lys Met Val His Lys Pro Ser Gly Gln 115 120 125 ATA ATG GCA GTT AAA AGA ATT CGG TCA ACA GTG GAT GAA AAA GAA CAA 435 Ile Met Ala Val Lys Arg Ile Arg Ser Thr Val Asp Glu Lys Glu Gln 130 135 140 AAA CAA CTT CTT ATG GAT TTG GAT GTA GTA ATG CGG AGT AGT GAT TGC 483 Lys Gln Leu Leu Met Asp Leu Asp Val Val Met Arg Ser Ser Asp Cys 145 150 155 CCA TAC ATT GTT CAG TTT TAT GGT GCA CTC TTC AGA GAG GGT GAC TGT 531 Pro Tyr Ile Val Gln Phe Tyr Gly Ala Leu Phe Arg Glu Gly Asp Cys 160 165 170 TGG ATC TGT ATG GAA CTC ATG TCT ACC TCG TTT GAT AAG TTT TAC AAA 579 Trp Ile Cys Met Glu Leu Met Ser Thr Ser Phe Asp Lys Phe Tyr Lys 175 180 185 190 TAT GTA TAT AGT GTA TTA GAT GAT GTT ATT CCA GAA GAA ATT TTA GGC 627 Tyr Val Tyr Ser Val Leu Asp Asp Val Ile Pro Glu Glu Ile Leu Gly 195 200 205 AAA ATC ACT TTA GCA ACT GTG AAA GCA CTA AAC CAC TTA AAA GAA AAC 675 Lys Ile Thr Leu Ala Thr Val Lys Ala Leu Asn His Leu Lys Glu Asn 210 215 220 TTG AAA ATT ATT CAC AGA GAT ATC AAA CCT TCC AAT ATT CTT CTG GAC 723 Leu Lys Ile Ile His Arg Asp Ile Lys Pro Ser Asn Ile Leu Leu Asp 225 230 235 AGA AGT GGA AAT ATT AAG CTC TGT GAC TTC GGC ATC AGT GGA CAG CTT 771 Arg Ser Gly Asn Ile Lys Leu Cys Asp Phe Gly Ile Ser Gly Gln Leu 240 245 250 GTG GAC TCT ATT GCC AAG ACA AGA GAT GCT GGC TGT AGG CCA TAC ATG 819 Val Asp Ser Ile Ala Lys Thr Arg Asp Ala Gly Cys Arg Pro Tyr Met 255 260 265 270 GCA CCT GAA AGA ATA GAC CCA AGC GCA TCA CGA CAA GGA TAT GAT GTC 867 Ala Pro Glu Arg Ile Asp Pro Ser Ala Ser Arg Gln Gly Tyr Asp Val 275 280 285 CGC TCT GAT GTC TGG AGT TTG GGG ATC ACA TTG TAT GAG TTG GCC ACA 915 Arg Ser Asp Val Trp Ser Leu Gly Ile Thr Leu Tyr Glu Leu Ala Thr 290 295 300 GGC CGA TTT CCT TAT CCA AAG TGG AAT AGT GTA TTT GAT CAA CTA ACA 963 Gly Arg Phe Pro Tyr Pro Lys Trp Asn Ser Val Phe Asp Gln Leu Thr 305 310 315 CAA GTC GTG AAA GGA GAT CCT CCG CAG CTG AGT AAT TCT GAG GAA AGG 1011 Gln Val Val Lys Gly Asp Pro Pro Gln Leu Ser Asn Ser Glu Glu Arg 320 325 330 GAA TTC TCC CCG AGT TTC ATC AAC TTT GTC AAC TTG TGC CTT ACG AAG 1059 Glu Phe Ser Pro Ser Phe Ile Asn Phe Val Asn Leu Cys Leu Thr Lys 335 340 345 350 GAT GAA TCC AAA AGG CCA AAG TAT AAA GAG CTT CTG AAA CAT CCC TTT 1107 Asp Glu Ser Lys Arg Pro Lys Tyr Lys Glu Leu Leu Lys His Pro Phe 355 360 365 ATT TTG ATG TAT GAA GAA CGT GCC GTT GAG GTC GCA TGC TAT GTT TGT 1155 Ile Leu Met Tyr Glu Glu Arg Ala Val Glu Val Ala Cys Tyr Val Cys 370 375 380 AAA ATC CTG GAT CAA ATG CCA GCT ACT CCC AGC TCT CCC ATG TAT GTC 1203 Lys Ile Leu Asp Gln Met Pro Ala Thr Pro Ser Ser Pro Met Tyr Val 385 390 395 GAT TGATATCGCT GCTACATCAG ACTCTAGAAA AAAGGGCTGA GAGGAAGCAA GACGTA 1262 Asp AAGAATTTTC ATCCCGTATC ACAGTGTTTT TATTGCTCGC CCAGACACCA TGTGCAATAA 1322 GATTGGTGTT CGTTTCCATC ATGTCTGTAT ACTCCTGTCA CCTAGAACGT GCATCCTTGT 1382 AATACCTGAT TGATCACACA GTGTTAGTGC TGGTCAGAGA GACCTCATCC TGCTCTTTTG 1442 TGATGAACAT ATTCATGAAA TGTGGAAGTC AGTACGATCA AGTTGTTGAC TGTGATTAGA 1502 TCACATCTTA AATTCATTTC TAGACTCAAA ACCTGGAGAT GCAGCTACTG GAATGGTGTT 1562 TTGTCAGACT TCCAAATCCT GGAAGGACAC AGTGATGAAT GTACTATATC TGAACATAGA 1622 AACTCGGGCT TGAGTGAGAA GAGCTTGCAC AGCCAACGAG ACACATTGCC TTCTGGAGCT 1682 GGGAGACAAA GGAGGAATTT ACTTTCTTCA CCAAGTGCAA TAGATTACTG ATGTGATATT 1742 CTGTTGCTTT ACAGTTACAG TTGATGTTTG GGGATCGATG TGCTCAGCCA AATTTCCTGT 1802 TTGAAATATC ATGTTAAATT AGAATGAATT TATCTTTACC AAAAACCATG TTGCGTTCAA 1862 AGAGGTGAAC ATTAAAATAT AGAGACAGGA CAGAATGTGT TCTTTTCTCC TCTACCAGTC 1922 CTATTTTTCA ATGGGAAGAC TCAGGAGTCT GCCACTTGTC AAAGAAGGTG CTGATCCTAA 1982 GAATTTTTCA TTCTCAGAAT TCGGTGTGCT GCCAACTTGA TGTTCCACCT GCCACAAACC 2042 ACCAGGACTG AAAGAAGAAA ACAGTACAGA AGGCAAAGTT TACAGATGTT TTTAATTCTA 2102 GTATTTTATC TGGAACAACT TGTAGCAGCT ATATATTTCC CCTTGGTCCC AAGCCTGATA 2162 CTTTAGCCAT CATAACTCAC TAACAGGGAG AAGTAGCTAG TAGCAATGTG CCTTGATTGA 2222 TTAGATAAAG ATTTCTAGTA GGCAGCAAAA GACCAAATCT CAGTTGTTTG CTTCTTGCCA 2282 TCACTGGTCC AGGTCTTCAG TTTCCGAATC TCTTTCCCTT CCCCTGTGGT CTATTGTCGC 2342 TATGTGACTT GCGCTTAATC CAATATTTTG CCTTTTTTCT ATATCAAAAA ACCTTTACAG 2402 TTAGCAGGGA TGTTCCTTAC CGAGGATTTT TAACCCCCAA TCTCTCATAA TCGCTAGTGT 2462 TTAAAAGGCT AAGAATAGTG GGGCCCAACC GATGTGGTAG GTGATAAAGA GGCATCTTTT 2522 CTAGAGACAC ATTGGACCAG ATGAGGATCC GAAACGGCAG CCTTTACGTT CATCACCTGC 2582 TAGAACCTCT CGTAGTCCAT CACCATTTCT TGGCATTGGA ATTCTACTGG AAAAAAATAC 2642 AAAAAGCAAA ACAAAACCCT CAGCACTGTT ACAAGAGGCC ATTTAAGTAT CTTGTGCTTC 2702 TTCACTTACC CATTAGCCAG GTTCTCATTA GGTTTTGCTT GGGCCTCCCT GGCACTGAAC 2762 CTTAGGCTTT GTATGACAGT GAAGCAGCAC TGTGAGTGGT TCAAGCACAC TGGAATATAA 2822 AACAGTCATG GCCTGAGATG CAGGTGATGC CATTACAGAA CCAAATCGTG GCACGTATTG 2882 CTGTGTCTCC TCTCAGAGTG ACAGTCATAA ATACTGTCAA ACAATAAAGG GAGAATGGTG 2942 CTGTTTAAAG TCACATCCCT GTAAATTGCA GAATTCAAAA GTGATTATCT CTTTGATCTA 3002 CTTGCCTCAT TTCCCTATCT TCTCCCCCAC GGTATCCTAA ACTTTAGACT TCCCACTGTT 3062 CTGAAAGGAG ACATTGCTCT ATGTCTGCCT TCGACCACAG CAAGCCATCA TCCTCCATTG 3122 CTCCCGGGGA CTCAAGAGGA ATCTGTTTCT CTGCTGTCAA CTTCCCATCT GGCTCAGCAT 3182 AGGGTCACTT TGCCATTATG CAAATGGAGA TAAAAGCAAT TCTGGCTGTC CAGGAGCTAA 3242 TCTGACCGTT CTATTGTGTG GATGACCACA TAAGAAGGCA ATTTTAGTGT ATTAATCATA 3302 GATTATTATA AACTATAAAC TTAAGGGCAA GGAGTTTATT ACAATGTATC TTTATTAAAA 3362 CAAAAGGGTG TATAGTGTTC ACAAACTGTG AAAATAGTGT AAGAACTGTA CATTGTGAGC 3422 TCTGGTTATT TTTCTCTTGT ACCATAGAAA AATGTATAAA AATTATCAAA AAGCTAATGT 3482 GCAGGGATAT TGCCTTATTT GTCTGTAAAA AATGGAGCTC AGTAACATAA CTGCTTCTTG 3542 GAGCTTTGGA ATATTTTATC CTGTATTCTT GTTT 3576 399 amino acids amino acid linear protein internal 10 Met Ala Ala Pro Ser Pro Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser 1 5 10 15 Gly Ser Gly Thr Pro Gly Pro Val Gly Ser Pro Ala Pro Gly His Pro 20 25 30 Ala Val Ser Ser Met Gln Gly Lys Arg Lys Ala Leu Lys Leu Asn Phe 35 40 45 Ala Asn Pro Pro Phe Lys Ser Thr Ala Arg Phe Thr Leu Asn Pro Asn 50 55 60 Pro Thr Gly Val Gln Asn Pro His Ile Glu Arg Leu Arg Thr His Ser 65 70 75 80 Ile Glu Ser Ser Gly Lys Leu Lys Ile Ser Pro Glu Gln His Trp Asp 85 90 95 Phe Thr Ala Glu Asp Leu Lys Asp Leu Gly Glu Ile Gly Arg Gly Ala 100 105 110 Tyr Gly Ser Val Asn Lys Met Val His Lys Pro Ser Gly Gln Ile Met 115 120 125 Ala Val Lys Arg Ile Arg Ser Thr Val Asp Glu Lys Glu Gln Lys Gln 130 135 140 Leu Leu Met Asp Leu Asp Val Val Met Arg Ser Ser Asp Cys Pro Tyr 145 150 155 160 Ile Val Gln Phe Tyr Gly Ala Leu Phe Arg Glu Gly Asp Cys Trp Ile 165 170 175 Cys Met Glu Leu Met Ser Thr Ser Phe Asp Lys Phe Tyr Lys Tyr Val 180 185 190 Tyr Ser Val Leu Asp Asp Val Ile Pro Glu Glu Ile Leu Gly Lys Ile 195 200 205 Thr Leu Ala Thr Val Lys Ala Leu Asn His Leu Lys Glu Asn Leu Lys 210 215 220 Ile Ile His Arg Asp Ile Lys Pro Ser Asn Ile Leu Leu Asp Arg Ser 225 230 235 240 Gly Asn Ile Lys Leu Cys Asp Phe Gly Ile Ser Gly Gln Leu Val Asp 245 250 255 Ser Ile Ala Lys Thr Arg Asp Ala Gly Cys Arg Pro Tyr Met Ala Pro 260 265 270 Glu Arg Ile Asp Pro Ser Ala Ser Arg Gln Gly Tyr Asp Val Arg Ser 275 280 285 Asp Val Trp Ser Leu Gly Ile Thr Leu Tyr Glu Leu Ala Thr Gly Arg 290 295 300 Phe Pro Tyr Pro Lys Trp Asn Ser Val Phe Asp Gln Leu Thr Gln Val 305 310 315 320 Val Lys Gly Asp Pro Pro Gln Leu Ser Asn Ser Glu Glu Arg Glu Phe 325 330 335 Ser Pro Ser Phe Ile Asn Phe Val Asn Leu Cys Leu Thr Lys Asp Glu 340 345 350 Ser Lys Arg Pro Lys Tyr Lys Glu Leu Leu Lys His Pro Phe Ile Leu 355 360 365 Met Tyr Glu Glu Arg Ala Val Glu Val Ala Cys Tyr Val Cys Lys Ile 370 375 380 Leu Asp Gln Met Pro Ala Thr Pro Ser Ser Pro Met Tyr Val Asp 385 390 395 393 amino acids amino acid linear protein 11 Met Pro Lys Lys Lys Pro Thr Pro Ile Gln Leu Asn Pro Ala Pro Asp 1 5 10 15 Gly Ser Ala Val Asn Gly Thr Ser Ser Ala Glu Thr Asn Leu Glu Ala 20 25 30 Leu Gln Lys Lys Leu Glu Glu Leu Glu Leu Asp Glu Gln Gln Arg Lys 35 40 45 Arg Leu Glu Ala Phe Leu Thr Gln Lys Gln Lys Val Gly Glu Leu Lys 50 55 60 Asp Asp Asp Phe Glu Lys Ile Ser Glu Leu Gly Ala Gly Asn Gly Gly 65 70 75 80 Val Val Phe Lys Val Ser His Lys Pro Ser Gly Leu Val Met Ala Arg 85 90 95 Lys Leu Ile His Leu Glu Ile Lys Pro Ala Ile Arg Asn Gln Ile Ile 100 105 110 Arg Glu Leu Gln Val Leu His Glu Cys Asn Ser Pro Tyr Ile Val Gly 115 120 125 Phe Tyr Gly Ala Phe Tyr Ser Asp Gly Glu Ile Ser Ile Cys Met Glu 130 135 140 His Met Asp Gly Gly Ser Leu Asp Gln Val Leu Lys Lys Ala Gly Arg 145 150 155 160 Ile Pro Glu Gln Ile Leu Gly Lys Val Ser Ile Ala Val Ile Lys Gly 165 170 175 Leu Thr Tyr Leu Arg Glu Lys His Lys Ile Met His Arg Asp Val Lys 180 185 190 Pro Ser Asn Ile Leu Val Asn Ser Arg Gly Glu Ile Lys Leu Cys Asp 195 200 205 Phe Gly Val Ser Gly Gln Leu Ile Asp Ser Met Ala Asn Ser Phe Val 210 215 220 Gly Thr Arg Ser Tyr Met Ser Pro Glu Arg Leu Gln Gly Thr His Tyr 225 230 235 240 Ser Val Gln Ser Asp Ile Trp Ser Met Gly Leu Ser Leu Val Glu Met 245 250 255 Ala Val Gly Arg Tyr Pro Ile Pro Pro Pro Asp Ala Lys Glu Leu Glu 260 265 270 Leu Met Phe Gly Cys Gln Val Glu Gly Asp Ala Ala Glu Thr Pro Pro 275 280 285 Arg Pro Arg Thr Pro Gly Arg Pro Leu Ser Ser Tyr Gly Met Asp Ser 290 295 300 Arg Pro Pro Met Ala Ile Phe Glu Leu Leu Asp Tyr Ile Val Asn Glu 305 310 315 320 Pro Pro Pro Lys Leu Pro Ser Gly Val Phe Ser Leu Glu Phe Gln Asp 325 330 335 Phe Val Asn Lys Cys Leu Ile Lys Asn Pro Ala Glu Arg Ala Asp Leu 340 345 350 Lys Gln Leu Met Val His Ala Phe Ile Lys Arg Ser Asp Ala Glu Glu 355 360 365 Val Asp Phe Ala Gly Trp Leu Cys Ser Thr Ile Gly Leu Asn Gln Pro 370 375 380 Ser Thr Pro Thr His Ala Ala Gly Val 385 390 400 amino acids amino acid linear protein 12 Met Leu Ala Arg Arg Lys Pro Val Leu Pro Ala Leu Thr Ile Asn Pro 1 5 10 15 Thr Ile Ala Glu Gly Pro Ser Pro Thr Ser Glu Gly Ala Ser Glu Ala 20 25 30 Asn Leu Val Asp Leu Gln Lys Lys Leu Glu Glu Leu Glu Leu Asp Glu 35 40 45 Gln Gln Lys Lys Arg Leu Glu Ala Phe Leu Thr Gln Lys Ala Lys Val 50 55 60 Gly Glu Leu Lys Asp Asp Asp Phe Glu Arg Ile Ser Glu Leu Gly Ala 65 70 75 80 Gly Asn Gly Gly Val Val Thr Lys Val Gln His Arg Pro Ser Gly Leu 85 90 95 Ile Met Ala Arg Lys Leu Ile His Leu Glu Ile Lys Pro Ala Ile Arg 100 105 110 Asn Gln Ile Ile Arg Glu Leu Gln Val Leu His Glu Cys Asn Ser Pro 115 120 125 Tyr Ile Val Gly Phe Tyr Gly Ala Phe Tyr Ser Asp Gly Glu Ile Ser 130 135 140 Ile Cys Met Glu His Met Asp Gly Gly Ser Leu Asp Gln Val Leu Lys 145 150 155 160 Glu Ala Lys Arg Ile Pro Glu Glu Ile Leu Gly Lys Val Ser Ile Ala 165 170 175 Val Leu Arg Gly Leu Ala Tyr Leu Arg Glu Lys His Gln Ile Met His 180 185 190 Arg Asp Val Lys Pro Ser Asn Ile Leu Val Asn Ser Arg Gly Glu Ile 195 200 205 Lys Leu Cys Asp Phe Gly Val Ser Gly Gln Leu Ile Asp Ser Met Ala 210 215 220 Asn Ser Phe Val Gly Thr Arg Ser Tyr Met Ala Pro Glu Arg Leu Gln 225 230 235 240 Gly Thr His Tyr Ser Val Gln Ser Asp Ile Trp Ser Met Gly Leu Ser 245 250 255 Leu Val Glu Leu Ala Val Gly Arg Tyr Pro Ile Pro Pro Pro Asp Ala 260 265 270 Lys Glu Leu Glu Ala Ile Phe Gly Arg Pro Val Val Asp Gly Glu Glu 275 280 285 Gly Glu Pro His Ser Ile Ser Pro Arg Pro Arg Pro Pro Gly Arg Pro 290 295 300 Val Ser Gly His Gly Met Asp Ser Arg Pro Ala Met Ala Ile Phe Glu 305 310 315 320 Leu Leu Asp Tyr Ile Val Asn Glu Pro Pro Pro Lys Leu Pro Asn Gly 325 330 335 Val Phe Thr Pro Asp Phe Gln Glu Phe Val Asn Lys Cys Leu Ile Lys 340 345 350 Asn Pro Ala Glu Arg Ala Asp Leu Lys Met Leu Thr Asn His Thr Phe 355 360 365 Ile Lys Arg Ser Glu Val Glu Glu Val Asp Phe Ala Gly Trp Leu Cys 370 375 380 Lys Thr Leu Arg Leu Asn Gln Pro Gly Thr Pro Thr Arg Thr Ala Val 385 390 395 400 367 amino acids amino acid linear protein 13 Gly Thr Thr Pro Arg Thr Gly Asn Ser Asn Asn Ser Asn Ser Gly Ser 1 5 10 15 Ser Gly Gly Gly Gly Leu Phe Ala Asn Phe Ser Lys Tyr Val Asp Ile 20 25 30 Lys Ser Gly Ser Leu Asn Phe Ala Gly Lys Leu Ser Leu Ser Ser Lys 35 40 45 Gly Ile Asp Phe Ser Asn Gly Ser Ser Ser Arg Ile Thr Leu Asp Glu 50 55 60 Leu Glu Phe Leu Asp Glu Leu Gly His Gly Asn Tyr Gly Asn Val Ser 65 70 75 80 Lys Val Leu His Lys Pro Thr Asn Val Ile Met Ala Thr Lys Glu Val 85 90 95 Arg Leu Glu Leu Asp Glu Ala Lys Phe Arg Gln Ile Leu Met Glu Leu 100 105 110 Glu Val Leu His Lys Cys Asn Ser Pro Tyr Ile Val Asp Phe Tyr Gly 115 120 125 Ala Phe Phe Ile Glu Gly Ala Val Tyr Met Cys Met Glu Tyr Met Asp 130 135 140 Gly Gly Ser Leu Asp Lys Ile Tyr Asp Glu Ser Ser Glu Ile Gly Gly 145 150 155 160 Ile Asp Glu Pro Gln Leu Ala Phe Ile Ala Asn Ala Val Ile His Gly 165 170 175 Leu Lys Glu Leu Lys Glu Gln His Asn Ile Ile His Arg Asp Val Lys 180 185 190 Pro Thr Asn Ile Leu Cys Ser Ala Asn Gln Gly Thr Val Lys Leu Cys 195 200 205 Asp Phe Gly Val Ser Gly Asn Leu Val Ala Ser Leu Ala Lys Thr Met 210 215 220 Asn Ile Gly Cys Gln Ser Tyr Met Ala Pro Glu Arg Ile Lys Ser Leu 225 230 235 240 Asn Pro Asp Arg Ala Thr Tyr Thr Val Gln Ser Asp Ile Trp Ser Leu 245 250 255 Gly Leu Ser Ile Leu Glu Met Ala Leu Gly Arg Tyr Pro Tyr Pro Pro 260 265 270 Glu Thr Tyr Asp Asn Ile Phe Ser Gln Leu Ser Ala Ile Val Asp Gly 275 280 285 Pro Pro Pro Arg Leu Pro Ser Asp Lys Phe Ser Ser Asp Ala Gln Asp 290 295 300 Phe Val Ser Leu Cys Leu Gln Lys Ile Pro Glu Arg Arg Pro Thr Tyr 305 310 315 320 Ala Ala Leu Thr Glu His Pro Trp Leu Val Lys Tyr Arg Asn Gln Asp 325 330 335 Val His Met Ser Glu Tyr Ile Thr Glu Arg Leu Glu Arg Arg Asn Lys 340 345 350 Ile Leu Arg Glu Arg Gly Glu Asn Gly Leu Ser Lys Asn Val Pro 355 360 365 23 base pairs nucleic acid single linear cDNA 14 TTYTAYGGNG CNTTYTTYAT HGA 23 20 base pairs nucleic acid single linear cDNA 15 ATBCTYTCNG GNGCCATKTA 20 8 amino acids amino acid linear peptide 16 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 1623 base pairs nucleic acid double linear cDNA Coding Sequence 281...1318 17 GGAAAGGCAG CCTCCTGTAG GTGAAAATTC TGTTCACTAC CTGGCCACCT GGCCTGACTG 60 ACCTTCACAG CTTGATCATC TTCCTGAAGA GGCATTCAGG ATTCCCTCCA TCCCTACCCC 120 TTCTGGACAA AGTCTTCCAC GTTTCCTTCC TGGGAGTTTC TTCCAGGAAC TGGAGATACC 180 CAGAGCCCTG CAACTCCCAC TGGCCAACGA TGGGGGCAGC CGCTCACCAT CCTCAGAGAG 240 CTCCCCACAG CACCCTACAC CCCCCACCCG GCCCCGCCAC ATG CTG GGG CTC CCA 295 Met Leu Gly Leu Pro 1 5 TCA ACC TTG TTC ACA CCG CGC AGT ATG GAG AGC ATC GAG ATT GAC CAG 343 Ser Thr Leu Phe Thr Pro Arg Ser Met Glu Ser Ile Glu Ile Asp Gln 10 15 20 AAG CTG CAG GAG ATC ATG AAG CAG ACA GGG TAC CTG ACT ATC GGG GGC 391 Lys Leu Gln Glu Ile Met Lys Gln Thr Gly Tyr Leu Thr Ile Gly Gly 25 30 35 CAG CGT TAT CAG GCA GAA ATC AAT GAC TTG GAG AAC TTG GGT GAG ATG 439 Gln Arg Tyr Gln Ala Glu Ile Asn Asp Leu Glu Asn Leu Gly Glu Met 40 45 50 GGC AGT GGT ACC TGT GGT CAG GTG TGG AAG ATG CGG TTC CGG AAG ACA 487 Gly Ser Gly Thr Cys Gly Gln Val Trp Lys Met Arg Phe Arg Lys Thr 55 60 65 GGC CAC ATC ATT GCT GTT AAG CAA ATG CGG CGC TCT GGG AAC AAG GAA 535 Gly His Ile Ile Ala Val Lys Gln Met Arg Arg Ser Gly Asn Lys Glu 70 75 80 85 GAG AAT AAG CGC ATT TTG ATG GAC CTG GAT GTA GTA CTC AAG AGC CAT 583 Glu Asn Lys Arg Ile Leu Met Asp Leu Asp Val Val Leu Lys Ser His 90 95 100 GAC TGC CCT TAC ATC GTT CAG TGC TTT GGC ACC TTC ATC ACC AAC ACA 631 Asp Cys Pro Tyr Ile Val Gln Cys Phe Gly Thr Phe Ile Thr Asn Thr 105 110 115 GAC GTC TTT ATT GCC ATG GAG CTC ATG GGC ACA TGT GCA GAG AAG CTG 679 Asp Val Phe Ile Ala Met Glu Leu Met Gly Thr Cys Ala Glu Lys Leu 120 125 130 AAG AAA CGA ATG CAG GGC CCC ATT CCA GAG CGA ATC CTG GGC AAC ATG 727 Lys Lys Arg Met Gln Gly Pro Ile Pro Glu Arg Ile Leu Gly Asn Met 135 140 145 ACT GTG GCG ATT GTG AAA GCA CTG TAC TAT CTG AAG GAG AAG CAT GGC 775 Thr Val Ala Ile Val Lys Ala Leu Tyr Tyr Leu Lys Glu Lys His Gly 150 155 160 165 GTC ATC CAT CGC GAT GTC AAA CCC TCC AAC ATC CTG CTA GAT GAG CGG 823 Val Ile His Arg Asp Val Lys Pro Ser Asn Ile Leu Leu Asp Glu Arg 170 175 180 GGC CAG ATC AAG CTC TGT GAC TTT GGC ATC AGT GGC CGC CTT GTT GAC 871 Gly Gln Ile Lys Leu Cys Asp Phe Gly Ile Ser Gly Arg Leu Val Asp 185 190 195 TCC AAA GCC AAA ACA CGG AGT GCT GGC TGT GCT GCC TAT ATG GCT CCC 919 Ser Lys Ala Lys Thr Arg Ser Ala Gly Cys Ala Ala Tyr Met Ala Pro 200 205 210 GAG CGC ATC GAC CCT CCA GAT CCC ACC AAG CCT GAC TAT GAC ATC CGA 967 Glu Arg Ile Asp Pro Pro Asp Pro Thr Lys Pro Asp Tyr Asp Ile Arg 215 220 225 GCT GAT GTG TGG AGC CTG GGC ATC TCA CTG GTG GAG CTG GCA ACA GGA 1015 Ala Asp Val Trp Ser Leu Gly Ile Ser Leu Val Glu Leu Ala Thr Gly 230 235 240 245 CAG TTC CCC TAT AAG AAC TGC AAG ACG GAC TTT GAG GTC CTC ACC AAA 1063 Gln Phe Pro Tyr Lys Asn Cys Lys Thr Asp Phe Glu Val Leu Thr Lys 250 255 260 GTC CTA CAG GAA GAG CCC CCA CTC CTG CCT GGT CAC ATG GGC TTC TCA 1111 Val Leu Gln Glu Glu Pro Pro Leu Leu Pro Gly His Met Gly Phe Ser 265 270 275 GGG GAC TTC CAG TCA TTT GTC AAA GAC TGC CTT ACT AAA GAT CAC AGG 1159 Gly Asp Phe Gln Ser Phe Val Lys Asp Cys Leu Thr Lys Asp His Arg 280 285 290 AAG AGA CCA AAG TAT AAT AAG CTA CTT GAA CAC AGC TTC ATC AAG CAC 1207 Lys Arg Pro Lys Tyr Asn Lys Leu Leu Glu His Ser Phe Ile Lys His 295 300 305 TAT GAG ATA CTC GAG GTG GAT GTC GCG TCC TGG TTT AAG GAT GTC ATG 1255 Tyr Glu Ile Leu Glu Val Asp Val Ala Ser Trp Phe Lys Asp Val Met 310 315 320 325 GCG AAG ACC GAG TCC CCA AGG ACT AGT GGA GTC CTG AGT CAG CAC CAT 1303 Ala Lys Thr Glu Ser Pro Arg Thr Ser Gly Val Leu Ser Gln His His 330 335 340 CTG CCC TTC TTC AGG TAGCCTCATG GCAGCGGCCA GCCCCGCAGG GGCCCCGGGC C 1359 Leu Pro Phe Phe Arg 345 ACGGCCACCG ACCCCCCCCC CAACCTGGCC AACCCAGCTG CCCATCAGGG GACCTGGGAC 1419 CTGGACGACT GCCAAGGACT GAGGACAGAA AGTAGGGGGT TCCCATCCAG CTCTGACTCC 1479 CTGCCTACCA GCTGTGGACA AAAGGGCATG CTGGTTCCTA ATCCCTCCCA CTCTGGGGTC 1539 AGCCAGCAGT GTGAGCCCCA TCCCACCCCG ACAGACACTG TGAACGGAAG ACAGCAGGCC 1599 AAAAAAAAAA AAAAAAAAAA AAAA 1623 346 amino acids amino acid linear protein internal 18 Met Leu Gly Leu Pro Ser Thr Leu Phe Thr Pro Arg Ser Met Glu Ser 1 5 10 15 Ile Glu Ile Asp Gln Lys Leu Gln Glu Ile Met Lys Gln Thr Gly Tyr 20 25 30 Leu Thr Ile Gly Gly Gln Arg Tyr Gln Ala Glu Ile Asn Asp Leu Glu 35 40 45 Asn Leu Gly Glu Met Gly Ser Gly Thr Cys Gly Gln Val Trp Lys Met 50 55 60 Arg Phe Arg Lys Thr Gly His Ile Ile Ala Val Lys Gln Met Arg Arg 65 70 75 80 Ser Gly Asn Lys Glu Glu Asn Lys Arg Ile Leu Met Asp Leu Asp Val 85 90 95 Val Leu Lys Ser His Asp Cys Pro Tyr Ile Val Gln Cys Phe Gly Thr 100 105 110 Phe Ile Thr Asn Thr Asp Val Phe Ile Ala Met Glu Leu Met Gly Thr 115 120 125 Cys Ala Glu Lys Leu Lys Lys Arg Met Gln Gly Pro Ile Pro Glu Arg 130 135 140 Ile Leu Gly Asn Met Thr Val Ala Ile Val Lys Ala Leu Tyr Tyr Leu 145 150 155 160 Lys Glu Lys His Gly Val Ile His Arg Asp Val Lys Pro Ser Asn Ile 165 170 175 Leu Leu Asp Glu Arg Gly Gln Ile Lys Leu Cys Asp Phe Gly Ile Ser 180 185 190 Gly Arg Leu Val Asp Ser Lys Ala Lys Thr Arg Ser Ala Gly Cys Ala 195 200 205 Ala Tyr Met Ala Pro Glu Arg Ile Asp Pro Pro Asp Pro Thr Lys Pro 210 215 220 Asp Tyr Asp Ile Arg Ala Asp Val Trp Ser Leu Gly Ile Ser Leu Val 225 230 235 240 Glu Leu Ala Thr Gly Gln Phe Pro Tyr Lys Asn Cys Lys Thr Asp Phe 245 250 255 Glu Val Leu Thr Lys Val Leu Gln Glu Glu Pro Pro Leu Leu Pro Gly 260 265 270 His Met Gly Phe Ser Gly Asp Phe Gln Ser Phe Val Lys Asp Cys Leu 275 280 285 Thr Lys Asp His Arg Lys Arg Pro Lys Tyr Asn Lys Leu Leu Glu His 290 295 300 Ser Phe Ile Lys His Tyr Glu Ile Leu Glu Val Asp Val Ala Ser Trp 305 310 315 320 Phe Lys Asp Val Met Ala Lys Thr Glu Ser Pro Arg Thr Ser Gly Val 325 330 335 Leu Ser Gln His His Leu Pro Phe Phe Arg 340 345 1465 base pairs nucleic acid double linear cDNA Coding Sequence 3...1169 19 GC ACG AGC CCT GCT CCT GCC CCG TCC CAG CGA GCA GCC CTG CAA CTC 47 Thr Ser Pro Ala Pro Ala Pro Ser Gln Arg Ala Ala Leu Gln Leu 1 5 10 15 CCA CTG GCC AAC GAT GGG GGC AGC CGC TCA CCA TCC TCA GAG AGC TCC 95 Pro Leu Ala Asn Asp Gly Gly Ser Arg Ser Pro Ser Ser Glu Ser Ser 20 25 30 CCA CAG CAC CCT ACA CCC CCC ACC CGG CCC CGC CAC ATG CTG GGG CTC 143 Pro Gln His Pro Thr Pro Pro Thr Arg Pro Arg His Met Leu Gly Leu 35 40 45 CCA TCA ACC TTG TTC ACA CCG CGC AGT ATG GAG AGC ATC GAG ATT GAC 191 Pro Ser Thr Leu Phe Thr Pro Arg Ser Met Glu Ser Ile Glu Ile Asp 50 55 60 CAG AAG CTG CAG GAG ATC ATG AAG CAG ACA GGG TAC CTG ACT ATC GGG 239 Gln Lys Leu Gln Glu Ile Met Lys Gln Thr Gly Tyr Leu Thr Ile Gly 65 70 75 GGC CAG CGT TAT CAG GCA GAA ATC AAT GAC TTG GAG AAC TTG GGT GAG 287 Gly Gln Arg Tyr Gln Ala Glu Ile Asn Asp Leu Glu Asn Leu Gly Glu 80 85 90 95 ATG GGC AGT GGT ACC TGT GGT CAG GTG TGG AAG ATG CGG TTC CGG AAG 335 Met Gly Ser Gly Thr Cys Gly Gln Val Trp Lys Met Arg Phe Arg Lys 100 105 110 ACA GGC CAC ATC ATT GCT GTT AAG CAA ATG CGG CGC TCT GGG AAC AAG 383 Thr Gly His Ile Ile Ala Val Lys Gln Met Arg Arg Ser Gly Asn Lys 115 120 125 GAA GAG AAT AAG CGC ATT TTG ATG GAC CTG GAT GTA GTA CTC AAG AGC 431 Glu Glu Asn Lys Arg Ile Leu Met Asp Leu Asp Val Val Leu Lys Ser 130 135 140 CAT GAC TGC CCT TAC ATC GTT CAG TGC TTT GGC ACC TTC ATC ACC AAC 479 His Asp Cys Pro Tyr Ile Val Gln Cys Phe Gly Thr Phe Ile Thr Asn 145 150 155 ACA GAC GTC TTT ATT GCC ATG GAG CTC ATG GGC ACA TGT GCA GAG AAG 527 Thr Asp Val Phe Ile Ala Met Glu Leu Met Gly Thr Cys Ala Glu Lys 160 165 170 175 CTG AAG AAA CGA ATG CAG GGC CCC ATT CCA GAG CGA ATC CTG GGC AAG 575 Leu Lys Lys Arg Met Gln Gly Pro Ile Pro Glu Arg Ile Leu Gly Lys 180 185 190 ATG ACT GTG GCG ATT GTG AAA GCA CTG TAC TAT CTG AAG GAG AAG CAT 623 Met Thr Val Ala Ile Val Lys Ala Leu Tyr Tyr Leu Lys Glu Lys His 195 200 205 GGC GTC ATC CAT CGC GAT GTC AAA CCC TCC AAC ATC CTG CTA GAT GAG 671 Gly Val Ile His Arg Asp Val Lys Pro Ser Asn Ile Leu Leu Asp Glu 210 215 220 CGG GGC CAG ATC AAG CTC TGT GAC TTT GGC ATC AGT GGC CGC CTT GTT 719 Arg Gly Gln Ile Lys Leu Cys Asp Phe Gly Ile Ser Gly Arg Leu Val 225 230 235 GAC TCC AAA GCC AAA ACA CGG AGT GCT GGC TGT GCT GCC TAT ATG GCT 767 Asp Ser Lys Ala Lys Thr Arg Ser Ala Gly Cys Ala Ala Tyr Met Ala 240 245 250 255 CCC GAG CGC ATC GAC CCT CCA GAT CCC ACC AAG CCT GAC TAT GAC ATC 815 Pro Glu Arg Ile Asp Pro Pro Asp Pro Thr Lys Pro Asp Tyr Asp Ile 260 265 270 CGA GCT GAT GTG TGG AGC CTG GGC ATC TCA CTG GTG GAG CTG GCA ACA 863 Arg Ala Asp Val Trp Ser Leu Gly Ile Ser Leu Val Glu Leu Ala Thr 275 280 285 GGA CAG TTC CCC TAT AAG AAC TGC AAG ACG GAC TTT GAG GTC CTC ACC 911 Gly Gln Phe Pro Tyr Lys Asn Cys Lys Thr Asp Phe Glu Val Leu Thr 290 295 300 AAA GTC CTA CAG GAA GAG CCC CCA CTC CTG CCT GGT CAC ATG GGC TTC 959 Lys Val Leu Gln Glu Glu Pro Pro Leu Leu Pro Gly His Met Gly Phe 305 310 315 TCA GGG GAC TTC CAG TCA TTT GTC AAA GAC TGC CTT ACT AAA GAT CAC 1007 Ser Gly Asp Phe Gln Ser Phe Val Lys Asp Cys Leu Thr Lys Asp His 320 325 330 335 AGG AAG AGA CCA AAG TAT AAT AAG CTA CTT GAA CAC AGC TTC ATC AAG 1055 Arg Lys Arg Pro Lys Tyr Asn Lys Leu Leu Glu His Ser Phe Ile Lys 340 345 350 CAC TAT GAG ATA CTC GAG GTG GAT GTC GCG TCC TGG TTT AAG GAT GTC 1103 His Tyr Glu Ile Leu Glu Val Asp Val Ala Ser Trp Phe Lys Asp Val 355 360 365 ATG GCG AAG ACC GAG TCC CCA AGG ACT AGT GGA GTC CTG AGT CAG CAC 1151 Met Ala Lys Thr Glu Ser Pro Arg Thr Ser Gly Val Leu Ser Gln His 370 375 380 CAT CTG CCC TTC TTC AGG TAGCCTCATG GCAGCGGCCA GCCCCGCAGG GGCCCCGG 1207 His Leu Pro Phe Phe Arg 385 GCCACGGCCA CCGACCCCCC CCCCAACCTG GCCAACCCAG CTGCCCATCA GGGGACCTGG 1267 GACCTGGACG ACTGCCAAGG ACTGAGGACA GAAAGTAGGG GGTTCCCATC CAGCTCTGAC 1327 TCCCTGCCTA CCAGCTGTGG ACAAAAGGGC ATGCTGGTTC CTAATCCCTC CCACTCTGGG 1387 GTCAGCCAGC AGTGTGAGCC CCATCCCACC CCGACAGACA CTGTGAACGG AAGACAGCAA 1447 AAAAAAAAAA AAAAAAAA 1465 389 amino acids amino acid linear protein internal 20 Thr Ser Pro Ala Pro Ala Pro Ser Gln Arg Ala Ala Leu Gln Leu Pro 1 5 10 15 Leu Ala Asn Asp Gly Gly Ser Arg Ser Pro Ser Ser Glu Ser Ser Pro 20 25 30 Gln His Pro Thr Pro Pro Thr Arg Pro Arg His Met Leu Gly Leu Pro 35 40 45 Ser Thr Leu Phe Thr Pro Arg Ser Met Glu Ser Ile Glu Ile Asp Gln 50 55 60 Lys Leu Gln Glu Ile Met Lys Gln Thr Gly Tyr Leu Thr Ile Gly Gly 65 70 75 80 Gln Arg Tyr Gln Ala Glu Ile Asn Asp Leu Glu Asn Leu Gly Glu Met 85 90 95 Gly Ser Gly Thr Cys Gly Gln Val Trp Lys Met Arg Phe Arg Lys Thr 100 105 110 Gly His Ile Ile Ala Val Lys Gln Met Arg Arg Ser Gly Asn Lys Glu 115 120 125 Glu Asn Lys Arg Ile Leu Met Asp Leu Asp Val Val Leu Lys Ser His 130 135 140 Asp Cys Pro Tyr Ile Val Gln Cys Phe Gly Thr Phe Ile Thr Asn Thr 145 150 155 160 Asp Val Phe Ile Ala Met Glu Leu Met Gly Thr Cys Ala Glu Lys Leu 165 170 175 Lys Lys Arg Met Gln Gly Pro Ile Pro Glu Arg Ile Leu Gly Lys Met 180 185 190 Thr Val Ala Ile Val Lys Ala Leu Tyr Tyr Leu Lys Glu Lys His Gly 195 200 205 Val Ile His Arg Asp Val Lys Pro Ser Asn Ile Leu Leu Asp Glu Arg 210 215 220 Gly Gln Ile Lys Leu Cys Asp Phe Gly Ile Ser Gly Arg Leu Val Asp 225 230 235 240 Ser Lys Ala Lys Thr Arg Ser Ala Gly Cys Ala Ala Tyr Met Ala Pro 245 250 255 Glu Arg Ile Asp Pro Pro Asp Pro Thr Lys Pro Asp Tyr Asp Ile Arg 260 265 270 Ala Asp Val Trp Ser Leu Gly Ile Ser Leu Val Glu Leu Ala Thr Gly 275 280 285 Gln Phe Pro Tyr Lys Asn Cys Lys Thr Asp Phe Glu Val Leu Thr Lys 290 295 300 Val Leu Gln Glu Glu Pro Pro Leu Leu Pro Gly His Met Gly Phe Ser 305 310 315 320 Gly Asp Phe Gln Ser Phe Val Lys Asp Cys Leu Thr Lys Asp His Arg 325 330 335 Lys Arg Pro Lys Tyr Asn Lys Leu Leu Glu His Ser Phe Ile Lys His 340 345 350 Tyr Glu Ile Leu Glu Val Asp Val Ala Ser Trp Phe Lys Asp Val Met 355 360 365 Ala Lys Thr Glu Ser Pro Arg Thr Ser Gly Val Leu Ser Gln His His 370 375 380 Leu Pro Phe Phe Arg 385 393 amino acids amino acid linear protein 21 Ser Ala Ser Ser Ser Ser Ser Ser Ala Ser Ala Phe Ala Ser Ala Ala 1 5 10 15 Pro Ala Thr Gly Thr Phe Gly Gly Thr Tyr Thr Pro Pro Thr Thr Arg 20 25 30 Val Ser Arg Ala Thr Pro Thr Leu Pro Met Leu Ser Ser Gly Pro Gly 35 40 45 Gly Gly Leu Asn Arg Thr Arg Pro Asn Ile Leu Pro Leu Pro Thr Pro 50 55 60 Pro His Pro Pro Val Ser Glu Thr Asp Met Lys Leu Lys Ile Ile Met 65 70 75 80 Glu Gln Thr Gly Lys Leu Asn Ile Asn Gly Arg Gln Tyr Pro Thr Asp 85 90 95 Ile Asn Asp Leu Lys His Leu Gly Asp Leu Gly Asn Gly Thr Ser Gly 100 105 110 Asn Val Val Lys Met Met His Leu Ser Ser Asn Thr Ile Ile Ala Val 115 120 125 Lys Gln Met Arg Arg Thr Gly Asn Ala Glu Glu Asn Lys Arg Ile Leu 130 135 140 Met Asp Leu Asp Val Val Leu Lys Ser His Asp Cys Lys Tyr Ile Val 145 150 155 160 Lys Cys Leu Gly Cys Phe Val Arg Asp Pro Asp Val Trp Ile Cys Met 165 170 175 Glu Leu Met Ser Met Cys Phe Asp Lys Leu Leu Lys Leu Ser Lys Lys 180 185 190 Pro Val Pro Glu Gln Ile Leu Gly Lys Val Thr Val Ala Thr Val Asn 195 200 205 Ala Leu Ser Tyr Leu Lys Asp Lys His Gly Val Ile His Arg Asp Val 210 215 220 Lys Pro Ser Asn Ile Leu Ile Asp Glu Arg Gly Asn Ile Lys Leu Cys 225 230 235 240 Asp Phe Gly Ile Ser Gly Arg Leu Val Asp Ser Lys Ala Lys Thr Arg 245 250 255 Ser Ala Gly Cys Ala Ala Tyr Met Ala Pro Glu Arg Ile Asp Pro Lys 260 265 270 Lys Pro Lys Tyr Asp Ile Arg Ala Asp Val Trp Ser Leu Gly Ile Thr 275 280 285 Leu Val Glu Leu Ala Thr Ala Arg Ser Pro Tyr Glu Gly Cys Asn Thr 290 295 300 Asp Phe Glu Val Leu Thr Lys Val Leu Asp Ser Glu Pro Pro Cys Leu 305 310 315 320 Pro Tyr Gly Glu Gly Tyr Asn Phe Ser Gln Gln Phe Arg Asp Phe Val 325 330 335 Ile Lys Cys Leu Thr Lys Asn His Gln Asp Arg Pro Lys Tyr Pro Glu 340 345 350 Leu Leu Ala Gln Pro Phe Ile Arg Ile Tyr Glu Ser Ala Lys Val Asp 355 360 365 Val Pro Asn Gln Ser Ile Lys Asp Asn Arg Leu Arg Ala Asn Gly Asp 370 375 380 Pro Thr Leu Gln Arg Leu Pro Asn Ser 385 390 405 amino acids amino acid linear protein 22 Ile Gly Gln Val Leu Pro Glu Ala Thr Thr Thr Ala Phe Glu Tyr Glu 1 5 10 15 Asp Glu Asp Gly Asp Arg Ile Thr Val Arg Ser Asp Glu Glu Met Lys 20 25 30 Ala Met Leu Ser Tyr Tyr Tyr Ser Thr Val Met Glu Gln Gln Val Asn 35 40 45 Gly Gln Leu Ile Glu Pro Leu Gln Ile Phe Pro Arg Ala Cys Lys Pro 50 55 60 Pro Gly Glu Arg Asn Ile His Gly Leu Lys Val Asn Thr Arg Ala Gly 65 70 75 80 Pro Ser Gln His Ser Ser Pro Ala Val Ser Asp Ser Leu Pro Ser Asn 85 90 95 Ser Leu Lys Lys Ser Ser Ala Glu Leu Lys Lys Ile Leu Ala Asn Gly 100 105 110 Gln Met Asn Glu Gln Asp Ile Arg Tyr Arg Asp Thr Leu Gly His Gly 115 120 125 Asn Gly Gly Thr Val Glu Lys Met Arg His Val Pro Ser Gly Lys Ile 130 135 140 Leu Ala Val Lys Val Ile Leu Leu Asp Ile Thr Leu Glu Leu Gln Lys 145 150 155 160 Gln Ile Met Ser Glu Leu Glu Ile Leu Ile Lys Cys Asp Ser Ser Tyr 165 170 175 Ile Ile Gly Phe Tyr Gly Ala Phe Phe Val Glu Asn Arg Ile Ser Ile 180 185 190 Cys Thr Glu Phe Met Asp Gly Gly Ser Leu Asp Asp Ile Gly Lys Met 195 200 205 Pro Glu His Val Leu Gly Arg Ile Ala Val Ala Val Val Lys Gly Leu 210 215 220 Thr Tyr Lys Gly Leu Thr Tyr Leu Trp Ser Leu Lys Ile Leu His Arg 225 230 235 240 Asp Val Lys Pro Ser Asn Met Val Asn Thr Arg Gly Gln Val Lys Leu 245 250 255 Cys Asp Phe Gly Val Ser Thr Gln Leu Val Asn Ser Ile Ala Lys Thr 260 265 270 Tyr Val Gly Thr Asn Ala Tyr Met Ala Pro Glu Arg Ile Ser Gly Glu 275 280 285 Gln Tyr Gly Ile His Ser Asp Val Trp Ser Leu Gly Ile Thr Met Ile 290 295 300 Glu Leu Ala Thr Gly Arg Phe Pro Tyr Pro Lys Trp Asn Ser Val Leu 305 310 315 320 Gln Leu Leu Gln Cys Ile Val Asp Glu Asp Ser Pro Val Leu Pro Val 325 330 335 Gly Glu Phe Ser Glu Pro Phe Val His Phe Ile Thr Gln Cys Met Arg 340 345 350 Thr Gln Pro Lys Glu Arg Pro Ala Pro Glu Glu Leu Met Gly His Pro 355 360 365 Phe Ile Val Gln Phe Asn Asp Gly Asn Ala Ala Val Val Ser Met Trp 370 375 380 Val Cys Arg Ala Leu Glu Glu Arg Arg Thr Ser Arg Gly Pro Arg Glu 385 390 395 400 Ala Ala Ala Gly His 405 18 base pairs nucleic acid single linear DNA 23 ATNGCNGTNA ARCARATG 18 20 base pairs nucleic acid single linear DNA 24 ATNCKYTCNG GNGCCATRTA 20 843 base pairs nucleic acid double linear cDNA Coding Sequence 62...841 25 TGTTTGTCTG CCGGACTGAC GGGCGGCCGG GCGGTGCGCG GCGGCGGTGG CGGCGGGGAA 60 G ATG GCG GCG TCC TCC CTG GAA CAG AAG CTG TCC CGC CTG GAA GCA AAG 109 Met Ala Ala Ser Ser Leu Glu Gln Lys Leu Ser Arg Leu Glu Ala Lys 1 5 10 15 CTG AAG CAG GAG AAC CGG GAG GCC CGG CGG AGG ATC GAC CTC AAC CTG 157 Leu Lys Gln Glu Asn Arg Glu Ala Arg Arg Arg Ile Asp Leu Asn Leu 20 25 30 GAT ATC AGC CCC CAG CGG CCC AGG CCC ACC CTG CAG CTC CCG CTG GCC 205 Asp Ile Ser Pro Gln Arg Pro Arg Pro Thr Leu Gln Leu Pro Leu Ala 35 40 45 AAC GAT GGG GGC AGC CGC TCG CCA TCC TCA GAG AGC TCC CCG CAG CAC 253 Asn Asp Gly Gly Ser Arg Ser Pro Ser Ser Glu Ser Ser Pro Gln His 50 55 60 CCC ACG CCC CCC GCC CGG CCC CGC CAC ATG CTG GGG CTC CCG TCA ACC 301 Pro Thr Pro Pro Ala Arg Pro Arg His Met Leu Gly Leu Pro Ser Thr 65 70 75 80 CTG TTC ACA CCC CGC AGC ATG GAG AGC ATT GAG ATT GAC CAG AAG CTG 349 Leu Phe Thr Pro Arg Ser Met Glu Ser Ile Glu Ile Asp Gln Lys Leu 85 90 95 CAG GAG ATC ATG AAG CAG ACG GGC TAC CTG ACC ATC GGG GGC CAG CGC 397 Gln Glu Ile Met Lys Gln Thr Gly Tyr Leu Thr Ile Gly Gly Gln Arg 100 105 110 TAC CAG GCA GAA ATC AAC GAC CTG GAG AAC TTG GGC GAG ATG GGC AGC 445 Tyr Gln Ala Glu Ile Asn Asp Leu Glu Asn Leu Gly Glu Met Gly Ser 115 120 125 GGC ACC TGC GGC CAG GTG TGG AAG ATG CGC TTC CGG AAG ACC GGC CAC 493 Gly Thr Cys Gly Gln Val Trp Lys Met Arg Phe Arg Lys Thr Gly His 130 135 140 GTC ATT GCC GTT AAG CAA ATG CGG CGC TCC GGG AAC AAG GAG GAG AAC 541 Val Ile Ala Val Lys Gln Met Arg Arg Ser Gly Asn Lys Glu Glu Asn 145 150 155 160 AAG CGC ATC CTC ATG GAC CTG GAT GTG GTG CTG AAG AGC CAC GAC TGC 589 Lys Arg Ile Leu Met Asp Leu Asp Val Val Leu Lys Ser His Asp Cys 165 170 175 CCC TAC ATC GTG CAG TGC TTT GGG ACG TTC ATC ACC AAC ACG GAC GTC 637 Pro Tyr Ile Val Gln Cys Phe Gly Thr Phe Ile Thr Asn Thr Asp Val 180 185 190 TTC ATC GCC ATG GAG CTC ATG GGC ACC TGC GCT GAG AAG CTC AAG AAG 685 Phe Ile Ala Met Glu Leu Met Gly Thr Cys Ala Glu Lys Leu Lys Lys 195 200 205 CGG ATG CAG GGC CCC ATC CCC GAG CGC ATT CTG GGC AAG ATG ACA GTG 733 Arg Met Gln Gly Pro Ile Pro Glu Arg Ile Leu Gly Lys Met Thr Val 210 215 220 GCG ATT GTG AAG GCG CTG TAC TAC CTG AAG GAG AAG CAC GGT GTC ATC 781 Ala Ile Val Lys Ala Leu Tyr Tyr Leu Lys Glu Lys His Gly Val Ile 225 230 235 240 CAC CGC GAC GTC AAG CCC TCC AAC ATC CTG CTG GAC GAG CGG GGC CAG 829 His Arg Asp Val Lys Pro Ser Asn Ile Leu Leu Asp Glu Arg Gly Gln 245 250 255 ATC AAG CTG TGC GA 843 Ile Lys Leu Cys 260 260 amino acids amino acid linear protein internal 26 Met Ala Ala Ser Ser Leu Glu Gln Lys Leu Ser Arg Leu Glu Ala Lys 1 5 10 15 Leu Lys Gln Glu Asn Arg Glu Ala Arg Arg Arg Ile Asp Leu Asn Leu 20 25 30 Asp Ile Ser Pro Gln Arg Pro Arg Pro Thr Leu Gln Leu Pro Leu Ala 35 40 45 Asn Asp Gly Gly Ser Arg Ser Pro Ser Ser Glu Ser Ser Pro Gln His 50 55 60 Pro Thr Pro Pro Ala Arg Pro Arg His Met Leu Gly Leu Pro Ser Thr 65 70 75 80 Leu Phe Thr Pro Arg Ser Met Glu Ser Ile Glu Ile Asp Gln Lys Leu 85 90 95 Gln Glu Ile Met Lys Gln Thr Gly Tyr Leu Thr Ile Gly Gly Gln Arg 100 105 110 Tyr Gln Ala Glu Ile Asn Asp Leu Glu Asn Leu Gly Glu Met Gly Ser 115 120 125 Gly Thr Cys Gly Gln Val Trp Lys Met Arg Phe Arg Lys Thr Gly His 130 135 140 Val Ile Ala Val Lys Gln Met Arg Arg Ser Gly Asn Lys Glu Glu Asn 145 150 155 160 Lys Arg Ile Leu Met Asp Leu Asp Val Val Leu Lys Ser His Asp Cys 165 170 175 Pro Tyr Ile Val Gln Cys Phe Gly Thr Phe Ile Thr Asn Thr Asp Val 180 185 190 Phe Ile Ala Met Glu Leu Met Gly Thr Cys Ala Glu Lys Leu Lys Lys 195 200 205 Arg Met Gln Gly Pro Ile Pro Glu Arg Ile Leu Gly Lys Met Thr Val 210 215 220 Ala Ile Val Lys Ala Leu Tyr Tyr Leu Lys Glu Lys His Gly Val Ile 225 230 235 240 His Arg Asp Val Lys Pro Ser Asn Ile Leu Leu Asp Glu Arg Gly Gln 245 250 255 Ile Lys Leu Cys 260 1643 base pairs nucleic acid double linear cDNA Coding Sequence 82...1338 27 AGCGCAGGCG CAGTGCGGTG TTTGTCTACC CCGGACTGAC GGGTGGCCTG GCGGTGAGCG 60 GCGGCAGCGG CGGCGGGGAA G ATG GCG GCG TCC TCC CTG GAG CAG AAG CTG 111 Met Ala Ala Ser Ser Leu Glu Gln Lys Leu 1 5 10 TCC CGC CTG GAA GCC AAG CTG AAG CAG GAG AAC CGT GAG GCC CGC AGG 159 Ser Arg Leu Glu Ala Lys Leu Lys Gln Glu Asn Arg Glu Ala Arg Arg 15 20 25 AGG ATC GAC CTC AAC TTG GAT ATC AGC CCA CAG CGG CCC AGG CCC ACC 207 Arg Ile Asp Leu Asn Leu Asp Ile Ser Pro Gln Arg Pro Arg Pro Thr 30 35 40 CTG CAA CTC CCA CTG GCC AAC GAT GGG GGC AGC CGC TCA CCA TCC TCA 255 Leu Gln Leu Pro Leu Ala Asn Asp Gly Gly Ser Arg Ser Pro Ser Ser 45 50 55 GAG AGC TCC CCA CAG CAC CCT ACA CCC CCC ACC CGG CCC CGC CAC ATG 303 Glu Ser Ser Pro Gln His Pro Thr Pro Pro Thr Arg Pro Arg His Met 60 65 70 CTG GGG CTC CCA TCA ACC TTG TTC ACA CCG CGC AGT ATG GAG AGC ATC 351 Leu Gly Leu Pro Ser Thr Leu Phe Thr Pro Arg Ser Met Glu Ser Ile 75 80 85 90 GAG ATT GAC CAG AAG CTG CAG GAG ATC ATG AAG CAG ACA GGG TAC CTG 399 Glu Ile Asp Gln Lys Leu Gln Glu Ile Met Lys Gln Thr Gly Tyr Leu 95 100 105 ACT ATC GGG GGC CAG CGT TAT CAG GCA GAA ATC AAT GAC TTG GAG AAC 447 Thr Ile Gly Gly Gln Arg Tyr Gln Ala Glu Ile Asn Asp Leu Glu Asn 110 115 120 TTG GGT GAG ATG GGC AGT GGT ACC TGT GGT CAG GTG TGG AAG ATG CGG 495 Leu Gly Glu Met Gly Ser Gly Thr Cys Gly Gln Val Trp Lys Met Arg 125 130 135 TTC CGG AAG ACA GGC CAC ATC ATT GCT GTT AAG CAA ATG CGG CGC TCT 543 Phe Arg Lys Thr Gly His Ile Ile Ala Val Lys Gln Met Arg Arg Ser 140 145 150 GGG AAC AAG GAA GAG AAT AAG CGC ATT TTG ATG GAC CTG GAT GTA GTA 591 Gly Asn Lys Glu Glu Asn Lys Arg Ile Leu Met Asp Leu Asp Val Val 155 160 165 170 CTC AAG AGC CAT GAC TGC CCT TAC ATC GTT CAG TGC TTT GGC ACC TTC 639 Leu Lys Ser His Asp Cys Pro Tyr Ile Val Gln Cys Phe Gly Thr Phe 175 180 185 ATC ACC AAC ACA GAC GTC TTT ATT GCC ATG GAG CTC ATG GGC ACA TGT 687 Ile Thr Asn Thr Asp Val Phe Ile Ala Met Glu Leu Met Gly Thr Cys 190 195 200 GCA GAG AAG CTG AAG AAA CGA ATG CAG GGC CCC ATT CCA GAG CGA ATC 735 Ala Glu Lys Leu Lys Lys Arg Met Gln Gly Pro Ile Pro Glu Arg Ile 205 210 215 CTG GGC AAG ATG ACT GTG GCG ATT GTG AAA GCA CTG TAC TAT CTG AAG 783 Leu Gly Lys Met Thr Val Ala Ile Val Lys Ala Leu Tyr Tyr Leu Lys 220 225 230 GAG AAG CAT GGC GTC ATC CAT CGC GAT GTC AAA CCC TCC AAC ATC CTG 831 Glu Lys His Gly Val Ile His Arg Asp Val Lys Pro Ser Asn Ile Leu 235 240 245 250 CTA GAT GAG CGG GGC CAG ATC AAG CTC TGT GAC TTT GGC ATC AGT GGC 879 Leu Asp Glu Arg Gly Gln Ile Lys Leu Cys Asp Phe Gly Ile Ser Gly 255 260 265 CGC CTT GTT GAC TCC AAA GCC AAA ACA CGG AGT GCT GGC TGT GCT GCC 927 Arg Leu Val Asp Ser Lys Ala Lys Thr Arg Ser Ala Gly Cys Ala Ala 270 275 280 TAT ATG GCT CCC GAG CGC ATC GAC CCT CCA GAT CCC ACC AAG CCT GAC 975 Tyr Met Ala Pro Glu Arg Ile Asp Pro Pro Asp Pro Thr Lys Pro Asp 285 290 295 TAT GAC ATC CGA GCT GAT GTG TGG AGC CTG GGC ATC TCA CTG GTG GAG 1023 Tyr Asp Ile Arg Ala Asp Val Trp Ser Leu Gly Ile Ser Leu Val Glu 300 305 310 CTG GCA ACA GGA CAG TTC CCC TAT AAG AAC TGC AAG ACG GAC TTT GAG 1071 Leu Ala Thr Gly Gln Phe Pro Tyr Lys Asn Cys Lys Thr Asp Phe Glu 315 320 325 330 GTC CTC ACC AAA GTC CTA CAG GAA GAG CCC CCA CTC CTG CCT GGT CAC 1119 Val Leu Thr Lys Val Leu Gln Glu Glu Pro Pro Leu Leu Pro Gly His 335 340 345 ATG GGC TTC TCA GGG GAC TTC CAG TCA TTT GTC AAA GAC TGC CTT ACT 1167 Met Gly Phe Ser Gly Asp Phe Gln Ser Phe Val Lys Asp Cys Leu Thr 350 355 360 AAA GAT CAC AGG AAG AGA CCA AAG TAT AAT AAG CTA CTT GAA CAC AGC 1215 Lys Asp His Arg Lys Arg Pro Lys Tyr Asn Lys Leu Leu Glu His Ser 365 370 375 TTC ATC AAG CAC TAT GAG ATA CTC GAG GTG GAT GTC GCG TCC TGG TTT 1263 Phe Ile Lys His Tyr Glu Ile Leu Glu Val Asp Val Ala Ser Trp Phe 380 385 390 AAG GAT GTC ATG GCG AAG ACC GAG TCC CCA AGG ACT AGT GGA GTC CTG 1311 Lys Asp Val Met Ala Lys Thr Glu Ser Pro Arg Thr Ser Gly Val Leu 395 400 405 410 AGT CAG CAC CAT CTG CCC TTC TTC AGG TAGCCTCATG GCAGCGGCCA GCCCCGC 1365 Ser Gln His His Leu Pro Phe Phe Arg 415 AGGGGCCCCG GGCCACGGCC ACCGACCCCC CCCCCAACCT GGCCAACCCA GCTGCCCATC 1425 AGGGGACCTG GGACCTGGAC GACTGCCAAG GACTGAGGAC AGAAAGTAGG GGGTTCCCAT 1485 CCAGCTCTGA CTCCCTGCCT ACCAGCTGTG GACAAAAGGG CATGCTGGTT CCTAATCCCT 1545 CCCACTCTGG GGTCAGCCAG CAGTGTGAGC CCCATCCCAC CCCGACAGAC ACTGTGAACG 1605 GAAGACAGCA GGCCAAAAAA AAAAAAAAAA AAAAAAAA 1643 419 amino acids amino acid linear protein internal 28 Met Ala Ala Ser Ser Leu Glu Gln Lys Leu Ser Arg Leu Glu Ala Lys 1 5 10 15 Leu Lys Gln Glu Asn Arg Glu Ala Arg Arg Arg Ile Asp Leu Asn Leu 20 25 30 Asp Ile Ser Pro Gln Arg Pro Arg Pro Thr Leu Gln Leu Pro Leu Ala 35 40 45 Asn Asp Gly Gly Ser Arg Ser Pro Ser Ser Glu Ser Ser Pro Gln His 50 55 60 Pro Thr Pro Pro Thr Arg Pro Arg His Met Leu Gly Leu Pro Ser Thr 65 70 75 80 Leu Phe Thr Pro Arg Ser Met Glu Ser Ile Glu Ile Asp Gln Lys Leu 85 90 95 Gln Glu Ile Met Lys Gln Thr Gly Tyr Leu Thr Ile Gly Gly Gln Arg 100 105 110 Tyr Gln Ala Glu Ile Asn Asp Leu Glu Asn Leu Gly Glu Met Gly Ser 115 120 125 Gly Thr Cys Gly Gln Val Trp Lys Met Arg Phe Arg Lys Thr Gly His 130 135 140 Ile Ile Ala Val Lys Gln Met Arg Arg Ser Gly Asn Lys Glu Glu Asn 145 150 155 160 Lys Arg Ile Leu Met Asp Leu Asp Val Val Leu Lys Ser His Asp Cys 165 170 175 Pro Tyr Ile Val Gln Cys Phe Gly Thr Phe Ile Thr Asn Thr Asp Val 180 185 190 Phe Ile Ala Met Glu Leu Met Gly Thr Cys Ala Glu Lys Leu Lys Lys 195 200 205 Arg Met Gln Gly Pro Ile Pro Glu Arg Ile Leu Gly Lys Met Thr Val 210 215 220 Ala Ile Val Lys Ala Leu Tyr Tyr Leu Lys Glu Lys His Gly Val Ile 225 230 235 240 His Arg Asp Val Lys Pro Ser Asn Ile Leu Leu Asp Glu Arg Gly Gln 245 250 255 Ile Lys Leu Cys Asp Phe Gly Ile Ser Gly Arg Leu Val Asp Ser Lys 260 265 270 Ala Lys Thr Arg Ser Ala Gly Cys Ala Ala Tyr Met Ala Pro Glu Arg 275 280 285 Ile Asp Pro Pro Asp Pro Thr Lys Pro Asp Tyr Asp Ile Arg Ala Asp 290 295 300 Val Trp Ser Leu Gly Ile Ser Leu Val Glu Leu Ala Thr Gly Gln Phe 305 310 315 320 Pro Tyr Lys Asn Cys Lys Thr Asp Phe Glu Val Leu Thr Lys Val Leu 325 330 335 Gln Glu Glu Pro Pro Leu Leu Pro Gly His Met Gly Phe Ser Gly Asp 340 345 350 Phe Gln Ser Phe Val Lys Asp Cys Leu Thr Lys Asp His Arg Lys Arg 355 360 365 Pro Lys Tyr Asn Lys Leu Leu Glu His Ser Phe Ile Lys His Tyr Glu 370 375 380 Ile Leu Glu Val Asp Val Ala Ser Trp Phe Lys Asp Val Met Ala Lys 385 390 395 400 Thr Glu Ser Pro Arg Thr Ser Gly Val Leu Ser Gln His His Leu Pro 405 410 415 Phe Phe Arg 1578 base pairs nucleic acid double linear cDNA Coding Sequence 281...1420 29 GGAAAGGCAG CCTCCTGTAG GTGAAAATTC TGTTCACTAC CTGGCCACCT GGCCTGACTG 60 ACCTTCACAG CTTGATCATC TTCCTGAAGA GGCATTCAGG ATTCCCTCCA TCCCTACCCC 120 TTCTGGACAA AGTCTTCCAC GTTTCCTTCC TGGGAGTTTC TTCCAGGAAC TGGAGATACC 180 CAGAGCCCTG CAACTCCCAC TGGCCAACGA TGGGGGCAGC CGCTCACCAT CCTCAGAGAG 240 CTCCCCACAG CACCCTACAC CCCCCACCCG GCCCCGCCAC ATG CTG GGG CTC CCA 295 Met Leu Gly Leu Pro 1 5 TCA ACC TTG TTC ACA CCG CGC AGT ATG GAG AGC ATC GAG ATT GAC CAG 343 Ser Thr Leu Phe Thr Pro Arg Ser Met Glu Ser Ile Glu Ile Asp Gln 10 15 20 AAG CTG CAG GAG ATC ATG AAG CAG ACA GGG TAC CTG ACT ATC GGG GGC 391 Lys Leu Gln Glu Ile Met Lys Gln Thr Gly Tyr Leu Thr Ile Gly Gly 25 30 35 CAG CGT TAT CAG GCA GAA ATC AAT GAC TTG GAG AAC TTG GGT GAG ATG 439 Gln Arg Tyr Gln Ala Glu Ile Asn Asp Leu Glu Asn Leu Gly Glu Met 40 45 50 GGC AGT GGT ACC TGT GGT CAG GTG TGG AAG ATG CGG TTC CGG AAG ACA 487 Gly Ser Gly Thr Cys Gly Gln Val Trp Lys Met Arg Phe Arg Lys Thr 55 60 65 GGC CAC ATC ATT GCT GTT AAG CAA ATG CGG CGC TCT GGG AAC AAG GAA 535 Gly His Ile Ile Ala Val Lys Gln Met Arg Arg Ser Gly Asn Lys Glu 70 75 80 85 GAG AAT AAG CGC ATT TTG ATG GAC CTG GAT GTA GTA CTC AAG AGC CAT 583 Glu Asn Lys Arg Ile Leu Met Asp Leu Asp Val Val Leu Lys Ser His 90 95 100 GAC TGC CCT TAC ATC GTT CAG TGC TTT GGC ACC TTC ATC ACC AAC ACA 631 Asp Cys Pro Tyr Ile Val Gln Cys Phe Gly Thr Phe Ile Thr Asn Thr 105 110 115 GAC GTC TTT ATT GCC ATG GAG CTC ATG GGC ACA TGT GCA GAG AAG CTG 679 Asp Val Phe Ile Ala Met Glu Leu Met Gly Thr Cys Ala Glu Lys Leu 120 125 130 AAG AAA CGA ATG CAG GGC CCC ATT CCA GAG CGA ATC CTG GGC AAG ATG 727 Lys Lys Arg Met Gln Gly Pro Ile Pro Glu Arg Ile Leu Gly Lys Met 135 140 145 ACT GTG GCG ATT GTG AAA GCA CTG TAC TAT CTG AAG GAG AAG CAT GGC 775 Thr Val Ala Ile Val Lys Ala Leu Tyr Tyr Leu Lys Glu Lys His Gly 150 155 160 165 GTC ATC CAT CGC GAT GTC AAA CCC TCC AAC ATC CTG CTA GAT GAG CGG 823 Val Ile His Arg Asp Val Lys Pro Ser Asn Ile Leu Leu Asp Glu Arg 170 175 180 GGC CAG ATC AAG CTC TGT GAC TTT GGC ATC AGT GGC CGC CTT GTT GAC 871 Gly Gln Ile Lys Leu Cys Asp Phe Gly Ile Ser Gly Arg Leu Val Asp 185 190 195 TCC AAA GCC AAA ACA CGG AGT GCT GGC TGT GCT GCC TAT ATG GCT CCC 919 Ser Lys Ala Lys Thr Arg Ser Ala Gly Cys Ala Ala Tyr Met Ala Pro 200 205 210 GAG CGC ATC GAC CCT CCA GAT CCC ACC AAG CCT GAC TAT GAC ATC CGA 967 Glu Arg Ile Asp Pro Pro Asp Pro Thr Lys Pro Asp Tyr Asp Ile Arg 215 220 225 GCT GAT GTG TGG AGC CTG GGC ATC TCA CTG GTG GAG CTG GCA ACA GGA 1015 Ala Asp Val Trp Ser Leu Gly Ile Ser Leu Val Glu Leu Ala Thr Gly 230 235 240 245 CAG TTC CCC TAT AAG AAC TGC AAG ACG GAC TTT GAG GTC CTC ACC AAA 1063 Gln Phe Pro Tyr Lys Asn Cys Lys Thr Asp Phe Glu Val Leu Thr Lys 250 255 260 GTC CTA CAG GAA GAG CCC CCA CTC CTG CCT GGT CAC ATG GGC TTC TCA 1111 Val Leu Gln Glu Glu Pro Pro Leu Leu Pro Gly His Met Gly Phe Ser 265 270 275 GGG GAC TTC CAG TCA TTT GTC AAA GAC TGC CTT ACT AAA GAT CAC AGG 1159 Gly Asp Phe Gln Ser Phe Val Lys Asp Cys Leu Thr Lys Asp His Arg 280 285 290 AAG AGA CCA AAG TAT AAT AAG CTA CTT GAA CAC AGC TTC ATC ATC AAG 1207 Lys Arg Pro Lys Tyr Asn Lys Leu Leu Glu His Ser Phe Ile Ile Lys 295 300 305 CAC TAT GAG ATA CTC GAG GTG GAT GTC GCG TCC TGG TTT AAG GAT GTC 1255 His Tyr Glu Ile Leu Glu Val Asp Val Ala Ser Trp Phe Lys Asp Val 310 315 320 325 ATG GCG AAG ACC GAG TCC CCA AGG ACT AGT GGA GTC CTG AGT CAG CAC 1303 Met Ala Lys Thr Glu Ser Pro Arg Thr Ser Gly Val Leu Ser Gln His 330 335 340 CAT CTG CCC TTC TTC AGT GGG AGT CTG GAG GAG TCT CCC ACT TCC CCA 1351 His Leu Pro Phe Phe Ser Gly Ser Leu Glu Glu Ser Pro Thr Ser Pro 345 350 355 CCT TCT CCC AAG TCC TTC CCT CTG TCA CCA GCC ATC CCT CAG GCC CAG 1399 Pro Ser Pro Lys Ser Phe Pro Leu Ser Pro Ala Ile Pro Gln Ala Gln 360 365 370 GCA GAG TGG GTC TCG GGC AGG TAGGGACCTG GAGTGGCCTG GTCCCACCCT CTGA 1454 Ala Glu Trp Val Ser Gly Arg 375 380 CCTCCTCCTC AGGCCACCAG TGTTGCCCTC TTCCCTTTTT AAAACAAAAT ACCCTTGTTT 1514 GTAAATCCTT AGACGCTTGA GAATAAAACC CTTCCCTTTT CTTCCGAAAA AAAAAAAAAA 1574 AAAA 1578 380 amino acids amino acid linear protein internal 30 Met Leu Gly Leu Pro Ser Thr Leu Phe Thr Pro Arg Ser Met Glu Ser 1 5 10 15 Ile Glu Ile Asp Gln Lys Leu Gln Glu Ile Met Lys Gln Thr Gly Tyr 20 25 30 Leu Thr Ile Gly Gly Gln Arg Tyr Gln Ala Glu Ile Asn Asp Leu Glu 35 40 45 Asn Leu Gly Glu Met Gly Ser Gly Thr Cys Gly Gln Val Trp Lys Met 50 55 60 Arg Phe Arg Lys Thr Gly His Ile Ile Ala Val Lys Gln Met Arg Arg 65 70 75 80 Ser Gly Asn Lys Glu Glu Asn Lys Arg Ile Leu Met Asp Leu Asp Val 85 90 95 Val Leu Lys Ser His Asp Cys Pro Tyr Ile Val Gln Cys Phe Gly Thr 100 105 110 Phe Ile Thr Asn Thr Asp Val Phe Ile Ala Met Glu Leu Met Gly Thr 115 120 125 Cys Ala Glu Lys Leu Lys Lys Arg Met Gln Gly Pro Ile Pro Glu Arg 130 135 140 Ile Leu Gly Lys Met Thr Val Ala Ile Val Lys Ala Leu Tyr Tyr Leu 145 150 155 160 Lys Glu Lys His Gly Val Ile His Arg Asp Val Lys Pro Ser Asn Ile 165 170 175 Leu Leu Asp Glu Arg Gly Gln Ile Lys Leu Cys Asp Phe Gly Ile Ser 180 185 190 Gly Arg Leu Val Asp Ser Lys Ala Lys Thr Arg Ser Ala Gly Cys Ala 195 200 205 Ala Tyr Met Ala Pro Glu Arg Ile Asp Pro Pro Asp Pro Thr Lys Pro 210 215 220 Asp Tyr Asp Ile Arg Ala Asp Val Trp Ser Leu Gly Ile Ser Leu Val 225 230 235 240 Glu Leu Ala Thr Gly Gln Phe Pro Tyr Lys Asn Cys Lys Thr Asp Phe 245 250 255 Glu Val Leu Thr Lys Val Leu Gln Glu Glu Pro Pro Leu Leu Pro Gly 260 265 270 His Met Gly Phe Ser Gly Asp Phe Gln Ser Phe Val Lys Asp Cys Leu 275 280 285 Thr Lys Asp His Arg Lys Arg Pro Lys Tyr Asn Lys Leu Leu Glu His 290 295 300 Ser Phe Ile Ile Lys His Tyr Glu Ile Leu Glu Val Asp Val Ala Ser 305 310 315 320 Trp Phe Lys Asp Val Met Ala Lys Thr Glu Ser Pro Arg Thr Ser Gly 325 330 335 Val Leu Ser Gln His His Leu Pro Phe Phe Ser Gly Ser Leu Glu Glu 340 345 350 Ser Pro Thr Ser Pro Pro Ser Pro Lys Ser Phe Pro Leu Ser Pro Ala 355 360 365 Ile Pro Gln Ala Gln Ala Glu Trp Val Ser Gly Arg 370 375 380 1598 base pairs nucleic acid double linear cDNA Coding Sequence 82...1440 31 AGCGCAGGCG CAGTGCGGTG TTTGTCTACC CCGGACTGAC GGGTGGCCTG GCGGTGAGCG 60 GCGGCAGCGG CGGCGGGGAA G ATG GCG GCG TCC TCC CTG GAG CAG AAG CTG 111 Met Ala Ala Ser Ser Leu Glu Gln Lys Leu 1 5 10 TCC CGC CTG GAA GCC AAG CTG AAG CAG GAG AAC CGT GAG GCC CGC AGG 159 Ser Arg Leu Glu Ala Lys Leu Lys Gln Glu Asn Arg Glu Ala Arg Arg 15 20 25 AGG ATC GAC CTC AAC TTG GAT ATC AGC CCA CAG CGG CCC AGG CCC ACC 207 Arg Ile Asp Leu Asn Leu Asp Ile Ser Pro Gln Arg Pro Arg Pro Thr 30 35 40 CTG CAA CTC CCA CTG GCC AAC GAT GGG GGC AGC CGC TCA CCA TCC TCA 255 Leu Gln Leu Pro Leu Ala Asn Asp Gly Gly Ser Arg Ser Pro Ser Ser 45 50 55 GAG AGC TCC CCA CAG CAC CCT ACA CCC CCC ACC CGG CCC CGC CAC ATG 303 Glu Ser Ser Pro Gln His Pro Thr Pro Pro Thr Arg Pro Arg His Met 60 65 70 CTG GGG CTC CCA TCA ACC TTG TTC ACA CCG CGC AGT ATG GAG AGC ATC 351 Leu Gly Leu Pro Ser Thr Leu Phe Thr Pro Arg Ser Met Glu Ser Ile 75 80 85 90 GAG ATT GAC CAG AAG CTG CAG GAG ATC ATG AAG CAG ACA GGG TAC CTG 399 Glu Ile Asp Gln Lys Leu Gln Glu Ile Met Lys Gln Thr Gly Tyr Leu 95 100 105 ACT ATC GGG GGC CAG CGT TAT CAG GCA GAA ATC AAT GAC TTG GAG AAC 447 Thr Ile Gly Gly Gln Arg Tyr Gln Ala Glu Ile Asn Asp Leu Glu Asn 110 115 120 TTG GGT GAG ATG GGC AGT GGT ACC TGT GGT CAG GTG TGG AAG ATG CGG 495 Leu Gly Glu Met Gly Ser Gly Thr Cys Gly Gln Val Trp Lys Met Arg 125 130 135 TTC CGG AAG ACA GGC CAC ATC ATT GCT GTT AAG CAA ATG CGG CGC TCT 543 Phe Arg Lys Thr Gly His Ile Ile Ala Val Lys Gln Met Arg Arg Ser 140 145 150 GGG AAC AAG GAA GAG AAT AAG CGC ATT TTG ATG GAC CTG GAT GTA GTA 591 Gly Asn Lys Glu Glu Asn Lys Arg Ile Leu Met Asp Leu Asp Val Val 155 160 165 170 CTC AAG AGC CAT GAC TGC CCT TAC ATC GTT CAG TGC TTT GGC ACC TTC 639 Leu Lys Ser His Asp Cys Pro Tyr Ile Val Gln Cys Phe Gly Thr Phe 175 180 185 ATC ACC AAC ACA GAC GTC TTT ATT GCC ATG GAG CTC ATG GGC ACA TGT 687 Ile Thr Asn Thr Asp Val Phe Ile Ala Met Glu Leu Met Gly Thr Cys 190 195 200 GCA GAG AAG CTG AAG AAA CGA ATG CAG GGC CCC ATT CCA GAG CGA ATC 735 Ala Glu Lys Leu Lys Lys Arg Met Gln Gly Pro Ile Pro Glu Arg Ile 205 210 215 CTG GGC AAG ATG ACT GTG GCG ATT GTG AAA GCA CTG TAC TAT CTG AAG 783 Leu Gly Lys Met Thr Val Ala Ile Val Lys Ala Leu Tyr Tyr Leu Lys 220 225 230 GAG AAG CAT GGC GTC ATC CAT CGC GAT GTC AAA CCC TCC AAC ATC CTG 831 Glu Lys His Gly Val Ile His Arg Asp Val Lys Pro Ser Asn Ile Leu 235 240 245 250 CTA GAT GAG CGG GGC CAG ATC AAG CTC TGT GAC TTT GGC ATC AGT GGC 879 Leu Asp Glu Arg Gly Gln Ile Lys Leu Cys Asp Phe Gly Ile Ser Gly 255 260 265 CGC CTT GTT GAC TCC AAA GCC AAA ACA CGG AGT GCT GGC TGT GCT GCC 927 Arg Leu Val Asp Ser Lys Ala Lys Thr Arg Ser Ala Gly Cys Ala Ala 270 275 280 TAT ATG GCT CCC GAG CGC ATC GAC CCT CCA GAT CCC ACC AAG CCT GAC 975 Tyr Met Ala Pro Glu Arg Ile Asp Pro Pro Asp Pro Thr Lys Pro Asp 285 290 295 TAT GAC ATC CGA GCT GAT GTG TGG AGC CTG GGC ATC TCA CTG GTG GAG 1023 Tyr Asp Ile Arg Ala Asp Val Trp Ser Leu Gly Ile Ser Leu Val Glu 300 305 310 CTG GCA ACA GGA CAG TTC CCC TAT AAG AAC TGC AAG ACG GAC TTT GAG 1071 Leu Ala Thr Gly Gln Phe Pro Tyr Lys Asn Cys Lys Thr Asp Phe Glu 315 320 325 330 GTC CTC ACC AAA GTC CTA CAG GAA GAG CCC CCA CTC CTG CCT GGT CAC 1119 Val Leu Thr Lys Val Leu Gln Glu Glu Pro Pro Leu Leu Pro Gly His 335 340 345 ATG GGC TTC TCA GGG GAC TTC CAG TCA TTT GTC AAA GAC TGC CTT ACT 1167 Met Gly Phe Ser Gly Asp Phe Gln Ser Phe Val Lys Asp Cys Leu Thr 350 355 360 AAA GAT CAC AGG AAG AGA CCA AAG TAT AAT AAG CTA CTT GAA CAC AGC 1215 Lys Asp His Arg Lys Arg Pro Lys Tyr Asn Lys Leu Leu Glu His Ser 365 370 375 TTC ATC ATC AAG CAC TAT GAG ATA CTC GAG GTG GAT GTC GCG TCC TGG 1263 Phe Ile Ile Lys His Tyr Glu Ile Leu Glu Val Asp Val Ala Ser Trp 380 385 390 TTT AAG GAT GTC ATG GCG AAG ACC GAG TCC CCA AGG ACT AGT GGA GTC 1311 Phe Lys Asp Val Met Ala Lys Thr Glu Ser Pro Arg Thr Ser Gly Val 395 400 405 410 CTG AGT CAG CAC CAT CTG CCC TTC TTC AGT GGG AGT CTG GAG GAG TCT 1359 Leu Ser Gln His His Leu Pro Phe Phe Ser Gly Ser Leu Glu Glu Ser 415 420 425 CCC ACT TCC CCA CCT TCT CCC AAG TCC TTC CCT CTG TCA CCA GCC ATC 1407 Pro Thr Ser Pro Pro Ser Pro Lys Ser Phe Pro Leu Ser Pro Ala Ile 430 435 440 CCT CAG GCC CAG GCA GAG TGG GTC TCG GGC AGG TAGGGACCTG GAGTGGCCTG 1460 Pro Gln Ala Gln Ala Glu Trp Val Ser Gly Arg 445 450 GTCCCACCCT CTGACCTCCT CCTCAGGCCA CCAGTGTTGC CCTCTTCCCT TTTTAAAACA 1520 AAATACCCTT GTTTGTAAAT CCTTAGACGC TTGAGAATAA AACCCTTCCC TTTTCTTCCG 1580 AAAAAAAAAA AAAAAAAA 1598 453 amino acids amino acid linear protein internal 32 Met Ala Ala Ser Ser Leu Glu Gln Lys Leu Ser Arg Leu Glu Ala Lys 1 5 10 15 Leu Lys Gln Glu Asn Arg Glu Ala Arg Arg Arg Ile Asp Leu Asn Leu 20 25 30 Asp Ile Ser Pro Gln Arg Pro Arg Pro Thr Leu Gln Leu Pro Leu Ala 35 40 45 Asn Asp Gly Gly Ser Arg Ser Pro Ser Ser Glu Ser Ser Pro Gln His 50 55 60 Pro Thr Pro Pro Thr Arg Pro Arg His Met Leu Gly Leu Pro Ser Thr 65 70 75 80 Leu Phe Thr Pro Arg Ser Met Glu Ser Ile Glu Ile Asp Gln Lys Leu 85 90 95 Gln Glu Ile Met Lys Gln Thr Gly Tyr Leu Thr Ile Gly Gly Gln Arg 100 105 110 Tyr Gln Ala Glu Ile Asn Asp Leu Glu Asn Leu Gly Glu Met Gly Ser 115 120 125 Gly Thr Cys Gly Gln Val Trp Lys Met Arg Phe Arg Lys Thr Gly His 130 135 140 Ile Ile Ala Val Lys Gln Met Arg Arg Ser Gly Asn Lys Glu Glu Asn 145 150 155 160 Lys Arg Ile Leu Met Asp Leu Asp Val Val Leu Lys Ser His Asp Cys 165 170 175 Pro Tyr Ile Val Gln Cys Phe Gly Thr Phe Ile Thr Asn Thr Asp Val 180 185 190 Phe Ile Ala Met Glu Leu Met Gly Thr Cys Ala Glu Lys Leu Lys Lys 195 200 205 Arg Met Gln Gly Pro Ile Pro Glu Arg Ile Leu Gly Lys Met Thr Val 210 215 220 Ala Ile Val Lys Ala Leu Tyr Tyr Leu Lys Glu Lys His Gly Val Ile 225 230 235 240 His Arg Asp Val Lys Pro Ser Asn Ile Leu Leu Asp Glu Arg Gly Gln 245 250 255 Ile Lys Leu Cys Asp Phe Gly Ile Ser Gly Arg Leu Val Asp Ser Lys 260 265 270 Ala Lys Thr Arg Ser Ala Gly Cys Ala Ala Tyr Met Ala Pro Glu Arg 275 280 285 Ile Asp Pro Pro Asp Pro Thr Lys Pro Asp Tyr Asp Ile Arg Ala Asp 290 295 300 Val Trp Ser Leu Gly Ile Ser Leu Val Glu Leu Ala Thr Gly Gln Phe 305 310 315 320 Pro Tyr Lys Asn Cys Lys Thr Asp Phe Glu Val Leu Thr Lys Val Leu 325 330 335 Gln Glu Glu Pro Pro Leu Leu Pro Gly His Met Gly Phe Ser Gly Asp 340 345 350 Phe Gln Ser Phe Val Lys Asp Cys Leu Thr Lys Asp His Arg Lys Arg 355 360 365 Pro Lys Tyr Asn Lys Leu Leu Glu His Ser Phe Ile Ile Lys His Tyr 370 375 380 Glu Ile Leu Glu Val Asp Val Ala Ser Trp Phe Lys Asp Val Met Ala 385 390 395 400 Lys Thr Glu Ser Pro Arg Thr Ser Gly Val Leu Ser Gln His His Leu 405 410 415 Pro Phe Phe Ser Gly Ser Leu Glu Glu Ser Pro Thr Ser Pro Pro Ser 420 425 430 Pro Lys Ser Phe Pro Leu Ser Pro Ala Ile Pro Gln Ala Gln Ala Glu 435 440 445 Trp Val Ser Gly Arg 450 44 amino acids amino acid linear protein 33 Met Xaa Ser Pro Ala Pro Ala Pro Ser Gln Arg Ala Ala Leu Gln Leu 1 5 10 15 Pro Leu Ala Asn Asp Gly Gly Ser Arg Ser Pro Ser Ser Glu Ser Ser 20 25 30 Pro Gln His Pro Thr Pro Pro Thr Arg Pro Arg His 35 40 77 amino acids amino acid linear protein 34 Glu Gly Gly Gly Val Lys His Met Ala Lys Leu Tyr Val Phe Tyr Gly 1 5 10 15 Ala Gly Cys Met Glu Met Ser Asp Ile Glu Leu Leu Leu His Arg Asp 20 25 30 Lys Pro Asn Leu Gly Lys Cys Asp Phe Gly Ser Gly Leu Ser Ala Gly 35 40 45 Tyr Met Pro Glu Arg Tyr Val Ser Asp Trp Ser Gly Glu Ala Arg Pro 50 55 60 Phe Leu Val Pro Leu Phe Phe Cys Leu Lys Arg Leu His 65 70 75

Claims

What is claimed is:

1. A substantially pure mammalian mitogen-activated protein kinase kinase (MKK) polypeptide having serine, threonine, and tyrosine kinase activity, and phosphorylating mitogen-activated protein (MAP) kinase JNK, but not p38.

2. A polypeptide of claim 1 comprising the amino acid sequence of SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 26, SEQ ID NO: 28, SEQ ID NO: 30, or SEQ ID NO: 32.

3. An isolated polynucleotide sequence encoding a polypeptide of claim 1.

4. An isolated polynucleotide sequence of claim 3 comprising the sequence of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31, or degenerate variants thereof, or a polynucleotide sequence fully complementary to the sequence of SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, or SEQ ID NO: 31, or degenerate variants thereof.

5. An isolated polynucleotide sequence of claim 3 comprising a polynucleotide sequence that hybridizes under stringent hybridization conditions to the sequence of SEQ ID NO: 17, SEQ ID NO: 19, or SEQ ID NO: 25, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, or a complement thereof.

6. A recombinant expression vector containing a polynucleotide sequence of claim 3.

7. A recombinant host cell comprising a polynucleotide sequence of claim 3.

8. A purified antibody that binds specifically to a polypeptide of claim 1.

9. A purified antibody that binds specifically to a polypeptide of claim 2.

10. A method of measuring the activity of a mitogen-activated protein kinase kinase (MKK7) in a biological test sample, said method comprising:

a) incubating said test sample with an MKK substrate for the MKK polypeptide of claim 1 and labeled phosphate under conditions sufficient to allow phosphorylation of said substrate; and

b) determining the rate of incorporation of labeled phosphate into said substrate, wherein said rate of incorporation is a measure of MKK7 activity.

11. A method of claim 10, wherein said MKK substrate is selected from the group consisting of JNK MAP kinases, activating transcription factor-2 (ATF2), ATFa, cAMP response element binding protein (CRE-BPa), and c-Jun.

12. A method of claim 10, wherein said biological test sample is fluid, cells, or tissue obtained from a mammal.

13. A method for measuring the synthesis of MKK7 in a biological test sample, the method comprising the steps of:

a) obtaining a biological sample;

b) contacting said biological sample with an antibody that specifically binds an MKK7 polypeptide of claim 1; and

c) detecting said antibody bound to MKK7 polypeptide, wherein the level of MKK7 synthesis is determined by the amount of bound antibody.

14. A method for measuring the level of expression of MKK7 in a test sample, the method comprising the steps of:

a) isolating total or polyadenylated RNA from the test sample;

b) incubating the RNA with a polynucleotide probe specific for an MKK7 polynucleotide of claim 3; and

c) determining the amount of said probe hybridized to the RNA, wherein the level of expression of MKK7 is directly related to the amount of MKK7 probe hybridized to the RNA.

15. A method for identifying a reagent that modulates MKK7 activity, said method comprising:

a) obtaining a test sample containing MKK7;

b) incubating said test sample with an MKK substrate for the MKK polypeptide of claim 1, a range of reagent concentrations, and labeled phosphate under conditions sufficient to allow phosphorylation of said subtrate when said reagent is not present;

c) detecting phosphorylation of said substrate; and

d) comparing the effect of said reagent on MKK7 activity relative to a control, wherein any variation compared to control indicates a reagent able to modulate MKK7 substrate phosphorylation.

16. A method of claim 15, wherein said MKK7 substrate is one or more of JNK, ATF2, ATFa, CRE-BPa, and c-Jun.

17. A method of claim 15 wherein said modulation is inhibition of MKK7 activity.

18. A method for identifying a reagent that modulates MKK7 synthesis, said method comprising:

a) providing a sample capable of MKK7 synthesis;

b) incubating said sample with a range of reagent concentrations under conditions that allow synthesis of MKK7 when said reagent is not present;

c) detecting an MKK7 polypeptide of claim 1; and

d) comparing the effect of said reagent on MKK7 synthesis relative to a control, wherein any variation compared to control indicates a reagent able to modulate MKK7.

19. A method of claim 18 wherein said modulation is inhibition of MKK7 synthesis.

20. A method for identifying a reagent that modulates MKK7 expression, said method comprising:

a) providing a sample capable of expressing MKK7;

b) incubating said sample with a range of concentrations of said reagent under conditions where MKK7 is expressed in the absence of said reagent;

c) isolating total or polyadenylated RNA from the sample;

d) incubating the RNA with a polynucleotide probe specific for a MKK7 nucleic acid of claim 3; and

e) comparing the effect of said reagent on MKK7 RNA expression relative to a control, wherein any variation compared to control indicates a reagent able to modulate MKK7 expression.

21. A method of treating an MKK7-mediated disorder in a patient, the method comprising administering to the patient a therapeutically effective amount of a reagent that modulates MKK7 activity.

22. The method of claim 21, wherein said MKK7-mediated disorder is selected from the group consisting of ischemic heart disease, kidney failure, oxidative liver damage, respiratory distress syndrome, heat and radiation burns, septic shock, rheumatoid arthritis, autoimmune disorders, and inflammatory diseases.

23. A method of treating an MKK7-associated disorder in a patient, comprising administering to the patient a therapeutically effective amount of an MKK7 polypeptide.

24. The method of claim 23, wherein said MKK7-associated disorder is ischemic heart disease, kidney failure, oxidative liver damage, respiratory distress syndrome, heat and radiation burns, septic shock, rheumatoid arthritis, autoimmune disorders, or inflammatory diseases.

25. A substantially pure human mitogen-activated protein kinase kinase (MKK) polypeptide having serine, threonine, and tyrosine kinase activity, and phosphorylating human mitogen-activated protein (MAP) kinase p38.

26. A polypeptide of claim 25 comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

27. A polypeptide of claim 25, further characterized in that said polypeptide phosphorylates human mitogen-activated protein (MAP) kinase JNK.

28. A polypeptide of claim 27, comprising the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10.

29. A purified antibody which binds specifically to a polypeptide of claim 25.

30. A purified antibody which binds specifically to a polypeptide of claim 26.

31. A purified antibody which binds specifically to a polypeptide of claim 27.

32. A purified antibody which binds specifically to a polypeptide of claim 28.

33. A method of measuring the activity of a mitogen-activated protein kinase kinase (MKK) in a biological test sample, said method comprising:

a) incubating said test sample with an MKK substrate for the MKK polypeptide of claim 25 and labeled phosphate under conditions sufficient to allow phosphorylation of said substrate, and

b) determining the rate of incorporation of labeled phosphate into said substrate, wherein said rate of incorporation is a measure of MKK activity.

34. A method of claim 33, wherein said MKK substrate is selected from the group consisting of p38 and JNK MAP kinases, activating transcription factor-2 (ATF2), kinases, activating transcription factor-2 (ATF2), ATFa, cAMP response element binding protein (CRE-BPa), and c-Jun.

35. A method of claim 33, wherein said biological test sample is fluid, cells, or tissue obtained from a mammal.

36. A method for measuring the synthesis of MKK in a biological test sample, comprising the steps of:

a) fractionating proteins present in said sample by gel electrophoresis;

b) transferring the proteins onto a membrane; and

c) probing the proteins with a labeled antibody specific to a MKK polypeptide of claim 25, wherein the level of MKK synthesis is determined by the amount of bound labeled antibody.

37. A method for measuring the level of expression of MKK in a test sample, comprising the steps of:

a) isolating polyadenylated RNA from the test sample;

b) incubating polyadenylated RNA with a polynucleotide probe specific for a MKK polypeptide of claim 25;

c) determining the amount of said probe hybridized said polyadenylated RNA, wherein the level of expression of MKK is directly related to the amount of MKK probe hybridized to said RNA.

38. A method for identifying a reagent which modulates MKK activity, said method comprising:

a) using the method of claim 33;

b) comparing the effect of said reagent on MKK activity relative to a control, wherein a reagent able to modulate MKK substrate phosphorylation is identified.

39. A method of claim 38, wherein said MKK substrate is one or more of p38, JNK, ATF2, ATFa, CRE-BPa, and c-Jun.

40. A method of claim 38, wherein said modulation is inhibition of MKK activity.

41. A method for identifying a reagent which modulates MKK synthesis, said method comprising:

a) using the method of claim 36;

b) comparing the effect of said reagent on MKK synthesis relative to a control, wherein a reagent able to modulate MKK synthesis is identified.

42. A method of claim 41, wherein said MKK substrate is one or more of p38, JNK, ATF2, ATFa, CRE-BPa, and c-Jun.

43. A method of claim 41, wherein said modulation is inhibition of MKK synthesis.

44. A method for identifying a reagent which modulates MKK expression, said method comprising:

a) using the method of claim 37;

b) comparing the effect of said reagent on MKK expression relative to a control, wherein a reagent able to modulate MKK expression is identified.

45. A method of treating an MKK-mediated disorder in a patient, comprising administering to the patient a therapeutically effective amount of a reagent that modulates MKK activity.

46. The method of claim 45, wherein said MKK-mediated disorder is selected from the group consisting of ischemic heart disease, kidney failure, oxidative liver damage, respiratory distress syndrome, heat and radiation burns, septic shock, rheumatoid arthritis, autoimmune disorders, and inflammatory diseases.