WO2006054770A1 - Nouvelle map kinase-kinase-kinase (map3k) - Google Patents

Nouvelle map kinase-kinase-kinase (map3k) Download PDF

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WO2006054770A1
WO2006054770A1 PCT/JP2005/021452 JP2005021452W WO2006054770A1 WO 2006054770 A1 WO2006054770 A1 WO 2006054770A1 JP 2005021452 W JP2005021452 W JP 2005021452W WO 2006054770 A1 WO2006054770 A1 WO 2006054770A1
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ask3
seq
activity
protein
amino acid
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PCT/JP2005/021452
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Japanese (ja)
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Hidenori Ichijo
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The University Of Tokyo
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/45—Transferases (2)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • C12Q1/485—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase involving kinase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G01N33/6896—Neurological disorders, e.g. Alzheimer's disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2510/00—Detection of programmed cell death, i.e. apoptosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2835—Movement disorders, e.g. Parkinson, Huntington, Tourette

Definitions

  • MAPKKK New MAP kinase kinase kinase
  • the present invention relates to providing a novel MAP kinase kinase kinase (MAPKKK) that induces apoptosis (cell death).
  • MAPKKK novel MAP kinase kinase kinase
  • the present invention also relates to a preventive / therapeutic agent for diseases associated with the promotion of apoptosis by inhibiting this enzyme.
  • MAP kinase The signal transduction cascade by mitogen-activated protein (MAP) kinase is a well-conserved intracellular signal transduction pathway from yeast to vertebrates, and includes MAP kinase (MAPK) and MAPK. It is composed of three different members of protein kinases including kinase (MAPKK) and MAPK K kinase (MAPKKK). These molecules in turn, MAPKKK phosphorylate MAPKK, which activates MAPKK force S phosphorylation, and activated MAPKK phosphorylates MAPK, which activates MA PK. It is known that activated MAPK moves to the cell nucleus and regulates the activity of transcription factors, thereby controlling the expression of various genes!
  • MAPK signaling pathways in mammalian cells include multiple signaling pathways involving p38, signaling pathways involving JNK, signaling pathways involving ERK, etc. -It became clear that the MAPK signaling pathway works! Studies using cultured cells suggest that these groups of kinases play important roles in proliferation, differentiation, cell death, and the like.
  • p38 and JNK are molecules that are positioned as stress-responsive because they are activated by stimuli such as high osmotic pressure, heating, ultraviolet irradiation, and inflammatory site force-in. .
  • the upstream signaling mechanism that activates stress-responsive MAPKs p38 and JNK includes MAPKK that functions in the p38 signaling pathway as MAPKK, MKK3 / MKK6, and MAPKK that functions in the JNK signaling pathway. MKK4 / MKK7 is known! /
  • ASK1 Apoptosis Signal-regulating Kinase 1
  • Roning was performed and the mechanism of action was clarified (Patent Document 1; Patent Document 2; Non-Patent Document 1; Non-Patent Document 2).
  • ASK1 has the ability to phosphorylate MKK3 / MKK6 or MKK4 / MKK7, resulting in the activation of ASK1 force JNK signaling pathway or p38 signaling pathway This suggests that it may promote apoptosis.
  • Non-patent Document 3 DDB J registration numbers AB167411 (human ASK2) and AB021861 (mouse ASK2)). It was also clarified that this ASK2 also has MAPKKK activity like ASK1 and can activate the JNK signaling pathway or the p38 signal transmission pathway (Non-patent Document 3).
  • Patent Document 1 Japanese Patent Laid-Open No. 10-93
  • Patent Document 2 WO02 / 38179
  • Non-patent literature l Ichijo et al., Science, 275, 90-94, 1997
  • Non-Patent Document 2 Nishitoh et al., Genes & Development, 16, 1345-1355, 2002
  • Non-Patent Document 3 Wang et al, Biochem. Biophys. Res. Commun. 253, 33-37, 1998 Disclosure of the Invention
  • An object of the present invention is to provide a novel MAPKKK molecule in human or mouse. Another object of the present invention is to provide a safer and more effective prophylactic / therapeutic agent for diseases associated with the promotion or suppression of apoptosis by inhibiting or promoting the activity of the novel MAPKKK molecule of the present invention.
  • the inventors have found a protein having a novel amino acid sequence including the serine Z threonine kinase region from cDNAs of human HEK-293 cells and mouse fetal fibroblasts, and detailed functions of this molecule in the cell.
  • the present invention has been completed based on the analysis results.
  • a protein having MAPKKK activity comprising: a DNA comprising a nucleotide sequence complementary to a DNA comprising a nucleotide sequence; and an amino acid sequence encoded by a DNA that hybridizes under stringent conditions and encodes a protein having MAPKKK activity. I will provide a.
  • the present invention further relates to at least 60%, more preferably at least 70%, more preferably at least 80%, most preferably at least at least between the amino acid sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4.
  • a protein having MAP KKK activity consisting of an amino acid sequence having 90% amino acid sequence homology is also provided.
  • the inventors relate to the novel MAPKKK described above.
  • DNA comprising a nucleotide sequence having 90% nucleotide sequence homology and encoding a protein having MAPKKK activity.
  • the MAPKKK activity described above is provided.
  • the present invention provides a pharmaceutical composition for treating or preventing a disease associated with promotion of apoptosis, comprising a MAPKKK inhibitor.
  • the present invention by promoting the function of the protein having the MAPKKK activity described above, stress-induced apoptosis involving the JNK or p38 pathway is promoted, resulting in suppression of apoptosis.
  • the present invention provides a pharmaceutical composition for treating or preventing a disease associated with suppression of apoptosis, comprising a MAPKKK promoter.
  • the present invention further provides an antibody that binds to the protein shown in SEQ ID NO: 2 or SEQ ID NO: 4.
  • This antibody does not bind to the known MAPKKK, ASK1 and ASK2, but may have the characteristics.
  • the present invention can provide a novel MAPKKK derived from humans or mice.
  • the present invention also inhibits or promotes the kinase activity of this novel protein, thereby inhibiting or promoting the JNK Zp38 signaling pathway and suppressing or promoting apoptosis in diseases associated with promoting or suppressing apoptosis, As a result, it is shown that such a disease can be treated and prevented, and as a result, a pharmaceutical composition containing an agent that inhibits or promotes the kinase activity of the novel protein can be provided.
  • FIG. 1 shows the nucleotide sequence of mouse ASK3 and the encoded amino acid sequence. The underlined in the sequence and the name and SEQ ID NO above it indicate the primer used.
  • FIG. 1 shows the nucleotide sequence of mouse ASK3 and the encoded amino acid sequence. The underlined in the sequence and the name and SEQ ID NO above it indicate the primer used.
  • FIG. 1 shows the nucleotide sequence of mouse ASK3 and the encoded amino acid sequence. Underlined in the sequence and the name and SEQ I above it D NO indicates the primer used.
  • FIG. 2-1 shows the nucleotide sequence of human ASK3 and the amino acid sequence encoded by it.
  • the underlined portion described in the sequence and the name described above and SEQ ID NO indicate the primer used.
  • Fig. 2 shows the nucleotide sequence of human ASK3 and the encoded amino acid sequence.
  • the underlined portion described in the sequence and the name described above and SEQ ID NO indicate the primer used.
  • FIG. 2 shows the nucleotide sequence of human ASK3 and the encoded amino acid sequence.
  • the underlined portion described in the sequence and the name described above and SEQ ID NO indicate the primer used.
  • FIG. 3 compares the amino acid sequence of SEQ ID NO: 2 derived from human and the amino acid sequence of SEQ ID NO: 4 derived from mouse with the amino acid sequences of known MAPKKK, ASKl and ASK2 derived from human and mouse.
  • FIG. 3 compares the amino acid sequence of SEQ ID NO: 2 derived from human and the amino acid sequence of SEQ ID NO: 4 derived from mouse with the amino acid sequences of known MAPKKK, ASKl and ASK2 derived from human and mouse.
  • FIG. 4 shows the expression of ASK3 mRNA and ASK3 protein of the present invention in mice using Northern blotting (FIG. 4A) and Western blotting (FIG. 4B and FIG. 4C), as opposed to ASK1. It is a figure which shows notably expressing in a kidney.
  • FIG. 5 is a diagram showing that ASK3 is a MAPKKK that specifically activates the JNK signaling pathway and the p38 signaling pathway in vitro.
  • Fig. 6 is a view showing the results of identification of active sites controlling ASK3 phosphate activity.
  • FIG. 7 is a diagram showing that ASK3 forms a complex with ASK1 in cells.
  • FIG. 8 is a diagram showing that ASK3 phosphorylates ASK1, ASK1 phosphorylates ASK3, and the two are in a mutually activated relationship.
  • FIG. 9 shows that ASK3 is phosphorylated by H 0 stimulation and mitoxin (MTX) stimulation
  • FIG. 10 is a view showing that ASK3 has an activity of inducing apoptosis and inducing apoptosis in a kinase activity-dependent manner.
  • FIG. 11 is a view showing that ASK3 has an activity of inducing production of cytoforce-in (TNF-a).
  • FIG. 12 shows that ASK1 is activated by hyperosmotic stimulation with 0.5 M sorbitol, whereas ASK3 is inactivated by hyperosmotic stimulation in contrast to ASK1. It is a figure which shows being done.
  • FIG. 13 shows that when ASK3 force high osmotic pressure stimulation is applied, dephosphorylation occurs, but when low osmotic pressure stimulation is applied, the degree of phosphate increase increases.
  • FIG. 14 is a diagram showing the active state of ASK3 examined using anti-phosphorylated ASK antibody, the expression level of ASK3 examined using anti-flag antibody (FIG. 14A), and osmotic pressure.
  • FIG. 14 is a diagram (FIG. 14B) showing the relationship with the amount of phosphate salt.
  • a plurality of PCR primers are designed and prepared, and cDNA prepared from mouse cells (eg, mouse fetal fibroblasts; MEF cells) is prepared.
  • PCR can be performed as a saddle type to obtain a part of the target unknown gene. Longer sequences can also be obtained by joining fragments of these genes based on overlapping sequence portions. It is also possible to design and prepare a primer inside the obtained sequence fragment and use it to carry out an amplification reaction for the purpose of terminal extension such as 5'RACE or 3'RACE.
  • human homologous Nucleic acids encoding new molecules can be obtained. That is, using the nucleotide sequence of the obtained mouse cDNA (FIG. 1, SEQ ID NO: 3), human genome sequences can be searched to confirm the presence of homologous genes. Furthermore, based on the results of such searches, primers for PCR were designed and prepared, and PCR was performed using cDNA prepared from human cells (for example, HEK-293 cells) as a homologous cDNA. Can be obtained.
  • nucleotide sequence (SEQ ID NO: 3) of the above-described novel mouse-derived molecule using a technique commonly used in the art such as PCR and RACE related thereto.
  • ORF open reading frame
  • DNA having the nucleotide sequence represented by SEQ ID NO: 1 and protein having the amino acid sequence represented by SEQ ID NO: 2 were analyzed at the nucleotide level and amino acid level, and human and mouse MAPKKK, ASK1 and The nucleotide sequence and amino acid sequence of ASK2 were compared.
  • a sequence comparison program used by those skilled in the art can be used for sequence homology analysis of amino acid sequences. For example, it can be determined by comparing with the sequence information using the BLAST program described in Altschul et al. (Nucl. Acids. Res. 25., p. 3389-3402, 1997).
  • a query base sequence can be input using the Nucleotide BLAST (BLASTN) program and collated with base sequence databases such as GenBank, EMBL, and DDBJ.
  • BLASTN Nucleotide BLAST
  • query amino acid sequences such as GenBank CDS, PDB, SwissProt, and PIR can be collated using the Protein BLAST (BLASTP) program.
  • the program can be used on the Internet from the National Center for Biotechnology Information (NCBI) or the DNA Data Bank of Japan (DDBJ) website.
  • NCBI National Center for Biotechnology Information
  • DDBJ DNA Data Bank of Japan
  • Various conditions (parameters) for homology search using the BLAST program are described in detail on the same site, and some settings can be changed as appropriate, but the search is usually performed using default values. Other than sequence comparison used by those skilled in the art These programs can also be used.
  • sequence homology of the amino acid sequence may be determined by visual inspection and mathematical calculation.
  • sequence homology between two protein sequences is based on the algorithm of Needleman and Wunsch (J. Mol Biol, 48: 443-453, 1970) and is available from the University of Wisconsin Genetics Computer Group (UWGCG) It may be determined by comparing the sequence information using the appropriate GAP computer program.
  • Preferable GAP program default parameters include: (1) Scoring 'matrix as described in Henikoffife and Henikoff (Proc. Natl. Acad. Sci. USA, 89: 10915-10919, 1992), (2) 12 gap weights; (3) 4 gap length weights; and (4) No penalty for end gaps.
  • sequence comparison software that can be used in the technical field, such as ClustalV, ClustalW, Jotun Hein Method and the like.
  • the amino acid level and nucleotides are determined by Clust alV for the protein consisting of the amino acid sequence of SEQ ID NO: 2 and the DNA consisting of the nucleotide sequence of SEQ ID NO: 1 encoding the protein.
  • the one with the highest sequence homology between the SEQ ID NO: 2 protein of the present invention and the DNA of SEQ ID NO: 1 was previously known as MAPKKK
  • the human and mouse ASK1 and ASK2 amino acid sequences and nucleotide sequences of the novel human MAPKKK and ASK3 obtained by the present invention are 56.5% and 49.5% of the amino acid sequence and nucleotide sequence of human ASK1, respectively.
  • the MAPKKK and ASK2 proteins and DNAs that are only homologous and obtained as ASK1 family molecules have 41.7% and 41.1% phases, respectively. It was found to have only a sex. From these facts, it was found that the protein of SEQ ID NO: 2 and the DNA of SEQ ID NO: 1 do not have significant sequence homology as a whole with known MAPKKK, AS K1, and ASK2, respectively.
  • the protein having the amino acid sequence ability shown in SEQ ID NO: 2 obtained from human and the SEQ ID NO obtained from mouse are used.
  • the protein having the amino acid sequence shown in 4 is a novel protein having a kinase activity similar to the serine Z threonine kinase activity of ASK1 and ASK2. It was expected to be of parcel quality. Therefore, the protein consisting of the amino acid sequence of SEQ ID NO: 2 was named “ASK3” and an attempt was made to specify its function.
  • MKK4 / MKK7 a MAPKK that functions in the p38 signaling pathway, was phosphorylated. From this, it was clarified that the protein has activity as MAPKKK consisting of the amino acid sequence of SEQ ID NO: 2 of the present invention.
  • a novel protein having MAPKKK activity a human-derived protein having the amino acid sequence of SEQ ID NO: 2, ASK3, or this A protein having a MAPKKK activity can be provided.
  • a "variant" t of the protein of SEQ ID NO: 2 in which case one or more amino acids are deleted, substituted or added in the amino acid sequence of the target protein.
  • a protein having MAPKKK activity comprising: (c) an amino acid sequence encoded by DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1; or (d) DNA consisting of a nucleotide sequence complementary to the DNA that also has the nucleotide sequence capability set forth in SEQ ID NO: 1.
  • a protein having a MAPKKK activity consisting of: an amino acid sequence that is hybridized under stringent conditions and encoded by a DNA encoding a protein having M APKKK activity; or
  • Amino acid sequence that has at least 60%, more preferably at least 70%, more preferably at least 80%, and most preferably at least 90% amino acid sequence homology with the amino acid sequence represented by SEQ ID NO: 2.
  • the present invention also provides derivatives of the above-described proteins.
  • protein derivative refers to an amino group at the amino terminal (N-terminal) of a protein, a functional group of each amino acid side chain (for example, amino group, carboxyl group, hydrogen group, thiol group, Part or all of the amide group, etc., and the carboxyl group (C-terminal) carboxyl group of the protein or the carboxyl group power of the side chain of each amino acid.
  • substituents Modification with other appropriate substituents is performed for the purpose of, for example, protecting functional groups present in proteins, improving safety and tissue migration, improving resistance to degradation, or enhancing activity. .
  • any substituent known in the art may be used.
  • protein derivatives include (1) a hydrogen atom, substitution or substitution of a part or all of the amino group at the amino terminal (N-terminal) of the protein or the side chain of each amino acid.
  • Unsubstituted alkyl group (which may be linear, branched or cyclic) (eg, methyl, ethyl, propyl, isopropyl, isobutyl, butyl, t-butyl, cyclopropyl, cyclopropyl) Hexyl group, benzyl group), substituted or unsubstituted isyl group (for example, formyl group, acetyl group, force propyl group, cyclohexyl carbo ol group, benzol group, phthaloyl group, tosyl group, nicotinyl group, pipette group) Lysine carboyl group), urethane type protecting group (for example, p-trobendioxy carbo
  • the present invention provides a DNA encoding a novel protein (SEQ ID NO: 2) derived from a human having MAPKKK activity (eg, SEQ ID NO: 1) or a variant encoding the variant thereof DNA can be provided. That is, in another aspect, the present invention relates to the above-described novel human MAPKKK.
  • DNA consisting of the nucleotide sequence described in SEQ ID NO: 1; or (d) DNA complementary to the nucleotide sequence described in SEQ ID NO: 1.
  • one or more means preferably 1 to 20, more preferably 1 to 10, most preferably 1 to 5.
  • “deletion”, “substitution”, and “added” occur in the amino acid sequence shown in SEQ ID NO: 2.
  • Deletion ”,“ substitution ”,“ addition ”, and“ deletion ”,“ substitution ”,“ addition ”protein powers As well as proteins that also have amino acid sequence capabilities described in SEQ ID NO: 2, A substance having MAPKKK activity.
  • DNA having the nucleotide sequence shown in SEQ ID NO: 1 “deletion”, “substitution”, and “added” mean that “deletion” occurs in the nucleotide sequence shown in S EQ ID NO: 1. ”,“ Substitution ”,“ addition ”and the protein encoded by the“ deletion ”,“ substitution ”,“ addition ”nucleotide sequence is the amino acid sequence capability described in SEQ ID NO: 2. Like protein, it has MAPK KK activity.
  • substitution for example, substitution of amino acids having similar properties, for example, substitution of one hydrophobic amino acid force with another hydrophobic amino acid, from one hydrophilic amino acid to another hydrophilic amino acid Substitution that does not significantly change the overall function of the protein, such as substitution with another acidic amino acid, substitution with another acidic amino acid, or substitution with another basic amino acid. It is.
  • the variant of the protein having the MAPKKK activity consisting of the amino acid sequence shown in SEQ ID NO: 2 is complementary to the DN A also having the nucleotide sequence ability shown in SEQ ID NO: 1.
  • D hybridizable with DNA under stringent conditions A protein having MAPKKK activity that also has the amino acid sequence ability encoded by NA is included.
  • stringent conditions mean that the target DNA is a DNA consisting of a nucleotide sequence encoding the protein shown in SEQ ID NO: 2 (for example, SEQ ID NO: 1) or a degenerate state thereof. It is a condition that can specifically hybridize with a base sequence in the relationship of. Hybridization conditions are forces determined in consideration of conditions such as temperature and ion concentration. Generally, it is known that the higher the temperature and the lower the ion concentration, the higher the stringency level. Such stringent conditions can be set based on the description of a person skilled in the art (for example, Samorook and Russel (Molecular Cloning: A Laboratory Manual, 3rd edition (2001)).
  • hybridization conditions such as 6 X SSC, 5 X Denhardt's, 0.1% SDS, 25 ° C or 68 ° C.
  • the hybridization temperature is preferably 45 ° C, 68 ° C (without formamide) or 25 ° C, and 50 ° C (50% formamide). it can.
  • the variant of the protein having MAPKKK activity of the present invention includes at least 60%, more preferably at least 70%, more preferably between the amino acid sequence shown in SEQ ID NO: 2. Included are proteins with MAPKKK activity that also have an amino acid sequence ability with at least 80%, most preferably at least 90% amino acid sequence homology.
  • the variant of DNA encoding the protein having the MAPKKK activity of the present invention also has at least 60%, more preferably at least 70%, more preferably at least 80% with respect to the nucleotide sequence shown in SEQ ID NO: 1. Most preferably, DNA having a nucleotide sequence having at least 90% nucleotide sequence homology and encoding a protein having MAPKKK activity is included.
  • a protein having MAPKKK activity comprising:
  • a protein having a MAPKKK activity consisting of: an amino acid sequence that is hybridized under stringent conditions and encoded by a DNA encoding a protein having M APKKK activity; or
  • Amino acid sequence that has at least 60%, more preferably at least 70%, more preferably at least 80%, and most preferably at least 90% amino acid sequence homology with the amino acid sequence represented by SEQ ID NO: 4. , A protein having MAPKKK activity;
  • the present invention further relates to a novel MAPKKK of this mouse
  • DNA consisting of the nucleotide sequence described in SEQ ID NO: 3; or (d) DNA complementary to the nucleotide sequence described in SEQ ID NO: 3; DNA that encodes a protein that hybridizes and has MAPKKK activity;
  • the protein ASK3 force of the present invention It was clarified by which action mechanism the phosphorylation activity is regulated. From the above-described sequence analysis and analysis of the kinase region motif, it was speculated that the protein ASK3 of the present invention has acupuncture activity. As a result of a detailed examination of this action, the protein ASK3 of the present invention is in an active relationship with each other by binding to ASK1 and phosphorylating each other, and functions in the ⁇ 38 signaling pathway. By activating KKKK 3/6 and MAPKK MKK4 / MKK7, which function in the JNK signaling pathway, through phosphorylation, JNKZP38 was finally activated.
  • the activation mechanism of the protein ASK3 of the present invention was regulated in detail.
  • the protein ASK3 of the present invention is activated by stimulation with peroxyhydrogen (0) and also activated by calcium stimulation (ie, , Phosphorylated).
  • These activation mechanisms are similar to those of ASK1, which has a similar sequence.
  • the protein ASK3 of the present invention changes its active state greatly in accordance with changes in osmotic pressure, apart from these activation mechanisms.
  • ASK3 is activated (phosphorylated) on the low osmotic pressure side, while ASK3 is inactivated (dephosphorylated) on the high osmotic pressure side, with normal osmotic pressure in the body as a boundary.
  • ASK3 is effective in hydrogen peroxide (H 0) stimulation, calcium stimulation and
  • MKK3 / a MAPKK that functions in the p38 signaling pathway, is activated (phosphorylated) or inactivated (dephosphorylated) by 2 2 Z or osmotic stimulation, and depending on its activated (phosphorylated) state.
  • MKK4 / MKK7 a MAPKK that functions in the MKK6 and JNK signaling pathways, it is possible to ultimately regulate the activity of JNKZP38 through activation / inactivation. Indicated.
  • the present invention provides a vector capable of replicating in a host cell, which contains a gene having the nucleotide sequence according to the present invention (eg, SEQ ID NO: 1) in a state in which a protein encoded by the sequence can be expressed. Can do.
  • the present invention also provides a host cell transformed with such a vector.
  • the host-vector system may be any host-vector system that can be used in the technical field, and is not particularly limited.
  • any vector conventionally used in the art may be used.
  • a plasmid vector for example, an expression vector in prokaryotic cells, yeast, insect cells, animal cells, etc.
  • Viral vectors eg, retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, Sendai virus vectors, HIV vectors, vaccinia virus vectors
  • ribosome vectors eg, cationic ribosomes) Vector
  • the vector construction procedure and method according to the present invention may be those commonly used in the field of genetic engineering.
  • Sambrook and Russel Molecular Cloning: A Laboratory Manual , 3rd edition (2001)
  • a host cell to be transformed a host cell compatible with the above-described vector is used.
  • host cells include, for example, E. coli, yeast, insect cells, and COS cells, mink lung epithelial cells, lymphocytes, fibroblasts, CHO cells, blood cells, and tumor cells (HEK-293 cells, HeLa cells) Animal cells such as) can be used.
  • antibodies against peptide regions characteristic of human protein (SEQ ID NO: 2) or mouse protein (SEQ ID NO: 4) having MAPKKK activity was made. That is, in one embodiment of the present invention, an antibody that binds to the protein represented by S EQ ID NO: 2 or SEQ ID NO: 4 can be provided. In this embodiment, it is preferable that the obtained antibody does not bind to human-derived MAPKKK, ASK1 and ASK2, more preferably, a protein having human or mouse-derived MAPKKK activity. Except for MAPKKK, ASK1 and ASK2 derived from all species (including humans)
  • An antibody according to the present invention can be obtained by using Delves (Antibody Production: Essential Techniques (1997)), Haward and SEQ ID NO: 2 or SEQ ID NO: 4 protein prepared as described above or a fragment thereof. It can be obtained according to known methods described in detail in Bethell (Basic Methods in Antibody Production and Practice (2000)), Kontermann and Dubel (Antibody Engineering: Springer Lab Manual (2001)).
  • the ability to react with the protein of SEQ ID NO: 2 or SEQ ID NO: 4 To prepare antibodies that do not react with the known MAPKKK, ASK1, and ASK2 of other species, it is preferred to use as an immunogen a polypeptide that also has an amino acid sequence that is characteristic of NO: 2 or SEQ ID NO: 4, but not present in the amino acid sequences of other species of AS K1 and ASK2.
  • the antibody of the present invention may be obtained as a polyclonal antibody, a monoclonal antibody or other type of antibody.
  • the polyclonal antibody can be obtained by immunizing a suitable immunized animal with the protein of SEQ ID NO: 2 or SEQ ID NO: 4, or a fragment thereof, and collecting the recovered serum.
  • Usual animals such as rabbits, hidges, goats, guinea pigs, and mice are generally used as immune animals, but are not limited thereto.
  • a monoclonal antibody is obtained by collecting antibody-producing cells of an animal immunized with the above-mentioned protein of SEQ ID NO: 2 or SEQ ID NO: 4, or a fragment thereof, and combining it with an appropriate fusion partner such as a myeloma cell. Cells are fused to obtain high-pridoma cells, and a clone producing an antibody with the necessary activity is cultured in a culture medium suitable for growth and then cloned. The culture supernatant strength can also be obtained by culturing with the above method. Furthermore, antibodies can also be produced by growing the thus obtained hybridoma cells in the peritoneal cavity of mammals.
  • the immunized animal for example, a mouse, a nude mouse, a rat, or a chicken is preferable.
  • the antibodies thus obtained are subjected to general isolation and purification methods such as centrifugation, dialysis, salting out with ammonium sulfate, ion exchange chromatography, gel filtration, and affinity chromatography. And can be purified.
  • the polypeptide region used to generate an antibody specific to ASK3 is similar to other proteins (for example, ASK1 and ASK2) as a result of sequence alignment. It can be anywhere as long as it is not, for example, an amino acid polypeptide consisting of amino acids 197 to 214 of SEQ ID NO: 2 (also called CES peptide; SEQU ID NO: 26) or SEQ ID NO: 2 16 amino acid polypeptide consisting of amino acids 913 to 928 (also referred to as RQV peptide; SEQ ID NO: 27) can be used as an immunogen.
  • the antibody of the present invention that can be obtained as described above can be used for purification, detection, activity inhibition and the like of a protein having MAPKKK activity.
  • the antibody of the present invention can be used after being made into F (ab ′) fragment or Fab ′ fragment by a known method. Also
  • a radioisotope eg, 3 or 3 H
  • an enzyme eg, horseradish peroxidase
  • an appropriate affinity is used. It can be labeled with a sex ligand (eg avidin-piotin).
  • the protein ASK3 of the present invention has MAPKKK activity, and is active by phosphorylating MKK3 / MKK6, a MAPKK that functions in the p38 signaling pathway, and MKK4 / MKK7, a MAPKK that functions in the JNK signaling pathway.
  • JNKZP38 is activated. It has been known so far that apoptosis is promoted by the activation of JNKZP38 in cells (Tibbies and Woodgett, Cell. Mol. Life Sci "55, 1230-1254, 1999).
  • ASK3, apoptosis can be regulated in diseases associated with abnormal apoptosis, and the prevention and treatment of such diseases are performed. Therefore, a pharmaceutical composition containing an ASK3 activity modulator can be provided.
  • the regulation of ASK3 kinase activity includes both inhibition of ASK3 kinase activity and promotion of ASK3 kinase activity, and abnormal apoptosis! /, In other cases, promotion of apoptosis and apoptosis. Both cases of suppression are included.
  • ASK3 kinase activity when ASK3 kinase activity is promoted in the body, MAPKK of JNK signaling pathway and p38 signaling pathway is activated, and phosphorylation of JNKZP38 occurs, thereby promoting apoptosis. Therefore, by inhibiting the kinase activity of the protein of the present invention, ASK3, apoptosis can be suppressed in diseases associated with the promotion of apoptosis, and such diseases can be prevented and treated. Therefore, a pharmaceutical composition containing an ASK3 activity inhibitor can be provided.
  • ASK3 activity inhibitor that can be used in the present invention, for example, a dominant negative mutant of ASK3, an antisense oligonucleotide, a double-stranded RNA, and a low molecular weight compound may be used. it can.
  • ASK3 kinase activity is inhibited in the body, JNK signaling pathway and p38 signaling pathway MAPKK are not activated, and JNKZP38 phosphorylation does not occur! / Is suppressed. Therefore, by promoting the kinase activity of the protein of the present invention, ASK3, apoptosis can be promoted in diseases associated with inhibition of apoptosis, and the prevention and treatment of such diseases can be performed.
  • a pharmaceutical composition containing an ASK3 activity promoter can be provided. As such diseases related to suppression of apoptosis, tumors, cancers, autoimmune diseases and the like are known.
  • ASK3 activity promoter that can be used in the present invention, for example, ASK3 overexpression by gene transfer, a low molecular weight compound, and the like can be used.
  • the gene can be introduced by any method commonly used in the art, for example, ribosome, adenovirus vector, lipofectin, particle gun, retrovirus vector, etc. Can do.
  • the ASK3 of the present invention can modify the apoptosis of a disease associated with the promotion or suppression of apoptosis by modifying the kinase activity of the protein of the present invention, ASK3.
  • a method of screening for an agent that modifies the kinase activity of the protein of the invention, ASK3, is also provided.
  • the present invention is a method for screening a compound that modifies the phosphate state of ASK3,
  • HEK-293 cells or mouse embryonic fibroblasts (MEF cells) into which the human ASK3 gene or mouse ASK3 gene of the present invention has been introduced are used as cells into which the ASK3 gene has been introduced.
  • ASK3 phosphorylation is due to the fact that the phosphorylation state of ASK3 is actually detected using the antibodies described in the present invention, such as Western blotting, immunoblotting, and ELISA. Measuring the change in mass of ASK3 using mass spectrometry; substituting the cells with [ 32 P] H PO
  • primers were prepared as follows:
  • mouse fetal fibroblast (MEF cell) cDNA was synthesized from total RNA of MEF cells using SUPER SCRIPT TM Preamplification System (GIBCO BRL). It was. Subsequently, PCR was carried out using the MEF cell cDNA as a cage.
  • MEF cell mouse fetal fibroblast
  • Taq DNA polymerase SIGMA
  • LA Taq Takara
  • iCycler BIO-RAD
  • PCR products were obtained with the primer combinations of S8 / AS7, S2 / AS2, and S3 / AS5.
  • PCR products are extracted using GENECLEAN II Kit (Q-Biogene) or Wizard SV Geland PCR Clean—Up System (Promega). And DH5a complex Itoda vesicles were transformed. Plasmid recovery was performed with Wizard Plus Minipreps DNA Purification Systems (Promega). The sequence was first determined using CEQ Quick Start Kit (Beckman) and then determined with CEQ 2000 (BECKMAN COULTER).
  • the PCR product obtained from the S8 / AS7, S2 / AS2, and S3 / AS5 primer combinations consisted of 193 bp, 1671 bp, and 2041 bp, respectively.
  • S8-AS5 CDNA sequence from S8 to AS5 (3745 bp; hereinafter referred to as “S8-AS5” fragment) (Fig. 1).
  • the lower case and upper case parts of the base sequence indicate the untranslated region and the translated region, respectively.
  • primer S27 was prepared on the 5'-most side of the sequence, and PCR primers were prepared as follows:
  • PCR was performed using the primer combinations of S27 / AS14, S27 / AS19, and S27 / AS22 under the same reaction conditions as the PCR conditions described in (1). Extraction of the obtained PCR product was performed by GENECLEAN II Kit (Q-Biogene) or Wizard SV Geland PCR Clean-Up System. The extracted DNA was subcloned into pGEM-T Easy Vector using pGEM-T Easy Vector System (Promega), and DH5a-competent cells were transformed. Plasmid recovery can be performed using Wizard Plus Minipreps DNA Purincation systems (Promega). First, PCR was performed with CEQ Quick Start Kit (Beckman) and then determined with CEQ 2000 (BECKM AN COULTER). As a result, it was revealed that the obtained PCR product had the sequence as expected from the RACE results (Fig. 1).
  • the lower case and upper case parts of the base sequence indicate the untranslated region and the translated region, respectively.
  • cDNA of HEK-293 cells derived from human fetal kidney was prepared from total RNA of HEK-293 cells using SUPER SCRIPT Preamplification System (GIBCO BRL). Using this SI / AS1 primer combination, PCR was carried out using the cDNA prepared from HEK-293 cells as a saddle, and the base sequence of the PCR product was determined using CEQ 2000 (BECKMAN COULTER). It was found that the cDNA was consistent with the sequence. So hi It was confirmed that ASK3 was expressed in HEK-293 cells.
  • primers S4, AS3, S6, and AS6 were designed and prepared.
  • the nucleotide sequences of these primers are as follows:
  • a sequence on the X chromosome similar to the 5 'end of mouse ASK3 was found on the human genome by a search using NCBI Human Genomic BLAST. Based on this sequence, primers having the following sequences, HS1 (SEQ ID NO: 24; sense) and G1AS2 (SEQ ID NO: 25; antisense) were prepared:
  • G1AS2 5 '-GTAGAAGAGGGAAGGCTGTCTGGAGA-3' (SEQ ID NO: 25)
  • the lower case part and human part of the base sequence indicate the untranslated region and the translated region, respectively.
  • HEK-293 cDNA was subjected to PCR in a saddle shape.
  • the PCR product (515 bp) contains a sequence on the human genome X chromosome, and has a predicted amino acid sequence mouse ASK It was found that it encodes a gene product homologous to 3 (Fig. 2).
  • an initiation codon and an upstream in-frame stop codon were confirmed on the 5 'side, and this PCR product was thought to encode the N-terminus of human ASK3 protein (Fig. 2).
  • mouse ASK3 has already been cleaved in full length, and the 5'-most ATG codon on cDNA functions as the start codon. It has been suggested.
  • nucleotide sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4 derived from mouse obtained in Example 1 and the nucleotide of SEQ ID NO: 1 of a similar molecule derived from human obtained in Example 2 The sequence and amino acid sequence of SEQ ID NO: 2 were compared to the previously known nucleotide sequence and amino acid sequence of MAPKKK, human and mouse ASK1 and ASK2. Comparison of nucleotide sequence of DNA and amino acid sequence of protein were both carried out using ClustalV program (Higgins and Sharp, CABIOS, Vol. 5, No. 2: pp. 151-153, 1989).
  • the amino acid sequence of SEQ ID NO: 2 of the present invention is 56.5%, 52.9 between the amino acid sequences of human A SKI, mouse ASK1, human ASK2, and mouse ASK2. %, 41.7%, and 41.3% sequence homology was found.
  • the nucleotide sequence of SEQ ID NO: 1 of the present invention is 49.5%, 50.6% between the nucleotide sequences encoding human ASK 1, mouse ASK1, human ASK2, and mouse ASK2, respectively. 41.1% and 41.7% sequence homology was found.
  • a novel human and mouse protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 was added to the amino acid sequences of ASK1 and ASK2 derived from humans and mice. I made it line. The results are shown in Fig. 3. As clearly shown in FIG. 3, amino acids 650 to 908 corresponding to the central region of the novel protein (SEQ ID NO: 2) derived from the human of the present invention are compared with other regions. In comparison, it was found that there is a portion with a high sequence homology. Specifically, this region of SEQ ID NO: 2 had 86.9% and 74.9% sequence homology, respectively, compared to the corresponding region of human ASK1 and human ASK2.
  • SEQ ID NO: 2 Of SEQ ID NO: 2 obtained in Example 1, a polypeptide of 18 amino acids consisting of amino acids 197 to 214 (CES peptide; SEQ ID NO: 26) (this sequence is represented by SEQ ID NO: 4) (Corresponding to amino acids 201 to 218) or a 16 amino acid polypeptide consisting of amino acids 909 to 924 of SEQ ID NO: 2 (RQV peptide; SEQ ID NO: 27) (this sequence is SEQ ID NO : Corresponds to amino acids 913 to 928 in 4) as an immunogen at a concentration of 150 g / ml and administered to the rabbit, using the same immunogen at a concentration of 300 g / ml every 14 days. Boosted 3 times, blood was collected 14 days after the final boost, and antiserum was obtained from that blood.
  • polypeptide sepharose bead column polypeptide sepharose bead column.
  • Polyclonal antibodies prepared using the polypeptide of SEQ ID NO: 26 are the anti-ASK3 (CES) antibody and polyclonal antibodies prepared using the polypeptide of SEQ ID NO: 27, respectively.
  • the antibody was named “anti-ASK3 (R QV) antibody”. These antibodies recognize ASK3 on the membrane during Western blotting, but cannot recognize other ASK1 and ASK2, and have the ability to immunoprecipitate ASK3 from cell extracts. (See Fig. 4).
  • Example 2 the amino acid sequence of SEQ ID NO: 4 obtained in Example 2 was used. The expression distribution of the proteins possessed was examined at the mRNA and protein levels.
  • Fig. 4A The results are shown in Fig. 4A. As is apparent from this figure, the mouse ASK3 mRNA is expressed in various tissues in mice, but especially in the heart and kidney, it is found that much mRNA is expressed. Wow.
  • ASK3 molecules were immunoprecipitated from anti-ASK3 (CES) antibody from a crushing extract sample prepared from Sarakuko and kidney, and the force was also used for the experiment.
  • an anti-ASK3 (CES) antibody preincubated with a CES peptide (SEQ ID NO: 26) was used as a negative control for immunoprecipitation.
  • immunoprecipitation samples were analyzed by Western blotting using an anti-ASK3 (RQV) antibody (FIG. 4C).
  • WB ASK3 (RQV)” or “WB: p38 (C-20G)” means anti-ASK3 (RQV) antibody and anti-p38 (C-20G) antibody, respectively.
  • IP ASK3 (CES)” is a notation indicating that immunoprecipitation was performed using an anti-ASK3 (CES) antibody. The same applies to the following figures in the book.
  • Example 6 Analysis of kinase activity of ASK3
  • ASK3 specifically has human and mouse ASK3 kinase activity in vitro and whether it specifically activates the JNK and p38 signaling pathways. I investigated.
  • Wild-type ASK3 (Flag-ASK3-WT) and a kinase-inactive mutant ASK3 (Flag-ASK3-KM) tagged with a Flag tag using pcDNA3 expression vector (Invitrogen) were combined with FuGENE6 transfusion reagent (Roche).
  • FuGENE6 transfusion reagent (Roche).
  • FuGENE6 transfusion reagent (Roche).
  • ERK ERK-specific kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase kinase antibody; Cell Signaling).
  • ASK1-WT wild type ASK1
  • epidermal growth factor EGF stimulation was used as a positive control to activate ERK.
  • FIG. 5A The results are shown in FIG. 5A.
  • ASK3-WT wild-type ASK3
  • ASK3-KM mutant ASK3
  • Activation of JNK and p38 MAP kinase was observed.
  • the activity of ERK was unacceptable. From these results, it was confirmed that ASK3 specifically activates the JNK and p38 MAP kinase pathways depending on the kinase activity.
  • coli BL21 was used as a substrate for MKK3 and MKK6 (Fig. 5B).
  • GST-JNK-KN (inactive mutant JNK) fusion protein purified from E. coli BL21 was used as a substrate (Fig. 5C), and in vitro kinase analysis was performed.
  • ASK1 was used as a positive control to activate all MKK molecules
  • ASK3 hardly activated MKK7 in cells (Fig. 4C), it activated MK K3, ⁇ 4, and ⁇ 6 to the same extent as ASK1 (Figs. 4 ⁇ and 4C).
  • ASK3 may function as a MAPKKK in the JNK and p38 MAP kinase pathways! /.
  • Wild-type ASK3 (Flag-ASK3 WT) to which Flag tag was added to HEK-293 cells, and mutant ASK3 in which 808 and 812 threonine residues of human ASK3 were substituted with alanine residues ( (T808A and T812A, respectively) are transiently expressed and anti-phosphorylated ASK Antibody (Toblume et al, J. Cell.
  • JNK1 JNK1 (FL), Santa Cruz, CA, USA) detected the activity state and expression level of ASK3 and downstream JNK, respectively.
  • HEK-293 Cell Strength Endogenous AS K3 was immunoprecipitated in the prepared cell extract with anti-ASK3 (CES) antibody.
  • an anti-ASK3 (CES) antibody that had been incubated with an antigen peptide, CES peptide, was used as a negative control for immunoprecipitation.
  • immunoprecipitation samples were separated by SDS polyacrylamide electrophoresis, transferred to PVDF membrane, and subjected to Western blotting using anti-ASK3 (RQV) antibody and anti-ASK1 antibody (F9; Santa Cruz). Endogenous ASK3 and endogenous ASK1 were detected.
  • ASK3 and ASK1 in cells were confirmed.
  • a wild-type ASK3 (6xMyc-ASK3-WT) with a Myc tag and a kinase-inactive mutant ASK1 (HA-ASK1-KM) with an HA tag were incorporated into a pcDNA3 expression vector, and FuGENE6 Using EXK reagent (Roche), leave the residue in HEK-293 cells.
  • a gene was introduced and expressed simultaneously.
  • kinase-inactive mutant ASK3 (6xMyc-ASK3-KM) with Myc tag and wild-type ASK1 (HA-ASK1-WT) with HA tag incorporated into pcDNA3 expression vector were used as FuGENE6 transfection reagent.
  • genes were introduced and expressed simultaneously. Incubate using DMEM medium containing 10% FBS. Use cell extracts obtained from both cells 24 hours after introduction. Use anti-phosphorylated ASK antibody to determine the activation state of ASK3 and ASK1, anti-Myc antibody and anti-HA antibody. Respectively, the expression levels of ASK3 and ASK1 were detected by Western blotting.
  • Flag-ASK3 incorporated into pcDNA3 expression vector was introduced into HEK-293 cells by FuGENE6 transfection reagent (Roche). 24 hours after introduction, 1 mM H 0, 1
  • ASK3 is activated by stimulation with hydrogen peroxide (H 0)
  • Example 9 ASK3 action to induce apoptosis
  • HEK-293 cells (2 X 10 5 cells / ml) were treated with wild-type ASK3 (Flag-ASK3) or kinase-inactive mutant ASK3 (Flag-ASK3-KM) incorporated into a pcDNA3 expression vector. Each gene was introduced and expressed using FuG ENE6 transfection reagent (Roche). 10 hours after introduction, cells (7 X 10 4 cells / ml) were seeded on a 96-well plate for Atsey, and 20 hours later, caspase3 / 7 activity (DEVDase activity) was measured using Caspase-Glo 3/7 Atsekit kit (Promega ) Is revised by 7).
  • Flag-ASK3 wild-type ASK3
  • Flag-ASK3-KM kinase-inactive mutant ASK3
  • ASK3-WT In cells overexpressing ASK3-WT, do not express the cells and activity! A significant increase in caspase3 / 7 activity was observed compared to cells expressing the mutant ASK3-KM (Fig. 10). This result indicates that ASK3 has an activity of inducing apoptosis and inducing apoptosis depending on the kinase activity.
  • Example 10 Effect of ASK3 on site-powered cows.
  • ASK3-WT wild-type ASK3
  • ASK3-KM active mutations
  • 5'-terminal Flag-tags were used in HEK-293 cells (2 X 10 5 cells / ml) using the pcDNA3 expression vector
  • Each type ASK3 was transduced with FuGEN E6 transfection reagent (Roche Diagnostics).
  • the concentration of TNF-a in the culture supernatant of the transformed cells 24 hours after introduction was quantified using a human TNF-a ELISA kit (TECHNE).
  • the expression level and activation of ASK3 were analyzed using Western blotting.
  • the above-mentioned disrupted extract sample of transformed cells 24 hours after introduction was prepared, separated by SDS polyacrylamide electrophoresis, transferred to a PVDF membrane, and anti-Flag antibody (M2 ASK3 expression was detected with an antibody (SIGMA), and ASK3 activity was detected with an anti-phosphorylated ASK recognition antibody that recognizes an active ASK molecule.
  • SIGMA anti-Flag antibody
  • ASK3 activity was detected with an anti-phosphorylated ASK recognition antibody that recognizes an active ASK molecule.
  • activation of endogenous p38 MAP kinase downstream of ASK3 was also detected with an anti-phosphorylated p38 recognition antibody (Cell Signaling) that recognizes an activated p38 molecule.
  • Example 11 Anti-acupuncture stimulation of ASK3 (1)
  • ASK3 is phosphorylated and activated by receiving a high osmotic pressure stimulus in ASK1, so that ASK3 of the present invention can It was confirmed whether it would react like this.
  • Wild-type ASK3 (Flag_ASK3-WT) and wild-type ASK1 (Flag-ASKl-WT) with a Flag tag incorporated into a pcDNA3 expression vector were combined with HEK-293 using FuGENE6 transfection reagent (Ro che). The cells were transfected and expressed. Cells 24 hours after introduction were subjected to hyperosmotic stimulation with 0.5 M sorbitol for the time shown in Fig. 12, Blast extract was prepared. With respect to this cell extract, the activity of ASK3 and ASK1 was detected using an anti-phosphorylated ASK antibody, and the expression levels of ASK3 and ASK 1 were detected using an anti-Flag antibody, respectively, by Western plotting. In addition, the activity of endogenous JNK and p38 was detected with an anti-phosphate recognition antibody (Cell Signaling) that recognizes activated kinase molecules.
  • Cell Signaling an anti-phosphate recognition antibody
  • Wild-type ASK3 (Flag_ASK3-WT) with a Flag tag incorporated into a pcDNA3 expression vector was introduced into HEK-293 cells and expressed using FuGENE6 transfection reagent (Roche).
  • Hyperosmotic buffer 500 mOsm / kg H 0 buffer; 130 mM NaCl, 2 mM KC1) for 1, 2, 5, 10, 30, 60, and 120 minutes after 24 hours of transfection
  • an isotonic buffer (300 mOsm / kg H 0 buffer; 130 mM NaCl, 2 mM KC1, 1 mM KH PO, 2 mM CaCl
  • the cell extract was prepared. From this cell extract, the protein was separated by SDS polyacrylamide electrophoresis, transferred to a PVDF membrane, the activity of ASK3 was determined using anti-phosphorylated ASK antibody, and the activity of ASK3 was determined using anti-Flag antibody. The expression level was detected by Western plot method.
  • FIG. In this figure, high osmotic pressure stimulation and low osmotic pressure stimulation respectively.
  • the upper row shows the active state of ASK3 examined using anti-phosphorylated ASK antibody
  • the lower row shows the expression level of ASK3 examined using anti-Flag antibody.
  • ASK3 is phosphorylated to some extent in an isosmotic state (300 mOs / kg H 0)! / ⁇
  • the extracellular solution was made from 100 mM to 190 mM NaCl set at 10 mM intervals, 4 mM KC1, 2 mM KH PO, 4 mM CaCl, 4 mM MgCl, 20 mM Na—HEPES pH 7.3, 20
  • Osmotic stimulation was performed for 20 minutes.
  • the other experimental conditions were the same as those described in Example 12, using the anti-phosphorylated ASK antibody to determine the active state of ASK3, and the anti-Flag antibody to ASK3.
  • the expression level was detected by Western blotting.
  • FIG. 14A The results are shown in FIG. In this figure, in the Western blot diagram (Fig. 14A), the upper row shows the activity state of ASK3 examined using anti-phosphorylated ASK antibody, and the lower row shows expression of ASK3 examined using anti-Flag antibody. The amount is indicated respectively.
  • the actual osmotic pressure measured by the osmometer showed 310 mOsm, which is considered to be almost isosmotic at a NaCl concentration of 130 mM.
  • ASK3 was phosphorylated to some extent at a NaCl concentration of 130 mM, which showed 310 mOsm, which is considered to be almost isotonic.
  • the value (times) obtained by dividing phosphorylated ASK3 (Phospho-ASK3 in Fig. 14A) by the total amount of ASK3 (Flag-ASK3) is defined as 1, and the results are plotted for each osmotic solution ( Figure 14B).
  • ASK3 showed a phosphorylation amount about 3 times the isosmotic pressure around 250 mOsm (100 mM NaCl), and was almost completely dephosphorylated around 350 mOsm (150 mM Na CI). .
  • ASK3 changes its activity from isosmotic pressure to ⁇ 50 mOsm with respect to osmotic pressure, and reacts most sensitively to changes near isosmotic pressure.
  • the present invention can provide a novel MAPKKK derived from human or mouse.
  • the present invention also inhibits or promotes the kinase activity of this novel protein, thereby inhibiting or promoting the JNK Zp38 signaling pathway and suppressing or promoting apoptosis in diseases associated with promoting or suppressing apoptosis, As a result, it is shown that such a disease can be treated and prevented, and as a result, a pharmaceutical composition containing an agent that inhibits or promotes the kinase activity of the novel protein can be provided.

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Abstract

La présente invention concerne une nouvelle MAP3K chez l’homme et la souris. Elle concerne également un agent prophylactique / un médicament présentant une meilleure innocuité et une efficacité importante contre une maladie liée à l’apoptose en inhibant la nouvelle MAP3K chez l’homme ou la souris ; à savoir, la présente invention concerne une nouvelle protéine provenant des hommes ou des souris et servant de MAP3K. En outre, il est indiqué que la voie de transduction du signal JNK/p38 est inhibée ou favorisée et, ainsi, l’apoptose peut être favorisée ou supprimée dans des maladies associées à l’apoptose en inhibant ou en favorisant l’activité kinase de la protéine mentionnée précédemment. Ainsi, la maladie peut être traitée / prévenue. Par conséquent, la présente invention concerne une composition médicinale contenant un agent en mesure d'inhiber ou de favoriser l’activité kinase de la nouvelle protéine décrite précédemment.
PCT/JP2005/021452 2004-11-22 2005-11-22 Nouvelle map kinase-kinase-kinase (map3k) WO2006054770A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001077338A2 (fr) * 2000-04-10 2001-10-18 Sugen, Inc. Nouvelles proteine kinases humaines et nouvelles enzymes de type proteine kinase
WO2002046384A2 (fr) * 2000-12-06 2002-06-13 Incyte Genomics, Inc. Kinases et phosphatases
WO2005000020A2 (fr) * 2003-06-23 2005-01-06 Cognis Ip Management Gmbh Vecteurs d'alcool d'alkoxylate pour principes actifs pesticides
WO2005044993A2 (fr) * 2003-11-04 2005-05-19 Metabolex, Inc. Compositions et procedes d'utilisation d'une kinase en relation avec la kinase de signalisation de l'apoptose (askrk)
WO2005083067A1 (fr) * 2004-02-26 2005-09-09 Bayer Healthcare Ag Nouvelle protéine kinase activée par les mitogènes, map3kx, et ses utilisations

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001077338A2 (fr) * 2000-04-10 2001-10-18 Sugen, Inc. Nouvelles proteine kinases humaines et nouvelles enzymes de type proteine kinase
WO2002046384A2 (fr) * 2000-12-06 2002-06-13 Incyte Genomics, Inc. Kinases et phosphatases
WO2005000020A2 (fr) * 2003-06-23 2005-01-06 Cognis Ip Management Gmbh Vecteurs d'alcool d'alkoxylate pour principes actifs pesticides
WO2005044993A2 (fr) * 2003-11-04 2005-05-19 Metabolex, Inc. Compositions et procedes d'utilisation d'une kinase en relation avec la kinase de signalisation de l'apoptose (askrk)
WO2005083067A1 (fr) * 2004-02-26 2005-09-09 Bayer Healthcare Ag Nouvelle protéine kinase activée par les mitogènes, map3kx, et ses utilisations

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] STRAUSBERG R L ET AL: "Generation and initial analysis of more than sequences.", Database accession no. (BC031147) *
PNAS USA., vol. 99, no. 26, 2002, pages 16899 - 16903, XP002309773 *

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