JPWO2006054770A1 - New MAP kinase kinase kinase (MAPKKKK) - Google Patents

Abstract

An object of the present invention is to provide a novel MAPKKK in humans and mice. Another object of the present invention is to provide a safer and more effective prophylactic / therapeutic agent for diseases related to apoptosis by inhibiting novel MAPKKK in humans and mice. The inventors of the present invention provide a novel protein that functions as MAPKKK derived from human or mouse, and also inhibits or promotes the kinase activity of this novel protein, thereby inhibiting the JNK / p38 signaling pathway Show that it is possible to suppress or promote apoptosis in diseases associated with the promotion or suppression of apoptosis, thereby treating and preventing such diseases and consequently inhibiting the kinase activity of novel proteins Alternatively, a pharmaceutical composition comprising an agent for promoting is provided.

Description

  The present invention relates to providing a novel MAP kinase kinase kinase (MAPKKK) that induces apoptosis (cell death). The present invention also relates to a preventive / therapeutic agent for diseases associated with the promotion of apoptosis by inhibiting this enzyme.

  The mitogen-activated protein (MAP) kinase signaling cascade is a well-preserved intracellular signaling pathway from yeast to vertebrates, including MAP kinase (MAPK), MAPK kinase (MAPKK) and It consists of three different members of protein kinases including MAPKK kinase (MAPKKK). In turn, these molecules phosphorylate MAPKK, which activates MAPKK by phosphorylating it, and activated MAPKK phosphorylates MAPK, thereby activating MAPK. 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 MAPKKK-MAPKK-MAPK signaling pathways, including signaling pathways involving p38, signaling pathways involving JNK, and signaling pathways involving ERK Was found to be functioning. Studies using cultured cells suggest that these groups of kinases play important roles in proliferation, differentiation, cell death, and the like. Among these MAPKs, p38 and JNK are molecules that are positioned as stress responsive types because they are activated by stimuli such as high osmotic pressure, heating, ultraviolet irradiation, and inflammatory cytokines.

  The upstream signaling mechanisms that activate p38 and JNK, which are stress-responsive MAPKs, include MKK3 / MKK6, a MAPKK that functions in the p38 signaling pathway as MAPKK, and MKK4 / MKK7, a MAPKK that functions in the JNK signaling pathway. It has been known. In addition, the inventors of the present invention cloned ASK1 (Apoptosis Signal-regulating Kinase 1) as an upstream MAPKKK that activates p38 and JNK, which are stress-responsive MAP kinases, and revealed the mechanism of action. (Patent Document 1; Patent Document 2; Non-Patent Document 1; Non-Patent Document 2). That is, according to these documents, ASK1 has the ability to phosphorylate MKK3 / MKK6 or MKK4 / MKK7, and ASK1 activates the JNK signaling pathway or p38 signaling pathway, resulting in apoptosis. It was suggested that it might be promoting.

Furthermore, the existence of ASK2 having homology to ASK1 was also clarified (Non-patent Document 3; DDBJ accession numbers AB167411 (human ASK2) and AB021861 (mouse ASK2)). And it was clarified that this ASK2 also has MAPKKK activity like ASK1 and can activate the JNK signaling pathway or the p38 signaling pathway (Non-patent Document 3).
Japanese Patent Laid-Open No. 10-93 WO02 / 38179 Ichijo et al., Science, 275, 90-94, 1997 Nishitoh et al., Genes & Development, 16, 1345-1355, 2002 Wang et al., Biochem. Biophys. Res. Commun. 253, 33-37, 1998

  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 serine / threonine kinase regions from cDNAs of human HEK-293 cells and mouse fetal fibroblasts, and have analyzed the detailed functions of the molecules in the cells. Based on the above, the present invention has been completed.

That is, the inventors, in one aspect,
(A) the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; or (b) the deletion of one or more amino acids in the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4. A protein having MAPKKK activity, or (c) an amino acid sequence encoded by DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3; or (D) a protein that hybridizes under stringent conditions with a DNA comprising a nucleotide sequence complementary to the DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3, and has a MAPKKK activity A protein having MAPKKK activity, comprising: an amino acid sequence encoded by the encoding DNA.

  The present invention further provides at least 60%, more preferably at least 70%, more preferably at least 80%, most preferably at least 90% of the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. A protein having MAPKKK activity consisting of an amino acid sequence having amino acid sequence homology is also provided.

In another embodiment, the present invention relates to the above-described novel MAPKKK: (a) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; or (b) SEQ ID DNA comprising any of NO: 2 or SEQ ID NO: 4 having a deletion, substitution, or addition of one or more amino acids and encoding a protein having MAPKKK activity Or (c) DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3; or (d) complementary to DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3. DNA which hybridizes under stringent conditions with DNA consisting of a specific nucleotide sequence and which encodes a protein having MAPKKK activity
Also provide.

  In the present invention, at least 60%, more preferably at least 70%, more preferably at least 80%, and most preferably at least 90% of the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 3 is used. DNA comprising a nucleotide sequence having nucleotide sequence homology and encoding a protein having MAPKKK activity is also provided.

  In yet another aspect of the present invention, by inhibiting the function of the above-mentioned protein having MAPKKK activity, stress-induced apoptosis involving JNK or p38 pathway is inhibited, and as a result, promotion of apoptosis is associated. Can treat and prevent diseases. That is, the present invention provides a pharmaceutical composition for treating or preventing a disease associated with promotion of apoptosis, comprising a MAPKKK inhibitor.

  In yet another aspect of the present invention, the function of the protein having the MAPKKK activity described above is promoted to promote stress-induced apoptosis involving the JNK or p38 pathway, resulting in inhibition of apoptosis. Can treat and prevent diseases. That is, 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 may have the characteristic of not binding to known MAPKKK, ASK1 and ASK2.

  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 / p38 signaling pathway and suppressing or promoting apoptosis in diseases associated with the promotion or suppression of 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. The underlined in the sequence and the name and SEQ ID NO above it indicate the primer used. FIG. 2 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 and SEQ ID NO described above indicate the primer used. FIG. 2 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 and SEQ ID NO described above indicate the primer used. FIG. 2 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 and SEQ ID NO described above indicate the primer used. FIG. 3 is a diagram comparing 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, ASK1, and ASK2 derived from human and mouse. . FIG. 4 confirms the expression of ASK3 mRNA and ASK3 protein of the present invention in mice using Northern blotting (FIG. 4A) and Western blotting (FIGS. 4B and 4C), prominent in the kidney as opposed to ASK1 It is a figure which shows having expressed. FIG. 5 shows that ASK3 is a MAPKKK that specifically activates the JNK signaling pathway and the p38 signaling pathway in vitro. FIG. 6 is a diagram showing the results of specifying an active site that controls the phosphorylation activity of ASK3. 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 and activated by H ₂ O ₂ stimulation and mitoxin (MTX) stimulation. FIG. 10 is a diagram showing that ASK3 has an activity of inducing apoptosis and inducing apoptosis in a kinase activity-dependent manner. FIG. 11 is a diagram showing that ASK3 has an activity of inducing production of cytokine (TNF-α). Figure 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. FIG. 13 shows that ASK3 is dephosphorylated when a high osmotic pressure stimulus is applied, but the degree of phosphorylation increases when a low osmotic pressure stimulus is applied. FIG. 14 is a diagram showing the activation state of ASK3 investigated using anti-phosphorylated ASK antibody, the expression level of ASK3 examined using anti-Flag antibody (FIG. 14A), and the relationship between osmotic pressure and phosphorylated level FIG. 14B is a diagram (FIG. 14B).

BEST MODE FOR CARRYING OUT THE INVENTION

Cloning of new molecules As a result of homology search using the nucleotide sequence of mouse ASK1 cDNA, two mouse-derived ESTs (Expressed Sequence Tags) that map onto the mouse X chromosome are obtained, and these ESTs are encoded. When the genomic sequence in the vicinity of the region was examined in detail, a base sequence having homology with the C-terminal region of ASK1 was found when converted to an amino acid.

  Based on these sequence information, in the present invention, first, a plurality of PCR primers are designed and prepared, and PCR is performed using cDNA prepared from mouse cells (for example, mouse fetal fibroblasts; MEF cells) as a template. A part of the target unknown gene can be obtained. Longer sequences can be obtained by joining fragments of some 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.

  Thus, using techniques commonly used in the art such as PCR and RACE, the present invention comprises an open reading frame (ORF) consisting of the nucleotide sequence of SEQ ID NO: 3, A novel nucleotide sequence from mouse can be obtained (FIG. 1).

  Based on the information of the above-described mouse-derived novel nucleic acid sequence (FIG. 1), a nucleic acid encoding a new human homologous molecule can be obtained. That is, using the obtained mouse cDNA base sequence (FIG. 1, SEQ ID NO: 3), the human genome sequence can be searched to confirm the presence of a homologous gene. Furthermore, based on the results of such searches, PCR primers can be designed and prepared, and PCR can be performed using cDNA prepared from human cells (eg, HEK-293 cells) as a template to obtain homologous cDNA. it can.

  In this way, using techniques commonly used in the art such as PCR and related RACE, the present invention is based on the nucleotide sequence (SEQ ID NO: 3) of the mouse-derived novel molecule described above. In the invention, it is possible to obtain a novel nucleotide sequence derived from human including an open reading frame (ORF) consisting of the nucleotide sequence of SEQ ID NO: 1 (FIG. 2).

Sequence analysis Next, SEQ ID NO: 1 DNA and SEQ ID having the nucleotide sequence shown in NO: a protein having an amino acid sequence represented by 2, and analyzed at the nucleotide level and amino acid level, human and mouse MAPKKK, ASK1 And compared to the nucleotide and amino acid sequences of ASK2.

  In the present invention, a sequence comparison program used by those skilled in the art can be used for sequence homology analysis of amino acid or base sequences. For example, it can be determined by comparing with sequence information using the BLAST program described in Altschul et al. (Nucl. Acids. Res. 25., p. 3389-3402, 1997). Specifically, in the case of nucleotide sequence analysis, it is possible to input a query base sequence with the Nucleotide BLAST (BLASTN) program and collate it with base sequence databases such as GenBank, EMBL, DDBJ. In the case of amino acid sequence analysis, a Query BLAST (BLASTP) program can be used to input a query amino acid sequence and collate with amino acid sequence databases such as GenBank CDS, PDB, SwissProt, and PIR. 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. 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 programs used by those skilled in the art of sequence comparison can also be used.

  In the present invention, the sequence homology of amino acid or base sequence may be determined by visual inspection and mathematical calculation. Alternatively, the sequence homology of 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). The determination may be made by comparing the sequence information using a computer program. Preferred default parameters for the GAP program include: (1) Scoring matrix, blosum62; as described in Henikoff and Henikoff (Proc. Natl. Acad. Sci. USA, 89: 10915-10919, 1992); ) 12 gap weights; (3) 4 gap length weights; and (4) No penalty for end gaps. In the present invention, it is also possible to utilize other sequence comparison software available in the art, such as ClustalV, ClustalW, Jotun Hein Method and the like.

  In the present invention, ClustalV was used to search for sequence homology at the amino acid level and at the nucleotide level 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. However, the highest sequence homology between the protein of SEQ ID NO: 2 and the DNA of SEQ ID NO: 1 of the present invention is the conventionally known MAPKKK, human and mouse ASK1 and ASK2. However, the amino acid sequence and nucleotide sequence of the novel human MAPKKK, ASK3 obtained in the present invention have only 56.5% and 49.5% homology with the amino acid sequence and nucleotide sequence of human ASK1, respectively, and ASK1 The MAPKKK and ASK2 proteins and DNA obtained as family molecules have only 41.7% and 41.1% homology, respectively. It was found. From these facts, it was found that the protein of SEQ ID NO: 2 and the DNA of SEQ ID NO: 1 did not have significant sequence homology as a whole with the known MAPKKK, ASK1, and ASK2, respectively.

  From the analysis of such nucleotide sequence and amino acid sequence, in the present invention, a protein comprising the amino acid sequence shown in SEQ ID NO: 2 obtained from human and the amino acid sequence shown in SEQ ID NO: 4 obtained from mouse The protein consisting of was predicted to be a novel protein having a kinase activity similar to the serine / threonine kinase activity of ASK1 and ASK2. 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.

  By transfecting mammalian cells with SEQ ID NO: 1 ASK3 DNA incorporated into the expression vector, cells were transformed and the phosphorylation status of endogenous JNK and p38, which were considered targets of ASK3, was anti-phosphorylated. Examination using an oxidation-recognizing antibody revealed that the ASK3 protein of SEQ ID NO: 2 produced by the mammalian cell has an activity to phosphorylate endogenous JNK and p38. In addition, we investigated which stage of endogenous JNK and p38 activation exerts kinase activity, and the ASK3 protein of SEQ ID NO: 2 of the present invention is a MAPKK that functions in the JNK signaling pathway MKK3 / MKK6 or p38 signal It was revealed that MKK4 / MKK7, a MAPKK that functions in the transmission pathway, was phosphorylated. This revealed that the protein consisting of the amino acid sequence of SEQ ID NO: 2 of the present invention has activity as MAPKKK.

  Therefore, based on these findings, in one embodiment of the present invention, as a novel protein having MAPKKK activity, a protein having the amino acid sequence described in SEQ ID NO: 2 derived from human, ASK3, or a variant thereof Thus, a protein having MAPKKK activity can be provided.

  In the present invention, the term “variant” of the protein of SEQ ID NO: 2 refers to an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence of the target protein; An amino acid sequence encoded by a base sequence capable of hybridizing under stringent conditions between a base sequence encoding the amino acid sequence of and a complementary base sequence; or between the amino acid sequence of the target protein An amino acid sequence having at least 60%, more preferably at least 70%, more preferably at least 80%, most preferably at least 90% amino acid sequence homology, including a protein having MAPKKK activity. means.

That is, the present invention more specifically,
(A) an amino acid sequence described in SEQ ID NO: 2; or (b) an amino acid sequence having a deletion, substitution, or addition of one or more amino acids in the sequence described in SEQ ID NO: 2. , A protein having MAPKKK activity;
(C) an amino acid sequence encoded by a DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1; or (d) a DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1. A protein having a MAPKKK activity consisting of: an amino acid sequence encoded by a DNA encoding a protein having a MAPKKK activity;
Consisting of an amino acid sequence having at least 60%, more preferably at least 70%, more preferably at least 80%, most preferably at least 90% amino acid sequence homology with the amino acid sequence shown in SEQ ID NO: 2. A protein having MAPKKK activity;
Can be provided.

  The present invention also provides derivatives of the proteins described above. In the present specification, the term “protein derivative” refers to the amino group at the amino terminal (N-terminal) of protein, the functional group of each amino acid side chain (for example, amino group, carboxyl group, hydrogen group, thiol group, amide group). Etc.) and one or more functional groups selected from the group consisting of a carboxyl group at the carboxyl terminus (C terminus) of the protein or a carboxyl group at the side chain of each amino acid, and other suitable substituents Refers to those modified by Modification with an appropriate other substituent is performed for the purpose of, for example, protecting a functional group present in a protein, improving safety and tissue migration, improving resistance to degradation, or enhancing activity. As such a substituent, any substituent known in the art may be used.

Specific examples of protein derivatives include: (1) an amino group at the amino terminus (N terminus) of a protein or a partial or all hydrogen atom of the amino group of the side chain of each amino acid is substituted or unsubstituted alkyl. Group (may be linear, branched or cyclic) (eg, methyl, ethyl, propyl, isopropyl, isobutyl, butyl, t-butyl, cyclopropyl, cyclohexyl, benzyl) ), A substituted or unsubstituted acyl group (for example, formyl group, acetyl group, caproyl group, cyclohexylcarbonyl group, benzoyl group, phthaloyl group, tosyl group, nicotinoyl group, piperidinecarbonyl group), urethane type protecting group (for example, p -Nitrobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, p-biphenylisopropylo Substituted with a xyloxycarbonyl group, t-butoxycarbonyl group) or a urea-type substituent (for example, methylaminocarbonyl group, phenylcarbonyl group, cyclohexylaminocarbonyl group), etc., and (2) the carboxyl terminal (C terminal) of the protein ) Or a part or all of the carboxyl group of the side chain of each amino acid has an ester type modification (for example, its hydrogen atom is methyl, ethyl, isopropyl, cyclohexyl, phenyl, benzyl, t- Butyl, substituted with 4-picolyl), modified with amide type (eg unsubstituted amide, C ₁ -C ₆ alkylamide (eg methylamide, ethylamide, isopropylamide) And (3) amino acids in the side chain of each amino acid of the protein And a functional group other than carboxyl group (e.g., hydrogen group, thiol group, amino group, etc.) a part or all of, such as those modified with such same substituents or trityl group and an amino group as described above may be mentioned.

In another embodiment, the present invention provides a mutant DNA encoding a novel protein (SEQ ID NO: 2) derived from a human having MAPKKK activity (SEQ ID NO: 1) or a variant thereof. Can be provided. That is, in another aspect, the present invention relates to the above-described novel human MAPKKK (a) DNA encoding a protein consisting of the amino acid sequence described in SEQ ID NO: 2; or (b) described in SEQ ID NO: 2. A DNA encoding a protein having one or more amino acid deletions, substitutions, or additions and having MAPKKK activity;
(C) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 1; or (d) DNA comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 1 under stringent conditions. A DNA that hybridizes and encodes a protein having MAPKKK activity; or
DNA comprising a nucleotide sequence having at least 60%, more preferably at least 70%, more preferably at least 80%, most preferably at least 90% nucleotide sequence homology with the nucleotide sequence represented by SEQ ID NO: 1 And a DNA encoding a protein having MAPKKK activity;
Can also be provided.

  Here, for example, “(a) DNA encoding a protein consisting of the amino acid sequence described in SEQ ID NO: 2” has a degenerate relationship with DNA consisting of the nucleotide sequence of SEQ ID NO: 1. Those skilled in the art can easily understand that DNA having a nucleotide sequence is included.

  Here, “one or more” means preferably 1 to 20, more preferably 1 to 10, and most preferably 1 to 5. In the case of a protein consisting of the amino acid sequence represented by SEQ ID NO: 2 of the present invention, “deletion”, “substitution”, and “added” occur in the amino acid sequence represented by SEQ ID NO: 2. In the same manner as the protein consisting of the amino acid sequence described in SEQ ID NO: 2, the MAPKKK is a “deletion”, “substitution”, “addition”. The one having activity. Further, in the case of DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1, "deletion", "substitution", "added" means "deletion" that occurs in the nucleotide sequence shown in SEQ ID NO: 1, A protein that is a “substitution”, “addition”, and that is encoded by a nucleotide sequence that is “deletion”, “substitution”, “addition” is the same as the protein consisting of the amino acid sequence set forth in SEQ ID NO: 2. , Which has MAPKKK activity.

  For example, in the case of “substitution” of amino acids, substitution between amino acids having similar properties, for example, substitution from one hydrophobic amino acid to another hydrophobic amino acid, substitution from one hydrophilic amino acid to another hydrophilic amino acid Substitution that does not significantly change the function of the whole protein, such as substitution from one acidic amino acid to another acidic amino acid, or substitution from one basic amino acid to another basic amino acid, is included.

  In order to create a protein having such “deletion”, “substitution”, “addition”, or a base sequence having “deletion”, “substitution”, “addition” as described above, site-specific Use various methods known in the technical field of the present invention, such as mutagenesis (Site Directed Mutagenesis), random mutagenesis using mutagen treatment and PCR amplification mistakes (Random Mutagenesis), and cassette mutagenesis (Cassette Mutagenesis). Can do.

  As described above, a variant of a protein having the MAPKKK activity consisting of the amino acid sequence shown in SEQ ID NO: 2 includes a DNA consisting of the nucleotide sequence shown in SEQ ID NO: 1 and a complementary DNA. And a protein having MAPKKK activity, which consists of an amino acid sequence encoded by DNA that can hybridize under stringent conditions.

  In the present invention, 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 relationship therewith. It refers to conditions that allow specific hybridization with a specific base sequence. Hybridization conditions are determined in consideration of conditions such as temperature and ion concentration. In general, 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 by those skilled in the art based on, for example, the description of Sambrook and Russel (Molecular Cloning: A Laboratory Manual, 3rd edition (2001)). As specific examples of these stringent conditions, it may be possible to use hybridization conditions such as 6 × SSC, 5 × Denhardt's, 0.1% SDS, 25 ° C. to 68 ° C., for example. In this case, the hybridization temperature is more preferably 45 ° C. to 68 ° C. (without formamide) or 25 ° C. to 50 ° C. (50% formamide).

  As mentioned above, variants of the protein having MAPKKK activity of the present invention include at least 60%, more preferably at least 70%, more preferably at least 80% between the amino acid sequence shown in SEQ ID NO: 2. , Most preferably a protein having MAPKKK activity consisting of an amino acid sequence having at least 90% amino acid sequence homology. A variant of DNA encoding a protein having MAPKKK activity of the present invention may also have at least 60%, more preferably at least 70%, more preferably at least 80% between 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.

  Furthermore, in the present invention, when cDNA derived from a mouse was examined based on the sequence of the protein described in SEQ ID NO: 2 or the DNA sequence described in SEQ ID NO: 1, which encodes the protein, SEQ ID NO: 3 DNA having the nucleotide sequence described in 1 was obtained, and the DNA encoded a protein consisting of the amino acid sequence described in SEQ ID NO: 4, and this protein was also found to have MAPKKK activity.

Based on this finding, in another aspect of the present invention,
(A) the amino acid sequence described in SEQ ID NO: 4; or (b) the amino acid sequence having one or more amino acid deletions, substitutions, or additions in the sequence described in SEQ ID NO: 4. , A protein having MAPKKK activity;
(C) an amino acid sequence encoded by DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 3; or (d) DNA consisting of a nucleotide sequence complementary to the DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 3. A protein having a MAPKKK activity consisting of: an amino acid sequence encoded by a DNA encoding a protein having a MAPKKK activity;
Consisting of an amino acid sequence having 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 shown in SEQ ID NO: 4. A protein having MAPKKK activity;
Can be provided.

The present invention further relates to a novel MAPKKK of this mouse: (a) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 4; or (b) one or more of the sequences set forth in SEQ ID NO: 4 A DNA encoding a protein having a deletion, substitution, or addition of one amino acid and having MAPKKK activity;
(C) DNA comprising the nucleotide sequence set forth in SEQ ID NO: 3; or (d) DNA comprising a nucleotide sequence complementary to the nucleotide sequence set forth in SEQ ID NO: 3 under stringent conditions. A DNA that hybridizes and encodes a protein having MAPKKK activity; or
DNA comprising a nucleotide sequence having at least 60%, more preferably at least 70%, more preferably at least 80%, most preferably at least 90% nucleotide sequence homology with the nucleotide sequence represented by SEQ ID NO: 3. And a DNA encoding a protein having MAPKKK activity;
Can also be provided.

Analysis whereas the kinase domain motifs, which in ASK1 and ASK2 of reported human and mouse up, serine / The structure of threonine kinase region has been preserved, also in the new MAPKKK, ASK3 of the present invention the serine The serine / threonine kinase region of human ASK3 is 86.9% at the amino acid sequence level and 70.9% at the nucleotide sequence level compared to the serine / threonine kinase region of human ASK1. Compared with the serine / threonine kinase region of human ASK2, it was revealed that the sequence homology was 74.9% at the amino acid sequence level and 64.7% at the nucleotide sequence level.

  Furthermore, using a known antibody that recognizes the 845th threonine residue, which is the phosphorylation site of mouse ASK1, and recognizes the phosphorylation state of mouse ASK1, which site the phosphorylation site of ASK3 exists in Further detailed analysis revealed that the kinase activity of human ASK3 is inhibited by changing the 808th threonine residue or 812th threonine residue of human ASK3 to another amino acid such as alanine, It can be seen that the phosphorylation site of human ASK3 exists at the 808th threonine residue, and that the 812th threonine residue plays an important role in phosphorylation of the 808th threonine residue. became.

Mechanism of action of ASK3 The mechanism of action of the protein ASK3 of the present invention was clarified. 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 MAPKKK activity. As a result of a detailed examination of this action, the protein ASK3 of the present invention has a relationship of mutual activation by binding to ASK1 and phosphorylating each other, and MKK3 /, a MAPKK that functions in the p38 signaling pathway. It was found that phosphorylating and activating MKK4 / MKK7, which is a MAPKK that functions in the MKK6 and JNK signaling pathways, ultimately activates JNK / p38.

On the other hand, the activation mechanism of the protein ASK3 of the present invention was also examined in detail. As a result, the protein ASK3 of the present invention is activated by hydrogen peroxide (H ₂ O ₂ ) stimulation and also activated (ie, phosphorylated) by calcium stimulation. These activation mechanisms are similar to those of ASK1 with similar sequences. However, the activation state of the protein ASK3 of the present invention varies greatly in accordance with changes in osmotic pressure, apart from these activation mechanisms. Specifically, ASK3 is activated (phosphorylated) on the low osmotic pressure side, and on the contrary, ASK3 is inactivated (dephosphorylated) on the high osmotic pressure side, with normal osmotic pressure in the living body. Has characteristics.

From these facts, ASK3 is activated (phosphorylated) or inactivated (dephosphorylated) by hydrogen peroxide (H ₂ O ₂ ) stimulation, calcium stimulation and / or osmotic pressure stimulation, and its activity (phosphorus). By regulating the phosphorylation state of MKK3 / MKK6, a MAPKK that functions in the p38 signaling pathway, and MKK4 / MKK7, a MAPKK that functions in the JNK signaling pathway, depending on the (oxidation) state, through activation / inactivation, Eventually it is shown to regulate the activity of JNK / p38.

Vector and transformant In the present invention, a vector capable of replicating in a host cell comprising a gene having a nucleotide sequence according to the present invention (for example, SEQ ID NO: 1) in a state capable of expressing a protein encoded by the sequence. Can be provided. The present invention also provides host cells transformed with such vectors. The host-vector system may be any host-vector system that can be used in the art, and is not particularly limited.

  As the vector, any vector conventionally used in the art may be used. For example, a plasmid vector (for example, an expression vector in prokaryotic cells, yeast, insect cell animal cells, etc.), viral vector Examples (eg, retrovirus vectors, adenovirus vectors, adeno-associated virus vectors, herpes virus vectors, Sendai virus vectors, HIV vectors, vaccinia virus vectors), liposome vectors (eg, cationic liposome vectors) and the like can be mentioned as examples. .

  As the vector construction procedure and method according to the present invention, those conventionally used in the field of genetic engineering can be used. For example, Sambrook and Russel (Molecular Cloning: A Laboratory Manual, 3rd edition) (2001)) can be used.

  As a host cell to be transformed, a host cell compatible with the above-described vector is used. For example, examples of host cells include Escherichia 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.

  By culturing a host cell transformed with the prepared vector in an appropriate medium, it is possible to obtain the protein according to the present invention from the culture and study signal transduction.

Antibody In the present invention, based on the results of these sequence analyses, against a peptide region characteristic of a human protein (SEQ ID NO: 2) or mouse protein (SEQ ID NO: 4) having MAPKKK activity Antibodies were made. That is, in one embodiment of the present invention, an antibody that binds to the protein shown in SEQ ID NO: 2 or SEQ ID NO: 4 can be provided. In this embodiment, the obtained antibody is preferably characterized in that it does not bind to human-derived MAPKKK, ASK1, and ASK2, more preferably, a human-derived or mouse-derived protein having MAPKKK activity is excluded. All are characterized by not binding to MAPKKK, ASK1 and ASK2 from species (including humans).

  The antibody according to the present invention can be obtained by using Delves (Antibody Production: Essential Techniques (1997)), Howard and Bethell (Basic Methods) using the protein of SEQ ID NO: 2 or SEQ ID NO: 4 or a fragment thereof prepared as described above. in Antibody Production and Characterization (2000)), Kontermann and Dubel (Antibody Engineering: Springer Lab Manual (2001)) and the like.

  In order to prepare an antibody that reacts with the protein of SEQ ID NO: 2 or SEQ ID NO: 4 but does not react with known MAPKKK, ASK1 and ASK2 of other species, the result of sequence analysis is SEQ ID NO: 2 Alternatively, a polypeptide consisting of an amino acid sequence characteristically present in SEQ ID NO: 4 but not present in the amino acid sequences of other species ASK1 and ASK2 is preferably used as an immunogen.

  The antibodies of the present invention may be obtained as polyclonal antibodies, monoclonal antibodies or other types of antibodies.

  Polyclonal antibodies can be obtained from sera collected by immunizing a suitable immunized animal with the protein of SEQ ID NO: 2 or SEQ ID NO: 4, or a fragment thereof. Rabbits, sheep, goats, guinea pigs, mice and the like are generally used as immunized animals, but are not limited thereto.

  A monoclonal antibody is obtained by recovering 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 cell-fusioning it with an appropriate fusion partner such as myeloma cells. By obtaining a hybridoma cell, culturing and cloning a clone producing an antibody having the necessary activity in a medium suitable for growth, and culturing the cloned hybridoma cell under a suitable condition, whereby the culture supernatant is obtained. Can be obtained from. Furthermore, antibodies can also be produced by growing the hybridoma cells thus obtained in the peritoneal cavity of mammals. As the immunized animal, for example, a mouse, a nude mouse, a rat, or a chicken is preferable. The antibody thus obtained can be purified using general isolation and purification methods such as centrifugation, dialysis, salting out with ammonium sulfate, ion exchange chromatography, gel filtration, affinity chromatography and the like. .

  Other types of antibodies that can be used in the present invention include chimeric antibodies composed of variable regions derived from non-human antibodies and constant regions derived from human antibodies, or CDRs (complementarity determining regions) derived from non-human antibodies and humans. An antibody-derived FR (framework region) and a humanized antibody consisting of a constant region can also be used.

  In the present invention, the polypeptide region used for producing an antibody specific for ASK3 may be any part as long as it is not similar to other proteins (for example, ASK1 and ASK2) as a result of sequence alignment. For example, from an amino acid polypeptide consisting of amino acids 197 to 214 of SEQ ID NO: 2 (also referred to as CES peptide; SEQ ID NO: 26) or amino acids 913 to 928 of SEQ ID NO: 2. The 16 amino acid polypeptide (also called 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 F (ab ′) ₂ fragment or Fab ′ fragment by a known method. When the antibody of the present invention is used for detection of a protein having DNA repair promoting activity, a radioisotope (eg, ³⁵ S or ³ H), an enzyme (eg, horseradish peroxidase), or an appropriate affinity It can be labeled with a ligand (eg, avidin-biotin).

Pharmaceutical composition Protein ASK3 of the present invention has MAPKKK activity and is activated 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 By doing so, JNK / p38 is activated. It has been known so far that apoptosis is promoted by activating JNK / p38 in cells (Tibbles and Woodgett, Cell. Mol. Life Sci., 55, 1230-1254, 1999).

  Therefore, by regulating the kinase activity of the protein of the present invention, ASK3, apoptosis can be regulated in diseases associated with abnormalities of apoptosis, and ASK3 activity for preventing and treating such diseases Pharmaceutical compositions containing the modulator can be provided. Here, the regulation of kinase activity of ASK3 includes both inhibition of kinase activity of ASK3 and promotion of kinase activity of ASK3, and abnormality of apoptosis refers to both promotion of apoptosis and suppression of apoptosis. Includes cases.

  That is, when ASK3 kinase activity is promoted in the body, MAPKK of JNK signaling pathway and p38 signaling pathway is activated and phosphorylation of JNK / p38 occurs, so that apoptosis is promoted. 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 ASK3 activity for preventing and treating such diseases Pharmaceutical compositions containing the inhibitor can be provided. As such diseases associated with the promotion of apoptosis, neurodegenerative diseases such as polyglutamine disease, Alzheimer's disease, Parkinson's disease, prion disease, and amyotrophic lateral sclerosis are known.

  As an ASK3 activity inhibitor that can be used in the present invention, for example, dominant negative mutants of ASK3, antisense oligonucleotides, double-stranded RNAs, and low molecular weight compounds can be used.

  Conversely, when the kinase activity of ASK3 is inhibited in the body, MAPKK of the JNK signaling pathway and p38 signaling pathway is not activated, and phosphorylation of JNK / p38 does not occur, thereby suppressing apoptosis. From this, 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 ASK3 activity for preventing and treating such diseases A pharmaceutical composition comprising an accelerator can be provided. As such diseases related to suppression of apoptosis, tumors, cancers, autoimmune diseases and the like are known.

  As an 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. In this case, the gene can be introduced by any method commonly used in the art, for example, liposome, adenovirus vector, lipofectin, particle gun, retrovirus vector, etc. it can.

Screening method As described above, ASK3 of the present invention can modify apoptosis in a disease associated with 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, ASK3, is also provided.

Specifically, the present invention is a screening method for a compound that modifies the phosphorylation state of ASK3,
(A) a step of administering a test sample to cells into which the ASK3 gene has been introduced,
The screening method includes (b) a step of detecting the phosphorylation state of ASK3, and (c) a step of selecting a compound that modifies the phosphorylation state of ASK3.

  For example, when using HEK-293 cells or mouse embryonic fibroblasts (MEF cells) introduced with the human ASK3 gene or mouse ASK3 gene of the present invention as cells into which the ASK3 gene has been introduced, these cells are cultured under culture conditions. In contrast, by administering a candidate compound to inhibit or promote ASK3 kinase activity by examining whether ASK3 phosphorylation is modified, more specifically, inhibited or promoted The agent to be screened can be screened.

The modification of ASK3 phosphorylation is a method of actually detecting the phosphorylation state of ASK3 using the above-described antibodies of the present invention, such as Western blotting, immunoblotting, ELISA, etc .; using mass spectrometry Measurement of the change in the mass of ASK3, a method in which cells are metabolically labeled with [ ³² P] H ₃ PO ₄ and detected by autoradiography, and the like.

  In the remainder of the description, examples are given for the purpose of illustrating the invention in more detail. The description of this example is not intended to limit the scope of the invention, but merely to explain the invention in more detail.

Example 1 Cloning of mouse ASK3 (1) Cloning of mouse ASK3 partial sequence As a result of homology search using the base sequence of mouse ASK1 cDNA, two mouse-derived ESTs (Expressed Sequence Tags) were obtained, both on the mouse X chromosome. It turns out that it is mapped to. Furthermore, when the genome sequence (NCBI contig NW # 042641) in the vicinity of the region encoding those ESTs was examined in detail, a base sequence having homology with the C-terminal region of ASK1 was found when converted to an amino acid.

  Based on this information, first, primers were prepared as follows:

In this example, first, cDNA synthesis of mouse fetal fibroblasts (MEF cells) was performed from total RNA of MEF cells using SUPER SCRIPT ^™ Preamplification System (GIBCO BRL). Subsequently, PCR was performed using the cDNA of MEF cells as a template. PCR was performed using Taq DNA polymerase (SIGMA) or LA Taq (Takara), and the iPCRr (BIO-RAD) was used for the following PCR reaction conditions: 1 cycle at 94 ° C for 1 minute. Dissociation reaction for 15 seconds; annealing at 60 ° C. for 30 seconds; and extension reaction at 72 ° C. for 1 minute] This is performed as 35 cycles, and finally the reaction is performed at 72 ° C. for 1 minute. did. Primers include S8 (SEQ ID NO: 7) / AS7 (SEQ ID NO: 10), S2 (SEQ ID NO: 5) / AS2 (SEQ ID NO: 8), S3 (SEQ ID NO: 6) / AS5 Each combination of (SEQ ID NO: 9) was used.

  As a result, 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 the extracted DNA is converted to pGEM-T Easy Vector using pGEM-T Easy Vector System (Promega) Subcloned and transformed DH5α competent cells. 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).

  As a result, the PCR products obtained with the S8 / AS7, S2 / AS2, and S3 / AS5 primer combinations consisted of 193 bp, 1671 bp, and 2041 bp base sequences, respectively. A cDNA sequence up to AS5 (3745 bp; hereinafter referred to as “S8-AS5” fragment) was obtained (FIG. 1).

  Later, a clone "similar to MAPKKK5 (ASK1)" created by automated computational analysis from mouse genome information was published on the database. Compared with the above cDNA (S8-AS5 fragment), this was a sequence containing a longer 3 ′ untranslated region, although it did not contain new information on the 5 ′ side.

  Therefore, we designed and created the sense primer S19 (SEQ ID NO: 11) in the S8-AS5 fragment already obtained, and the antisense primer AS21 (SEQ ID NO: 12) in the predicted sequence. PCR was performed using the cDNA of MEF cells as a template. The nucleotide sequences of both primers are as follows:

  As a result of determining the base sequence of the PCR product using CEQ 2000 (BECKMAN COULTER), it was found to be a cDNA matching the sequence on the database. Based on the above, it was confirmed that the nucleotide sequence published on the mouse genome database was actually expressed as a gene in MEF cells, and cDNA sequence information considered to be almost the full length of the gene product was obtained (S8-AS21). Equivalent). Thereafter, this gene product was named ASK3, and further cloning proceeded.

(2) Cloning of the 5 'end region of mouse ASK3
Since it was thought that the S8-AS21 region did not contain the start codon of mouse ASK3, we decided to attempt cloning of the 5 'end region by RACE. Therefore, using Mouse Kidney Marathon-Ready ^™ cDNA or SMART RACE cDNA Amplification Kit (both Clonetech) as a template, AS10 (SEQ ID NO: 13) for primary PCR and AS14 (SEQ ID NO: 14 for secondary PCR). ) Was used as a primer for 5'-RACE. At that time, Advantage-GC 2 Polymerase Mix (Clonetech) was used as an enzyme. As a result, a RACE product containing the 5 ′ sequence of the existing mouse ASK3 sequence was obtained. The primer sequences used here are as follows:

  Based on the nucleotide sequence determined by RACE, primer S27 was prepared on the 5′-most side of the sequence, and PCR primers were prepared as follows:

  PCR was performed under the same reaction conditions as the PCR conditions described in (1) with a combination of S27 / AS14, S27 / AS19, and S27 / AS22 primers. Extraction of the obtained PCR product was performed with 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 DH5α competent cells were transformed. Plasmid recovery was performed with Wizard Plus Minipreps DNA Purification Systems (Promega). The sequence was first determined by PCR with CEQ Quick Start Kit (Beckman) and then with CEQ 2000 (BECKMAN COULTER). As a result, it was revealed that the obtained PCR product had the sequence as expected from the RACE results (FIG. 1).

  Moreover, since the amino acid sequence predicted from the base sequence was homologous when compared with ASK1, the result of RACE was judged to be appropriate. Next, when 5′-RACE was performed again using AS19 as a primer for primary PCR and AS22 as a primer for secondary PCR based on the sequences obtained here, about 50 bp on the 5 ′ side was newly found ( Figure 1).

  The base sequence obtained by the cloning so far was considered to be the full-length mouse ASK3 (FIG. 1).

Example 2 Cloning of human ASK3 (1) Cloning of human ASK3 partial sequence In the mouse ASK3 sequence described above (FIG. 1), there are a plurality of candidates for the start codon, which is one of the grounds for determining it. Since there was no upstream in-frame stop codon, the initiation codon could not be determined. Since it was considered difficult to obtain more mouse ASK3 nucleotide sequence information with 5'-RACE, in this example, the 5 'terminal region of human ASK3 was also cloned and the human and mouse ASK3 nucleotide sequences were cloned. An attempt was made to determine the start codon from the similarity.

  Using the obtained mouse ASK3 cDNA base sequence (FIG. 1), the human genome sequence was searched, and it was found that the ASK3 gene is also present on the X chromosome in humans. Therefore, based on this sequence, the following PCR primers, S1 and AS1, were prepared:

First, cDNA of human fetal kidney-derived HEK-293 cells was prepared from total RNA of HEK-293 cells using SUPER SCRIPT ^™ Preamplification System (GIBCO BRL). Using this S1 / AS1 primer combination, PCR was performed using cDNA prepared from HEK-293 cells as a template, and the base sequence of the PCR product was determined using CEQ 2000 (BECKMAN COULTER). It was found to be a matched cDNA. Therefore, it was confirmed that human ASK3 is expressed in HEK-293 cells.

  Subsequently, S4, AS3, S6, and AS6 primers were designed and prepared based on such sequences. The nucleotide sequences of these primers are as follows:

  Using these primers in combination with S4 / AS3 and S6 / AS6 primers, PCR was performed under the same conditions as described above, and human ASK3 cDNA sequences from S4 to AS6 were obtained (Figure 2). .

(2) Cloning of the 5 ′ end region of human ASK3 A sequence on the X chromosome similar to the 5 ′ end of mouse ASK3 was found on the human genome by searching using NCBI Human Genomic BLAST. Based on the sequence, primers having the following sequences, HS1 (SEQ ID NO: 24; sense) and G1AS2 (SEQ ID NO: 25; antisense) were prepared:

  Using these primers, PCR was performed using HEK-293 cDNA as a template. The PCR product (515 bp) contained a sequence on the human genome X chromosome, and the amino acid predicted sequence was found to encode a gene product homologous to mouse ASK3 (FIG. 2). Furthermore, an initiation codon and an upstream in-frame termination codon were confirmed on the 5 ′ side, and this PCR product was considered to encode the N-terminus of human ASK3 protein (FIG. 2). In addition, comparing the base sequence and predicted amino acid sequence of mouse ASK3 and human ASK3, the full length of mouse ASK3 has already been cloned, and the 5'-most ATG codon on the cDNA functions as the start codon. It was suggested.

Example 3: Analysis of full-length sequence of the nucleotide sequence of SEQ ID NO: 3 derived from mouse and amino acid sequence of SEQ ID NO: 4 obtained in Example 1 and similar human-derived molecule obtained in Example 2 The nucleotide sequence of SEQ ID NO: 1 and the amino acid sequence of SEQ ID NO: 2 were compared with the previously known nucleotide and amino acid sequences of MAPKKK, human and mouse ASK1 and ASK2. Comparison of nucleotide sequence of DNA and amino acid sequence of protein were both performed using ClustalV program (Higgins and Sharp, CABIOS, Vol. 5, No. 2: pp. 151-153, 1989).

  As a result, at the protein level, the amino acid sequence of SEQ ID NO: 2 of the present invention is 56.5%, 52.9%, 41.7% among the amino acid sequences of human ASK1, mouse ASK1, human ASK2, and mouse ASK2. And 41.3% sequence homology was found. In addition, at the DNA level, the nucleotide sequence of SEQ ID NO: 1 of the present invention is 49.5%, 50.6%, 41.1% between nucleotide sequences encoding human ASK1, mouse ASK1, human ASK2, and mouse ASK2, respectively. % And 41.7% sequence homology.

  From the results of these analyses, it was revealed that the novel protein obtained in the present invention is a more closely related molecular species to ASK1 than ASK2.

  Furthermore, when the amino acid sequence and nucleotide sequence of a novel protein derived from human were compared with the amino acid sequence and nucleotide sequence of a novel protein derived from mouse, respectively, it was found that they had 86.3% and 69.7% sequence homology. became.

  The novel human and mouse proteins consisting of the amino acid sequences of SEQ ID NO: 2 or SEQ ID NO: 4 were then aligned with the amino acid sequences of ASK1 and ASK2 from human and mouse. The results are shown in FIG. As is apparent from FIG. 3, amino acids 650 to 908 corresponding to the central region of the novel human-derived protein (SEQ ID NO: 2) of the present invention are superior to other regions. It became clear that there is a part with 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. This sequence region has already been revealed to be a serine / threonine kinase region in the sequence analysis of ASK1 and ASK2 (Non-patent document 1; Non-patent document 3). The protein consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4 obtained in the invention was named “ASK3”.

Example 4: Preparation of antibody against ASK3 Among SEQ ID NO: 2 obtained in Example 1, an 18 amino acid polypeptide consisting of amino acids 197 to 214 (CES peptide; SEQ ID NO: 26) (this (The sequence corresponds to amino acids 201 to 218 of SEQ ID NO: 4) or a polypeptide of 16 amino acids consisting of amino acids 909 to 924 of SEQ ID NO: 2 (RQV peptide; SEQ ID NO: 27) (This sequence corresponds to amino acids 913 to 928 of SEQ ID NO: 4) as an immunogen at a concentration of 150 μg / ml and administered to rabbits at the same concentration of 300 μg / ml every 14 days. Boosted 3 times with the immunogen, blood was collected 14 days after the final boost, and antiserum was obtained from the blood.

  Subsequently, the IgG class was purified from the antiserum using an affinity column (peptide Sepharose bead column). The antibodies thus obtained are polyclonal antibodies prepared using the polypeptide of SEQ ID NO: 26, respectively, “anti-ASK3 (CES) antibody”, and polyclonal antibodies prepared using the polypeptide of SEQ ID NO: 27. The antibody was named “anti-ASK3 (RQV) 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 Figure 4).

Example 5: Expression analysis of mouse ASK3 In this example, the expression distribution of the protein having the amino acid sequence of SEQ ID NO: 4 obtained in Example 2 was measured at the mRNA level and protein level. Examined.

First, in order to analyze the expression at the mRNA level, a cDNA of about 100 bp encoding a part of the N-terminal region of mouse ASK3 was labeled with [α- ³² P] -dCTP. Using this probe, hybridization was performed with Mouse Multiple Tissue Blot (Clontech). For hybridization, ExpressHyb solution (Clontech) was used, and hybridization and washing conditions were in accordance with the attached protocol. After washing, analysis was performed with a STORM imaging system 840 (Amersham).

  The results are shown in FIG. 4A. As is apparent from this figure, it was found that the mouse ASK3 mRNA is expressed in various tissues in the mouse, but the mRNA is particularly expressed in the heart and kidney.

  Subsequently, in order to examine the production of ASK3 at the protein level, homogenized in 1% Triton X-100 / 10 mM Tris-HCl (pH 7.5) solution from kidney and liver extracted from mice, Samples were prepared. The expression of endogenous ASK3 protein and endogenous p38 MAP kinase protein in this disrupted extract sample was compared with the anti-ASK3 (RQV) antibody prepared in Example 4 using the RQV peptide (SEQ ID NO: 27) as an immunogen, and Analysis was performed using Western blotting using a commercially available anti-p38 antibody (C-20; Santa Cruz) (FIG. 4B). Furthermore, endogenous ASK3 molecules were immunoprecipitated from a crushing extract sample prepared from the kidney using an anti-ASK3 (CES) antibody and concentrated before being subjected to the experiment. At that time, an anti-ASK3 (CES) antibody preincubated with a CES peptide (SEQ ID NO: 26) was used as a negative control for immunoprecipitation. Thereafter, immunoprecipitation samples were analyzed by Western blotting using anti-ASK3 (RQV) antibody (FIG. 4C). In FIG. 4B and FIG. 4C, “WB: ASK3 (RQV)” or “WB: p38 (C-20G)” means an anti-ASK3 (RQV) antibody and an 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 this specification.

  As a result of these Western blots, it was confirmed that the endogenous ASK3 protein having the molecular weight (about 150 kDa) predicted from the amino acid sequence of SEQ ID NO: 4 was expressed at least in the kidney. In addition, the expression of ASK3 protein in the kidney was higher than that in the liver, and a correlation was found between the expression level of mRNA in each tissue shown in FIG. 4A.

Example 6: Analysis of kinase activity of ASK3 In this example, whether ASK3 has the kinase activity of human and mouse ASK3 in vitro and specifically the JNK and p38 signaling pathways It was investigated whether or not it was activated.

  Using wild-type ASK3 (Flag-ASK3-WT) and kinase-inactive mutant ASK3 (Flag-ASK3-KM) with a Flag tag using pcDNA3 expression vector (Invitrogen), using FuGENE6 transfection reagent (Roche) Then, the gene was introduced into HEK-293 cells derived from human fetal kidney. Recognize ASK3 expression with an anti-Flag antibody (M2 antibody; SIGMA) and an activated kinase molecule using a cell extract obtained from cells 24 hours after culturing using DMEM medium containing 10% FBS Anti-phosphorylated-JNK antibody (phosphorylated-SAPK / JNK (Thr183 / Tyr185) rabbit polyclonal antibody), anti-phosphorylated-p38 antibody (phosphorylated-p38 MAPK (Thr180 / Tyr182) rabbit polyclonal antibody), and anti-phosphorylated ERK Endogenous JNK in HEK-293 cells using three anti-phosphorylation-recognizing antibodies (both Cell Signaling) of antibodies (phosphorylated-p44 / 42 MAPK (T202 / Y204) (E10) mouse mAb), p38 , ERK activation (phosphorylation) was detected. The expression level of ERK was detected by Western blotting using an anti-ERK antibody (p44 / 42 MAP kinase antibody; Cell Signaling). ASK1-WT (wild type ASK1) was used as a positive control for activating JNK and p38, and epidermal growth factor EGF stimulation was used as a positive control for activating ERK.

  The results are shown in FIG. 5A. In cells overexpressing wild-type ASK3 (ASK3-WT), JNK and p38 MAP were compared to cells not expressing or expressing mutant ASK3 (ASK3-KM) without kinase activity. Kinase activation was observed. On the other hand, activation of ERK was not recognized. From this result, it was confirmed that ASK3 specifically activates the JNK and p38 MAP kinase pathways depending on the kinase activity.

  Subsequently, one of Flag-MKK3, Flag-MKK6, Flag-MKK4, Flag-MKK7 and ASK3 (HA-ASK3) to which an HA tag is attached are incorporated into a pcDNA3 expression vector, and FuGENE6 transfection reagent (Roche) is added. The gene was introduced into HEK-293 cells and expressed simultaneously. Each MKK molecule was immunoprecipitated using an anti-Flag antibody (M2 antibody; SIGMA) from a cell extract obtained from cells 24 hours after introduction cultured in DMEM medium containing 10% FBS. Using this immunoprecipitated sample, a GST-p38-KN (kinase inactive mutant p38) fusion protein (purified from E. coli BL21) was used as a substrate for MKK3 and MKK6 (Figure 5B), and for MKK4 and MKK7. In vitro kinase analysis was performed using GST-JNK-KN (inactive mutant JNK) fusion protein (purified from E. coli BL21) as a substrate (FIG. 5C). ASK1 was used as a positive control to activate all MKK molecules.

  Although ASK3 hardly activated MKK7 in the cells (FIG. 4C), it activated MKK3, MKK4, and MKK6 to the same extent as ASK1 (FIGS. 4B and 4C). Thus, ASK3 appears to function as a MAPKKK in the JNK and p38 MAP kinase pathways.

  Information about phosphorylation sites in ASK1 and ASK2, more specifically, the region called the activation loop that exists between subdomains VII and VIII of the kinase region is very important for the activity control as a kinase, more specifically Shows that the threonine residues 845 and 849 of mouse ASK1 (Thr845 and Thr849) are necessary for maintaining the kinase activity of ASK1, in particular, Thr845 is a phosphorylation site necessary for activation by stimulation of ASK1, Based on the fact that an antibody that specifically recognizes phosphorylation at this site (anti-phosphorylated ASK antibody) can monitor the activation of ASK1 sensitively, this example further controls the phosphorylation activity of ASK3. An attempt was made to identify active sites.

  First, the sequence alignment (Figure 3) confirmed that the amino acid residues corresponding to Thr845 / Thr849 of ASK1, Thr808 / Thr812, and their surrounding amino acid sequences were also highly conserved in human ASK3. . Therefore, it was confirmed whether or not this portion was actually phosphorylated (FIG. 6A).

  In HEK-293 cells, wild type ASK3 (Flag-ASK3 WT) to which a Flag tag was added, and mutant ASK3 in which 808 and 812 threonine residues of human ASK3 were substituted with alanine residues (T808A, respectively) T812A is expressed transiently, anti-phosphorylated ASK antibody (Toblume et al., J. Cell. Physiol., 191, 1091-1099, 2002), anti-Flag antibody (M2 antibody; SIGMA), anti- ASK3 and downstream by Western blotting using phosphorylated JNK antibody (phosphorylated-SAPK / JNK (Thr183 / Tyr185) antibody, Cell Signaling), anti-JNK antibody (JNK1 (FL), Santa Cruz, CA, USA) The activation state and expression level of each JNK were detected.

  Wild-type ASK3 activates JNK, whereas two types of Thr808 and Thr812 alanine substitution mutants (T808A, T812A) have almost completely failed to activate JNK, so Thr808 and Thr812 Is essential for maintaining the kinase activity of ASK3, and comparison of the amino acid sequence with ASK1 (see Figure 6A) shows that Thr808 is a phosphorylation site required for activation by stimulation of human ASK3. It was suggested. Further, since it was confirmed that the anti-phosphorylated ASK antibody recognizes WT-ASK3 and does not recognize T808A, it was considered that this antibody can monitor the activation of ASK3 in the same manner as ASK1.

Example 7: Analysis of the interaction of ASK3 with other molecules In this example, in order to clarify the mechanism of action of ASK3 and to activate the JNK / p38 signaling pathway, We tried to analyze the interaction with molecules.

  Anti-ASK3 (CES) antibody was added to the cell extract prepared from HEK-293 cells to immunoprecipitate endogenous ASK3. At that time, an anti-ASK3 (CES) antibody preincubated with CES peptide as an antigen peptide was used as a negative control for immunoprecipitation. Specifically, immunoprecipitation samples were separated by SDS polyacrylamide electrophoresis, transferred to PVDF membrane, and endogenous by Western blotting using anti-ASK3 (RQV) antibody and anti-ASK1 antibody (F9; Santa Cruz). ASK3 and endogenous ASK1 were detected.

  As a result, it was clarified that endogenous ASK1 was present in a sample obtained by immunoprecipitation of a cell extract of HEK-293 cells using an anti-ASK3 (CES) antibody (FIG. 7). Therefore, it was shown that ASK3 forms a complex with ASK1 in the intracellular steady state.

  Furthermore, the interaction between ASK3 and ASK1 in cells was confirmed. First, the wild-type ASK3 with Myc tag (6xMyc-ASK3-WT) and the kinase-inactive mutant ASK1 with HA tag (HA-ASK1-KM) were incorporated into the pcDNA3 expression vector and FuGENE6 transfection reagent (Roche) was used for gene transfer into HEK-293 cells and expression at the same time. On the other hand, 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 with FuGENE6 transfection reagent. In the same manner, the gene was introduced and expressed simultaneously. Using cell extracts obtained from both cells 24 hours after culturing using 10% FBS-containing DMEM medium, the activation state of ASK3 and ASK1 with anti-phosphorylated ASK antibody, anti-Myc antibody and anti-HA antibody Respectively, the expression levels of ASK3 and ASK1 were detected by Western blotting.

  Compared with cells expressing ASK1-KM alone, cells co-expressing ASK1-KM and ASK3-WT showed an increase in the degree of phosphorylation of ASK1-KM depending on the expression level of ASK3-WT. (FIG. 8A). On the other hand, compared to cells expressing ASK3-KM alone, cells co-expressing ASK3-KM and ASK1-WT increased the degree of phosphorylation of ASK3-KM depending on the expression level of ASK1-WT. Was observed (FIG. 8B). From these results, it was shown that ASK3 phosphorylates Thr845 of ASK1 and ASK1 phosphorylates Thr808 of ASK3, thereby activating each other.

Example 8: Analysis of activation mechanism of ASK3 In this example, in order to examine the mechanism by which ASK3 is activated, hydrogen peroxide which is already known as the activation mechanism of ASK1 is used. It was examined whether ASK3 was activated by (H ₂ O ₂ ) stimulation and calcium stimulation.

Flag-ASK3 incorporated into the pcDNA3 expression vector was introduced into HEK-293 cells using FuGENE6 transfection reagent (Roche). Twenty-four hours after introduction, 1 mM H ₂ O ₂ and 1 mM mitoxin (MTX) were used for stimulation for the times shown in FIGS. 9A and 9B, respectively, and then cell extract samples were prepared. In an experiment in which H ₂ O ₂ stimulation was performed (FIG. 9A), Flag-ASK1 was also used as a control.

  After protein separation by SDS polyacrylamide electrophoresis, it was transferred to a PVDF membrane, ASK1 and ASK3 active state was detected with anti-phosphorylated ASK antibody, and ASK1 and ASK3 expression was detected with anti-Flag antibody (M2 antibody), respectively. It detected using. In order to confirm that the target stimulus is applied to the cells, the activation state of endogenous p38 MAP kinase was also determined using anti-p38 recognition antibody (c-20; Santa Cruz) and anti-phosphorylated p38 antibody (phosphorylated-p38). MAP kinase (Thr180 / Tyr182) antibody; Cell Signaling) was similarly detected.

An increase in the degree of activation of ASK3 by stimulation with H ₂ O ₂ was observed from 10 minutes after stimulation, and continued activation until 30 minutes after stimulation, but a rapid decrease in the degree of activation was observed after 60 minutes (FIG. 9A). On the other hand, an increase in the activation level of ASK1 by stimulation with H ₂ O ₂ was also observed from 10 minutes after stimulation, but the activation level was maintained continuously even after 120 minutes without decreasing ( Figure 9A). Therefore, ASK3 is activated by stimulation with hydrogen peroxide (H ₂ O ₂ ), and the activation is considered to be more transient than ASK1.

  By injecting calcium into the cell from the outside by administration of MTX (mitoxin), an increase in the degree of activation of ASK3 was observed from 2 minutes after the stimulation, and the activation was sustained until 10 minutes. Therefore, it can be seen that ASK3 is also activated by calcium stimulation.

Example 9: Action of ASK3 on apoptosis In this example, the activity of ASK3 on apoptosis was examined based on the past finding that ASK1 has an action of promoting apoptosis.

First, HEK-293 cells (2 × 10 ⁵ cells / ml) were transfected with wild-type ASK3 (Flag-ASK3) or kinase-inactive mutant ASK3 (Flag-ASK3-KM) incorporated into pcDNA3 expression vector, FuGENE6 Each gene was introduced using a reagent (Roche) and expressed. 10 hours after introduction, cells (7 × 10 ⁴ cells / ml) are seeded in a 96-well plate for assay. After 20 hours, caspase 3/7 activity (DEVDase activity) is expressed in the Caspase-Glo 3/7 assay kit (Promega). Quantified by

  In cells overexpressing ASK3-WT, a significant increase in caspase3 / 7 activity was observed compared to cells not expressing and cells expressing mutant ASK3-KM having no activity (FIG. 10). ). From this result, it is shown that ASK3 has an activity of causing caspase activation in a kinase activity-dependent manner and inducing apoptosis.

Example 10: Effect of ASK3 on cytokine production In this example, the activity of ASK3 on cytokine production was examined.

HEK-293 cells (2 × 10 ⁵ cells / ml) with ASK3-WT (wild-type ASK3) and ASK3-KM (inactive mutant ASK3) with 5′-end Flag-tag using pcDNA3 expression vector Each gene was introduced with FuGENE6 transfection reagent (Roche Diagnostics). The concentration of TNF-α in the culture supernatant of the transformed cells 24 hours after introduction was quantified using a human TNF-α ELISA kit (TECHNE). The expression level and activation of ASK3 were analyzed using Western blotting.

  More specifically, the above-described disrupted extract sample of transformed cells 24 hours after introduction is prepared, separated by SDS polyacrylamide electrophoresis, transferred to a PVDF membrane, and anti-Flag antibody (M2 antibody; SIGMA) And ASK3 expression were detected, and activation of ASK3 was detected with an anti-phosphorylated ASK recognition antibody that recognizes activated ASK molecules. Similarly, 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. Similarly, activation of endogenous p38 MAP kinase downstream of ASK3 was detected with an anti-phosphorylated p38 recognition antibody (Cell Signaling) that recognizes an activated p38 molecule, and expression of p38 with an anti-p38 antibody (Santa Cruz) The amount was also confirmed.

  In the cells overexpressing ASK3-WT, an increase in the amount of TNF-α produced was observed as compared with cells not expressing and cells expressing mutant ASK3-KM having no activity (FIG. 11). Up). At this time, the expression levels of ASK3 were similar in both ASK3-WT and ASK3-KM, and it was confirmed that the activation of ASK3 itself and its downstream endogenous p38 was actually caused only by ASK3-WT expression ( Figure 11 bottom). From this result, it was shown that ASK3 has an activity to induce cytokine production.

Example 11: Response of ASK3 to osmotic pressure stimulation (1)
In this example, it is known that ASK1 is phosphorylated and activated by receiving hyperosmotic stimulation, so what kind of response does ASK3 of the present invention respond to hyperosmotic stimulation? Confirmed what to do.

  Wild-type ASK3 (Flag-ASK3-WT) and wild-type ASK1 (Flag-ASK1-WT) with Flag tag incorporated into pcDNA3 expression vector are transferred to HEK-293 cells using FuGENE6 transfection reagent (Roche) Introduced and expressed. The cells 24 hours after introduction were subjected to high osmotic pressure stimulation with 0.5 M sorbitol for the time shown in FIG. 12, and then a cell extract was prepared. About this cell extract, the activation state of ASK3 and ASK1 was detected using an anti-phosphorylated ASK antibody, and the expression levels of ASK3 and ASK1 were detected using an anti-Flag antibody, respectively, by Western blotting. In addition, activation of endogenous JNK and p38 was detected with an anti-phosphorylation recognition antibody (Cell Signaling) that recognizes an activated kinase molecule.

  In cells expressing ASK1-WT, ASK1 activation was observed by high osmotic stimulation with 0.5 M sorbitol, whereas in cells expressing ASK3-WT, ASK3 inactivation was induced by hyperosmotic stimulation. It was revealed that ASK1 and ASK3 showed a completely contrasting response to hyperosmotic stimulation (FIG. 12).

Example 12: Response of ASK3 to osmotic stimulation (2)
Next, in order to clarify in more detail the response of ASK3 of the present invention to osmotic pressure stimulation, the response to low osmotic pressure stimulation was compared with the above-described response to high osmotic pressure stimulation.

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 ₂ O buffer; 130 mM NaCl, 2 mM KCl, 1 mM) for 1, 2, 5, 10, 30, 60, and 120 minutes on cells 24 hours after transfection Hyperosmotic stimulation and hypotonic buffer (205 mOsm / kg H with KH ₂ PO ₄ , 2 mM CaCl ₂ , 2 mM MgCl ₂ , 10 mM Na-Hepes pH 7.3, 10 mM glucose, and 220 mM mannitol) ₂ O buffer; 90 mM NaCl, 2 mM KCl, 1 mM KH ₂ PO ₄ , 2 mM CaCl ₂ , 2 mM MgCl ₂ , 10 mM Na-Hepes pH 7.3, and 10 mM glucose) After performing, a cell extract was prepared. As a control, an isosmotic buffer (300 mOsm / kg H ₂ O buffer; 130 mM NaCl, 2 mM KCl, 1 mM KH ₂ PO ₄ , 2 mM CaCl ₂ , 2 mM MgCl ₂ , 10 mM Na-Hepes pH 7.3, 10 mM glucose and 20 mM mannitol) were used for isoosmotic stimulation, and a cell extract was prepared. For this cell extract, after separating the protein by SDS polyacrylamide electrophoresis, it is transferred to a PVDF membrane, the activation state of ASK3 using anti-phosphorylated ASK antibody, and the expression level of ASK3 using anti-Flag antibody, respectively. Were detected by Western blotting, respectively.

  The results are shown in FIG. In this figure, in each figure of high osmotic pressure stimulation and low osmotic pressure stimulation, the upper row shows the activation state of ASK3 examined using anti-phosphorylated ASK antibody, and the lower row shows ASK3 examined using anti-Flag antibody. The expression levels of are shown respectively.

ASK3 was phosphorylated to some extent in the isosmotic state (300 mOs / kg H ₂ O), indicating that it has phosphorylation activity. On the other hand, when hyperosmotic stimulation was given, ASK3 was dephosphorylated after 2 minutes and a decrease in activity was observed. The decrease in activity lasted up to 120 minutes. Conversely, when low osmotic pressure stimulation was applied, ASK3 showed an increase in the degree of activation (phosphorylation) after 2 minutes, and continued activation until 30 minutes. Thereafter, phosphorylation activity decreased gradually (FIG. 13).

  Therefore, ASK3 is controlled by the osmotic pressure of the extracellular solution, activated by low osmotic pressure, and inactivated by high osmotic pressure. I understood that I have it.

Example 13: Response of ASK3 to osmotic stimulation (3)
In this example, the reactivity of ASK3 activity to the osmotic pressure of various extracellular solutions was examined for the purpose of examining the relationship between ASK3 activity and osmotic pressure in more detail.

The extracellular solution was made from 100 mM to 190 mM NaCl set at 10 mM intervals, as well as 4 mM KCl, 2 mM KH ₂ PO ₄ , 4 mM CaCl ₂ , 4 mM MgCl ₂ , 20 mM Na-HEPES pH 7.3 And a solution having an osmotic pressure of about 250 to 430 mOs / kg H ₂ O containing 20 mM glucose and 40 mM mannitol. The osmotic pressure of the solution was measured with an osmotic pressure measuring device. The specific relationship between the NaCl concentration and the osmotic pressure of the solution is as shown in the following table.

  The osmotic pressure stimulation was performed for 20 minutes. The other experimental conditions were the same as those described in Example 12, the activation state of ASK3 using an anti-phosphorylated ASK antibody, and the expression level of ASK3 using an anti-Flag antibody, respectively. Were detected by Western blotting, respectively.

  The results are shown in FIG. In this figure, in the Western blot diagram (FIG. 14A), the upper row shows the activation state of ASK3 examined using the anti-phosphorylated ASK antibody, and the lower row shows the expression level of ASK3 examined using the anti-Flag antibody, Each is shown. The actual osmotic pressure measured by an osmotic pressure measuring instrument showed 310 mOsm, which is considered to be almost isotonic 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 ( FIG. 14B). As a result, ASK3 showed a phosphorylation amount about three times the isosmotic pressure around 250 mOsm (100 mM NaCl), and was almost completely dephosphorylated around 350 mOsm (150 mM NaCl).

  From this, it was found that ASK3 changes the activity between osmotic pressure and ± 50 mOsm with respect to osmotic pressure, and reacts most sensitively to changes near the osmotic 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 / p38 signaling pathway and suppressing or promoting apoptosis in diseases associated with the promotion or suppression of 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.

Claims (12)

(A) the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; or (b) the deletion of one or more amino acids in the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4. A novel MAP kinase kinase kinase (MAPKKK) consisting of; (C) an amino acid sequence encoded by DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3, or (d) from the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3. A novel MAPKKK comprising: an amino acid sequence encoded by a DNA comprising a nucleotide sequence complementary to the obtained DNA and a DNA that hybridizes under stringent conditions and encodes a protein having MAPKKK activity; (A) DNA encoding a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4; or (b) in the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 4, DNA comprising any one of: a DNA having a plurality of amino acid deletions, substitutions or additions and encoding a protein having MAPKKK activity. (C) DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3; or (d) complementary to DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3. A DNA comprising any one of: a DNA that hybridizes with a DNA comprising a nucleotide sequence under stringent conditions and encodes a protein having MAPKKK activity. An antibody that binds to the protein shown in SEQ ID NO: 2 or SEQ ID NO: 4. 8. The antibody according to claim 7, which does not bind to human-derived MAPKKK, ASK1, and ASK2. Includes ASK3 activity modulators for the prevention and treatment of diseases associated with abnormalities of apoptosis by regulating the kinase activity of ASK3 having the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 4. Pharmaceutical composition. 8. The pharmaceutical composition according to claim 7, wherein the ASK3 activity modulator inhibits ASK3 kinase activity and, as a result, prevents or treats a disease associated with promotion of apoptosis. 9. The pharmaceutical composition according to claim 8, wherein the ASK3 activity modulator is selected from the group consisting of a dominant negative mutant of ASK3, an antisense oligonucleotide, a double-stranded RNA, and a low molecular weight compound. 8. The pharmaceutical composition according to claim 7, wherein the ASK3 activity modulator promotes ASK3 kinase activity and, as a result, prevents or treats a disease associated with inhibition of apoptosis. 11. The pharmaceutical composition according to claim 10, wherein the ASK3 activity promoter is selected from the group consisting of ASK3 overexpression by gene transfer and a low molecular weight compound. A screening method for a compound that modifies the phosphorylation state of ASK3,
(A) a step of administering a test sample to cells into which the ASK3 gene has been introduced,
(B) detecting the phosphorylation state of ASK3, and (c) selecting a compound that modifies the phosphorylation state of ASK3.