JP2003135070A - TAK1-TAB1 fusion protein - Google Patents

Abstract

(57) [Problem] To provide a constitutively activated mutant TAK1. Also, a novel TAK1 utilizing the mutant TAK1
Methods for assaying, identifying and screening for inhibitors are provided. The present invention includes a partial polypeptide containing a region responsible for the protein kinase activity of TAK1 (TGF-β activated kinase 1) and a region responsible for the “function of activating TAK1” of TAB1 (TAK1 binding protein 1). A partial polypeptide is fused with a linker peptide if necessary, and a constitutively active mutant T
TAK1 and TAB1 that function as AK1
And fusion proteins. Also, a nucleic acid encoding the fusion protein. Also, a recombinant vector and a host cell containing the same. In addition, a method for assaying the effect of a test compound on the activity or function of TAK1 using the fusion protein or cells expressing the fusion protein.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an active form of TAK1.
It relates to a fusion protein of TAK1 and TAB1 (TAK1 binding protein 1) which functions as (TGF-β activated kinase 1). In addition, T using the fusion protein
The present invention relates to a method for assaying AK1 inhibitory action.

[0002]

PRIOR ART TGF-β activated kinase 1
(Transforming growth factor-β-activated kinase
1; also referred to as TAK1) is a mammalian MAPKKK
(Mitogen-activated protein kinase kinase kinase)
It was found as one of the following (Yamaguchi et al., Sci
ence, Vol. 270, pp. 2008-2011, 1995; JP-A-9-
163990). TAK1 is TGF-β (transformi
ng growth factor-β) regulates PAI-1
Activate the promoter. Further, since it is activated by TGF-β as the origin of its name, it has been considered that it acts in the intracellular transduction pathway of signals by members of the TGF-β superfamily.

TAK1 is a TAK1 binding protein 1
(TAK1 binding protein 1; also referred to as TAB1) becomes active by binding (interacting) with TAK1
Is known to function as MAPKKK in the signal transduction pathway in which P. alba is involved (Shibuya et al., Science,
272, pp. 1179-1182, 1996).

Furthermore, according to recent findings, the complex of TAK1 and TAB1 not only acts on the intracellular transduction pathway of TGF-β signaling, but also closely affects the pathological conditions of autoimmune diseases and inflammatory diseases. It has been clarified that it also acts on the signal transduction pathway involved in saccharomyces (JP 2000-
197500; Sakurai et al., Biochem. Biophys. Res. Co.
mmun., Vol. 243, pp. 545-549, 1998; Sakurai
FEBS Letters, 474, 141-145, 2000).

From such a background, the active mutant of TAK1 is used for the functional analysis of TAK1, the elucidation of the onset mechanism of autoimmune diseases and inflammatory diseases in which TAK1 is thought to be involved, and the screening of TAK1 inhibitors. And was considered to be useful for identification and the like.

[0006] In addition, TAK1 inhibitors can be used for elucidating the pathogenic mechanism of autoimmune diseases and inflammatory diseases, and are also expected as therapeutic agents for autoimmune diseases and inflammatory diseases. The assay system for screening or identifying the inhibitor of TAK1 includes TAK1 and TAB1.
It was necessary to make both proteins coexist, and the assay was complicated and the assay results were difficult to stabilize.
It was believed that more efficient assays would be possible by using active variants of AK1.

[0007] Previous reports (Shibuya et al., Science, No. 1)
272, pp. 1179-1182, 1996).
Mutant TAK1 in which 22 amino acid residues on the N-terminal side of Escherichia coli have been deleted
Was said to function as the active form even in the absence of TAB1. However, according to the study by the present inventors, the presence of TAB1 was essential for activation of TAK1 in which 22 amino acid residues on the N-terminal side were deleted (Sakurai et al., FEBSLetters, Vol. 474, 141-145,
the year of 2000).

[0008]

The object of the present invention is to provide a constitutively activated mutant TAK1.
Another object of the present invention is to provide a novel TAK1 inhibitor assay method, identification method and screening method using the mutant TAK1.

[0009]

The present inventors have found that human TA
K1 cDNA and human TAB1 cDNA were isolated, and the N-terminal partial polypeptide of TAK1 and TAB1 were isolated.
As a result of rigorous studies on the properties of such a fusion protein by ligating the C-terminal partial polypeptide of Escherichia coli, it was found that such a fusion protein functions as a constitutively active mutant TAK1 and completed the present invention. It was

That is, the present invention relates to TAK1 (TGF-
partial polypeptide containing a region responsible for the protein kinase activity of β-activated kinase 1), and TAB1
(TAK1-binding protein 1), a partial polypeptide containing a region responsible for the “function of activating TAK1”, is fused via a linker peptide, if necessary, and is a constitutively active mutant TAK1. Which is a fusion protein of TAK1 and TAB1.

A nucleic acid encoding the above fusion protein. Also, a recombinant vector and a host cell containing the same.

Further, it is a method of assaying the effect of a test compound on the activity or function of TAK1 using the fusion protein or cells expressing the fusion protein.

SEQ ID NO: 1 in the sequence listing below is human TAK1
Represents the nucleotide sequence of the cDNA of SEQ ID NO: 2, and SEQ ID NO: 2 is human TAK.
1 represents the protein amino acid sequence of 1. SEQ ID NO: 3 is human T
It represents the nucleotide sequence of the cDNA of AB1, and SEQ ID NO: 4 represents the protein amino acid sequence of human TAB1. SEQ ID NO: 5 is T
This shows an example of the nucleotide sequence of a cDNA encoding a partial polypeptide containing a region responsible for the protein kinase activity of AK1, and SEQ ID NO: 6 shows an example of the amino acid sequence of the partial polypeptide. SEQ ID NO: 7 represents an example of a nucleotide sequence of a cDNA encoding a partial polypeptide containing a region responsible for the “function of activating TAK1” of TAB1, SEQ ID NO: 8
Represents an example of the amino acid sequence of the partial polypeptide. SEQ ID NO: 9 represents an example of a nucleotide sequence of a cDNA encoding a linker peptide, and SEQ ID NO: 10 represents an example of an amino acid sequence of the linker peptide.

[0014]

BEST MODE FOR CARRYING OUT THE INVENTION TAK1 is intracellularly associated with TAB1.
It is activated by interacting (binding) with (TAK1-binding protein 1), and protein kinase activity (MAP
KKK activity), the T-type in the present invention
The fusion protein of AK1 and TAB1 is constitutively active.

A fusion protein of TAK1 and TAB1 of the present invention (hereinafter referred to as TAK1-TAB1 fusion protein)
Is a partial polypeptide (1) containing a region responsible for the protein kinase activity of TAK1 and a partial polypeptide (2) containing a region responsible for "the function of activating TAK1" of TAB1. ) Through the fusion.

In the present invention, TAK serving as a source of each partial polypeptide constituting the TAK1-TAB1 fusion protein
1 and TAB1 may be derived from any species, and examples thereof include mammals such as human, mouse, rat, rabbit, pig, dog, monkey and guinea pig. Of these, human-derived ones are preferably used for use in research and development of human therapeutic agents.

The cDNA and amino acid sequences of TAK1 are already known ((mouse) Genbank / EMBL database Accession No. D76446; Yamaguchi et al., Scie.
nce, 270, 2008-2011, 1995; (human) Genb
ank / EMBL Database Accession No.AB009356, No.AB
009357, No.AB009358, No.AF218074; Sakurai et al., Bio
Chem. Biophys. Res. Commun., Volume 243, Volume 545-549.
P., 1998; Dempsey et al., Biochem. Biophys. Acta, 15
17: 46-52, 2000). The region responsible for the protein kinase activity of TAK1 (protein kinase region) is presumed to exist on the N-terminal side (within the region corresponding to the amino acid residues from the 34th position to the 291st position) (Yamaguchi et al., Science, Volume 270, 2008-2
011 p. 1995).

In the fusion protein of the present invention, the structure of the partial polypeptide containing the region responsible for the protein kinase activity of TAK1 (protein kinase region) can be designed based on the above information.

The partial polypeptide containing the protein kinase region of TAK1 may have the same protein kinase activity as TAK1 (more specifically, MAPKKK activity).

Examples of such partial polypeptides include those consisting of the amino acid sequence shown in SEQ ID NO: 6. Further, the amino acid sequence represented by SEQ ID NO: 6 includes an amino acid sequence in which one or more amino acids are deleted, substituted or added. Amino acid deletions, substitutions or additions may be carried out to the extent that protein kinase activity (more specifically, MAPKKK activity) is not lost, and usually 1 to about 60, preferably 1
To about 30, more preferably 1 to about 15. That is, as such a partial polypeptide, in addition to the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6, one or more conservative amino acid substitutions (cons
Polypeptides having ervative amino acid substitutions).

Such partial polypeptides include partial polypeptides of mutant proteins found in nature, artificially modified partial polypeptides, partial polypeptides derived from different organisms, and the like. That is, such a partial polypeptide includes a conservative substitution variant of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6.
stitution variants) and naturally occurring allelic variants (natu
rally occuring allelic variants) are included.

Such a partial polypeptide has a homology of about 80% or more, preferably about 90% or more, more preferably about 95% or more with the amino acid sequence represented by SEQ ID NO: 6 at the amino acid level.

More specifically, such partial polypeptides (partial polypeptides containing the protein kinase active region of TAK1) include, for example, the following (A) to (E):
The following are listed. (A) A polypeptide having the amino acid sequence represented by SEQ ID NO: 6. (B) 1 in the amino acid sequence shown by SEQ ID NO: 6
Alternatively, a polypeptide consisting of an amino acid sequence in which a plurality of amino acids are deleted, substituted or added. (C) A polypeptide having one or more conservative amino acid substitutions as compared with the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6. (D) A polypeptide having an amino acid level homology of about 90% or more with the amino acid sequence represented by SEQ ID NO: 6. (E) A polypeptide encoded by a nucleic acid capable of hybridizing with a nucleic acid having the nucleotide sequence represented by SEQ ID NO: 5 under stringent conditions or a complement thereof.

Further, such a partial polypeptide (TAK1
The nucleic acid (DNA or RNA) encoding the partial polypeptide containing the protein kinase active region of
An example thereof includes a nucleic acid having the nucleotide sequence represented by SEQ ID NO: 5. In addition, a nucleic acid or a complement thereof (that is, a nucleic acid having a complementary sequence) capable of hybridizing with a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 5 under stringent conditions (more preferably under high stringent conditions) Some include. In such a hybridizable nucleic acid, the polypeptide encoded by the nucleic acid has a protein kinase activity (more specifically, MAPKKK).
As long as it has activity).

Such a nucleic acid has a homology of usually about 70% or more, preferably about 80% or more, more preferably about 90% or more with the base sequence shown in SEQ ID NO: 5. Such nucleic acids include nucleic acids corresponding to mutant genes found in nature, artificially modified nucleic acids, nucleic acids derived from different organisms (orthologs), and the like.

More specifically, examples of the nucleic acid encoding such a partial polypeptide (partial polypeptide containing the protein kinase active region of TAK1) include the following (a) and (b). (A) A nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 5. (B) A nucleic acid which hybridizes with a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 5 under stringent conditions or its complement.

The cDNA and amino acid sequences of TAB1 have also been reported ((human) Genbank / EMBL database Accession No. U49928, No. E14752; Shibuya.
Et al., Science, Vol. 272, pp. 1179-1182, 1996; JP-A-10-004976).

The "region responsible for the function of activating TAK1" of TAB1 (TAK1 activating region) is the C
It is presumed to exist at the terminal side (region corresponding to amino acid residues 436 to 504) (Shib
uya et al., Science, 272, 1179-1182, 1996;
Sakurai et al., FEBS Letters, 474, 141-145, 2
000 years).

In the fusion protein of the present invention, the structure of the partial polypeptide containing the "region responsible for the function of activating TAK1" of TAB1 (TAK1 activating region) can be designed based on the above information. TAB1 TAK1
The partial polypeptide containing the activation region may be one having a function of activating TAK1.

Examples of such partial polypeptides include those consisting of the amino acid sequence shown in SEQ ID NO: 8. In addition, the amino acid sequence represented by SEQ ID NO: 8 includes an amino acid sequence in which one or more amino acids are deleted, substituted or added. Amino acid deletions, substitutions or additions may be carried out to the extent that the TAK1 activating ability is not lost, and are usually 1 to about 15, preferably 1 to about 7, and more preferably 1 to about 4. That is, as such a partial polypeptide, one or more conservative amino acid substitutions (conservative) compared to the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8 and the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8. Polypeptides with amino acid substitutions).

Such partial polypeptides include partial polypeptides of mutant proteins found in nature, artificially modified partial polypeptides, partial polypeptides derived from different organisms, and the like. That is, such a partial polypeptide includes a conservative substitution variant of the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8.
stitution variants) and naturally occurring allelic variants (natu
rally occuring allelic variants) are included.

Such a partial polypeptide has a homology of about 80% or more, preferably about 90% or more, more preferably about 95% or more with the amino acid sequence represented by SEQ ID NO: 8 at the amino acid level.

More specifically, examples of such partial polypeptides (partial polypeptides containing the TAK1 activation region of TAB1) include the following (A) to (E). (A) A polypeptide having the amino acid sequence shown by SEQ ID NO: 8. (B) 1 in the amino acid sequence shown in SEQ ID NO: 8
Alternatively, a polypeptide consisting of an amino acid sequence in which a plurality of amino acids are deleted, substituted or added. (C) A polypeptide having one or more conservative amino acid substitutions as compared with the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8. (D) A polypeptide having an amino acid sequence homology of about 90% or more with the amino acid sequence represented by SEQ ID NO: 8. (E) A polypeptide encoded by a nucleic acid capable of hybridizing with a nucleic acid having the nucleotide sequence represented by SEQ ID NO: 7 under stringent conditions or a complement thereof.

Further, such a partial polypeptide (TAB1
No. 7 as a nucleic acid (DNA or RNA) encoding a partial polypeptide containing the TAK1 activation region of
Examples include those containing a nucleic acid having the base sequence shown in. In addition, a nucleic acid or a complement thereof (that is, a nucleic acid having a complementary sequence) capable of hybridizing with a nucleic acid comprising the nucleotide sequence represented by SEQ ID NO: 7 under stringent conditions (more preferably under high stringent conditions) Some include. In such a hybridizable nucleic acid, the polypeptide encoded therein is TAK1.
Any substance having an activation ability may be used.

Such a nucleic acid has a homology of usually about 70% or more, preferably about 80% or more, more preferably about 90% or more with the base sequence shown in SEQ ID NO: 7. Such nucleic acids include nucleic acids corresponding to mutant genes found in nature, artificially modified nucleic acids, nucleic acids derived from different organisms (orthologs), and the like.

More specifically, a nucleic acid encoding such a partial polypeptide (a partial polypeptide containing the TAK1 activation region of TAB1) is, for example, the following (c).
And (d). (C) A nucleic acid having the nucleotide sequence represented by SEQ ID NO: 7. (D) A nucleic acid that hybridizes with a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 7 under stringent conditions or a complement thereof.

In the TAK1-TAB1 fusion protein of the present invention, a partial polypeptide (1) containing a region responsible for the protein kinase activity of TAK1 and "TAK1 of TAB1.
The partial polypeptide (2) containing a region responsible for the function of activating "may be in the form of direct binding,
A form in which a linker peptide is interposed between the two may be used. Such a linker peptide preferably has an amino acid sequence rich in glycine residues having a high degree of structural freedom. The length of the linker peptide portion is not particularly limited, but it may be appropriately designed usually with a length of about 5 to 30 amino acid residues. Specific examples of such a linker peptide sequence include the following (A) to (C). (A) A peptide consisting of the amino acid sequence shown by SEQ ID NO: 10. (B) in the amino acid sequence shown by SEQ ID NO: 10,
A polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added. (C) About 90% of the amino acid sequence represented by SEQ ID NO: 10
A polypeptide having the above amino acid-level homology.

In the TAC1-TAB1 fusion protein of the present invention, a partial polypeptide (1) containing a region responsible for the protein kinase activity of TAC1 and "TAK1" of TAB1.
The positional relationship with the partial polypeptide (2) containing a region having a function of activating
By designing (2) to be present on the C-terminal side of (1), a fusion protein that can function properly can be obtained.

In the present invention, hybridization under stringent conditions is usually carried out in a hybridization solution having a salt concentration of 6 × SSC or a salt equivalent thereto under the temperature condition of 50 to 60 ° C. for about 16 hours. And 6 × SSC or a solution having a salt concentration equivalent to 6 × SSC, if necessary, and then prewashed, and then 1 ×
It can be carried out by washing in SSC or a solution having a salt concentration equivalent thereto. Further, under conditions having higher stringency (high stringent conditions), the washing can be carried out by performing the washing in 0.1 × SSC or a solution having a salt concentration equivalent thereto.

A nucleic acid encoding the fusion protein of the present invention is
A nucleic acid encoding a partial polypeptide containing the protein kinase active region of TAK1 and a nucleic acid encoding a partial polypeptide containing the TAK1 activating region of TAB1
If necessary, along with the nucleic acid encoding the linker peptide,
It can be obtained by sequentially connecting by a usual gene recombination technique.

The nucleic acid encoding the linker peptide is D
It can be obtained by chemical synthesis using an NA synthesizer or the like.

"Nucleic acid encoding a partial polypeptide containing a region responsible for TAK1 protein kinase activity" and "nucleic acid encoding a partial polypeptide containing a TAK1 activating region of TAB1" are both known sequence information or the present specification. Design primers and probes based on the sequence information disclosed in the Sequence Listing of
lymerase chain reaction) method, colony hybridization method, and plaque hybridization method can be selected and obtained by appropriately combining them.

For example, cDNA is synthesized from mRNA prepared from mammalian cells or tissues, and this is used as a template.
TAK1 and TAB1 cDNAs are obtained by the PCR method. It is also possible to obtain a full-length cDNA by screening a cDNA library by a colony hybridization method or a plaque hybridization method using the cDNA obtained by the PCR method as a probe. The desired partial polypeptide can be obtained by further performing PCR using the full-length cDNA as a template. Examples of mammals used as gene sources include non-human animals such as dogs, cows, horses, goats, sheep, monkeys, pigs, rabbits, rats and mice, as well as humans. Of these, it is desirable to use human-derived cells or tissues as a gene source for the construction of fusion proteins used for screening and identification of TAK1 inhibitors that can be therapeutic agents for humans.

The cDNA library is, for example, "Mo
"Regular Cloning" (Sambrook, J., Fr
itsch, EF and Maniatis, T., Cold Spring Harbo
r Laboratory Press, published in 1989). Alternatively, if a commercially available library is available, this may be used.

By determining the base sequence of the obtained cDNA, it can be confirmed that the base sequence encodes the target partial polypeptide.

The fusion protein of the present invention or a partial polypeptide thereof can be produced by overexpression by a conventional gene recombination technique. Also,
It can also be expressed and produced in a form in which a tag peptide (6xHis, hemagglutinin antigen peptide, Xpress antigen peptide, etc.) is added.

A cell in which a fusion protein or a partial polypeptide thereof is overexpressed can be obtained, for example, as follows. First, a DNA encoding a protein or polypeptide is inserted into a vector so as to be linked downstream of an appropriate promoter to construct an expression vector. Then, the obtained expression vector is introduced into a host cell.

Examples of the expression system (host-vector system) include expression systems for bacteria, yeast, insect cells and mammalian cells. Among these, in order to obtain a protein whose function is well conserved, insect cells (Spodoptera frugiperd
a SF9, SF21, etc.) and mammalian cells (monkey COS-7)
Cells, Chinese hamster CHO cells, human HeL
It is preferable to use a cell) as a host.

As a promoter for expressing a protein or polypeptide, in the case of a mammalian cell system, S is used.
In the case of an insect cell system such as V40 promoter, LTR promoter, and elongation 1α promoter, polyhedrin promoter and the like can be used.

As the vector, a retrovirus type vector, papilloma virus vector, vaccinia virus vector, SV40 type vector or the like can be used in the case of mammalian cell system, and a baculovirus vector or the like can be used in the case of insect cell type.

D encoding a protein or polypeptide
NA is a cDNA corresponding to mRNA that exists in nature.
Can be used, but is not limited thereto. It is also possible to design and use a DNA corresponding to the amino acid sequence of the target protein. In this case, 1 to 6 types of codons encoding one amino acid are known, and the selection of the codon to be used may be arbitrary. However, for example, considering the frequency of codon usage of the host used for expression, the expression efficiency can be further improved. Higher sequences can be designed. D with designed nucleotide sequence
NA can be obtained by chemical synthesis of DNA, fragmentation and binding of the above-mentioned cDNA, partial modification of the nucleotide sequence, and the like. The artificial partial modification and mutation of the nucleotide sequence is carried out by a PCR method using a primer composed of a synthetic oligonucleotide encoding a desired modification or a site-specific mutagenesis method (site specifi).
c mutagenesis) (Mark et al., Proceedings of National A
cademy of Sciences, Vol. 81, pages 5662-5666, 1984
Years) etc.

By partially modifying or introducing mutations in the artificial base sequence, desired addition / deletion / substitution of the amino acid sequence of the corresponding polypeptide can be introduced.

The fusion protein of the present invention, its partial polypeptide and the like can be purified from a culture of cells into which an expression vector has been introduced by a known purification method (salting out with an inorganic salt, fractional precipitation with an organic solvent, an ion exchange resin column). Chromatography, affinity column chromatography, gel filtration method, etc.) can be appropriately combined for separation and purification.

The fusion protein of the present invention (or TAK1
The partial polypeptide that is presumed to contain the protein kinase domain of Escherichia coli has a protein kinase activity similar to that of TAK1 (more specifically, MAPKKK activity), for example, MKK6 (MAPKK6) (Raingeaud
Et al., Molecular and Cellular Biology, Vol. 16, 1247.
~ 1255, 1996; Moriguchi et al., Journal of Biolog.
ical Chemistry, Vol 271, 13675-13679, 1996
), Human MKK3 (Derijard et al., Science, vol. 267,
682-685, 1995), etc. MAPKK (mitogen acti)
vated protein kinase kinase) as a substrate protein and is used in the literature (Sakurai et al., J. Biol. Chem., Vol. 274, Vol. 106.
41-10648, 1999), the kinase reaction is carried out in a solution containing ATP (which is labeled with RI if necessary), and the activity is measured using the phosphorylation of the substrate protein as an index. It can be confirmed by doing.

The fact that the partial polypeptide containing the TAK1 activation region of TAB1 has the TAK1 activating ability is described in the literature (Sakurai et al., FEBS Letters, Volume 474, 141-145).
Page, 2000) and can be confirmed, for example, as follows. That is, the protein kinase activity (MAPKKK activity) of TAK1 is measured under the condition that the partial polypeptide coexists with TAK1.
It is compared with the protein kinase activity of TAK1 in the absence of the partial polypeptide. In the presence of the partial polypeptide, TAK1 protein kinase activity (MA
If the PKKK activity) is increased, the partial polypeptide is judged to have TAK1 activating ability.

Since the fusion protein of the present invention functions as a constitutively active mutant TAK1, the inhibitory effect of a test compound on TAK1 can be suitably assayed using this. Such an assay is
It can be suitably used for identifying or screening a compound having a K1 inhibitory action.

Examples of such an assay include those including the following steps (i), (ii) and (iii). (I) contacting the fusion protein of the present invention with a test compound,
Measuring the protein kinase activity of the fusion protein in the presence of a test compound; (ii) the protein kinase of the fusion protein in the absence of the test compound, or in the presence to a lesser extent than in (i) A step of measuring the activity; and (iii) comparing the activity measurement results in step (i) and step (ii), and based on the result, the test compound
A step of determining whether or not it has an action of inhibiting the activity of AK1, or the degree thereof.

Alternatively, the effect of the test compound on the function of TAK1 is assayed by using the test cells expressing the fusion protein of the present invention and culturing the cells in the presence or absence of the test substance. You can also TAK
The effect of test compounds on the function of 1 can also be assayed. Such an assay includes, for example, an assay including the following steps (i), (ii) and (iii). (I) a step of bringing test cells expressing the fusion protein of the present invention into contact with a test compound, and detecting activation of a signal transduction system in the test cells in the presence of the test compound; (ii) test compound In the absence of, or in the presence of to a lesser extent than in (i); and (iii) detecting in steps (i) and (ii) The results are compared, and based on the results, the test compound is TAK
A step of determining whether or not it has an action of inhibiting the activity of 1. or its degree. The "activation of the signal transduction system" detected here may be the activation of the signal transduction system known to be involved in TAK1. Specific examples of the activation of such a signal transduction system include JNK (c-Ju
n N-terminal kinase), activation of MAPK such as p38, and activation of IKK (IkB kinase). Alternatively, it includes induction of production of inflammatory cytokines such as IL-6, IL-1 and TNF.

The activation of MAPKs such as JNK and p38 and the activation of IKK are described in the literature (Sakurai et al., J. Biol. Chem., No. 274) using changes in MAPK activity and IKK activity as indicators.
Vol. 10641-10648, 1999).

Induction of the production of inflammatory cytokines such as IL-6, IL-1, TNF, etc. is described in the literature (Tsuji et al., Immunol. Let) using the amount of cytokine secreted in the medium as an index.
t., 68, 275-279, 1999). The inflammatory cytokine can be quantified by an immunochemical quantification method, a bioassay, or the like.

As cells to be used in the test, cell lines derived from mammals such as humans can be preferably used. For example, human H
Examples include eLa cells, human Jurkat cells, human THP-1 cells, monkey COS-7 cells, Chinese hamster CHO cells, among which human HeLa cells and human Ju cells.
Rkat cells, human THP-1 cells and the like are preferable.

Regarding the test substance which has been found to have an inhibitory effect on the activity or function of TAK1 by the above-mentioned assay method, it is further tested in a known pathological model (in vitro or in vivo) of an autoimmune disease or a refractory disease exhibiting inflammatory symptoms. The therapeutic and / or preventive effect may be confirmed.

As a known pathological model (in vitro or in vivo) of an autoimmune disease or a refractory disease exhibiting inflammatory symptoms, a PHA-induced IL-2 production model using a human T cell line (Jurkat cells) (Wacholtz et al., Cell Immunology,
135, 285-298, 1991), LPS + IFN-γ-induced iNOs production model using human macrophage cell line RAW264.7 (Xie et al., Science, 256, 2).
25-228, 1992) and TN using human HeLa cells.
In vitro model such as F-α-induced IL-6 production model, rat adjuvant arthritis model (Connor et al., European J
ournal of Pharmacology, Volume 273, pp. 15-24, 1995
, Trinitrobenzenesulfonic acid-induced colitis model (Kiss et al., European Journal of Pharmacology, No. 336).
Vol., Pp. 219-224, 1997) and rat model of Masugi nephritis (Sakurai et al., Biochimica Biophysica Acta, vol. 1316,
132-138, 1996) and the like in vivo models.

Hereinafter, the present invention will be described in more detail with reference to examples, but these examples do not limit the present invention.

In the following examples, each operation is described as "Molecular cloning" unless otherwise specified.
lar Cloning) "(Sambrook, J., Fritsch, EF and Ma
niatis, T., Cold Spring Harbor Laboratory Press
Published in 1989), or when using commercially available reagents and kits, they were used according to the instructions for the commercially available products.

[0066]

Example 1 Preparation of cDNA encoding fusion protein of TAK1 and TAB1 (1) Preparation of cDNA of human TAK1 gene Reference (JP 2000-197500; Sakurai et al., Biochem. Biophy
s. Res. Commun. 243, 545-549, 1998; Gen
The cDNA of human TAK1 (TAK1a) was obtained as follows with reference to the sequence information and the method described in the bank / EMBL database Accession No. AB009356). That is, the human uterine cervical cancer-derived cell line HeLa (ATCC C
Poly (A) RNA was prepared from CL2), and single-stranded cDNA was prepared using this as a template. Obtained single-stranded cDNA
PCR (polymerase chain reaction) using A as a template
Thus, a cDNA containing the entire translation region of human TAK1 (TAK1a) was obtained. The nucleotide sequence of the cDNA containing the full-length TAK1 translation region (hTAK1a-cDNA) and the protein amino acid sequence of TAK1 (TAK1a) encoded by the cDNA are shown in SEQ ID NOS: 1 and 2 of the Sequence Listing below.

(2) Preparation of cDNA of human TAB1 gene Reference (Shibuya et al., Science, Vol. 272, pp. 1179-1182,
1996; Genbank / EMBL database Accession No.U4
9928), the cDNA of human TAB1 was obtained as follows with reference to the sequence information and method described in 9928). That is, poly (A) RNA was prepared from a human uterine cervical cancer-derived cell line HeLa (ATCC CCL2), and this was used as a template for RT.
-PCR (reverse transcript-polymerase chain rea
ction), c containing the entire translation region of human TAB1
DNA was obtained. The nucleotide sequence of cDNA containing the entire TAB1 translation region and TAB1 encoded by the nucleotide sequence
The amino acid sequence of the protein is shown in SEQ ID NOs: 3 and 4 in the sequence listing below.

(3) Preparation of cDNA encoding the protein kinase region of TAK1 Human TAK1 consists of 579 amino acids, but human TAK
Regarding the region responsible for the protein kinase activity (protein kinase region) of 1, it was considered that a kinase domain exists at the N-terminal side from the homology search with other protein kinases. Therefore, the protein kinase region of human TAK1 is located on the N-terminal side (1st to 30th).
(In the partial polypeptide consisting of the third amino acid residue)
Is assumed to exist in the
The A fragment was obtained as follows.

Using the cDNA fragment containing the full-length translation region of human TAK1 (human TAK1a) obtained in (1) above as a template, PCR was performed to analyze the first to N-terminal side of human TAK1.
C that encodes a portion consisting of the 303rd amino acid residue
A DNA fragment was obtained. Among the primers used for PCR, a 30-mer primer (SEQ ID NO: 13) consisting of a sequence containing a restriction enzyme BamHI recognition site, a translation initiation codon of TAK1 cDNA and a sequence downstream thereof was used as a sense primer. As the antisense primer, a 30-mer primer (SEQ ID NO: 14) consisting of a sequence containing a restriction enzyme EcoRI recognition site, a codon corresponding to the 303rd amino acid residue of TAK1 and a sequence complementary to the upstream sequence thereof was used. Using.

The obtained PCR product was digested with the restriction enzyme BamHI.
And digested with EcoRI to obtain a cDNA fragment encoding a partial polypeptide containing the protein kinase protein kinase region of TAK1.

(4) Preparation of cDNA encoding the TAC1 activation region of TAB1 Human TAB1 consists of 504 amino acids,
A region of B1 that has a function of activating TAK1 (T
The following is known about the AK1 activation region). That is, yeast or human cultured cells HeL
From the analysis using a, 68 amino acids at the C-terminal side of TAB1 are necessary and sufficient for binding to TAK1 and activation of TAK1, and therefore, the TAK1 activation domain is 68 amino acids at the C-terminal side. (Reference: Shibuya
Et al., Science, Volume 272, pp. 1179-1182, 1996; Sakur
ai et al., FEBS Letters, Volume 474, Pages 141-145, 2000.
Year).

From the above, it is presumed that the TAK1 activation region of human TAB1 is present on the C-terminal side (in the partial polypeptide consisting of the 437th to 504th amino acid residues), and that portion A cDNA fragment coding for was obtained as follows.

Using the cDNA fragment containing the full-length translated region of human TAB1 obtained in (2) above as a template, the C-terminal side of human TAB1 (the portion consisting of the 437th to 504th amino acid residues) was encoded by PCR. Was obtained. Among the primers used for PCR, as a sense primer, a 44-mer primer (sequence consisting of a sequence containing a restriction enzyme EcoRI recognition site, a linker sequence, a codon corresponding to the 437th amino acid residue of TAB1 and a sequence downstream thereof (sequence) No. 15) was used.
(The sequence of this linker portion was designed to encode a glycine-rich amino acid with a high degree of structural freedom.)
As the antisense primer, the restriction enzyme Sa
A 30-mer primer (SEQ ID NO: 16) consisting of a sequence containing the 11 recognition site, the C-terminal stop codon of TAB1 and a sequence complementary to the upstream sequence thereof was used.

The obtained PCR product was digested with the restriction enzyme EcoRI.
And cleaved with SalI to obtain a cDNA fragment encoding a partial polypeptide containing the linker portion and the TAK1 activation region of TAB1.

(5) Preparation of cDNA encoding fusion protein of human TAK1 and TAB1 and recombinant plasmid containing the same cDNA encoding partial peptide containing protein kinase region of human TAK1 obtained in the above (3) and The linker portion obtained in (4) above and TA of human TAB1
CDNA encoding a partial peptide containing the K1 activation region
A and the vector pBluescriptII-SK
(+) (Stratagene) was inserted into the BamHI-SalI site. Thus, a fusion protein containing the protein kinase region of human TAK1 and the TAK1 activation region of TAB1 (hereinafter referred to as TAK1-TAB1 fusion protein)
A cDNA coding for and a recombinant plasmid containing the cDNA (referred to as pBS / TAK1-TAB1) were obtained. In addition, this was cleaved with restriction enzymes BamHI and SalI to obtain cDNA encoding TAK1-TAB1 fusion protein.
A fragment was obtained.

A schematic diagram of this TAK1-TAB1 fusion protein is shown in FIG. The cDNA base sequence of TAK1-TAB1 fusion protein (whole) (including restriction enzyme recognition sites attached upstream and downstream) is shown in SEQ ID NO: 11 and the amino acid sequence is shown in SEQ ID NO: 12. In addition, these sequences are shown together in FIG. Also, this TAK1-TAB
The cDNA nucleotide sequence of the portion containing the protein kinase region derived from TAK1 in 1 fusion protein is shown in SEQ ID NO: 5,
The amino acid sequence is shown in SEQ ID NO: 6. Also, TAK1-
The cDNA base sequence of the portion containing the TAB1 activation region derived from TAB1 in the TAB1 fusion protein is shown in SEQ ID NO: 7.
The amino acid sequence is shown in SEQ ID NO: 8. Also, TAK
C corresponding to the linker portion in the 1-TAB1 fusion protein
The DNA base sequence is shown in SEQ ID NO: 9 and the amino acid sequence is shown in 10. This linker portion has a glycine-rich amino acid sequence having a high degree of structural freedom.

(6) Preparation of vector for expressing TAK1-TAB1 fusion protein cDNA fragment encoding TAK1-TAB1 fusion protein (BamHI-SalI fragment obtained in (5) above)
Was inserted into the BamHI-XhoI site of the vector plasmid pcDNA3.1-His (manufactured by Invirogen). Here, the vector plasmid pcDNA3.1-
His (manufactured by Invirogen) is a vector for expressing a protein having 6 × His and Xpress tag peptides added to the N-terminus as tag peptides in mammalian cells. Thus, TAK1-TA with 6xHis and Xpress tag peptides added to the N-terminus
An expression vector for expressing the B1 fusion protein was obtained.

Further, a cDNA fragment encoding the TAK1-TAB1 fusion protein was ligated to the vector plasmid pcDN.
It was inserted into the BamHI-XhoI site of A3.1-HA. Here, the vector plasmid pcDNA3.1-H
A is a vector for expressing a protein having a hemagglutinin antigen added to the N-terminus in mammalian cells,
The pcDNA3.1-His nucleotide sequence numbers 923 to 1012 are removed by HindIII-KpnI treatment, and DN encoding a hemagglutinin antigen tag is further removed.
It was obtained by incorporating a base sequence containing A (SEQ ID NO: 17). Thus, an expression vector for expressing the TAK1-TAB1 fusion protein having a hemagglutinin antigen added to the N-terminus was obtained.

A cDNA fragment encoding a partial polypeptide containing the protein kinase region of human TAK1 (BamHI-EcoRI fragment obtained in (3) above)
Is the Ba of the vector plasmid pcDNA3.1-HA.
By inserting into the mHI-EcoRI site, an expression vector for expressing the TAK1 protein kinase region having a hemagglutinin antigen added to the N-terminus was obtained.

In addition, a cDNA fragment encoding a partial polypeptide containing the TAK1 activation region of human TAB1 (EcoRI-SalI fragment obtained in the above (4)) was added to p
EGFP-C1 (manufactured by Clontech) was inserted into the multi-cloning site, and EGFP (Enhanced g
reen fluorescence protein) and a linker sequence were added to obtain an expression vector for expressing the TAK1 activation region of TAB1. (Where vector plasmid p
EGFP-C1 is a vector for expressing a protein in which EGFP protein is fused at the N-terminus in mammalian cells. ) Example 2 MAP of fusion protein of TAK1 and TAB1
Detection of KKK activity (1) Transfection Various expression vectors such as the vector for expressing TAK1-TAB1 fusion protein obtained in (6) of Example 1 above were transfected into human uterine cancer-derived cultured cells HeLa as follows. (Transient transfection; transien
TAK1-TAB1 to which a tag peptide (hemagglutinin antigen, etc.) has been added by transfection
A cell overexpressing the fusion protein and the like was obtained. (As a control, cells and the like were obtained in which the partial polypeptide containing the protein kinase region of TAK1 to which the tag peptide was added and / or the partial polypeptide containing the TAK1 activation region of TAB1 to which the tag peptide was added were overexpressed. ) First, about 1 × 10 ⁶ HeLa cells were placed in a 6 cm diameter culture dish (IW
AKI Co., Ltd.) and cultivated for 24 hours and used for transfection. The HeLa cells were cultured in Dulbecco-Eagle medium (Gibco) containing high glucose supplemented with 10% fetal bovine serum at 37 ° C. under 5% CO ₂ . Transfection is carried out using cationic liposomes for transfection (trade name: LipofectAMINE, Life
The technology was used as follows. That is, 1 mg of each plasmid DNA was added to 100 ml of medium (OPTI
-MEM-I medium; made by Life Technologies). Also, add 15 ml of LipofectAMINE reagent to 100 ml of medium (OPTI
-MEM-I medium). Both solutions were mixed and allowed to stand at room temperature for 30 minutes. 2 ml of medium (OPTI-MEM-I medium) was added and mixed by pipetting to obtain a transfection solution. This transfection solution was directly added to the cells previously washed with 2 ml of medium (OPTI-MEM-I medium). After culturing for 5 hours, the transfection solution was removed and the medium was replaced with a culture medium (high glucose-containing Dulbecco-Eagle medium supplemented with 10% fetal bovine serum), and the culture was further continued.

(2) Preparation of cell extract About 24 hours after the transfection carried out as described in (1) above, the cells were collected and the cell lysate containing the overexpressed protein was prepared as follows. (Cell lysate) was prepared. First, the medium was removed, and the cells were diluted with 1 ml of ice-cooled physiological phosphate buffer (PBS; phosphate buffered salin).
Washed in e). Directly add 0.5 ml of whole cell lysis buffer (25 mM HEP) to the cells on the culture dish.
ES-NaOH (pH7.7), 0.3 M NaCl, 1.5 mM MgCl ₂ , 0.2 mM
EDTA, 0.1% Triton X-100, 1 mM DTT, 20 mM b-glycero
phosphate, 1 mM sodium orthovanadate, 0.5 mM PMSF,
10 μg / ml aprotinin, 10 μg / ml leupeptin) was added. The cells were collected in an Eppendorf tube with a cell scraper (Sumitomo Bakelite Co., Ltd.), vigorously stirred for 10 seconds, and then centrifuged (15000 rpm, 10 minutes, 4 ° C.), and the obtained supernatant was used as a cell extract. The obtained cell extract was-
It was stored at 80 ° C and melted before use.

(3) Western blotting and immunoprecipitation In a cell extract of a protein (TAK1-TAB1 fusion protein with a tag peptide added) overexpressed by Western blotting using the cell extract obtained in (2) above. Confirmed the existence of.

Western blotting was performed as follows. That is, the cell extract or immunoprecipitated fraction is SD
After separation by S-PAGE, nylon membrane (Immobilon-P, M
(manufactured by illipore). The membrane was blocked overnight using Block Ace (Dainippon Pharmaceutical Co., Ltd.).
After washing with PBS + 0.1% Tween-20, 1-2 at room temperature in PBS + 0.1% Tween-20 containing 1/1000 volume of antibody solution
Reacted for hours. After washing 3 times with PBS + 0.1% Tween-20, 1/10000 volume of secondary antibody (peroxydase-conjugated)
PBS + 0. with anti-rabit IgG, manufactured by Jackson).
The reaction was carried out in 1% Tween-20 at room temperature for 0.5 to 1 hour. PB
After washing 3 times with S + 0.1% Tween-20, ECL chemiluminesc
The specific band was detected using the ence reagent (Amersham Pharmacia Biotechnology).

Immunoprecipitation was performed as follows. That is, in the cell extract obtained above, an equal volume of the dilution buffer (20 mM HEPES-NaOH (pH 7.6), 2.5 mM MgCl ₂ , 0.1 mM E
DTA, 0.05% Triton X-100, 1 mM DTT, 20 mM b-glycero
phosphate, 1 mM sodium orthovanadate, 0.5 mM PMSF,
10 μg / ml aprotinin, 10 μg / ml leupeptin],
It was allowed to stand on ice for 10 minutes. Centrifuge (15000 rpm, 10 minutes, 4 ℃)
After that, the supernatant was separated, 1/100 volume of the antibody solution was added thereto, and the mixture was allowed to stand on ice for 1.5 hours. Then, a washing buffer (the cell lysis buffer and the dilution buffer were mixed at a volume ratio of 1: 1).
Protein G washed twice with (mixed in)
-Cephalose (trade name, Amersham Pharmacia Biotech
nology) was added as a bead volume of about 20 μL, and the temperature was 4 ° C.
The mixture was rotated for 1.5 hours and gently mixed to adsorb the immune complex to Protein G sepharose beads. The beads were washed 3 times with 500 μL of a washing buffer and used as an immunoprecipitation fraction in the following operation.

The antibodies used in immunoprecipitation and Western blotting were as follows depending on the protein of interest to be immunoprecipitated or detected or the tag peptide added thereto. Anti-hemagglutinin antibody (trade name Y11, Santa Cr
uz Biotechnology), anti-GFP antibody (Clontech), anti-Xpress antibody (product name Omni-probe M-2)
1, Santa Cruz Biotechnology), anti-JNK1 antibody (trade name FL, Santa Cruz Biotechnology), anti-I
KKα antibody (trade name: H-744, Santa Cruz Biotechnolog
y), anti-phosphorylated p38 antibody (New England Biolabs
Anti-p38 antibody (C-20, Santa Cruz Biotechnolo)
(manufactured by gy) (4) Kinase assay When the protein kinase activity (MAPKKK activity) of TAK1 is measured, the immunoprecipitate obtained in (3) above is used, and 6 × His-MKK is used as a substrate protein.
The assay was performed as follows using 6 (human MKK6 with 6xHis added). That is, 30 μl of the kinase assay buffer (2
0 mM HEPES-NaOH (pH 7.6), 20 mM MgCl ₂ , 2 mM DTT, 2
0 mM b-glycerophosphate, 0.1 mM sodium orthovanad
ate, 20 mM PNPP, 20 μM ATP, 3 μCi [γ- ³² P] AT
P, 1-5 μg substrate protein) was added and reacted at 30 ° C. for 30 minutes. 30 μL of 2 × SDS-PAGE sample buffer (50 mM Tris-HCl (pH 6.8), 10% glycerol, 2%
The reaction was stopped by the addition of SDS, 1 mg / ml BPB). After heating this at 95 ° C for 2 minutes, 10% SD
After subjecting to S-PAGE, the gel after electrophoresis was subjected to autoradiography using an X-ray film (manufactured by Fuji Film Co., Ltd.) to detect a phosphorylated substrate protein band.

In addition, IKK (IκB kinase) and J
When the protein kinase activity of NK (c-Jun N-terminal kinase) is measured, the N-terminal partial polypeptide of IκBα (GST-IκB) to which GST (glutathione S-transferase) is added as a substrate protein, respectively.
α- (1-54)) and GST-added c-Jun N-terminal partial polypeptide (GST-c-Jun (1-79)) were used to perform a kinase assay in the same manner as described above.

The activation (phosphorylation) of p38 protein kinase was detected by Western blotting using anti-phosphorylated p38 antibody in the cell extract.

6 × His-MKK6 used as a substrate protein
Is a reference (Japanese Patent Laid-Open No. 2000-197500; Sakurai
Et al., FEBS Letters 474, 141-145, 2000). First, the method of Moriguchi et al. (Journal of Biological Chemis
try, Vol. 271, pp. 13675-13679, 1996), and sequence information on human MKK6 (Genbank / EMBL database Accession No. U39656 and U39657; Raingeaud
Et al., Molecular and Cellular Biology, Vol. 16, 1247.
Pp. 1255, 1996) and designed a PCR method using them to obtain a cDNA containing the entire translation region of human MKK6 or a cDNA containing a sequence near the amino acid residue phosphorylated by TAK1. get. Using these cDNAs, an E. coli expression vector pQE-30 (QIAGEN) or pGEX-2T containing an appropriate DNA sequence designed to add a tag peptide (6 × His or glutathione-S-transferase).
(Manufactured by Pharmacia) to insert into a multi-cloning site to obtain a human MKK6 expression plasmid. The resulting plasmid was introduced into a host Escherichia coli (JM109 strain, etc.) and the resulting transformed cell was cultured to give 6 as a tag peptide.
XHis-added human MKK6 (6xHis-MKK6) was expressed, and 6xHis-MK was extracted from the cell extract by affinity chromatography for the tag peptide.
K6 was purified and acquired.

GST-IκBα used as a substrate protein
(1-54) and GST-c-Jun (1-79) are published in the literature (Sakurai
Et al., Journal of Biological Chemistry, Volumes 274, 10
641-10648, 1999) and prepared.

(5) M of TAK1-TAB1 fusion protein
Results of detection of APKK activity From the cells overexpressing the TAK1-TAB1 fusion protein to which the tag peptide was added (obtained in the same manner as in (1) above), the cell extract was obtained by the method described in (2) above. Was prepared.

Then, the cell extract was subjected to Western blotting using an antibody against the tag peptide by the method described in (3) above, and as a result, the overexpressed protein, that is, TAK1 to which the tag peptide was added, was added.
-It was confirmed that the TAB1 fusion protein was present in the cell extract.

From these cell extracts, a tag peptide-added TAK1- was prepared using an antibody against the tag peptide.
An immunoprecipitated fraction containing the TAB1 fusion protein was obtained. Using this immunoprecipitated fraction, as a substrate protein, 6 × His-
The TAK1 protein kinase activity was measured using MKK6 in the same manner as in (4) above.

As a result, as shown in FIG. 3, phosphorylation of the substrate protein MKK6 was detected in the anti-hemagglutinin immunoprecipitated fraction obtained from cells overexpressing the TAK1-TAB1 fusion protein to which the hemagglutinin antigen tag was added. , Strong MAPKKK activity was observed.

On the other hand, as a control, human TAK1
In the immunoprecipitated fraction obtained from cells overexpressing a partial polypeptide containing only the protein kinase region of TAB1 (not including the TAK1 activation region of TAB1)
No phosphorylation of KK6 was detected and no MAPKKK activity was observed.

Further, a partial polypeptide containing only the protein kinase region of human TAK1 (TAK1 of TAB1
The immunoprecipitated fraction obtained from cells overexpressing both the human TAB1 and the polypeptide containing only the TAK1 activation region of human TAB1 (not including the protein kinase region of TAK1) contained the substrate protein Phosphorylation of MKK6 was detected and MAPKKK activity was observed.

From these facts, each domain (partial polypeptide containing only the protein kinase region of human TAK1) in the TAK1-TAB1 fusion protein was identified as TAK.
It was confirmed that it was necessary and sufficient for the expression of the activity of 1. Further, it was revealed that the TAK1-TAB1 fusion protein constitutively has the same activity as that of activated TAK1. That is, the TAK1-TAB1 fusion protein was considered to function as a mutant TAK1 that is constitutively active.

In the TAK1-TAB1 fusion protein, a mutation was introduced at the ATP binding site in the protein kinase region derived from TAK1, and the 63rd Lys residue was replaced with Tr.
The protein substituted with p (referred to as KW) is MAPKKK
Since it showed no activity, it was confirmed that the protein kinase activity of the TAK1-TAB1 fusion protein depends on the function of the protein kinase region derived from TAK1.

Example 3 Assay of Action of Test Substance on MAPKKK Activity of TAK1-TAB1 Fusion Protein Using the same system as in Example 2 (5) above, TAK1-TA
The effect of the test substance on the MAPKKK activity of the B1 fusion protein is assayed. That is, using the human TAK1-TAB1 fusion protein as an enzyme (MAPKKK), human MK
Using K6 as a substrate, an enzyme reaction is carried out in the presence or absence of a test substance to measure MAPKKK activity.
Then, MAP in the presence and absence of the test substance
Compare KKK activity. From this result, whether or not the test substance has an action of inhibiting the MAPKKK activity of active TAK1 (or its degree) is assayed.

Example 4 Interaction between protein kinase domain and TAK1 activation domain in human TAC1-TAB1 fusion protein The protein kinase domain of TAK1 is TA of TAB1.
It is activated by interacting (binding) with the K1 activation region. Therefore, whether the protein kinase region and the TAK1 activation region in the human TAK1-TAB1 fusion protein interact with each other in the molecule (bond) or interact with each other in the molecule (bond) is described above. The same immunoprecipitation assay method as in 2 was used for the examination.

As a result, as shown in FIG. 4, Xpres
In the anti-hemagglutinin immunoprecipitated fraction obtained from cells in which both the s-tagged TAK1-TAB1 fusion protein and the hemagglutinin antigen-tagged TAK1 kinase region-containing partial polypeptide were both overexpressed, Xpr
The TAK1-TAB1 fusion protein to which the ess tag was added was co-precipitated.

However, in the anti-hemagglutinin immunoprecipitated fraction obtained from cells in which both the Xpress-tagged TAK1-TAB1 fusion protein and the hemagglutinin-antigen-tagged TAK1-TAB1 fusion protein were both overexpressed, TAK with Xpress tag
The 1-TAB1 fusion protein has not been coprecipitated.

From these results, it was considered that the protein kinase region and TAK1 activation region in the TAK1-TAB1 fusion protein interact in the molecule, not in the molecule.

Example 5 Activation Assay of Signal Transduction System by TAK1-TAB1 Fusion Protein (1) Activation of Signal Transduction System by TAK1-TAB1 Fusion Protein In contrast to the signal transduction system involving TAK1, TAK1-
Whether the TAB1 fusion protein has a function of transmitting information, for example, JNK, p38, and IKK of MAP kinase system
Whether or not it has the function of activating the above was examined using the same immunoprecipitation assay method as in Example 2 above.

An anti-JNK immunoprecipitation fraction was obtained from a cell extract of cells overexpressing the TAK1-TAB1 fusion protein (or both TAK1 and TAB1). GST-c-Jun was obtained from this immunoprecipitation fraction. The kinase activity of JNK as a substrate protein was detected. The assay of JNK kinase activity was performed in the same manner as in the method described in item (4) of Example 2.

The result (upper part of FIG. 5), TAK1-TA
In cells overexpressing the B1 fusion protein, anti-JN
JNK kinase activity was observed in the K immunoprecipitated fraction.
It was confirmed that NK activation was occurring. TAK
The JNK kinase activity in cells overexpressing the 1-TAB1 fusion protein was similar to the JNK kinase activity in cells overexpressing both TAK1 and TAB1, and the same degree of JNK activation occurred. I found out that

In the same manner as described above, cells (or TAK1 and T) which overexpressed the TAK1-TAB1 fusion protein were overexpressed.
Activation of p38 and IKK was detected in cells overexpressing both AB1). As a result, as shown in the middle of FIG. 5, p38 phosphorylation (activation) in cells overexpressing the TAK1-TAB1 fusion protein.
Was similar to the phosphorylation (activation) of p38 in cells in which both TAK1 and TAB1 were overexpressed, and it was found that the same degree of p38 activation occurred.

As shown in the lower part of FIG. 5, TAK
The IKK kinase activity in cells overexpressing the 1-TAB1 fusion protein was similar to the IKK kinase activity in cells overexpressing both TAK1 and TAB1, and the same degree of IKK activation occurred. I found out that

A mutation was introduced into the ATP-binding site in the protein kinase region derived from TAK1 in the TAK1-TAB1 fusion protein, and the 63rd Lys residue was replaced with Tr.
In cells overexpressing the p-substituted protein (KW), activation of these signal transduction systems (JNK, p38 and IKK) was not observed, so TA
It was confirmed that the protein kinase activity of the K1-TAB1 fusion protein depends on the function of the protein kinase region derived from TAK1.

(2) Assay of the action of the test substance Using the same system as in the above (1), the action of the test substance on the activation of JNK, p38 or IKK kinase by the TAK1-TAB1 fusion protein can be assayed. That is, cells that overexpress the TAK1-TAB1 fusion protein are obtained, and the cells are cultured in the presence or absence of the test substance. Regarding the cells after culturing, JN
The kinase activity of K, p38 or IKK is measured.
The protein kinase activity in the presence and absence of the test substance is compared, and from the results, whether the test substance has an action of inhibiting the activation of the signal transduction system involving TAK1 (or The degree). Example 6 IL-by TAK1-TAB1 fusion protein
Activation Assay for 6 Production (1) Activation of IL-6 Production by TAK1-TAB1 Fusion Protein interleukin-6 (IL-6) is a cytokine whose expression is induced by activation of a signal transduction system involving TAK1. . It was confirmed as follows whether the TAK1-TAB1 fusion protein has an action of inducing IL-6 production.

First, as in the second embodiment, TAK1-
An expression vector for the TAB1 fusion protein (or TAK1 and TAB1) was transfected into HeLa cells. The medium was collected 24 hours after transfection, and the IL-6 produced in the medium was analyzed using a commercially available ELISA kit (G
It was investigated by using "enzymeTECHNE".

As a result of measuring the amount of IL-6 produced, as shown in FIG. 6, in the cells in which the TAK1-TAB1 fusion protein was overexpressed, IL was almost the same as that in the cells in which both TAK1 and TAB1 were overexpressed. Induction of −6 production was observed. From this fact, the TAK1-TAB1 fusion protein is IL-induced by activation of the signal transduction system.
It was confirmed that it also has an action of inducing the expression of 6.

(2) Assay of the action of the test substance Using the same system as in the above (1), the action of the test substance on the induction of IL-6 production by the TAK1-TAB1 fusion protein can be assayed. That is, TAK1-TAB
A cell overexpressing 1 fusion protein is obtained, and this is cultured in the presence or absence of a test substance. The amount of IL-6 produced in the cultured cells is measured in the same manner as above. The IL-6 production induction in the presence and absence of the test substance was compared, and from the results, the test substance showed the activation of the signal transduction system involving TAK1 (IL-6 production induction). It is tested whether it has an inhibitory effect (or its degree).

[0113]

INDUSTRIAL APPLICABILITY The TAK1-TAB1 fusion protein of the present invention is a constitutively active TAK even in the absence of TAB1.
It has the same activity and function as 1. Because of this property,
By using the TAK1-TAB1 fusion protein of the present invention, an efficient and stable TAK1 inhibitor assay method, identification method, and screening method can be carried out.

Further, the TAK1-TAB1 fusion protein of the present invention and the nucleic acid encoding the same are useful for functional analysis of TAK1 and elucidation of the onset mechanism of autoimmune diseases and inflammatory diseases in which TAK1 is considered to be involved. .

"Sequence listing free text" Free texts of SEQ ID NOs: 1 to 3 and 8 to 13 <223> Sequences of artificially synthesized primers (Artificia
lly Synthesized Primer Sequence) Free text of SEQ ID NOS: 11 and 12 <223> Kinase region of human TAK1 and human TAB1
Fusion protein comprising kinase domain of huma
n TAK1 and TAK1 activating domain of human TAB1)

[Sequence list]                                SEQUENCE LISTING        <110> Tanabe Seiyaku Co., Ltd. <120> TAK1-TAB1 fusion protein <130> A00-4771 <140> <141> <160> 17 <170> PatentIn Ver. 2.0 <210> 1 <211> 2785 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (163) .. (1899) <400> 1 ggacacggct gtggccgctg cctctacccc cgccacggat cgccgggtag taggactgcg 60 cggctccagg ctgagggtcg gtccggaggc gggtgggcgc gggtctcacc cggattgtcc 120 gggtggcacc gttcccggcc ccaccgggcg ccgcgaggga tc atg tct aca gcc 174                                                Met Ser Thr Ala                                                  1 tct gcc gcc tcc tcc tcc tcc tcg tct tcg gcc ggt gag atg atc gaa 222 Ser Ala Ala Ser Ser Ser Ser Ser Ser Ser Ala Gly Glu Met Ile Glu   5 10 15 20 gcc cct tcc cag gtc ctc aac ttt gaa gag atc gac tac aag gag atc 270 Ala Pro Ser Gln Val Leu Asn Phe Glu Glu Ile Asp Tyr Lys Glu Ile                  25 30 35 gag gtg gaa gag gtt gtt gga aga gga gcc ttt gga gtt gtt tgc aaa 318 Glu Val Glu Glu Val Val Gly Arg Gly Ala Phe Gly Val Val Cys Lys              40 45 50 gct aag tgg aga gca aaa gat gtt gct att aaa caa ata gaa agt gaa 366 Ala Lys Trp Arg Ala Lys Asp Val Ala Ile Lys Gln Ile Glu Ser Glu          55 60 65 tct gag agg aaa gcg ttt att gta gag ctt cgg cag tta tcc cgt gtg 414 Ser Glu Arg Lys Ala Phe Ile Val Glu Leu Arg Gln Leu Ser Arg Val      70 75 80 aac cat cct aat att gta aag ctt tat gga gcc tgc ttg aat cca gtg 462 Asn His Pro Asn Ile Val Lys Leu Tyr Gly Ala Cys Leu Asn Pro Val  85 90 95 100 tgt ctt gtg atg gaa tat gct gaa ggg ggc tct tta tat aat gtg ctg 510 Cys Leu Val Met Glu Tyr Ala Glu Gly Gly Ser Leu Tyr Asn Val Leu                 105 110 115 cat ggt gct gaa cca ttg cca tat tat act gct gcc cac gca atg agt 558 His Gly Ala Glu Pro Leu Pro Tyr Tyr Thr Ala Ala His Ala Met Ser             120 125 130 tgg tgt tta cag tgt tcc caa gga gtg gct tat ctt cac agc atg caa 606 Trp Cys Leu Gln Cys Ser Gln Gly Val Ala Tyr Leu His Ser Met Gln         135 140 145 ccc aaa gcg cta att cac agg gac ctg aaa cca cca aac tta ctg ctg 654 Pro Lys Ala Leu Ile His Arg Asp Leu Lys Pro Pro Asn Leu Leu Leu     150 155 160 gtt gca ggg ggg aca gtt cta aaa att tgt gat ttt ggt aca gcc tgt 702 Val Ala Gly Gly Thr Val Leu Lys Ile Cys Asp Phe Gly Thr Ala Cys 165 170 175 180 gac att cag aca cac atg acc aat aac aag ggg agt gct gct tgg atg 750 Asp Ile Gln Thr His Met Thr Asn Asn Lys Gly Ser Ala Ala Trp Met                 185 190 195 gca cct gaa gtt ttt gaa ggt agt aat tac agt gaa aaa tgt gac gtc 798 Ala Pro Glu Val Phe Glu Gly Ser Asn Tyr Ser Glu Lys Cys Asp Val             200 205 210 ttc agc tgg ggt att att ctt tgg gaa gtg ata acg cgt cgg aaa ccc 846 Phe Ser Trp Gly Ile Ile Leu Trp Glu Val Ile Thr Arg Arg Lys Pro         215 220 225 ttt gat gag att ggt ggc cca gct ttc cga atc atg tgg gct gtt cat 894 Phe Asp Glu Ile Gly Gly Pro Ala Phe Arg Ile Met Trp Ala Val His     230 235 240 aat ggt act cga cca cca ctg ata aaa aat tta cct aag ccc att gag 942 Asn Gly Thr Arg Pro Pro Leu Ile Lys Asn Leu Pro Lys Pro Ile Glu 245 250 255 260 agc ctg atg act cgt tgt tgg tct aaa gat cct tcc cag cgc cct tca 990 Ser Leu Met Thr Arg Cys Trp Ser Lys Asp Pro Ser Gln Arg Pro Ser                 265 270 275 atg gag gaa att gtg aaa ata atg act cac ttg atg cgg tac ttt cca 1038 Met Glu Glu Ile Val Lys Ile Met Thr His Leu Met Arg Tyr Phe Pro             280 285 290 gga gca gat gag cca tta cag tat cct tgt cag tat tca gat gaa gga 1086 Gly Ala Asp Glu Pro Leu Gln Tyr Pro Cys Gln Tyr Ser Asp Glu Gly         295 300 305 cag agc aac tct gcc acc agt aca ggc tca ttc atg gac att gct tct 1134 Gln Ser Asn Ser Ala Thr Ser Thr Gly Ser Phe Met Asp Ile Ala Ser     310 315 320 aca aat acg agt aac aaa agt gac act aat atg gag caa gtt cct gcc 1182 Thr Asn Thr Ser Asn Lys Ser Asp Thr Asn Met Glu Gln Val Pro Ala 325 330 335 340 aca aat gat act att aag cgc tta gaa tca aaa ttg ttg aaa aat cag 1230 Thr Asn Asp Thr Ile Lys Arg Leu Glu Ser Lys Leu Leu Lys Asn Gln                 345 350 355 gca aag caa cag agt gaa tct gga cgt tta agc ttg gga gcc tcc cgt 1278 Ala Lys Gln Gln Ser Glu Ser Gly Arg Leu Ser Leu Gly Ala Ser Arg             360 365 370 ggg agc agt gtg gag agc ttg ccc cca acc tct gag ggc aag agg atg 1326 Gly Ser Ser Val Glu Ser Leu Pro Pro Thr Ser Glu Gly Lys Arg Met         375 380 385 agt gct gac atg tct gaa ata gaa gct agg atc gcc gca acc aca ggc 1374 Ser Ala Asp Met Ser Glu Ile Glu Ala Arg Ile Ala Ala Thr Thr Gly     390 395 400 aac gga cag cca aga cgt aga tcc atc caa gac ttg act gta act gga 1422 Asn Gly Gln Pro Arg Arg Arg Ser Ile Gln Asp Leu Thr Val Thr Gly 405 410 415 420 aca gaa cct ggt cag gtg agc agt agg tca tcc agt ccc agt gtc aga 1470 Thr Glu Pro Gly Gln Val Ser Ser Arg Ser Ser Ser Pro Ser Val Arg                 425 430 435 atg att act acc tca gga cca acc tca gaa aag cca act cga agt cat 1518 Met Ile Thr Thr Ser Gly Pro Thr Ser Glu Lys Pro Thr Arg Ser His             440 445 450 cca tgg acc cct gat gat tcc aca gat acc aat gga tca gat aac tcc 1566 Pro Trp Thr Pro Asp Asp Ser Thr Asp Thr Asn Gly Ser Asp Asn Ser         455 460 465 atc cca atg gct tat ctt aca ctg gat cac caa cta cag cct cta gca 1614 Ile Pro Met Ala Tyr Leu Thr Leu Asp His Gln Leu Gln Pro Leu Ala     470 475 480 ccg tgc cca aac tcc aaa gaa tct atg gca gtg ttt gaa cag cat tgt 1662 Pro Cys Pro Asn Ser Lys Glu Ser Met Ala Val Phe Glu Gln His Cys 485 490 495 500 aaa atg gca caa gaa tat atg aaa gtt caa aca gaa att gca ttg tta 1710 Lys Met Ala Gln Glu Tyr Met Lys Val Gln Thr Glu Ile Ala Leu Leu                 505 510 515 tta cag aga aag caa gaa cta gtt gca gaa ctg gac cag gat gaa aag 1758 Leu Gln Arg Lys Gln Glu Leu Val Ala Glu Leu Asp Gln Asp Glu Lys             520 525 530 gac cag caa aat aca tct cgc ctg gta cag gaa cat aaa aag ctt tta 1806 Asp Gln Gln Asn Thr Ser Arg Leu Val Gln Glu His Lys Lys Leu Leu         535 540 545 gat gaa aac aaa agc ctt tct act tac tac cag caa tgc aaa aaa caa 1854 Asp Glu Asn Lys Ser Leu Ser Thr Tyr Tyr Gln Gln Cys Lys Lys Gln     550 555 560 cta gag gtc atc aga agt cag cag cag aaa cga caa ggc act tca 1899 Leu Glu Val Ile Arg Ser Gln Gln Gln Lys Arg Gln Gly Thr Ser 565 570 575 tgattctctg ggaccgttac attttgaaat atgcaaagaa agactttttt tttaaggaaa 1959 ggaaaacctt ataatgacga ttcatgagtg ttagcttttt ggcgtgttct gaatgccaac 2019 tgcctatatt tgctgcattt ttttcattgt ttattttcct tttctcatgg tggacataca 2079 attttactgt ttcattgcat aacatggtag catctgtgac ttgaatgagc agcactttgc 2139 aacttcaaaa cagatgcagt gaactgtggc tgtatatgca tgctcattgt gtgaaggcta 2199 gcctaacaga acaggaggta tcaaactagc tgctatgtgc aaacagcgtc cattttttca 2259 tattagaggt ggaacctcaa gaatgacttt attcttgtat ctcatctcaa aatattaata 2319 atttttttcc caaaagatgg tatataccaa gttaaagaca gggtattata aatttagagt 2379 gattggtggt atattacgga aatacggaac ctttagggat agttccgtgt aagggctttg 2439 atgccagcat ccttggatca gtactgaact cagttccatc cgtaaaatat gtaaaggtaa 2499 gtggcagctg ctctatttaa tgaaagcagt tttaccggat tttgttagac taaaatttga 2559 ttgtgataca ttgaacaaaa tggaactcat ttttttttaa ggagtaaaga tttttaattc 2619 tgtgattgtg tgtatgtgtg ttgaaactgt aaagctttta tgactctaat attaatctct 2679 taaatgaaat taaaaggcaa aagaacatga ttgagcttaa atgatcattt cttcctgcag 2739 tgattcttgg attgttttct catgtatttg aaaaaaaaaa aaaaaa 2785 <210> 2 <211> 579 <212> PRT <213> Homo sapiens <400> 2 Met Ser Thr Ala Ser Ala Ala Ser Ser Ser Ser Ser Ser Ser Ala Gly   1 5 10 15 Glu Met Ile Glu Ala Pro Ser Gln Val Leu Asn Phe Glu Glu Ile Asp              20 25 30 Tyr Lys Glu Ile Glu Val Glu Glu Val Val Gly Arg Gly Ala Phe Gly          35 40 45 Val Val Cys Lys Ala Lys Trp Arg Ala Lys Asp Val Ala Ile Lys Gln      50 55 60 Ile Glu Ser Glu Ser Glu Arg Lys Ala Phe Ile Val Glu Leu Arg Gln  65 70 75 80 Leu Ser Arg Val Asn His Pro Asn Ile Val Lys Leu Tyr Gly Ala Cys                  85 90 95 Leu Asn Pro Val Cys Leu Val Met Glu Tyr Ala Glu Gly Gly Ser Leu             100 105 110 Tyr Asn Val Leu His Gly Ala Glu Pro Leu Pro Tyr Tyr Thr Ala Ala         115 120 125 His Ala Met Ser Trp Cys Leu Gln Cys Ser Gln Gly Val Ala Tyr Leu     130 135 140 His Ser Met Gln Pro Lys Ala Leu Ile His Arg Asp Leu Lys Pro Pro 145 150 155 160 Asn Leu Leu Leu Val Ala Gly Gly Thr Val Leu Lys Ile Cys Asp Phe                 165 170 175 Gly Thr Ala Cys Asp Ile Gln Thr His Met Thr Asn Asn Lys Gly Ser             180 185 190 Ala Ala Trp Met Ala Pro Glu Val Phe Glu Gly Ser Asn Tyr Ser Glu         195 200 205 Lys Cys Asp Val Phe Ser Trp Gly Ile Ile Leu Trp Glu Val Ile Thr     210 215 220 Arg Arg Lys Pro Phe Asp Glu Ile Gly Gly Pro Ala Phe Arg Ile Met 225 230 235 240 Trp Ala Val His Asn Gly Thr Arg Pro Pro Leu Ile Lys Asn Leu Pro                 245 250 255 Lys Pro Ile Glu Ser Leu Met Thr Arg Cys Trp Ser Lys Asp Pro Ser             260 265 270 Gln Arg Pro Ser Met Glu Glu Ile Val Lys Ile Met Thr His Leu Met         275 280 285 Arg Tyr Phe Pro Gly Ala Asp Glu Pro Leu Gln Tyr Pro Cys Gln Tyr     290 295 300 Ser Asp Glu Gly Gln Ser Asn Ser Ala Thr Ser Thr Gly Ser Phe Met 305 310 315 320 Asp Ile Ala Ser Thr Asn Thr Ser Asn Lys Ser Asp Thr Asn Met Glu                 325 330 335 Gln Val Pro Ala Thr Asn Asp Thr Ile Lys Arg Leu Glu Ser Lys Leu             340 345 350 Leu Lys Asn Gln Ala Lys Gln Gln Ser Glu Ser Gly Arg Leu Ser Leu         355 360 365 Gly Ala Ser Arg Gly Ser Ser Val Glu Ser Leu Pro Pro Thr Ser Glu     370 375 380 Gly Lys Arg Met Ser Ala Asp Met Ser Glu Ile Glu Ala Arg Ile Ala 385 390 395 400 Ala Thr Thr Gly Asn Gly Gln Pro Arg Arg Arg Ser Ile Gln Asp Leu                 405 410 415 Thr Val Thr Gly Thr Glu Pro Gly Gln Val Ser Ser Arg Ser Ser Ser             420 425 430 Pro Ser Val Arg Met Ile Thr Thr Ser Gly Pro Thr Ser Glu Lys Pro         435 440 445 Thr Arg Ser His Pro Trp Thr Pro Asp Asp Ser Thr Asp Thr Asn Gly     450 455 460 Ser Asp Asn Ser Ile Pro Met Ala Tyr Leu Thr Leu Asp His Gln Leu 465 470 475 480 Gln Pro Leu Ala Pro Cys Pro Asn Ser Lys Glu Ser Met Ala Val Phe                 485 490 495 Glu Gln His Cys Lys Met Ala Gln Glu Tyr Met Lys Val Gln Thr Glu             500 505 510 Ile Ala Leu Leu Leu Gln Arg Lys Gln Glu Leu Val Ala Glu Leu Asp         515 520 525 Gln Asp Glu Lys Asp Gln Gln Asn Thr Ser Arg Leu Val Gln Glu His     530 535 540 Lys Lys Leu Leu Asp Glu Asn Lys Ser Leu Ser Thr Tyr Tyr Gln Gln 545 550 555 560 Cys Lys Lys Gln Leu Glu Val Ile Arg Ser Gln Gln Gln Lys Arg Gln                 565 570 575 Gly Thr Ser                                                                   <210> 3 <211> 3096 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (21) .. (1532) <400> 3 gcccgcaggg ttcctccaag atg gcg gcg cag agg agg agc ttg ctg cag agt 53                       Met Ala Ala Gln Arg Arg Ser Leu Leu Gln Ser                         1 5 10 gag cag cag cca agc tgg aca gat gac ctg cct ctc tgc cac ctc tct 101 Glu Gln Gln Pro Ser Trp Thr Asp Asp Leu Pro Leu Cys His Leu Ser              15 20 25 ggg gtt ggc tca gcc tcc aac cgc agc tac tct gct gat ggc aag ggc 149 Gly Val Gly Ser Ala Ser Asn Arg Ser Tyr Ser Ala Asp Gly Lys Gly          30 35 40 act gag agc cac ccg cca gag gac agc tgg ctc aag ttc agg agt gag 197 Thr Glu Ser His Pro Pro Glu Asp Ser Trp Leu Lys Phe Arg Ser Glu      45 50 55 aac aac tgc ttc ctg tat ggg gtc ttc aac ggc tat gat ggc aac cga 245 Asn Asn Cys Phe Leu Tyr Gly Val Phe Asn Gly Tyr Asp Gly Asn Arg  60 65 70 75 gtg acc aac ttc gtg gcc cag cgg ctg tcc gca gag ctc ctg ctg ggc 293 Val Thr Asn Phe Val Ala Gln Arg Leu Ser Ala Glu Leu Leu Leu Gly                  80 85 90 cag ctg aat gcc gag cac gcc gag gcc gat gtg cgg cgt gtg ctg ctg 341 Gln Leu Asn Ala Glu His Ala Glu Ala Asp Val Arg Arg Val Leu Leu              95 100 105 cag gcc ttc gat gtg gtg gag agg agc ttc ctg gag tcc att gac gac 389 Gln Ala Phe Asp Val Val Glu Arg Ser Phe Leu Glu Ser Ile Asp Asp         110 115 120 gcc ttg gct gag aag gca agc ctc cag tcg caa ttg cca gag gga gtc 437 Ala Leu Ala Glu Lys Ala Ser Leu Gln Ser Gln Leu Pro Glu Gly Val     125 130 135 cct cag cac cag ctg cct cct cag tat cag aag atc ctt gag aga ctc 485 Pro Gln His Gln Leu Pro Pro Gln Tyr Gln Lys Ile Leu Glu Arg Leu 140 145 150 155 aag acg tta gag agg gaa att tcg gga ggg gcc atg gcc gtt gtg gcg 533 Lys Thr Leu Glu Arg Glu Ile Ser Gly Gly Ala Met Ala Val Val Ala                 160 165 170 gtc ctt ctc aac aac aag ctc tac gtc gcc aat gtc ggt aca aac cgt 581 Val Leu Leu Asn Asn Lys Leu Tyr Val Ala Asn Val Gly Thr Asn Arg             175 180 185 gca ctt tta tgc aaa tcg aca gtg gat ggg ttg cag gtg aca cag ctg 629 Ala Leu Leu Cys Lys Ser Thr Val Asp Gly Leu Gln Val Thr Gln Leu         190 195 200 aac gtg gac cac acc aca gag aac gag gat gag ctc ttc cgt ctt tcg 677 Asn Val Asp His Thr Thr Glu Asn Glu Asp Glu Leu Phe Arg Leu Ser     205 210 215 cag ctg ggc ttg gat gct gga aag atc aag cag gtg ggg atc atc tgt 725 Gln Leu Gly Leu Asp Ala Gly Lys Ile Lys Gln Val Gly Ile Ile Cys 220 225 230 235 ggg cag gag agc acc cgg cgg atc ggg gat tac aag gtt aaa tat ggc 773 Gly Gln Glu Ser Thr Arg Arg Ile Gly Asp Tyr Lys Val Lys Tyr Gly                 240 245 250 tac acg gac att gac ctt ctc agc gct gcc aag tcc aaa cca atc atc 821 Tyr Thr Asp Ile Asp Leu Leu Ser Ala Ala Lys Ser Lys Pro Ile Ile             255 260 265 gca gag cca gaa atc cat ggg gca cag ccg ctg gat ggg gtg acg ggc 869 Ala Glu Pro Glu Ile His Gly Ala Gln Pro Leu Asp Gly Val Thr Gly         270 275 280 ttc ttg gtg ctg atg tcg gag ggg ttg tac aag gcc cta gag gca gcc 917 Phe Leu Val Leu Met Ser Glu Gly Leu Tyr Lys Ala Leu Glu Ala Ala     285 290 295 cat ggg cct ggg cag gcc aac cag gag att gct gcg atg att gac act 965 His Gly Pro Gly Gln Ala Asn Gln Glu Ile Ala Ala Met Ile Asp Thr 300 305 310 315 gag ttt gcc aag cag acc tcc ctg gac gca gtg gcc cag gcc gtc gtg 1013 Glu Phe Ala Lys Gln Thr Ser Leu Asp Ala Val Ala Gln Ala Val Val                 320 325 330 gac cgg gtg aag cgc atc cac agc gac acc ttc gcc agt ggt ggg gag 1061 Asp Arg Val Lys Arg Ile His Ser Asp Thr Phe Ala Ser Gly Gly Glu             335 340 345 cgt gcc agg ttc tgc ccc cgg cac gag gac atg acc ctg cta gtg agg 1109 Arg Ala Arg Phe Cys Pro Arg His Glu Asp Met Thr Leu Leu Val Arg         350 355 360 aac ttt ggc tac ccg ctg ggc gaa atg agc cag ccc aca ccg agc cca 1157 Asn Phe Gly Tyr Pro Leu Gly Glu Met Ser Gln Pro Thr Pro Ser Pro     365 370 375 gcc cca gct gca gga gga cga gtg tac cct gtg tct gtg cca tac tcc 1205 Ala Pro Ala Ala Gly Gly Arg Val Tyr Pro Val Ser Val Pro Tyr Ser 380 385 390 395 agc gcc cag agc acc agc aag acc agc gtg acc ctc tcc ctt gtc atg 1253 Ser Ala Gln Ser Thr Ser Lys Thr Ser Val Thr Leu Ser Leu Val Met                 400 405 410 ccc tcc cag ggc cag atg gtc aac ggg gct cac agt gct tcc acc ctg 1301 Pro Ser Gln Gly Gln Met Val Asn Gly Ala His Ser Ala Ser Thr Leu             415 420 425 gac gaa gcc acc ccc acc ctc acc aac caa agc ccg acc tta acc ctg 1349 Asp Glu Ala Thr Pro Thr Leu Thr Asn Gln Ser Pro Thr Leu Thr Leu         430 435 440 cag tcc acc aac acg cac acg cag agc agc agc tcc agc tct gac gga 1397 Gln Ser Thr Asn Thr His Thr Gln Ser Ser Ser Ser Ser Ser Asp Gly     445 450 455 ggc ctc ttc cgc tcc cgg ccc gcc cac tcg ctc ccg cct ggc gag gac 1445 Gly Leu Phe Arg Ser Arg Pro Ala His Ser Leu Pro Pro Gly Glu Asp 460 465 470 475 ggt cgt gtt gag ccc tat gtg gac ttt gct gag ttt tac cgc ctc tgg 1493 Gly Arg Val Glu Pro Tyr Val Asp Phe Ala Glu Phe Tyr Arg Leu Trp                 480 485 490 agc gtg gac cat ggc gag cag agc gtg gtg aca gca ccg tagggcagcc 1542 Ser Val Asp His Gly Glu Gln Ser Val Val Thr Ala Pro             495 500 ggaggaatgc agcccaagca gggcctggca tggggcagga cagggtccag ccttttccta 1602 acatctgcct gtgccacaac ggccagcagg tgccccatcc tctgcccaca gcagactctg 1662 tcccatggct ctccgggcag tagagtgtgt gagtgcagac tggacctgtg gttcatacct 1722 tgtcaccacc cgggaagctg aaggccactt cctcccagat ggcctcagcc aggaccatcg 1782 ccctttctca gagcagaggg ccaggtaggg aaaccgcagt gggcctgcaa gccgcccgag 1842 cctccccagc agcctcctac agagcaggaa gaggcgccct gtgaaccctg tagtgttgca 1902 ggcccagcag accctgctgt cccaagccca cccctcctcc caccatcacc tccctcacct 1962 cgggacagta gccctccact tctccagcct ctcagccctg tgctcctgta tccagagtgg 2022 aacccaggct ggtgtccgta tctgtccctg ggccccaccc ctggacctgc cttggttgtg 2082 tcatctgttg taaacattcc aggaggacca ggggagcatc tggggcctgg gatggccaca 2142 gaaggggcag gccaggtgga aaggagccag ggggaagtgg tctaagagac ctggaactgc 2202 cagaggatgg cggcctgggc ttccccagag ccaggcgtgc gggagaggtg aggactggcc 2262 ccggtgggct gaggcagggg ccgctgtcgt caggcctgag ccagggtgag ctggtgcctg 2322 ccttgcttct tccttctggt gctgtgaaga ccataggctg gcaggcagct gagatgaact 2382 gtctttacca ctgatgaggg gcctctgccg gctgagggta gcaagcaggg gttgtgagtc 2442 aggctggggg acttgtttga aagaaagagg agttggaatg tggttcccag gagggaagag 2502 gttcctttga gacacagtaa ccctgggagg cataggagaa gggtcgggcc agcccagccc 2562 agggcctgag ttagactatt tcccacatgt tctctgcctt cagtggggag ggggtgccac 2622 cagggctgtc ggccaggatt gccactcctg tttcagagga agcaggccga gagacttgca 2682 ccttggacaa gccacacaat cagtggggca gccagagctc agacctgagc cattgtgtca 2742 gtatccagga ccccccggat tctccacgcc ctccccatct cccagtctcc ctgcccccca 2802 tgccccagac cggcccacca gggactagcc gctgtcgcac agcctctggg gtgcttggtc 2862 tctgcaaagt caaaggcctg acagctctgt ggcctgggaa tccattttcc tgcgggagag 2922 cagggcctgg tgtggaacca gggagctgtg ggaagccaca gcagaaatgg aagaaaaaca 2982 ggtctcagcc cagggtcctc gctcactccc tcactcccca ctttgaagcc atctctgttc 3042 tgcaggtgag aggatttaaa gtcagtcaca aaggcttggg aacaaaagga attc 3096 <210> 4 <211> 504 <212> PRT <213> Homo sapiens <400> 4 Met Ala Ala Gln Arg Arg Ser Leu Leu Gln Ser Glu Gln Gln Pro Ser   1 5 10 15 Trp Thr Asp Asp Leu Pro Leu Cys His Leu Ser Gly Val Gly Ser Ala              20 25 30 Ser Asn Arg Ser Tyr Ser Ala Asp Gly Lys Gly Thr Glu Ser His Pro          35 40 45 Pro Glu Asp Ser Trp Leu Lys Phe Arg Ser Glu Asn Asn Cys Phe Leu      50 55 60 Tyr Gly Val Phe Asn Gly Tyr Asp Gly Asn Arg Val Thr Asn Phe Val  65 70 75 80 Ala Gln Arg Leu Ser Ala Glu Leu Leu Leu Gly Gln Leu Asn Ala Glu                  85 90 95 His Ala Glu Ala Asp Val Arg Arg Val Leu Leu Gln Ala Phe Asp Val             100 105 110 Val Glu Arg Ser Phe Leu Glu Ser Ile Asp Asp Ala Leu Ala Glu Lys         115 120 125 Ala Ser Leu Gln Ser Gln Leu Pro Glu Gly Val Pro Gln His Gln Leu     130 135 140 Pro Pro Gln Tyr Gln Lys Ile Leu Glu Arg Leu Lys Thr Leu Glu Arg 145 150 155 160 Glu Ile Ser Gly Gly Ala Met Ala Val Val Ala Val Leu Leu Asn Asn                 165 170 175 Lys Leu Tyr Val Ala Asn Val Gly Thr Asn Arg Ala Leu Leu Cys Lys             180 185 190 Ser Thr Val Asp Gly Leu Gln Val Thr Gln Leu Asn Val Asp His Thr         195 200 205 Thr Glu Asn Glu Asp Glu Leu Phe Arg Leu Ser Gln Leu Gly Leu Asp     210 215 220 Ala Gly Lys Ile Lys Gln Val Gly Ile Ile Cys Gly Gln Glu Ser Thr 225 230 235 240 Arg Arg Ile Gly Asp Tyr Lys Val Lys Tyr Gly Tyr Thr Asp Ile Asp                 245 250 255 Leu Leu Ser Ala Ala Lys Ser Lys Pro Ile Ile Ala Glu Pro Glu Ile             260 265 270 His Gly Ala Gln Pro Leu Asp Gly Val Thr Gly Phe Leu Val Leu Met         275 280 285 Ser Glu Gly Leu Tyr Lys Ala Leu Glu Ala Ala His Gly Pro Gly Gln     290 295 300 Ala Asn Gln Glu Ile Ala Ala Met Ile Asp Thr Glu Phe Ala Lys Gln 305 310 315 320 Thr Ser Leu Asp Ala Val Ala Gln Ala Val Val Asp Arg Val Lys Arg                 325 330 335 Ile His Ser Asp Thr Phe Ala Ser Gly Gly Glu Arg Ala Arg Phe Cys             340 345 350 Pro Arg His Glu Asp Met Thr Leu Leu Val Arg Asn Phe Gly Tyr Pro         355 360 365 Leu Gly Glu Met Ser Gln Pro Thr Pro Ser Pro Ala Pro Ala Ala Gly     370 375 380 Gly Arg Val Tyr Pro Val Ser Val Pro Tyr Ser Ser Ala Gln Ser Thr 385 390 395 400 Ser Lys Thr Ser Val Thr Leu Ser Leu Val Met Pro Ser Gln Gly Gln                 405 410 415 Met Val Asn Gly Ala His Ser Ala Ser Thr Leu Asp Glu Ala Thr Pro             420 425 430 Thr Leu Thr Asn Gln Ser Pro Thr Leu Thr Leu Gln Ser Thr Asn Thr         435 440 445 His Thr Gln Ser Ser Ser Ser Ser Ser Asp Gly Gly Leu Phe Arg Ser     450 455 460 Arg Pro Ala His Ser Leu Pro Pro Gly Glu Asp Gly Arg Val Glu Pro 465 470 475 480 Tyr Val Asp Phe Ala Glu Phe Tyr Arg Leu Trp Ser Val Asp His Gly                 485 490 495 Glu Gln Ser Val Val Thr Ala Pro             500 <210> 5 <211> 909 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (909) <400> 5 atg tct aca gcc tct gcc gcc tcc tcc tcc tcc tcg tct tcg gcc ggt 48 Met Ser Thr Ala Ser Ala Ala Ser Ser Ser Ser Ser Ser Ser Ala Gly   1 5 10 15 gag atg atc gaa gcc cct tcc cag gtc ctc aac ttt gaa gag atc gac 96 Glu Met Ile Glu Ala Pro Ser Gln Val Leu Asn Phe Glu Glu Ile Asp              20 25 30 tac aag gag atc gag gtg gaa gag gtt gtt gga aga gga gcc ttt gga 144 Tyr Lys Glu Ile Glu Val Glu Glu Val Val Gly Arg Gly Ala Phe Gly          35 40 45 gtt gtt tgc aaa gct aag tgg aga gca aaa gat gtt gct att aaa caa 192 Val Val Cys Lys Ala Lys Trp Arg Ala Lys Asp Val Ala Ile Lys Gln      50 55 60 ata gaa agt gaa tct gag agg aaa gcg ttt att gta gag ctt cgg cag 240 Ile Glu Ser Glu Ser Glu Arg Lys Ala Phe Ile Val Glu Leu Arg Gln  65 70 75 80 tta tcc cgt gtg aac cat cct aat att gta aag ctt tat gga gcc tgc 288 Leu Ser Arg Val Asn His Pro Asn Ile Val Lys Leu Tyr Gly Ala Cys                  85 90 95 ttg aat cca gtg tgt ctt gtg atg gaa tat gct gaa ggg ggc tct tta 336 Leu Asn Pro Val Cys Leu Val Met Glu Tyr Ala Glu Gly Gly Ser Leu             100 105 110 tat aat gtg ctg cat ggt gct gaa cca ttg cca tat tat act gct gcc 384 Tyr Asn Val Leu His Gly Ala Glu Pro Leu Pro Tyr Tyr Thr Ala Ala         115 120 125 cac gca atg agt tgg tgt tta cag tgt tcc caa gga gtg gct tat ctt 432 His Ala Met Ser Trp Cys Leu Gln Cys Ser Gln Gly Val Ala Tyr Leu     130 135 140 cac agc atg caa ccc aaa gcg cta att cac agg gac ctg aaa cca cca 480 His Ser Met Gln Pro Lys Ala Leu Ile His Arg Asp Leu Lys Pro Pro 145 150 155 160 aac tta ctg ctg gtt gca ggg ggg aca gtt cta aaa att tgt gat ttt 528 Asn Leu Leu Leu Val Ala Gly Gly Thr Val Leu Lys Ile Cys Asp Phe                 165 170 175 ggt aca gcc tgt gac att cag aca cac atg acc aat aac aag ggg agt 576 Gly Thr Ala Cys Asp Ile Gln Thr His Met Thr Asn Asn Lys Gly Ser             180 185 190 gct gct tgg atg gca cct gaa gtt ttt gaa ggt agt aat tac agt gaa 624 Ala Ala Trp Met Ala Pro Glu Val Phe Glu Gly Ser Asn Tyr Ser Glu         195 200 205 aaa tgt gac gtc ttc agc tgg ggt att att ctt tgg gaa gtg ata acg 672 Lys Cys Asp Val Phe Ser Trp Gly Ile Ile Leu Trp Glu Val Ile Thr     210 215 220 cgt cgg aaa ccc ttt gat gag att ggt ggc cca gct ttc cga atc atg 720 Arg Arg Lys Pro Phe Asp Glu Ile Gly Gly Pro Ala Phe Arg Ile Met 225 230 235 240 tgg gct gtt cat aat ggt act cga cca cca ctg ata aaa aat tta cct 768 Trp Ala Val His Asn Gly Thr Arg Pro Pro Leu Ile Lys Asn Leu Pro                 245 250 255 aag ccc att gag agc ctg atg act cgt tgt tgg tct aaa gat cct tcc 816 Lys Pro Ile Glu Ser Leu Met Thr Arg Cys Trp Ser Lys Asp Pro Ser             260 265 270 cag cgc cct tca atg gag gaa att gtg aaa ata atg act cac ttg atg 864 Gln Arg Pro Ser Met Glu Glu Ile Val Lys Ile Met Thr His Leu Met         275 280 285 cgg tac ttt cca gga gca gat gag cca tta cag tat cct tgt cag 909 Arg Tyr Phe Pro Gly Ala Asp Glu Pro Leu Gln Tyr Pro Cys Gln     290 295 300 <210> 6 <211> 303 <212> PRT <213> Homo sapiens <400> 6 Met Ser Thr Ala Ser Ala Ala Ser Ser Ser Ser Ser Ser Ser Ala Gly   1 5 10 15 Glu Met Ile Glu Ala Pro Ser Gln Val Leu Asn Phe Glu Glu Ile Asp              20 25 30 Tyr Lys Glu Ile Glu Val Glu Glu Val Val Gly Arg Gly Ala Phe Gly          35 40 45 Val Val Cys Lys Ala Lys Trp Arg Ala Lys Asp Val Ala Ile Lys Gln      50 55 60 Ile Glu Ser Glu Ser Glu Arg Lys Ala Phe Ile Val Glu Leu Arg Gln  65 70 75 80 Leu Ser Arg Val Asn His Pro Asn Ile Val Lys Leu Tyr Gly Ala Cys                  85 90 95 Leu Asn Pro Val Cys Leu Val Met Glu Tyr Ala Glu Gly Gly Ser Leu             100 105 110 Tyr Asn Val Leu His Gly Ala Glu Pro Leu Pro Tyr Tyr Thr Ala Ala         115 120 125 His Ala Met Ser Trp Cys Leu Gln Cys Ser Gln Gly Val Ala Tyr Leu     130 135 140 His Ser Met Gln Pro Lys Ala Leu Ile His Arg Asp Leu Lys Pro Pro 145 150 155 160 Asn Leu Leu Leu Val Ala Gly Gly Thr Val Leu Lys Ile Cys Asp Phe                 165 170 175 Gly Thr Ala Cys Asp Ile Gln Thr His Met Thr Asn Asn Lys Gly Ser             180 185 190 Ala Ala Trp Met Ala Pro Glu Val Phe Glu Gly Ser Asn Tyr Ser Glu         195 200 205 Lys Cys Asp Val Phe Ser Trp Gly Ile Ile Leu Trp Glu Val Ile Thr     210 215 220 Arg Arg Lys Pro Phe Asp Glu Ile Gly Gly Pro Ala Phe Arg Ile Met 225 230 235 240 Trp Ala Val His Asn Gly Thr Arg Pro Pro Leu Ile Lys Asn Leu Pro                 245 250 255 Lys Pro Ile Glu Ser Leu Met Thr Arg Cys Trp Ser Lys Asp Pro Ser             260 265 270 Gln Arg Pro Ser Met Glu Glu Ile Val Lys Ile Met Thr His Leu Met         275 280 285 Arg Tyr Phe Pro Gly Ala Asp Glu Pro Leu Gln Tyr Pro Cys Gln     290 295 300 <210> 7 <211> 204 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1) .. (204) <400> 7 caa agc ccg acc tta acc ctg cag tcc acc aac acg cac acg cag agc 48 Gln Ser Pro Thr Leu Thr Leu Gln Ser Thr Asn Thr His Thr Gln Ser   1 5 10 15 agc agc tcc agc tct gac gga ggc ctc ttc cgc tcc cgg ccc gcc cac 96 Ser Ser Ser Ser Ser Asp Gly Gly Leu Phe Arg Ser Arg Pro Ala His              20 25 30 tcg ctc ccg cct ggc gag gac ggt cgt gtt gag ccc tat gtg gac ttt 144 Ser Leu Pro Pro Gly Glu Asp Gly Arg Val Glu Pro Tyr Val Asp Phe          35 40 45 gct gag ttt tac cgc ctc tgg agc gtg gac cat ggc gag cag agc gtg 192 Ala Glu Phe Tyr Arg Leu Trp Ser Val Asp His Gly Glu Gln Ser Val      50 55 60 gtg aca gca ccg 204 Val Thr Ala Pro  65 <210> 8 <211> 68 <212> PRT <213> Homo sapiens <400> 8 Gln Ser Pro Thr Leu Thr Leu Gln Ser Thr Asn Thr His Thr Gln Ser   1 5 10 15 Ser Ser Ser Ser Ser Asp Gly Gly Leu Phe Arg Ser Arg Pro Ala His              20 25 30 Ser Leu Pro Pro Gly Glu Asp Gly Arg Val Glu Pro Tyr Val Asp Phe          35 40 45 Ala Glu Phe Tyr Arg Leu Trp Ser Val Asp His Gly Glu Gln Ser Val      50 55 60 Val Thr Ala Pro  65 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized primer sequence <220> <221> CDS <222> (1) .. (21) <400> 9 gaa ttc gga gga gga gga gga 21 Glu Phe Gly Gly Gly Gly Gly   1 5 <210> 10 <211> 7 <212> PRT <213> Artificial Sequence <400> 10 Glu Phe Gly Gly Gly Gly Gly   1 5 <210> 11 <211> 1149 <212> DNA <213> Artificial Sequence <220> <223> Fusion protein comprising kinase domain of human       TAK1 and TAK1 activating domain of human TAB1 <220> <221> CDS <222> (7) .. (1140) <400> 11 ggatcc atg tct aca gcc tct gcc gcc tcc tcc tcc tcc tcg tct tcg 48        Met Ser Thr Ala Ser Ala Ala Ser Ser Ser Ser Ser Ser Ser          1 5 10 gcc ggt gag atg atc gaa gcc cct tcc cag gtc ctc aac ttt gaa gag 96 Ala Gly Glu Met Ile Glu Ala Pro Ser Gln Val Leu Asn Phe Glu Glu  15 20 25 30 atc gac tac aag gag atc gag gtg gaa gag gtt gtt gga aga gga gcc 144 Ile Asp Tyr Lys Glu Ile Glu Val Glu Glu Val Val Gly Arg Gly Ala                  35 40 45 ttt gga gtt gtt tgc aaa gct aag tgg aga gca aaa gat gtt gct att 192 Phe Gly Val Val Cys Lys Ala Lys Trp Arg Ala Lys Asp Val Ala Ile              50 55 60 aaa caa ata gaa agt gaa tct gag agg aaa gcg ttt att gta gag ctt 240 Lys Gln Ile Glu Ser Glu Ser Glu Arg Lys Ala Phe Ile Val Glu Leu          65 70 75 cgg cag tta tcc cgt gtg aac cat cct aat att gta aag ctt tat gga 288 Arg Gln Leu Ser Arg Val Asn His Pro Asn Ile Val Lys Leu Tyr Gly      80 85 90 gcc tgc ttg aat cca gtg tgt ctt gtg atg gaa tat gct gaa ggg ggc 336 Ala Cys Leu Asn Pro Val Cys Leu Val Met Glu Tyr Ala Glu Gly Gly  95 100 105 110 tct tta tat aat gtg ctg cat ggt gct gaa cca ttg cca tat tat act 384 Ser Leu Tyr Asn Val Leu His Gly Ala Glu Pro Leu Pro Tyr Tyr Thr                 115 120 125 gct gcc cac gca atg agt tgg tgt tta cag tgt tcc caa gga gtg gct 432 Ala Ala His Ala Met Ser Trp Cys Leu Gln Cys Ser Gln Gly Val Ala             130 135 140 tat ctt cac agc atg caa ccc aaa gcg cta att cac agg gac ctg aaa 480 Tyr Leu His Ser Met Gln Pro Lys Ala Leu Ile His Arg Asp Leu Lys         145 150 155 cca cca aac tta ctg ctg gtt gca ggg ggg aca gtt cta aaa att tgt 528 Pro Pro Asn Leu Leu Leu Val Ala Gly Gly Thr Val Leu Lys Ile Cys     160 165 170 gat ttt ggt aca gcc tgt gac att cag aca cac atg acc aat aac aag 576 Asp Phe Gly Thr Ala Cys Asp Ile Gln Thr His Met Thr Asn Asn Lys 175 180 185 190 ggg agt gct gct tgg atg gca cct gaa gtt ttt gaa ggt agt aat tac 624 Gly Ser Ala Ala Trp Met Ala Pro Glu Val Phe Glu Gly Ser Asn Tyr                 195 200 205 agt gaa aaa tgt gac gtc ttc agc tgg ggt att att ctt tgg gaa gtg 672 Ser Glu Lys Cys Asp Val Phe Ser Trp Gly Ile Ile Leu Trp Glu Val             210 215 220 ata acg cgt cgg aaa ccc ttt gat gag att ggt ggc cca gct ttc cga 720 Ile Thr Arg Arg Lys Pro Phe Asp Glu Ile Gly Gly Pro Ala Phe Arg         225 230 235 atc atg tgg gct gtt cat aat ggt act cga cca cca ctg ata aaa aat 768 Ile Met Trp Ala Val His Asn Gly Thr Arg Pro Pro Leu Ile Lys Asn     240 245 250 tta cct aag ccc att gag agc ctg atg act cgt tgt tgg tct aaa gat 816 Leu Pro Lys Pro Ile Glu Ser Leu Met Thr Arg Cys Trp Ser Lys Asp 255 260 265 270 cct tcc cag cgc cct tca atg gag gaa att gtg aaa ata atg act cac 864 Pro Ser Gln Arg Pro Ser Met Glu Glu Ile Val Lys Ile Met Thr His                 275 280 285 ttg atg cgg tac ttt cca gga gca gat gag cca tta cag tat cct tgt 912 Leu Met Arg Tyr Phe Pro Gly Ala Asp Glu Pro Leu Gln Tyr Pro Cys             290 295 300 cag gaa ttc gga gga gga gga gga caa agc ccg acc tta acc ctg cag 960 Gln Glu Phe Gly Gly Gly Gly Gly Gln Ser Pro Thr Leu Thr Leu Gln         305 310 315 tcc acc aac acg cac acg cag agc agc agc tcc agc tct gac gga ggc 1008 Ser Thr Asn Thr His Thr Gln Ser Ser Ser Ser Ser Ser Asp Gly Gly     320 325 330 ctc ttc cgc tcc cgg ccc gcc cac tcg ctc ccg cct ggc gag gac ggt 1056 Leu Phe Arg Ser Arg Pro Ala His Ser Leu Pro Pro Gly Glu Asp Gly 335 340 345 350 cgt gtt gag ccc tat gtg gac ttt gct gag ttt tac cgc ctc tgg agc 1104 Arg Val Glu Pro Tyr Val Asp Phe Ala Glu Phe Tyr Arg Leu Trp Ser                 355 360 365 gtg gac cat ggc gag cag agc gtg gtg aca gca ccg taggtcgac 1149 Val Asp His Gly Glu Gln Ser Val Val Thr Ala Pro             370 375 <210> 12 <211> 378 <212> PRT <213> Artificial Sequence <400> 12 Met Ser Thr Ala Ser Ala Ala Ser Ser Ser Ser Ser Ser Ser Ala Gly   1 5 10 15 Glu Met Ile Glu Ala Pro Ser Gln Val Leu Asn Phe Glu Glu Ile Asp              20 25 30 Tyr Lys Glu Ile Glu Val Glu Glu Val Val Gly Arg Gly Ala Phe Gly          35 40 45 Val Val Cys Lys Ala Lys Trp Arg Ala Lys Asp Val Ala Ile Lys Gln      50 55 60 Ile Glu Ser Glu Ser Glu Arg Lys Ala Phe Ile Val Glu Leu Arg Gln  65 70 75 80 Leu Ser Arg Val Asn His Pro Asn Ile Val Lys Leu Tyr Gly Ala Cys                  85 90 95 Leu Asn Pro Val Cys Leu Val Met Glu Tyr Ala Glu Gly Gly Ser Leu             100 105 110 Tyr Asn Val Leu His Gly Ala Glu Pro Leu Pro Tyr Tyr Thr Ala Ala         115 120 125 His Ala Met Ser Trp Cys Leu Gln Cys Ser Gln Gly Val Ala Tyr Leu     130 135 140 His Ser Met Gln Pro Lys Ala Leu Ile His Arg Asp Leu Lys Pro Pro 145 150 155 160 Asn Leu Leu Leu Val Ala Gly Gly Thr Val Leu Lys Ile Cys Asp Phe                 165 170 175 Gly Thr Ala Cys Asp Ile Gln Thr His Met Thr Asn Asn Lys Gly Ser             180 185 190 Ala Ala Trp Met Ala Pro Glu Val Phe Glu Gly Ser Asn Tyr Ser Glu         195 200 205 Lys Cys Asp Val Phe Ser Trp Gly Ile Ile Leu Trp Glu Val Ile Thr     210 215 220 Arg Arg Lys Pro Phe Asp Glu Ile Gly Gly Pro Ala Phe Arg Ile Met 225 230 235 240 Trp Ala Val His Asn Gly Thr Arg Pro Pro Leu Ile Lys Asn Leu Pro                 245 250 255 Lys Pro Ile Glu Ser Leu Met Thr Arg Cys Trp Ser Lys Asp Pro Ser             260 265 270 Gln Arg Pro Ser Met Glu Glu Ile Val Lys Ile Met Thr His Leu Met         275 280 285 Arg Tyr Phe Pro Gly Ala Asp Glu Pro Leu Gln Tyr Pro Cys Gln Glu     290 295 300 Phe Gly Gly Gly Gly Gly Gln Ser Pro Thr Leu Thr Leu Gln Ser Thr 305 310 315 320 Asn Thr His Thr Gln Ser Ser Ser Ser Ser Ser Asp Gly Gly Leu Phe                 325 330 335 Arg Ser Arg Pro Ala His Ser Leu Pro Pro Gly Glu Asp Gly Arg Val             340 345 350 Glu Pro Tyr Val Asp Phe Ala Glu Phe Tyr Arg Leu Trp Ser Val Asp         355 360 365 His Gly Glu Gln Ser Val Val Thr Ala Pro     370 375 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized primer sequence <400> 13 attaggatcc atgtctacag cctctgccgc 30 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized primer sequence <400> 14 gccggaattc ctgacaagga tactgtaatg 30 <210> 15 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized primer sequence <400> 15 atgaattcgg aggaggagga ggacaaagcc cgaccttaac cctg 44 <210> 16 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized primer sequence <400> 16 attagtcgac ctacggtgct gtcaccacgc 30 <210> 17 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Artificially synthesized primer sequence <400> 17 tacccttatg atgtgccgga ttatgcg 27

[Brief description of drawings]

FIG. 1 TAB, a protein kinase region of TAK1
1 shows a schematic diagram of the TAK1 activation region of No. 1 and a fusion protein of TAK1 and TAB1. In the figure, "TAK1kd-TA
"B1ad" represents a fusion protein containing the protein kinase domain of TAK1 and the TAK1 activation domain of TAB1.

FIG. 2 is a view showing an amino acid sequence of a fusion protein of human TAK1 and human TAB1 and a nucleotide sequence of cDNA encoding the same. In the figure, "TAK1kd (1-303)" is T
It represents a portion containing a protein kinase region consisting of the 1st to 303rd amino acid residues of AK1. "TAB1ad
"(437-504)" represents a portion containing the TAK1 activation region consisting of amino acid residues 437 to 504 of TAB1.

FIG. 3 is a cell in which a fusion protein of human TAK1 and human TAB1 (and / or a partial polypeptide containing the protein kinase region of human TAK1 and / or a partial polypeptide containing the TAK1 activation region of human TAB1) is overexpressed. The figure showing the result of Western blotting of the cell extract obtained from and the result of detecting the protein kinase activity in the immunoprecipitated fraction. In the figure, "HA-
“TAK1kd-TAB1ad” is a protein kinase region of TAK1 and a T that have added a hemagglutinin antigen tag.
1 represents a fusion protein containing the TAK1 activation region of AB1.
“HA-TAK1kd” represents a partial polypeptide containing a protein kinase region of TAK1 to which a hemagglutinin antigen tag is added. "EGFP-TAB1ad" is EG
2 shows a partial polypeptide containing the TAK1 activation region of TAB1 fused with FP protein. "KW" is TAK1-T
This represents an inactive mutant protein in which the 63rd Lys residue of the AB1 fusion protein was replaced with Trp.

FIG. 4 shows Western blotting in cell extracts and immunoprecipitated fractions obtained from cells overexpressing a fusion protein of human TAK1 and human TAB1 (and / or a partial polypeptide containing the protein kinase region of human TAK1). The figure showing a result. In the figure, "HA-TAK1kd-
TAB1ad ”is a T that has a hemagglutinin antigen tag added.
Protein kinase region of AK1 and TA of TAB1
1 represents a fusion protein containing the K1 activation region. "XP-TA
"K1kd-TAB1ad" is a protein kinase region of TAK1 to which an Xpress antigen tag is added and TAB1.
3 shows a fusion protein containing the TAK1 activation region of Escherichia coli. "HA
-TAK1kd "represents a partial polypeptide containing the protein kinase region of TAK1 to which a hemagglutinin antigen tag is added.

FIG. 5: Fusion protein of human TAC1 and human TAB1 (and / or human TAK1 and / or human TAB
1) Activation of signal transduction system in cell extract and immunoprecipitation fraction obtained from cells overexpressing (1)
Or the figure which shows the result of having detected the protein kinase activity of IKK, and phosphorylation of p38. In the figure, "TAK1kd
-TAB1ad "represents a fusion protein containing the protein kinase domain of TAK1 and the TAK1 activation domain of TAB1. “KW” represents an inactive mutant protein in which the 63rd Lys residue of the TAK1-TAB1 fusion protein was replaced with Trp. "P-p38" represents phosphorylated p38.

FIG. 6: Fusion protein of human TAK1 and human TAB1 (and / or human TAK1 and / or human TAB
The figure which showed the result of having measured the production induction of IL-6 in the cell which overexpressed 1). In the figure, "TAK1kd-TAB
1ad ”is the protein kinase region of TAK1 and T
1 represents a fusion protein containing the TAK1 activation region of AB1.
“KW” represents an inactive mutant protein in which the 63rd Lys residue of the TAK1-TAB1 fusion protein was replaced with Trp.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) C12N 9/12 C12Q 1/48 Z C12Q 1/02 G01N 33/15 Z 1/48 33/50 Z G01N 33 / 15 33/53 D 33/50 M 33/53 33/566 C12N 15/00 ZNAA 33/566 5/00 AF Term (reference) 2G045 AA34 AA35 AA40 BA11 BB50 DA12 DA13 DA14 DA36 FB02 4B024 AA01 AA11 BA10 DA03 EA04 FA02 GA11 HA01 4B050 CC05 DD11 LL01 LL05 4B063 QA18 QQ27 QR77 QS31 4B065 AA93X AA99Y AB01 BA02 CA29

Claims (22)

[Claims] 1. A partial polypeptide comprising a region responsible for the protein kinase activity of TAK1 (TGF-β activated kinase 1) and a region responsible for the “function of activating TAK1” of TAB1 (TAK1 binding protein 1). The partial T-containing polypeptide is fused with a linker peptide, if necessary, and is a constitutively active mutant T
TAK1 and TAB1 that function as AK1
Fusion protein with. 2. TAB1 is attached to the C-terminal side of a partial polypeptide containing a region responsible for the protein kinase activity of TAK1.
2. The fusion protein according to claim 1, wherein the partial polypeptide containing a region responsible for the “function of activating TAK1” is fused via a linker peptide, if necessary. 3. TAK1 and TAB1 are human TAK1
And the fusion protein according to claim 1, which is human TAB1. 4. A partial polypeptide containing a region responsible for the protein kinase activity of TAK1 has the following (A) to
The fusion protein according to claim 1, 2 or 3, which is a polypeptide selected from (E). (A) A polypeptide having the amino acid sequence represented by SEQ ID NO: 6. (B) 1 in the amino acid sequence shown by SEQ ID NO: 6
Alternatively, a polypeptide consisting of an amino acid sequence in which a plurality of amino acids are deleted, substituted or added. (C) A polypeptide having one or more conservative amino acid substitutions as compared with the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 6. (D) A polypeptide having an amino acid level homology of about 90% or more with the amino acid sequence represented by SEQ ID NO: 6. (E) A polypeptide encoded by a nucleic acid capable of hybridizing with a nucleic acid having the nucleotide sequence represented by SEQ ID NO: 5 under stringent conditions or a complement thereof. 5. The partial polypeptide containing a region responsible for the “function of activating TAK1” of TAB1 is a polypeptide selected from the following (A) to (E): The fusion protein according to 3 or 4. (A) A polypeptide having the amino acid sequence shown by SEQ ID NO: 8. (B) 1 in the amino acid sequence shown in SEQ ID NO: 8
Alternatively, a polypeptide consisting of an amino acid sequence in which a plurality of amino acids are deleted, substituted or added. (C) A polypeptide having one or more conservative amino acid substitutions as compared with the polypeptide consisting of the amino acid sequence represented by SEQ ID NO: 8. (D) A polypeptide having an amino acid sequence homology of about 90% or more with the amino acid sequence represented by SEQ ID NO: 8. (E) A polypeptide encoded by a nucleic acid capable of hybridizing with a nucleic acid having the nucleotide sequence represented by SEQ ID NO: 7 under stringent conditions or a complement thereof. 6. The linker peptide comprises the following (A) to
3. The method according to claim 1, wherein the method is selected from (C).
The fusion protein according to 3, 4 or 5. (A) A peptide consisting of the amino acid sequence shown by SEQ ID NO: 10. (B) in the amino acid sequence shown by SEQ ID NO: 10,
A polypeptide comprising an amino acid sequence in which one or more amino acids are deleted, substituted or added. (C) About 90% of the amino acid sequence represented by SEQ ID NO: 10
A polypeptide having the above amino acid-level homology. 7. The protein kinase activity of TAK1 is M
The fusion protein according to claim 1, which has APKKK activity. 8. The fusion protein according to claim 1, wherein the “function of activating TAK1” of TAB1 is a function of phosphorylating TAK1 to convert it into an active form having MAPKKK activity. 9. A nucleic acid encoding the fusion protein according to claim 1. 10. A nucleic acid encoding a fusion protein of TAK1 and TAB1 which functions as a constitutively active mutant TAK1, which encodes a partial polypeptide containing a region responsible for the protein kinase activity of TAK1. , A nucleic acid encoding a partial polypeptide containing a region responsible for the “function of activating TAK1” of TAB1, if necessary via a nucleic acid encoding a linker peptide. 11. A nucleic acid encoding a partial polypeptide containing a region having a “function of activating TAK1” of TAB1 is provided downstream of a nucleic acid encoding a partial polypeptide containing a region having a protein kinase activity of TAK1. The nucleic acid according to claim 10, which is bound via a nucleic acid encoding a linker peptide, if necessary. 12. A nucleic acid encoding a partial polypeptide containing a region responsible for TAK1 protein kinase activity is selected from the following (a) and (b):
11. The nucleic acid according to claim 10, wherein the nucleic acid encoding the partial polypeptide containing the region responsible for the "TAK1 activating function" of B1 is selected from the following (c) and (d). (A) A nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 5. (B) A nucleic acid which hybridizes with a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 5 under stringent conditions or its complement. (C) A nucleic acid having the nucleotide sequence represented by SEQ ID NO: 7. (D) A nucleic acid that hybridizes with a nucleic acid consisting of the nucleotide sequence represented by SEQ ID NO: 7 under stringent conditions or a complement thereof. 13. A recombinant vector containing the nucleic acid according to claim 9 or 10. 14. The recombinant vector according to claim 13, which is an expression vector. 15. A host cell into which the recombinant vector according to claim 13 has been introduced. 16. A nucleic acid encoding the fusion protein according to claim 1 or an expression vector containing the nucleic acid is introduced into a cell, the cell is cultured, and the fusion protein is collected from the culture. The method for producing the fusion protein according to claim 1. 17. Use of the fusion protein according to claim 1,
A method for assaying the effect of a test compound on the activity of AK1. 18. T using the fusion protein according to claim 1.
A method for assaying the effect of a test compound on the function of AK1, comprising the following steps (i), (ii) and (iii). (I) contacting the fusion protein with a test compound and measuring the protein kinase activity of the fusion protein in the presence of the test compound; (ii) more than in the absence of the test compound or in (i) Measuring the protein kinase activity of the fusion protein in the presence of a small amount; and (iii) comparing the activity measurement results of step (i) and step (ii), and based on the results, the test compound T
A step of determining whether or not it has an action of inhibiting the activity of AK1, or the degree thereof. 19. A method for assaying the effect of a test compound on the function of TAK1 using the test cells expressing the fusion protein according to claim 1. 20. A method for assaying the action of a test compound on the function of TAK1 using the test cell in which the fusion protein according to claim 1 is expressed, which comprises the following (i):
A method comprising the steps (ii) and (iii). (I) contacting a test cell expressing the fusion protein according to claim 1 with a test compound, in the presence of the test compound,
A step of detecting the activation of the signal transduction system in the test cell; (ii) the activity of the signal transduction system in the test cell in the absence of the test compound or in the presence to a lesser extent than in (i) And (iii) comparing the detection results of step (i) and step (ii), and based on the result, whether the test compound has an action of inhibiting the activity of TAK1, or The process of determining the degree. Here, the activation of the signal transduction system is the activation of the signal transduction system involving TAK1. 21. Activation of the signal transduction system is caused by TAK1.
The activation of a signal transduction system involved in, which is selected from the following (A) and (B):
The method described in 0. (A) Activation of JNK, p38 or IKK; and (B) Induction of production of inflammatory cytokines selected from IL-6, IL-1 and TNF. 22. The method according to claim 17, which is used for identifying or screening a compound having a TAK1 inhibitory effect.
22. The method according to claim 1.