WO1999040202A1 - NF-λB ACTIVATION INHIBITORS TARGETING ON TAK1 AND METHOD FOR IDENTIFYING THE SAME - Google Patents

Abstract

Nuclear factor kappa B (NF-λB) activation inhibitors focusing on a novel transfer molecule; preventives/remedies for autoimmune diseases, etc.; and novel methods for identifying or screening the same. A method for identifying or screening NF-λB activation inhibitors which involves the step of examining the effect of a test substance of modulating the function of TGF-β activated kinase 1 (TAK1); a method for identifying or screening remedies and/or preventives for autoimmune diseases or intractable diseases with inflammation which involves the step of examining the effect of a test substance of modulating the function of TAK1 in the NF-λB activation pathway; and novel NF-λB activation inhibitors, and remedies/preventives for autoimmune diseases, intractable diseases with inflammation, etc. which are screened or identified by the above methods.

Description

 Specification

NF-κΒ activation inhibitor targeting TAK 1 and method for identifying the same

 The present invention relates to an NF- (Nuclear Factor kappa B) activation inhibitor, and an agent for treating or preventing an autoimmune disease or an intractable disease exhibiting inflammatory symptoms. Further, the present invention relates to these novel screening methods and identification methods. Background art

 NF- / B, known as one of the transcription factors, plays an important role in the transcriptional regulation of various genes involved in inflammatory and immune responses. Normally, in the cytoplasm, NF— is a force that exists as an inactive complex that binds to the regulatory protein I Β と. When a certain stimulus is given to cells, I / c Β modifies and degrades. It is activated by detaching from the receiving complex. The NF-II thus activated is translocated into the nucleus, and the specific nucleotide sequence (NF-/-consisting of about 10 bases) present in the upstream region (enhansa-one region) of various genes on the genome DN- cB binding sequence) to activate gene transcription. The NF- / cB binding sequence is present not only in the immunoglobulin gene but also in the upstream region of genes such as IL-1 and inflammatory cytokines such as tumor necrosis factor, interferon, and cell adhesion factor. A: B is involved in inflammation and immune response through induction of expression of these genes.

 NF- is also involved in the pathogenesis of autoimmune diseases and inflammatory diseases. Drugs that inhibit the activation of NF_ / cB are used in autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, systemic scleroderma) Disease, Behcet's disease, periarteritis nodosa, ulcerative colitis, glomerulonephritis, etc., intractable diseases with inflammatory symptoms (osteoarthritis, atherosclerosis, psoriasis, atopic dermatitis, etc.) It is known to be effective in treating and preventing various diseases such as viral diseases, endotoxin shock, and sepsis. For the development of prophylactic drugs for the treatment of these diseases, research and development of novel NF- / cB activation inhibitors is underway (Kopp et al., Science, 265, 956, 1994). Baeuerle et al., Advances in Immunology, Vol. 65, pp. 111-137, 1997; JP-A-7-291859; and JP-A-9-227561).

Conventional screening studies for NF-A: B activation inhibitors involve screening or identifying cells in vitro in the presence (or absence) of a stimulus, in the presence of a test drug, or in the presence of a test drug. A method of detecting the activity of NF-AB by culturing in the absence of NF-AB is generally used. However, the signal transduction pathway from the time when a cell receives a certain stimulus (signal) to the activation of NF- / cB is the existence of many steps involving various transduction molecules such as protein kinases. it is conceivable that. Therefore, for more efficient drug discovery research, it is desirable to clarify the key molecules that play a key role and to establish a new drug screening method that focuses on them. However, the mechanism of activation of NF- / B is based on several transmission factors (TRAF 2 (TNF-a receptor associated factor 2) and MAP KKK (mitogen-activated protein kinase kinase kinase) NIK ( NF-κΒ-inducing kinase), I / cB kinase (IKK), ubiquitin-conjugating enzyme, 26S proteosome, etc. are being identified, but little by little (Nikolai et al., Nature, 385, 540-544; Maniatis, Science, 278, 818-819, 1997; Baeuerle et al., Advances in Immunology 65, 111-137, 1997); There are many unclear points, so a more advanced mechanism was elucidated, and a screening method focused on new transmitting molecules was desired.

 On the other hand, TGF-activated kinase 1 (Transforming growth factor-β-activated kinase 1; also referred to as “TAK 1”) was discovered as one of mammalian MAP KKKs (Yamaguchi et al. Science, vol. 270, pp. 2008-2011, 1995; JP-A-9-163990). TAK 1 is TGF— /?

 (transforming growth factor-β) Activates the 1 ΡΑ-1 promoter. In addition, since it is activated by TGF-, which is the origin of the nomenclature, it has been thought that TGF- acts in the intracellular signal transduction pathway by a member of the superfamily. .

 In addition, TAK1 becomes active by binding (interacting) with TAK1 binding protein 1 (TAB1), and it can function as MAPKKK in the signal transduction pathway. It is known (Shibuya et al., Science, Vol. 272, Vol. 1179-: 1182, 1996). However, nothing was known about the association between TAK1 and NF— / cB activation.

 An object of the present invention is to provide a method for identifying an NF-activation inhibitor and a method for screening, which focus on a novel transmitting molecule. Another object of the present invention is to provide a novel identification method and a screening method for therapeutic and prophylactic agents for autoimmune diseases, intractable diseases exhibiting inflammatory symptoms, and the like.

It is still another object of the present invention to provide a novel NF- / cB activity inhibitory drug obtained by the above method, and a therapeutic or prophylactic drug for autoimmune diseases, intractable diseases showing inflammatory symptoms, and the like. The present inventors isolated three allelic variants of human TAK1 cDNA, and furthermore, in a study using these, increased expression of human TAK1 together with TAB1 (over expression). In addition, TAK1 interacts with TAB1 and interacts with IKK (I / cB kinase) complex to activate it. In addition, it was found that the mutant TAK1 which has lost kinase activity inhibits NF- / B activation. Based on these findings, TAK1 force ^s , an important signaling molecule in the signal transduction pathway (NF- / B activation pathway) leading to NF-κB activation, and drugs that suppress the function of TAK1 DISCLOSURE OF THE INVENTION that led to completion of the present invention, which was found to be a NF- / B activation inhibitor

 That is, the present invention provides a method for identifying or screening for a NF—; cB activation inhibitor, which comprises a step of assaying the modulatory effect of a test substance on the function of TAK 1 (TGF—; reactive kinase 1 :). Is the way.

 Further, the present invention provides a therapeutic and / or prophylactic agent for an autoimmune disease or an intractable disease exhibiting inflammatory symptoms, which comprises a step of assaying a modulatory effect of a test substance on the function of TAK1 in the NF-κΒ activation pathway. An identification method or a screening method. Furthermore, the present invention is a novel NF-activation inhibitor selected or identified by the above-mentioned method, and a therapeutic or prophylactic agent for an autoimmune disease, an intractable disease exhibiting inflammatory symptoms, or the like. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 shows a comparison of the amino acid sequences of mouse TAK1 (mTAK1) and three kinds of human ΤΔ1 (hTAKla, hTAK1b and hTAK1c); (C) A diagram showing the results of electrophoresis showing NF- / cB activation (nuclear translocation of NF-KB in gel shift assay) in cells in which TAK1 and TAB1 have been enhanced in expression;

 Fig. 3 shows NF- / cB activation (luciferase activity in reporter attase) of cells in which expression of human TAK1 and TAB1 was enhanced; Fig. 4 shows mutant humans. Diagram showing suppression of NF-KB activation in cells expressing TAK1 (results from gel shift to (A) and reporter to (B));

 Figure 5 shows the results of immunoblotting of immunoprecipitated fractions containing TAK1 obtained from cells with enhanced expression of human TAK1 (intracellular interaction between TAK1 and TAB1).

Replacement form (Rule 26) P

 Four

Figure);

 Figure 6 shows the results of kinase assay of the immunoprecipitated fraction containing TAK1 obtained from cells with enhanced expression of human TAK1 (autophosphorylation of TAB1 by TAK1). ;

 Fig. 7 shows the results of immunoplating of immunoprecipitated fractions containing TAK1 and cell lysates obtained from cells with enhanced expression of human TAK1 (interaction between TAK1 and IKK in cells). Figure showing;

 Fig. 8 shows the results of IKK kinase assay (activation of the IKK complex by TAK1) of immunoprecipitated fractions containing IKK obtained from cells with enhanced expression of human TAK1. Figure; and

 FIG. 9 is a schematic diagram showing the function of TAK 1 in the NF-κB activation pathway (in the figure, TRAF2 indicates TNF — << receptor 1 * association factor-12, IKK indicates I / B Kinase, NIK is NF- / B-inducing kinase, NEMO is NF-ΛΓ ΛΓEssential. 11:? 11 111: Complex 'associated' protein, Respectively). BEST MODE FOR CARRYING OUT THE INVENTION

 TAK1 used in the present invention may be derived from any species, for example, derived from mammals such as human, mouse, rat, peacock, bush, dog, monkey, guinea pig and the like. No. Of these, the use of human-derived drugs is preferred for use in research and development of human therapeutics.

 The cDNA and amino acid sequences of TAK 1 have already been reported

 (Genbank / EMBL database Accession No. D76446; Yamaguchi et al., Science, Vol. 270, 2008-2011, 1995). In addition, SEQ ID NOs: 3, 4, and 5 in the sequence listing below include the DNA sequences of three allelic variants of human TAK1 cDNA newly discovered by the inventors and the TAK encoded by them. The amino acid sequence of 1 was shown.

 As described above, according to the findings uniquely found by the inventors, TAK1 functions as a main transmitter molecule in the NF- / cB activation pathway.

TAK 1 is activated by interacting (binding) with TAB 1 (TAK 1 binding protein 1) in cells, and becomes an active form showing protein kinase activity (MAPKKK activity). And phosphorylation of TAB1. TAK 1 also interacts functionally with the IKK complex. Activated TAK1 is thought to activate the IKK complex, function as a transfer molecule in the NF- activation pathway, and induce NF- / cB activation. Replacement form (Rule 26) 4/1

 FIG. 9 shows a schematic diagram of the function of TAK1 in the NF-KB activation pathway. In the present invention, the function of the test substance (particularly, the inhibitory or inhibitory action) is assayed by focusing on the function of TAK1 as described above (particularly, the function in the activation pathway of NF-KB).

Replacement form (Rule 26) Five

You. More specifically, such functions include, for example,

 (1) Interaction (binding) between TAK 1 and TAB 1

 (2) TAK 1 protein kinase activity,

 (3) Activation of IKK complex by TAK1 in cells,

 (4) NF-KB activation induced by intracellular TAK1,

And the like. The methods for assaying the effects of test substances on these functions are described below.

 (1) Test for effects on the interaction (binding) between TAK1 and TAB1

 For example, a method for directly detecting the binding between TAK1 and TAB1, a method for detection by co-immunoprecipitation, or a two-hybrid system (U.S. Pat. No. 283,173, P ぉ p ぴ 1 ^^ &1; 1 ^^ & Sci. USA, Vol. 88, pp. 9578-9958, 1991).

 When detecting the binding between TAK 1 and TAB 1, TAK 1 and TAB 1 may be used in their entirety, or at least a partial polypeptide containing a region involved in the binding of both. Good. Alternatively, use an appropriate tag label (glutathione-S-transferase, 6XHis, protein A, galactosidase, maltose-binding protein, flag antigen, Xpress antigen, HA antigen, Myc antigen, etc.) A fusion protein to which a partial polypeptide or the like has been added may be used.

 To directly detect the binding between TAK 1 and TAB 1, for example, use TAK 1 (or TAB 1) labeled with RI, etc., and attach an appropriate tag label to TAB 1 (or TAK 1) as necessary. The binding to the added fusion protein is detected directly in the presence of the test substance.

 In the case of co-immunoprecipitation, for example, an antibody that recognizes TAK1, TAB1, or a tag label added thereto is used for detection. First, a cell lysate is prepared from cells expressing TAK1 and TAB1, and the protein in the cell lysate is immunoprecipitated using an antibody that recognizes one of the proteins. By detecting the presence of the other protein in the immunoprecipitated fraction by a method such as immunoblotting, the interaction (coupling) of both proteins in the cell can be detected.

 The two-hybrid system is a method using marker expression of a reporter as a marker (US Pat. No. 5,283,173 and roc.Natl.Acad.Sci. USA, Vol. 88, 9578-9958). P. 1991).

When using the two-hybrid system, specifically, for example, (i) transcription 6

A gene encoding the first fusion protein consisting of the first region of the factor (DNA binding region or transcription activation region) and TAK1, (ii) the second region of the transcription factor (transcription activation region or DNA binding region) And (iii) a response element capable of binding to a DNA binding region of a transcription factor and a reporter gene linked downstream thereof, using a test cell comprising: This is incubated with the test substance, and the effect of the test substance on the binding of TAK1 and TAB1 is assayed using the expression of the reporter gene as an index. If the test substance inhibits the binding of TAK1 to TAB1, a decrease in reporter activity is observed in the presence of the test substance.

 The genes encoding the first and second fusion proteins can be designed and constructed using conventional gene recombination techniques.

 Host cells include, for example, yeast cells, insect cells, mammalian cells, and the like. Among them, yeast cells are advantageous in that they can be cultured easily and quickly, and that it is easy to apply genetic recombination techniques such as introduction of a foreign gene.

 The transcription factor may be any as long as it functions in the host cell. For example, yeast GAL4 protein (Keegan et al., Science, 231: 699-704, 1986, Ma et al., Cell, 48) Pp. 847-853, 1987), GCN4 protein (Hope et al., Cell, Vol. 46, 885-894, 1986), ADR1 protein (Thukral et al., Molecular and

Cellular Biology, Vol. 9, pp. 2360-2369, 1989).

 The response element may be a response element corresponding to a transcription factor. For example, when GAL4 is used as a transcription element, the response element may be UASg (upper region of galactose metabolism). Site: A GAL4-specific DNA sequence called upstream activation site of galactose genes) can be used.

 The reporter gene is also not particularly limited. For example, stable and active genes such as the E. coli-derived /? Galactosidase gene (1 ac Z), the bacterial transposon-derived chloramphenicol acetyltransferase gene (CAT), and the firefly-derived luciferase gene (Luc). An enzyme gene or the like which can be easily measured quantitatively can be suitably used.

 (2) Assay of the effect of TAK 1 on protein kinase activity

For example, a solution containing TAK 1 and TAB 1 and a solution containing ATP (labeled with RI or the like as necessary) are added to the solution containing the substrate protein, and the solution is added in the presence or absence of the test substance. An enzymatic reaction is performed, and protein kinase activity is measured using the incorporation of phosphoric acid into the substrate protein as an index to test the effect of the test substance. As for Chohachi 1: 1 and Choha81, those expressed in a suitable host cell (eg, a yeast cell, an insect cell, or a mammalian cell) by a gene recombination technique can be used. 7

In addition, the N-terminal region of TAK1 is involved in binding to TAB1, and TAK1 in which the N-terminus (22 amino acids on the N-terminal side) has been deleted has an active signal even when it does not bind to TAB1. Since it is known to act as a transfer molecule (Yamaguchi et al., And Shibuya et al.), Instead of using both TAK1 and TAB1, an active mutation that deletes the N-terminus and shows TAB1-independent activity You can use type TAK 1.

 As the substrate protein, TAK1 itself, TAB1, or a partial peptide thereof can be used. In addition, a molecule functionally interacting with IKK and the IKK complex or a partial peptide thereof can also be used as a substrate protein. In addition, XMEK 2 (SEK 1) of African Megafrog (Shibuya et al., Science, Vol. 272, No. 1179-: 1182, 1996), human MKK3 (Derijard et al., Science, Vol. 267, No. 682) ~ 685, 1995), Human MKK6 (MAPKK6)

 (Raingeaud et al., Molecular and Cellular Biology, Vol. 16, pp. 1247-1255, 1996; Moriguchi et al., Journal of Biological Chemistry, Vol. 271, pp. 13675-13679, 1996). Protein kinase kinase) and their partial peptides can also be used as substrates. When MAP KK is used as a substrate, TAK 1 protein kinase activity can be measured using MAP KK activation (increase in phosphorylation activity against MAPK (mitogen-activated protein kinase)) as an index.

 (3) Assay for the effect of intracellular TAK1 on IKK complex activation For example, use cells overexpressing TAK1 (more specifically, active TAK1) as test cells . Examples of such test cells include cells having enhanced expression of both TAK1 and TAB1, and can be obtained by introducing a vector for expression of TAK1 and TAB1 into an appropriate host cell. Alternatively, cells in which the N-terminus is deleted and the expression of an active mutant TAK1 exhibiting TAB1-independent activity is enhanced may be used.

 The test cells are cultured, for example, in the presence or absence of a test substance. A fraction containing the IKK complex is obtained from the cultured cells by immunoprecipitation or the like, and is used to perform an IKK kinase reaction, measure the activity of the IKK complex, and determine the effect of the test substance. Is tested.

 (4) Investigation of effects on intracellular TAK1-induced NF- / cB activation

 For example, as in (3) above, cells that enhance the expression of active TAK1 are used as test cells, and cultured in the presence or absence of a test substance. NF- / cB activation is detected by gel shift assay and the effect of the test substance is assayed.

Activated TAK1 expression-enhancing cells are control cells. 8

Expression of TAK1, which acts as a signal transduction molecule, is increased as compared to that of cells that have been transformed. Therefore, it is suitable as a test cell for selecting a test drug that acts on TAK1. For example, if the activity of suppressing the NF-κB activation is observed in both the cells in which the expression of activated TAK1 has been enhanced and the control cells, due to the presence of the test substance, the action point of the test substance is TAK1 Is likely to be

 In the above methods (1) to (4), as the cells used for the expression test, cell lines derived from mammals such as humans can be suitably used. For example, human HeLa cells, human Jurkat cells, and human cells Examples include THP-1 cells, monkey COS-7 cells, Chinese Hamster CHO cells, and the like. Of these, human HeLa cells, human Jurkat cells, and human THP-1 cells are preferred.

 In the above methods (1) to (4), when expression of TAK1, TAB1, or a fusion protein thereof is enhanced, it can be carried out using known sequence information and ordinary gene recombination techniques.

 The sequence information of TAK1 is as described above, and the cDNA sequence and amino acid sequence of TAB1 have also been reported (Genbank / EMBL database Accession No. U49928; Shibuya et al., Science, Vol. 1179-; 1182, 1996). TAB1 may be derived from any species, and includes, for example, those derived from mammals such as human, mouse, rat, porcupine, pig, dog, monkey, and guinea pig. Of these, the use of human-derived drugs is preferred for use in research and development of human therapeutics.

 CDNAs or genes such as TAK 1 and TAB 1 can be synthesized using primers and probes designed and synthesized based on information on known amino acid sequences and base sequences, using ordinary PCR (Polymerase Chain Reaction) and RT-PCR. It can be isolated by a method or screening from a DNA library. These can be incorporated into an appropriate vector to construct an expression vector.

The vector of all, a suitable promoter (eg, CMV promoter, SV 40 promoter, LTR promoter, Eronge one Chillon 1 _alpha promoter one, etc.) vector for animal cells (e.g., retroviral-based vectors, Papi port - Mavirus vector, vaccinia virus vector, SV40 vector, etc.) can be used.

For the test substances that show an inhibitory effect or an inhibitory effect on the function of TAK1 by the assay methods described in (1) to (4) above, the inhibitory effect on NF- / B activation can be further confirmed. I just need. Alternatively, treatment and / or prognosis in a known disease state model (in vitro or in vivo) of an autoimmune disease or a refractory disease exhibiting inflammatory symptoms. 9

What is necessary is just to check the prevention effect.

 NF- «B activation is performed by the known gel shift assay (Sakurai et al., Journal of Neurochemistry Vol. 59, pp. 2067-2075, 1992; Sakurai et al., Biochimica Biophysica Acta, Vol. 1316, pp. 132-138) , 1996), reporter method (Tanaka et al., Journal of Veterinary Medical Science, Vol. 59, pp. 575-579, 1997; EP-6252920-A; JP-A-7-2) (Japanese Patent Application Laid-Open No. 9-158559; Japanese Patent Application Laid-Open No. Hei 9-2217561).

 As a known pathological model (in vitro or in vivo) of an autoimmune disease or an intractable disease exhibiting inflammatory symptoms, a PHA-induced IL-12 production model using a human T cell line (Jurkat cells) (Wacholtz et al., Cell Immunology 135, pp. 285-298, 1991), LP S + IFN-y-induced iNOs production model using human macrophage cell line RAW2 64.7 (Xie et al., Science, vol. 256, vol. 225-228, 1992) and in vitro models such as TNF-induced IL-6 production model using human HeLa cells, rat adjuvant arthritis model (Connor et al., European Journal of Pharmacology, vol. 273). Pp. 15-24, 1995), Trinitrobenzenesulfonate-induced colitis model (Kiss et al., European Journal of Pharmacology, 336, 219-224, 1997) and rat Masugi nephritis model (Sakurai). Et al., Biochimica Biophysica Acta, Vol. 1316, 132-: 138, 1996).

 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, unless otherwise specified, each operation is referred to as “Molecular Cloning” (Sambrook, J., Fritsch, E.F. and Maniatis,

T., published by the Cold Spring Harbor Laboratory Press in 1989), or according to the instructions for a commercial product when using a commercially available reagent kit. Example

Example 1 Isolation of cDNA for human TAK 1 and TAB 1

 (1) Isolation of cDNA for human TAK1

 Poly (A) RNA was prepared from human cervical carcinoma-derived cell line HeLa (ATCC CCL2). This was designated as 錡, and a single-stranded cDNA was prepared using an oligo dT primer.

The single-stranded cDNA obtained above was designated as type III, and a human TAK1 cDNA fragment was obtained by PCR (polymerase chain reaction). Used for PCR Ten

The primer was the cDNA sequence of mouse TAK1 (Genbank / EMBL database Accession No. D76446; Yamaguchi, Science Vol. 270, pp. 2008-2011,

1995) and synthesized using a DNA synthesizer. As the sense primer, a 30-mer synthetic primer consisting of a sequence containing a recognition site for restriction enzyme cleavage (10 bases) and a translation initiation codon of mouse TAK1 cDNA and a downstream sequence (20 bases) (described later) Using SEQ ID NO: 1) in the sequence listing, as an antisense primer, a sequence containing a recognition site for restriction enzyme cleavage (10 bases) and a termination codon of mouse TAK1 cDNA and its upstream complementary sequence A 30-mer synthetic primer (SEQ ID NO: 2 in the sequence listing below) consisting of (20 bases) was used.

 Using the product obtained from the PCR (a mixture of cDNA fragments of about 1.7 kb) as a probe and screening a human lung cDNA library (Clontech), two types of human TAK CDNAs (hTAK1a-cDNA and hTAK1b-cDNA) containing the entire coding region of No. 1 were obtained.

 HeLa mRNA prepared in the same manner as described above was converted into type III, and cDNA (h) containing the entire coding region of human TAK1 was separately separated by RT-PCR (Reverse transcript-polymerase chain reaction). TAK1c—cDNA) was obtained. As a primer, the same synthetic primer as described above was used.

 The DNA sequences of the three obtained cDNAs were determined by the dideoxy method. For each cDNA (hTAK1a-cDNA, hTAK1b-cDNA and hTAK1c-cDNA), the DNA sequence of the region including the coding region and the human TAK1 (hTAK1 The amino acid sequences of 1, hTAK lb and hTAK lc) are shown in SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 5 in the sequence listing below.

 The cDNA sequences of hTAK la, hTAK lb and hTAK lc show homology in the coding regions of 91.7%, 87.6% and 86.8%, respectively, as compared to the cDNA sequence of mouse TAK1. Atsuta.

 hTAK1a consists of 579 amino acid residues. Compared to mouse TAK1, 4 amino acid substitutions were observed, and the homology in the amino acid sequence was 99.3%. hTAKlb is composed of 606 amino acid residues, and has an insertion of 27 amino acids which seems to be caused by a splicing mutation at the C-terminal side as compared with hTAK1a. HTAK1c is composed of 567 amino acid residues. Compared to hTAK1a, hTAK1b has a 27-amino acid insertion at the C-terminal, similar to hTAK1b, and 3 A 9 amino acid deletion was found.

A comparison of the amino acid sequences of the three human TAK1 and mouse TAK1 is shown in FIG. 11

 In addition, TAK1 derived from human T cell line Jurkat described in SEQ ID NO: 5 of JP-A-9-1163990 has one amino acid substitution (the 372nd A amino acid) when compared with the amino acid sequence of hTAK1a. rg → His), which is considered to be an allelic variant.

 (2) Isolation of cDNA for human TAB1

 Poly (A) RNA prepared from HeLa in the same manner as in (1) above was used as type III, and cDNA of human TAB1 was obtained by RT-PCR. The primer was designed with reference to the reported cDNA sequence of human TAB1 (Genbank / EMBL database Accession No. U49928; Shibuya et al., Science. Vol. 272, pp. 1179-1182, 1996). And synthesized on a DNA synthesizer. As a sense primer, a 30-mer synthetic primer consisting of a sequence containing a recognition site for restriction enzyme cleavage (10 bases) and a translation initiation codon of TAB1 cDNA and a sequence downstream thereof (20 bases) (later). Using SEQ ID NO: 6) in the Sequence Listing, the antisense primer includes a sequence containing a recognition site for restriction enzyme cleavage (10 bases) and the termination codon of TAB1 cDNA and its upstream complementary sequence (20 bases). A 30-mer synthetic primer consisting of bases) (SEQ ID NO: 7 in the sequence listing below) was used.

 The DNA sequence of the obtained cDNA fragment was determined, and it was confirmed that it contained the entire coding region of known human TAB1. Example 2 Detection of NF-A: B Activity in Cells with Enhanced TAK1 Expression (1) Obtaining Cells with Enhanced Human TAK1 Expression

 Using the three types of human TAK1c DNAs obtained in (1) of Example 1 described above, partial fragments containing their coding regions (EcoRI-Nhel fragments of hTAK1a-cDNA, hTAK1b-c EcoRI-Nhel fragment of DNA and EcoRI-Xbal fragment of hTAK1c-cDNA) were digested with EcoRI-Xbal of vector eukaryotic expression vector pcDNA3.1 (+) (Invitrogen). The recombinant plasmid for expression of TAK1 was prepared by integrating into the site.

 In addition, using the human TAB1cDNA obtained in (2) of Example 1 described above, a partial fragment (Hindlll-EcoRI fragment) containing the coding region was ligated into the expression vector plasmid PCDNA3.1 (+). Recombinant plasmid for TAB1 expression was prepared by incorporating it into the Hindlll-EcoRI cleavage site.

The recombinant plasmid for TAK1 expression was transfected (transient transfection) into HeLa cells together with the recombinant plasmid for TAB1 expression or alone. At this time, the transfection was performed using a cationic ribosome for transfection (trade name: LipofectAMINE, manufactured by Life Technologies). 12

 Thus, cells with enhanced TAK1 expression or cells with enhanced TAK1-TAB1 expression were obtained. These cells were cultured in Dulbecco's Eagle's medium (Gibco) containing high glucose supplemented with 10% fetal serum, penicillin (100 units Zml) and streptomycin (100 gZml). .

 (2) Gersif Totsusee

 Using the TAK1 expression-enhanced cells and TAK1-TAB1 coexpression-enhanced cells obtained in (1) above, literature (Sakurai et al., Journal of Neurochemistry Vol. 59, pp. 2067-2075, 1992; Sakurai et al. , Biochim. Biophys. Acta, Vol. 1316, pp. 132-138, 1996), and gel-shifting was performed as follows. That is, after transfection, the cells were cultured, and 24 hours later, a nuclear extract was prepared from the cells.

 This nuclear extract (5 / g) and a detection probe labeled with RI were combined with a binding buffer (20 mM HEPES (pH 7.9), 0.3 mM EDTA, 0.2 mM EGTA, 80 mM NaCl, 10% glycerol, 2 g After binding reaction at room temperature for 30 minutes in / ml poly [dI-dC]), the reaction solution was subjected to polyacrylamide gel electrophoresis. After the gel was dried under reduced pressure, NF- / cB bound to the probe was detected by autoradiography. In addition, as a control, 0ct-] (Octamer-1) is a constitutively expressed transcription factor.

(Verrijzer et al., Genes and Developments Vol. 4, pp. 1964-1974, 1990).

As a detection probe, a double-stranded synthetic DNA labeled with ³² P was used. As the sequence of the NF-κNF detection probe, the same sequence as the NF-A: B binding sequence present in the LTR (Long Terminal Repeat) of HIV was used. In addition, oligo nucleotides containing the consensus sequence AGCTAAAT were used as the sequence of the Oct-1 detection probe.

 As described above, as a result of examining NF-Λ: B activation by gel shift assay using NF- / cB nuclear translocation as an index, as shown in FIG. 2, human TAK1 (hTAKla, hTAKl When b or hTAKlc) was enhanced with TAB1, expression of NF-<< B into the nucleus was observed, and activation of NF- / cB was observed. Such a result was observed when any of hTAKla, hTAKlb and hTAK1c was used as human TAK1, but the activation of NF-zcB was particularly remarkable in hTAK1b. .

On the other hand, in cells in which expression of only human TAK1 was enhanced, NF- / cB activation was not observed. In addition, 0 ct-1 detected as a control protein was not affected by the enhanced expression of TAK1 and / or TAB1, and was expressed in a straightforward manner. 13

 As described above, activation of NF- / cB was observed in association with enhancement of the action of human TAK1, and thus TAK1 was expressed in the signal transduction pathway leading to the activation of NF-κB. It turned out to play a major role as a transfer molecule.

 (3) Reporter Atsushi (Luciferase Atsee)

 According to the method described in the literature of Tanaka et al. (Tanaka et al., Journal of Veterinary Medical Science, Vol. 59, pp. 575-579, 1997), a reporter assay (Lucifera zetasii) was performed as follows.

 First, a reporter plasmid (p (kB) 4-Luc) was prepared by incorporating an oligonucleotide linked to four NF-KB binding sequences (GGGGACTTTCC) upstream of the firefly luciferase gene (Luc).

 Next, the recombinant plasmid for TAK1 expression, together with the recombinant plasmid for TAB1 expression, if necessary, is transfected into HeLa cells (transient) according to the method described in (1) above. Transfection (transient transfection). However, the reporter plasmid (p (kB) 4-Luc) obtained above was used for the transfusion.

 Thus, transfectants containing reporter plasmid and recombinant plasmid for TAK1 expression (and recombinant plasmid for TAB1 expression) were obtained. After culturing the obtained transfectant for 48 hours, the luciferase activity was measured for the extract prepared by lysing the cells. The luciferase activity was measured using a luciferase zea kit, Pitka Gene (trade name, manufactured by Toyo Ink Co., Ltd.) and a chemiluminescence measuring device (trade name: MicroLumant LB96P, manufactured by Berthold Japan KK).

 As a result, as shown in FIG. 3, cells expressing only human TAK1 (hTAKla, hTAKlb or hTAKlc) had enhanced luciferase activity compared to cells containing only the vector. Little increase (ie, activation of NF- / cB) was observed. However, cells with enhanced expression of human TAK1 together with TAB1 showed a marked increase in luciferase activity (ie, the ability of NF-κΒ to activate) as compared to cells containing only vector-1. s accepted.

Thus, similar to the gel shift assay described above, the reporter assay (Lucifera assay) also increases the activity of ΝF—Κ with the enhanced action of human TAK1. observed, TAK 1 ^force? this and the force that is the major acts as a signaling ^molecule? has been confirmed.

In addition, it is thought that the effect of the test drug on TAK 1 and the effect on NF-Κ Β activation of the test drug can be simultaneously assayed by the reporter-Assy system using TAK 1 expression enhancing cells and control cells. Can be 14

Example 3 Binding system between TAK1 and TAB1 using a two-hybrid system

 The translation region of the human TAK1 cDNA obtained in (1) of Example 1 was cut out and ligated with the DNA binding region of the transcription factor GAL4 (amino acid residues 1 to 147 of GAL4). Into the multiple cloning site of the expression vector pGBT9 (Clontech, a vector for the yeast two-hybrid system) containing the DNA to be expressed. As a result, a plasmid PGBT9-TAK1 for expressing a fusion protein of the DNA binding region of GAL4 and human TAK1 is obtained.

 The translation region of the human TAB cDNA obtained in (2) of Example 1 was cut out, and this was ligated to a DNA encoding the transcriptional activation region of GAL4 (amino acid residue at position 768 to 881 of GAL4). Into the multiple cloning site of the expression vector pGAD424 (Clontech, vector for yeast two-hybrid system). Thereby, a plasmid pGAD424-TAB1 for expressing a fusion protein of the transcriptional activation site of GAL4 and TAB1 is obtained.

 The fusion protein expression plasmids pGBT9-TAKl and pGAD424-TABl obtained above are introduced into a host yeast cell strain SFY526 (manufactured by Clontech). The cell line SFY526 is a cell line in which the fusion gene of GAL1 and lacZ is integrated into the chromosome and has a deletion mutation in the GAL4 gene (Bartel et al., BioTechniques, Vol. 14, pp. 920-924). , 1993). Transformation is carried out by culturing in a synthetic medium lacking tryptophan and leucine, which are selective markers for each plasmid, to obtain a transformant in which both plasmids have been introduced.

 The yeast transformant obtained above is cultured in a liquid medium. At the time of culturing, the test substance is added (or not added) to the medium. After culturing for 4 to 5 hours, the yeast cells are recovered by centrifugation, and the binding (interaction) between TAK1 and TAB1 is detected using the /?-Galactosidase activity as an index.

 If the addition of the test substance decreases the concentration of -galactosidase activity in a concentration-dependent manner, the test substance is considered to have an effect of inhibiting the binding between TAK1 and TAB1. Example 4 TAK 1 MAP KKK Activity Detection System

Human TAK1 (or human TAK1 lacking the N-terminus (22 amino acids)) is expressed and purified in an insect cell system as follows. That is, an appropriate DNA sequence designed to add a tag peptide (6XHis or glutathione-S-transferase) using the translation region of human TAK1 cDNA obtained in (1) of Example 1 above. Baculovirus expression vector containing pAc HLT or DAC GH Fifteen

 Insert it into the multicloning site of LT (Pharmingen) to obtain human TAK1 expression plasmid. The resulting plasmid is introduced into host insect cells SF21, and the resulting transformed cells are cultured to express human peptide-tagged human TAK1 (or N-terminal-deleted human TAK1). Then, the extract is purified from the cell extract by affinity chromatography using the added tag peptide.

 In the same manner as described above, human TAB1 is expressed and purified in an insect cell system. In addition, human MKK3 and human MKK6 are expressed and purified as follows. First, according to the method of Moriguchi et al. (Journal of Biological Chemistry, Vol. 271, pp. 13675-13679, 1996), sequence information on human MKK3 (Genbank / EMBL database Accession No. L36719) Derijard et al., Science, 267, 682-685, 1995) and sequence information on human MKK6.

 (Genbank / EMBL database Accession Nos. U39656 and 39657;

Raingeaud et al., Molecular and Cellular Biology, Vol. 16, No. 1247-: p. 1255, 1996) and designed the primers by PCR method using these primers, and the whole translation region of human MKK3 and human MKK6. Or a cDNA containing a sequence in the vicinity of an amino acid residue phosphorylated by TAK1. Using these cDNAs, an E. coli expression vector pQE-30 (QIAGEN) containing an appropriate DNA sequence designed to add a tag peptide (6XHis or glutathione-S-transferase) was added. Or pGEX-2T (Pharmacia) to obtain a human MKK3 expression plasmid and a human MKK6 expression plasmid. The resulting plasmid is introduced into host Escherichia coli (such as the strain JM109), and the resulting transformed cells are cultured to express human MKK3 and human MKK6 to which the tag peptide has been added, respectively. The solution is purified by affinity chromatography using the added tag peptide.

Using human TAK 1 (or N-terminal-deleted human TAK 1) obtained above as an enzyme (MAPKKK) in combination with human TAB 1 as necessary, using human MKK3 or human MKK6 as a substrate, Perform the enzymatic reaction in the presence or absence of the test substance. Substrate protein is used immobilized by the pre-plate, the reaction is Tris buffer containing ³² P or ³³ P-labeled ATP100 M (20m Tris - HC1, pH7.5, 2mM EGTA, lOmM MgCl 2) in 30 ° C Do it. After the enzymatic reaction, the plate is washed, and the incorporation of ³² P or ³³ P-labeled ATP is measured by a scintillation counter to measure the enzymatic activity and determine the presence or absence of inhibition by the test substance. Example 5 Suppression of NF— / cB Activation in Cells Expressing Mutant TAK 1 Mutant TAK 1 (or wild-type TAK lacking kinase activity) 16

 The presence or absence of NF- / cB activation was detected using cells with enhanced expression of 1).

 (1) Construction of TAK1 and TAB1 expression vectors and transfection vector plasmid pFLAG—CMV2 is a vector for expressing flag antigen-tagged proteins in mammalian cells. is there. By incorporating the full-length human TAK1 (human TAK1a) cDNA into the EcoRI-XbaI restriction enzyme cleavage site of pFLAG-CMV2 (manufactured by Kodak), the flagged wild-type TAK1 ( An expression vector of F1ag-TAK1) was obtained.

 Also, using a mutation specialist kit (trade name: QuickChange site-directed mutagenesis kit; manufactured by Stratagene), mutagenesis was performed into the TAK1 translation region of the F1ag-TAK1 expression vector to perform various mutations. The mutant expression vector was obtained and the nucleotide sequence was determined. Thus, an expression vector of the flagged mutant TAK1 (F1ag-TAK1K63W) was obtained. Is the mutant TAK1 expressed by this expression vector the lysine residue at position 63 of wild-type TAK1? It has been replaced by a tributofan residue, and has lost the kinase activity of TAK 1.

 The flag-added wild-type or mutant TAK1 (Flag-TAK1 or F1ag-TAK1K63W) expression vector is transfected into HeLa cells alone or together with the TAB1 expression vector. It was clarified and transiently expressed. As a control, only the vector was used instead of the TAK1 expression vector. The transfection was carried out using a ribofectamine reagent (manufactured by Life Technologies), and the same TAB1 expression vector as that in Example 2 (1) was used.

 (2) Gersif Totsusee

 Using cells obtained by enhancing the expression of the flagged mutant TAK1 (or wild-type TAK1) obtained in the above (1) together with TAB1, a gel shift assay was performed in the same manner as in Example 2 (2). .

 As a result, as shown in (A) of FIG. 4, compared to cells transfected with the vector alone, the expression of wild-type TAK1 (F1ag-TAKl) together with TAB1 in cells with enhanced NF- / B nuclear translocation ability NF- / B activation was observed. In the case of mutant TAK1 lacking kinase activity (Flag-TAK1 K63W), expression with TAB1 did not enhance nuclear translocation of NF-KB.

 (3) Reporter Atsushi (Luciferase Atsee)

The expression vector of the mutant TAK1 (F1ag-TAK1K63W) obtained in (1) above was transfected into HeLa cells. However, the amount of F 1 ag—TAK 1 K63W expression vector used for transfection is 0 g, 0.03〃 g, and 0.1 / g, and the total DNA amount is the same (0. 1 / g) 17

It was adjusted with Vector plasmid as follows.

 In addition, in the case of transfection, the reporter plasmid (NF-A: p (kB) 4-Luc containing the NF-A: B binding sequence and the firefly luciferase gene) obtained in (3) of Example 2 was used. At the same time.

 Twenty-four hours after transfection, TNF-— was added to the medium to a final concentration of 20 ng / ml (control was without TNF-α). Further, after culturing for 5 hours, the cells were lysed and the luciferase activity was measured in the same manner as in Example 2, (3).

 The results are shown in Fig. 4 (Β). In the figure, the unmarked, 10, and ++ of TAK1 K63W indicate the amounts of F1ag-TAK1163W expression vector added at 0 g, 0.03 / g and 0.1 / g.) As shown in Fig. 4 (B), the increase in luciferase activity (activation of NF-) induced by the TNF- "stimulation was dependent on the dose of the mutant TAK1-expressing vector used in the transfectants. Dependent and suppressed.

 These results indicate that mutant TAK1 lacking kinase activity suppresses NF_ / cB activation when expressed in cells.

 This confirms that TAK1 plays a major role in the NF- / cB activation pathway, as well as the result of (2) above, and also inhibits TAK1 kinase activity and TAK1 activation. This strongly supports that the inhibiting drug suppresses the activation of NF-κB. Example 6 Interaction between TAK 1 and TAB 1 in cells

 As described below, the interaction (binding) between TAK1 and TAB1 in cells was detected by immunoprecipitation using cells in which TAK1 expression was enhanced together with TAB1.

 (1) Transfection of cells

 First, in the same manner as in Example 5, the expression vector of the wild-type TAK1 (Flag-TAK1) or the mutant TAK1 (F1ag-TAK1K63W) to which the flag was added was used alone. Alternatively, it was transfected into HeLa cells together with the TAB1 expression vector.

 (2) Immunoprecipitation and immunoplotting

Twenty-four hours after the transfection, the cells were collected, and a cell lysate was prepared as follows. That is, cells were dissolved in a cell lysis buffer (25 mM HEPES (pH 7.7), 0.3 M NaCl, 1.5 mM gCl ₂ , 0.2 mM EDTA, 0.1% Triton X-100, 20 mM β-glycerophosphate, O.lmM sodium orthovanadate, 0.5 mM 18

 After dissolving with PMSF, lmMDTT, 10 μg / ml aprotinin, 10 μg / ml leupeptine), the mixture was diluted three-fold, and cooled on ice for 10 minutes. After centrifugation, the supernatant was collected and used as a cell lysate in the following procedure.

The cell lysate obtained above was incubated with an anti-flag antibody (M5, manufactured by Kodak) for 1.5 hours on ice and further added with Protein G Sepharose (manufactured by Pharmacia) at 4 ° C for 1.5 hours. With gentle mixing, the immune complexes were adsorbed to the Protein G Sepharose beads. After collecting the beads by centrifugation, the beads are washed five times with a washing buffer (20 mM HEPES (pH 7.7), 50 mM NaCl, 2.5 mM MgCl ₂ , O.lmM EDTA, 0.05% Triton X-100). Was used in the following procedure as an immunoprecipitated fraction.

 The beads (immunoprecipitated fraction) were subjected to SDS-polyacrylamide gel electrophoresis, transferred to a PVDF (polyvinylidene difluoride) membrane, subjected to immunoblotting, and analyzed for TAB1 and TAB1 present in the immunoprecipitated fraction. TAK 1 was detected. Antibodies for detecting TAK 1 and TAB 1 include anti-TAK 1 antibody (M-17)

 (Manufactured by Santa Cruz Biotechnology) and an anti-TAB1 antibody (N-19) (manufactured by Santa Cruz Biotechnology) were used.

 FIG. 5 shows the results of immunoplating of the anti-flag immunoprecipitated fraction. The upper row shows the detection results with the anti-TAB1 antibody, and the lower row shows the detection results with the anti-TAK1 antibody.

 As shown in FIG. 5, TAB1 coexisted in the anti-flag immunoprecipitated fraction of cells in which wild-type TAK1 (F1ag-TAKl) was enhanced. In addition, TAB1 was also co-present in the immunoprecipitated fraction in cells in which the expression of mutant TAK1 (Flag-TAKlK63W) was enhanced in place of the wild type.

 Thus, TAB1 was co-immunoprecipitated with TAK1 (wild type and mutant type), indicating that TAK1 and TAB1 interact in cells.

 In addition, wild-type TAK 1 and TAB 1 both showed a slight decrease in mobility in SDS-polyacrylamide gel electrophoresis when co-expressed, but mutants without kinase activity In the case of type TAK 1, no such decrease in mobility was observed. This decrease in mobility was thought to reflect that both proteins were phosphorylated by functional interaction.

 (3) Test for the effect of the test substance

In the same manner as in the above (1), cells in which TAK1 and TAB1 have been enhanced are obtained, and cultured in the presence or absence of the test substance. For the cultured cells, the interaction (binding) between TAK1 and TAB1 is detected by immunoprecipitation in the same manner as in (2) above. Co-immunoprecipitation of TAK 1 and TAB 1 due to presence of test substance 19

The effect of the test substance on the TAK1 and TAB1 interaction (binding) of the test substance is determined by determining whether the test substance is reduced. Example 7 Autophosphorylation by TAK1 and TAB1 phosphorylation

 TAK1 immunoprecipitated from cells with enhanced expression of TAK1 together with TAB1 was subjected to kinase assay to detect autophosphorylation by TAK1 and phosphorylation of TAB1 as follows. .

 (1) Cell transfection and immunoprecipitation

 First, in the same manner as in Example 5, the expression vector of the flagged wild-type TAK1 (Flag-TAK1) or mutant TAK1 (F1ag-TAK1K63W) was used alone or in TAB. The cells were transfected into HeLa cells together with one expression vector. A cell lysate was prepared from the cells 24 hours after the transfusion in the same manner as in Example 6, and immunoprecipitated with an anti-flag antibody.

 (2) Kinase Atsushi

 Using the anti-flag immunoprecipitation fraction obtained above, an in vitro kinase reaction was performed as follows.

 That is, the immunoprecipitated fraction was treated with a 30/1 kinase buffer (20 mM HEPES (pH

7.6), 20 mM MgCl _{2 2} mM DTT, 20 μM ATP, 20 mM β-glycerophosphate,

In addition to 20 mM disodium p-nitrophenylphosphate and O.lmM sodium orthovanadate ^ 3 / Ci [y- ^: 32P] ATP), the mixture was incubated at 30 ° (:, 30 minutes.) After the reaction was completed, the reaction solution was subjected to SDS-polyacrylamide gel. The gel was subjected to electrophoresis, and the gel after electrophoresis was subjected to autoradiography.

 As a result, as shown in Fig. 6, wild-type TAK 1 (Flag-TAK1) and T

In the anti-flag immunoprecipitation fraction of cells in which both expression of AB1 was enhanced, phosphorylation of TAK1 (autophosphorylation) and phosphorylation of TAB1 were observed. However, in the immunoprecipitated fraction of cells in which the expression of only wild-type TAK 1 was enhanced, TAK 1 and TAB

No phosphorylation of 1 was observed. In addition, mutant T lacking kinase activity

Regarding AK1, phosphorylation was not observed even when expression was enhanced together with TAB1.

 From these results, it was considered that TAK1 was activated by coexisting with TAB1, and that autophosphorylation of TAK1 and phosphorylation of TAB1 by TAK1 occurred. Example 8 Interaction between TAK 1 and IKK in cells

Use cells with enhanced expression of TAK1 together with IKK as described below. 20

The interaction (binding) between TAK1 and IKK in the cells was detected by the epidemicipitation method.

 (1) Cell transfection

 First, the expression vector of IKK was integrated by incorporating the cDNAs of human IΚΚ "and human IΚΚ /? Into vector-plasmid pcDNA3.1 (+) HisB (Invitrogen). Human IKKa (Genbank / EMBL accession No. AF 012890; Cell, Vol. 90, pp. 373-383, 1997), and human IΚΚ /? (Genbank / EMBL accession No. AF029684; Science) , 278, 866-869, 1997) was obtained from human monocyte-derived cell line (THP-1) mRNA by reverse transcription PCR (Reverse transcriptase-polymerase chain reaction). Was used.

 By these IΚΚ expression vectors (IΚΚα expression vector and I I /? Expression vector), IKK (Xpress-IKKα or Xpress-IΚΚ / ?) Can be expressed.

 Next, in the same manner as in Example 5, the expression vector of the flagged wild-type TAK1 (Flag-TAK1) was transfected into HeLa cells alone or together with the TAB1 expression vector. At this time, the expression vector of IKK (Xpress-ΙΔα or Xpress-IKK / 3) obtained above was simultaneously added (or not added) and transfection was performed.

 (2) Immunoprecipitation and immunoplotting

 A cell lysate was prepared from the cells 24 hours after the transfusion and immunoprecipitated with an anti-flag antibody in the same manner as in Example 6. After immunoprecipitation fraction and cell lysate were subjected to SDS-polyacrylamide electrophoresis, immunoblotting was performed to detect IKK and TAK1.

 Antibodies for detecting IKK (Xpress—IKK «and /?) And ΤAΚ1 include anti-Xpress antibody (M—21) (manufactured by Santa Cruz Biotechnology) and anti-TAK1 antibody (M-17) (manufactured by Santa Cruz Biotechnology) was used.

 FIG. 7 shows the results of immunoblotting of the anti-flag immunoprecipitated fraction.

The upper row shows the results of detection of the anti-flag immunoprecipitated fraction with anti-Xpress antibody, the middle row shows the results of detection of the cell lysate with anti-Xpress antibody, and the lower row shows the detection of anti-flag immunoprecipitated fraction with anti-TAK1 antibody The result.

As shown in FIG. 7, in cells where expression of TAK1 (Flag-TAK1) and IKK (Xpress-IKKH or Xpress-IKK / 9) was enhanced and TAB1 was not enhanced, IKK was detected in the anti-flag immunoprecipitated fraction. this twenty one

Thus, IKK (IKK. And /?) Was co-immunoprecipitated with TAK1, indicating that TAK1 and IKK (IKK "and /?) Interact intracellularly. However, IKK was not detected in the anti-flag immunoprecipitated fraction in cells that had enhanced expression of TAB1 together with TAK1 and IKK, indicating that TAK1 was not activated. It was considered that in the state, the ability to generate a stable bond with IKK in the cell, and in the state activated by TAB 1, the stable bond with the bond to IKK in the cell was not observed.

 In addition, as a result of immunoblotting of the immune extract of the cell lysate, TAK1 and IAK1

In cells in which TAB1 expression was enhanced along with KK, the mobility of IKK (IKKa and β) in SDS-polyacrylamide gel electrophoresis tended to decrease slightly. On the other hand, such a tendency was not observed in cells in which the expression of TAB1 was not enhanced.

 These results suggest that both subunits of IKK (11:11 and) undergo phosphorylation in cells by the presence of TAK1 activated by TAB1. That is, TAK 1 is NIK (Regnier et al., 1997; Woronicz et al,

As in 1997), NF- / cB activation is promoted by phosphorylating IKK (or a molecule that interacts functionally with the IKK complex) to promote kinase activity of IKK. It is considered to induce. Example 9 Activation of IKK complex by TAK1

 A kinase reaction using I / cB as a substrate (IKK kinase) was performed on the IKK complex immunoprecipitated from cells in which TAK1 and TAB1 were enhanced in the following manner. Activation of the complex was detected.

 (1) Cell transfection and immunoprecipitation

 First, in the same manner as in Example 5, expression of the flagged wild-type TAK 1 (Flag—TAK 1 K) or mutant TAK 1 (Flag—TAK 1 K63W) alone or The cells were transfected into HeLa cells together with the TAB1 expression vector.

 In the system for enhancing the expression of exogenous IKK, as in Example 8, the expression vector of XKK-tagged IKK (Xpress-Ιαα or Xpress-IKKβ) was added at the same time as transfection. I made an action.

A cell lysate was prepared from the cells 24 hours after the transfusion in the same manner as in Example 6, and immunoprecipitation was performed. However, the antibody used for immunoprecipitation uses an anti-I «« antibody (H-744) (manufactured by Santa Cruz Bio-technology) to immunoprecipitate the endogenous Ι Κ Κ complex. Anti-X pres for immunoprecipitation twenty two

 s antibody (M-21) (manufactured by Santa Cruz Biotechnology) was used. The anti-I K antibody used recognizes I ΚΚ as well as I ΚΚ ".

 (2) ΚΚ ΚΚKinase Atsui

 The immunoprecipitation fraction obtained above was subjected to an in vitro kinase reaction in the same manner as in Example 7. However, as a substrate, recombinant I / cB (2.5 g) was added to the reaction system. After completion of the reaction, the reaction solution was subjected to SDS-polyacrylamide gel electrophoresis, and the gel after the electrophoresis was subjected to photoradiography.

 Recombinant I / cB used as a reaction substrate includes a partial polysaccharide comprising the first to 54th amino acid residues of human IA: B at the C-terminus of GST (glutathione-S-transferase). Peptide-linked fusion peptides (hereinafter, GST-I / cB «1-54) were used.

 Recombinant I was prepared from a culture of a transformant in which the expression vector for GST-I / cB «1-54 was introduced into an E. coli host. The expression vector for GST-I «Βα 1-54 is the cDNA of human I κ Β« (Genbank / EMBL accession No. M69043; Cell, Vol. 65, 1281-: 1289, 1991). Created by inserting the cDNA portion encoding the 1st to 54th amino acid residues into the BamHI-Ec0RI cleavage site of vector-plasmid pGEX-2T (Pharmacia) did.

 The results of IKK kinase assay are shown in FIG. (A) shows the results of kinase assay of endogenous I KK complex (immunoprecipitated fraction with anti-IΚΚα antibody), and (Β) shows the results of exogenous IΚΚ (immunoprecipitated fraction with anti-Xpress antibody). ) This is the result of the kinase assay.

 As shown in Fig. 8 (A), when expression of both the flagged wild type TAK1 (Flag-TAK1) and TAB1 was enhanced, the IKK kinase activity of the endogenous IKK complex was remarkable. Increased. On the other hand, mutant TAK1 lacking kinase activity (F1ag-TAK1K63W) did not promote IΚΚ activity.

Also in cells expressing exogenous I Kappakappa, as shown in Figure 8 (beta), when expressed enhance wild-type TAK 1 with TAB 1, exogenous _I ΚΚ α and /? Of I Kappakappa Kinase activity Although greatly increased, mutant TAK1 did not increase IKK kinase activity even when expression was enhanced together with TAB1.

 These results confirm that TAK1 activated by TAB1 activates NF- / cB by activating ΚΚα and IKΚβ.

 (3) Test for the effect of the test substance

Using the same system as described above, the effect of the test substance on IKK complex activity due to TAK1 can be assayed. That is, cells in which the expression of TAK1 and TAB1 are enhanced are obtained, and cultured in the presence or absence of the test substance. After culture twenty three

For the cells, immunoprecipitate the IKK complex fraction in the same manner as described above, measure the IΚ kinase activity of the immunoprecipitated fraction, and determine whether the presence of the test substance reduces the IΚ kinase activity I do. Industrial applicability

 The method of the present invention provides a method for identifying and screening NF—ΚΚ activation inhibitors that focuses on new transmitter molecules. According to the present invention, a new type of NF-κB activity inhibitor having an action point at T A Κ1 can be obtained. In addition, the method of the present invention is also useful as a method for identifying and screening Z or prophylactic drugs for diseases such as autoimmune diseases and intractable diseases exhibiting inflammatory symptoms. The drug selected or identified by the method of the present invention has an obvious point of action, which is advantageous for development as a pharmaceutical.

 In addition, drugs that have the effect of inhibiting or suppressing the function of TAK1 include new types of NF-κB activation inhibitors, autoimmune diseases (rheumatoid arthritis, systemic lupus erythematosus, systemic scleroderma) , Pettiet's disease, periarteritis nodosa, ulcerative colitis, glomerulonephritis, etc., intractable diseases with inflammatory symptoms (osteoarthritis, atherosclerosis, psoriasis, atopic dermatitis, etc.) It is a therapeutic and / or prophylactic agent for diseases such as viral diseases, endotoxin shock, and sepsis.

Claims

24 Scope of reading I. Activation of nucleus factor kappa B (NF 1 / B), including the step of assaying the modulatory effect of the test substance on the function of TGF- / activator kinase 1 (TAK 1) A method for determining or screening an inhibitor. 2. The method according to claim 1, wherein the modulating effect of the test substance is an effect of inhibiting or suppressing the function of TAK1. 3. TAK 1 features  (1) interaction between TAK1 and TAK1-binding protein 1,  (2) TAK 1 protein kinase activity,  (3) Activation of I «Β kinase (I KK) complex by intracellular TAK 1, and (4) NF- / cB activation induced by intracellular TAK1 3. The method according to claim 2, wherein the method is selected from:  4. The method according to claim 2, wherein the function of TAK1 is TAK1 protein kinase activity.  5. The method according to claim 2, wherein the function of TAK1 is activation of an IKK complex by TAK1 in a cell.  6. The method according to claim 1, comprising using a test cell in which expression of TAK1 and TAK1-binding protein 1 is enhanced, and allowing the test cell to coexist with a test substance. 7. NF-II: any one of claims 1 to 6, wherein the B activation inhibitor is a therapeutic drug and / or a prophylactic drug for an intractable disease exhibiting an autoimmune disease or an inflammatory symptom at the same time. The described method.  8. An NF- / cB activation inhibitor selected or identified by the method according to any one of claims 1 to 6. 9. An NF- / cB activation inhibitor whose main component is a drug that modulates the function of TAK1. 10. A method for identifying a therapeutic drug and a Z or prophylactic drug for an autoimmune disease or an intractable disease exhibiting inflammatory symptoms, comprising a step of assaying the modulating effect of a test substance on the function of TAK 1 in the NF-κB activation pathway. Screening method.  I I. A therapeutic or Z or prophylactic agent for an autoimmune disease or an intractable disease exhibiting inflammatory symptoms, selected or identified by the method according to claim 10.  12. A therapeutic and / or prophylactic agent for an autoimmune disease or an intractable disease exhibiting inflammatory symptoms, comprising as a main component a drug having an action of inhibiting or suppressing the function of TAK1 in the NF-κΒ activation pathway.