AU2004205144B2 - Human TAK1 DNA encoding the same - Google Patents

Description

AUSTRALIA

PATENTS ACT 1990 DIVISIONAL APPLICATION NAME OF APPLICANT: Chugai Seiyaku Kabushiki Kaisha ADDRESS FOR SERVICE: DAVIES COLLISON CAVE Patent Attorneys 1 Nicholson Street Melbourne, 3000.

INVENTION TITLE: "Human TAK1 DNA encoding the same" The following statement is a full description of this invention, including the best method of performing it known to us/me: Q:\OPER\JEH\2004July\I 2481510 210.doc 19/8/04 P:\OPERAJH\Specs\ 2481510 Mod 06-12-07 doc 7/12107 0-1- KINASE IN TGF-p FAMILY SIGNAL TRANSDUCTION SYSTEM

O

BACKGROUND OF THE INVENTION 1. Field of Invention Ni3 The present invention relates to a kinase which is activated by transforming 0 growth factory-3 (TGF-P) and functions in the TGF-0 family signal transduction system (transforming growth factor-activated kinase: TAK1), to a process for production thereof, and to a gene coding for it. TAK 1, also known as an activator of MAPK kinase (AMKis an enzyme (a kinase) which is activated by TGF-0 and BMP (bone morphogenetic protein) and in turn activates MAPK kinase by phosphorylation.

2. Related Art The receptors of TGF-P superfamily comprise Ser/Thr kinases in the cytoplasmic region, and are classified into type I which has a repeating sequence of Gly-Ser (GS box) at the amino terminal proximal to the transmembrane domain, and type II which does not have the GS box. It is believed that, in the case of TGF-P, ligands form complexes with type I receptors after binding to type II receptors, and the kinases constitutively active type II receptors phosphorylate the type I receptors in the vicinity of the GS box, thus activating the type I receptors for transduce the signal from those ligands into the cell.

However, virtually nothing is known about the signal-transducing molecules downstream from these receptors.

According to the known signal transducing cascade from extracellular mating pheromones to mating in the eukaryotic budding yeast Saccharomyces cerevisiae, G protein is activated by the mating pheromone, G protein activates MAPKK kinase (MAPKKK) (Stell), the activated MAPKKK activates MAPK kinase (MAPKK) by phosphorylation, the thus activated MAPKK (Ste7) in turn activates MAP kinase (mitogen-activated protein kinase: MAPK) by phosphorylation, and finally MAPK activates FUSI protein, which initiates mating of the cells.

P:\OPER\AJH\Spccs\l2481510 Mod 06-12-07 doc 7/12/07 -2- SUMMARY OF INVENTION It is an object of the present invention to provide a novel factor which is located downstream of the mammalian receptor of the TGF-3 receptor signal transducing system Sand contributes to transduce those signals, a gene coding for it, and a method of producing Sthe gene.

0In attempting to achieve this object, the present inventors have succeeded in

(N

introducing mouse-derived DNA into Saccharomyces cerevisiae yeast lacking activity of MAPKKK (Stell) in the above-mentioned mating pheromone signaling cascade, screening for cDNA capable of complementing the activity-lacking MAPKKK, and cloning the cDNA capable of complementing the activity-lacking MAPKKK, thus completing the present invention.

Thus, the present invention provides DNA coding for a polypeptide with kinase activity which is activated by TGF-3 and comprises the amino acid sequence of SEQ ID NO:1 from Ser at position 23 to Ser at position 579, or an amino acid sequence which has a modification of the amino acid sequence wherein one to a few amino acids are added, deleted and/or substituted with other amino acids. According to one embodiment thereof, the DNA has the nucleotide sequence of SEQ ID NO:1 from T at position 223 to A at position 1893.

The present invention further provides DNA coding for a polypeptide with kinase activity which is activated by TGF-P and comprises the amino acid sequence of SEQ ID NO:1 from Met at position 1 to Ser at position 579, or an amino acid sequence which has a modification of said amino acid sequence wherein one to a few amino acids are added, deleted and/or substituted with other amino acids. According to one embodiment thereof, the DNA has the nucleotide sequence of SEQ ID NO:1 from A at position 157 to A at position 1893.

The present invention further provides DNA coding for a polypeptide with kinase activity which is activated by TGF-3 and has at least 80% homology with the nucleotide sequence of SEQ ID NO:1. The invention still further provides DNA coding for a polypeptide with kinase activity which is activated by TGF-P and is capable of P:\OPER\AJH\Specs\2481510 Mod 06-12-07 doc 7/12/07 -3hybridizing with the nucleotide sequence of SEQ ID NO:1 under conditions of 60° C., S0.1xSSC and 0.1% SDS.

The present invention provides DNA coding for a polypeptide with kinase activity which is activated by TGF-P and comprises the amino acid sequence of SEQ ID from Ser at position 23 to Ser at position 579, or an amino acid sequence which has a modification of said amino acid sequence wherein one to a few amino acids are added, deleted and/or substituted with other amino acids. According to one embodiment thereof, 0the DNA has the nucleotide sequence of SEQ ID NO:5 from T at position 249 to A at position 1919.

The present invention further provides DNA coding for a polypeptide with kinase activity which is activated by TGF-P and comprises the amino acid sequence of SEQ ID from Met at position 1 to Ser at position 579, or an amino acid sequence which has a modification of said amino acid sequence wherein one to a few amino acids are added, deleted and/or substituted with other amino acids. According to one embodiment thereof, the DNA has the nucleotide sequence of SEQ ID NO:5 from A at position 183 to A at position 1919.

The present invention further provides DNA coding for a polypeptide with kinase activity which is activated by TGF-0 and has at least 80% homology with the nucleotide sequence of SEQ ID NO:5. The present invention still further provides DNA coding for a polypeptide with kinase activity which is activated by TGF-P and is capable of hybridizing with the nucleotide sequence of SEQ ID NO:5 under conditions of 600 C., 0.1xSSC and 0.1% SDS.

The present invention still further provides a method of producing a polypeptide with kinase activity which is activated by TGF-P, characterized by culturing host cells transformed with a vector comprising any of the abovementioned DNA, and recovering the expressed product from the culture. The invention still further provides a polypeptide with kinase activity which is activated by TGF-P and is produced by this method. The polypeptide is expected to have the amino acid sequence of SEQ ID NO:1 from Ser at position 23 to Ser at position 579 or the amino acid sequence of SEQ ID NO:5 from Ser at position 23 to Ser at position 579. Thus, the invention still further provides a kinase enzyme which is activated by TGF-P and has these amino acid sequence.

P:\OPERH\AJ\Specs\I2481510 Mod 06-12-07 doc 7/12/07 -4- 0 BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the yeast expression vector pNV 1.

FIG. 2 is a graph showing a result of studying the effect of TGF-0 addition on expression of different TAKI genes, using the luciferase gene as a reporter gene.

FIG. 3 is a graph showing a result of measuring the effect of TGF-0 and BMP-4 Son activity of the TAKI gene in MC3T3 cells, by an immunoprecipitation method and a 0 coupled kinase method.

FIG. 4 is a graph showing the effect of various concentrations of TGF-0 and BMP-4 on TAKI kinase activity in cells transfected with the HA-TAK1 gene. The bar denoted as TAKIAN represents a result for cells transfected with the TAKIAN gene but not stimulated with TGF-0 or BMP-4.

FIGS. 5A to 5E compare nucleotide sequence of mouse TAK1 and that of human TAK1.

FIGS. 6A to 6B compare amino acid sequence of mouse TAK1 and that of human TAK1.

P \OPER\AJH\Spccs1 2481510 Mod 06-12-07doc 7/12/07 DETAILED DESCRIPTION According to the present invention, a gene may be cloned, for example, by introducing an expression vector containing mammalian cDNA into yeast which lacks SMAPKKK activity and has a reporter gene which is easily detectable at the end of the Scascade, and detecting whether cDNA which complements the lacking MAPKKK activity 0has been inserted based on expression of the reporter gene. Also other yeast, for example, lacking Ssk2/Ssk22 and Shol activities which works under a high osmotic pressure stress signal transducing system can be used.

The detection system used may be the MAPK cascade which transduces the intracellular signaling for the mating pheromone in Saccharomyces cerevisiae (I.

Herskowitz, Cell, Vol.80, 187 (1995); D. E. Lein et., Curr. Opin. Cell Biol. Vol. 7, 197 (1995); J. Schulz et al., Curr. Opin. Gene Dev., Vol. 5, 31 (1995)). The normal signaling cascade in this system includes Ste 11 kinase, Ste 7 kinase and Fus 3/Kss 1 kinase, which correspond to MAPKKK, MAPKK and MAPK, respectively. Ste 11, Ste 7 and Fus 3/Kss 1 act in succession to transduce signals to the transcription factor Ste 12, and Ste 12 in turn activates transcription of mating specific genes such as FUS 1.

For screening the cDNA, there may be used a cascade including a functional mutation of Ste 7 (STE7 P 368 and a deletion mutation of Ste 11 (StellA) from the abovementioned cascades Irie et al, Science Vol. 265, 1716 (1994)), and it has been confirmed that when this system is monitored based on the histidine-expressing phenotype imparted by the reporter gene FUSlp::HIS3 corresponding to the mating pathway, the activated form of either mammalian Raf or MEKK (RafAN and MEKKAN, respectively) can replace Ste 11 activity deficiency in an STE7P 368 dependent manner.

Thus, by introducing test cDNA into yeast with the above-mentioned mutated cascade and detecting the histidine-expressing phenotype, it is possible to select cDNA capable of complementing Ste 11A (MAPKKK deficiency).

The test cDNA library used may be any mammalian-derived cDNA library, an example of which is a cDNA expression library from a mouse cell line, such as the mouse cell line BAF-B03. This cDNA library is obtained by cloning cDNA corresponding to P \OPER\AJH\Specs\124B1510 Mod 06-12-07.doc 7/12107 -6- Spoly(A)-RNA from the mouse IL-3-dependent pro-P cell line BAF-B03, under the control O of the TDH3 promoter of the yeast expression vector pNV 11. Another example of the test cDNA library used may be a cDNA expression library from a human cell line, such as the human cell line Jurkat.

The above-mentioned cDNA library was screened with the screening system Sdescribed above to obtain a positive clone. The nucleotide sequence of the cDNA of this clone and the amino acid sequence encoded thereby correspond to nucleotides 223-1893 Sand amino acids 23-579 of SEQ ID NO:1. The cDNA library from a human cell line may be screened according to the above-mentioned screening system. Alternatively, the cDNA library from a human cell line may be screened using as a probe a mouse cDNA obtained as described above. The nucleotide sequence of the cDNA of another positive clone and the amino acid sequence encoded thereby correspond to nucleotides 249-1919 and amino acids 23-579 of SEQ ID To obtain longer cDNA (full-length cDNA), the above-mentioned cDNA was used as a probe for screening of the cDNA library to obtain positive clones. These clones had a portion of about 230 bp with respect to the cDNA. The cDNA with the elongated end was named TAKI cDNA, and the originally cloned cDNA without the elongated end was named TAK1AN cDNA. The nucleotide sequence of TAKI cDNA is represented by nucleotides 1 to 2443 of SEQ ID NO:1, and the amino acid sequence encoded thereby is represented by amino acids 1 to 579 of SEQ ID NO:1. The protein and polypeptide represented by this amino acid sequence are referred to as the TAKI protein and polypeptide. The protein and polypeptide represented by the amino acid sequence encoded by TAK1AN cDNA are referred to as TAK1AN protein and polypeptide. In addition, the nucleotide sequence of human TAKI cDNA is represented by nucleotides 1 to 2656 of SEQ ID NO:5, and the amino acid sequence encoded thereby is represented by amino acids 1 to 579 of SEQ ID The primary amino acid sequence of TAK1 protein suggests that the protein has an N-terminal protein kinase catalitic domain and a C-terminal domain of approximately 300 amino acid residues. The catalitic domain includes a consensus sequence corresponding to the protein kinase subdomains I-XI K. Hanks et al., Science 241, 42 (1988)). This catalyst domain has approximately 30% homology with the amino acid P:\OPER\AJH\Specs\l2481510 Mod 06-12-07 doc- 7/12/07 0 -7- Q sequence of the catalyst domains of Raf-1 I. Bonner et al., Nucleic Acids Res., Vol.14, O 1009 (1986)) and MEKK A. Langer-Carter et al., Science Vol. 260, 315 (1993)). The sequence of 300 C-terminal amino acid residues continuing from the catalyst domain has no significant homology with other proteins.

The TAK1AN cDNA lacking the codons for 22 N-terminal amino acids may be 0 introduced into yeast with the StellA mutation to complement the StellA mutation S(MAPKKK deficiency), but when the full-length TAKI cDNA is introduced into the

O

0 Stel 1A mutant it does not complement the Stel 1A mutation. Consequently it is believed that TAK1 kinase is activated by removal of the 22 N-terminal amino acids.

Thus, the present invention provides DNA coding for a polypeptide comprising the amino acid sequence of SEQ ID NO:1 from Met at position 1 to Ser at position 579.

This DNA includes, as typical examples, DNA coding for the polypeptide comprising the amino acid sequence from the 23rd amino acid Ser to the 579th amino acid Ser, and DNA coding for the polypeptide comprising the amino acid sequence from the 30th amino acid Glu to the 295th amino acid Asp. However, the DNA of the present invention is not limited to these, and also encompasses DNAs coding for polypeptides consisting of amino acid sequences from any of the amino acids between the 1st Met to the 30th Glu, to the 295th amino acid Asp. It will be readily recognized that even DNA coding for a polypeptide with an elongated N-terminus may be used to obtain an active enzyme by processing of the polypeptide after expression, and that lack of a C-terminal region other than the kinase will still give the same kinase activity.

Also, the present invention provides DNA coding for a polypeptide comprising an amino acid sequence of SEQ ID NO:5 from Ser at position 23 to Ser at position 579. This DNA includes, as typical examples, DNA coding for a polypeptide having an amino acid sequence from the first Ser to the 579th amino acid Ser and DNA coding for a polypeptide having an amino acid sequence from 23rd amino acid Ser to the 579th amino acid Ser.

The present invention also encompasses DNAs coding for polypeptides which have modifications of the polypeptides with the various amino acid sequences mentioned above, and maintain kinase activity by activation by TGF-P (referred to as TAK1 activity). Such a modification is intended to mean that the aforementioned amino acid sequences of various lengths from the amino acid sequence represented by SEQ ID NO:1 P \OPER\AJH\Specs\12481510 Mod 06-12-07doc 7/12/07 -8- Sor SEQ ID NO:5 may have one to a few, such as about 1 to 10 or 1 to 5, amino acids O added, deleted and/or substituted with other amino acids. In a more general sense, the present invention encompasses DNA coding for a polypeptide with an amino acid sequence which has at least 80%, preferably at least 90% and more preferably at least 95% homology with the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:5 and Smaintains TAK activity.

The present invention also encompasses DNA coding for a polypeptide which is Scapable of hybridizing with the nucleotide sequence represented by SEQ ID NO: 1 or SEQ ID NO:5 under conditions of, for example, 600 0.1xSSC and 0.1% SDS, and maintains TAKI activity. Here, the 0.1xSSC may be a 200-fold dilution of 20xSSC comprising 3 M NaCI and 0.3 M sodium citrate.

The present invention further provides polypeptides or proteins with amino acid sequences corresponding to the various DNA nucleotide sequences mentioned above, and particularly to polypeptides and proteins which maintain TAK1 activity. As a more concrete example, the present invention relates to a polypeptide or protein expressed by introduction of any of the aforementioned DNA into host cells, which may be animal cells or microorganic cells, as an insertion into, for example, a vector, particularly an expression vector, and particularly to polypeptides and proteins with TAKI activity.

A polypeptide or protein according to the invention typically has an amino acid sequence selected from the amino acid sequence represented by SEQ ID NO:1 or SEQ ID starting from any of the amino acids between the 1st amino acid Met and the 23rd amino acid Ser (inclusive) and continuing to the 579th amino acid Ser.

The present invention also encompasses polypeptides and proteins with amino acid sequences based on the above-mentioned amino acid sequence and modified by one to a few, such as about 1 to 10 or 1 to 5 amino acids added, deleted and/or substituted with other amino acids. They preferably have TAK1 activity. The invention further relates to polypeptides and proteins with amino acid sequences having at least preferably at least 90% and more preferably at least 95% homology with all or a portion of the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:5, and which maintain TAK1 activity.

As described above, cDNA coding for human TAK1 can be obtained by using that P\OPER\AJlPSpccs\12481S10 Mod 06-12-07.doc 7/12/07 -9- Sof the mouse TAKI and Examples 5 and 6 demonstrate the isolation of cDNA coding for 0 human TAK1.

The various DNA according to the invention may be cloned from animal cells as cDNA by the method described in Example 2, for example. DNA which has been mutated or modified with respect to the native cDNA may be prepared by common means Ssuch as, for example, using the native cDNA as a template for PCR amplification, site- Sspecific mutagenic treatment, etc.

0A polypeptide or protein according to the invention may be obtained by expressing the corresponding DNA in a suitable host. The host in this case may be a eukaryote such as cultured cells of higher eukaryotic organisms including humans, monkeys, mice, hamsters, frogs, etc., examples of which include THP-1 cells, MC3T3-El cells, XTC cells, MvlLu cells, CHO cells and COS cells; lower eukaryotic cells including filamentous fungi such as Aspergillus, such as Aspergillus niger; and yeast such as Saccharomyces, such as Saccharomyces cerevisiae. The host cells may also be prokaryotic cells, for example bacteria such as Escherichia coli.

When the desired DNA is expressed in such hosts, a promoter and other expression regulating sequences suitable for the host are used. For example, for expression in animal cells, plasmids with various promoters, e.g. pCDM8, pSV, pEF, etc.

are used, whereas in yeast hosts a plasmid such as pNVII is used and in E. coli a plasmid such as pGEMEX or pUEX is used.

The culturing of the transformed host may be carried out by a conventional method. The recovery and purification of the polypeptide or protein from the cultured product may be accomplished by any method commonly employed for the purification of enzymes, such as centrifugation, filtration, gel filtration chromatography, affinity chromatography, or the like.

Kinases activated by TGF-p, i.e. kinases in the TGF-3 family signal transduction system, are useful for the detection of agents which inhibit or promote signal transduce by TGF-P and its super family, which are known to be involved in a large number of disorders.

P \OPER\AJH\Specs\12481510 Mod 06-2-07doc- 7112/07 O EXAMPLES The present invention will now be explained in more detail by way of the following examples.

Example 1 SPreparation of cDNA library A conventional method was used to synthesize cDNA from poly(A)-RNA of a mouse IL-3-dependent cell line BAF-B03, which was then introduced into a yeast expression vector pNVI shown in FIG. 1 (Ninomiya-Tsuji, J. et al., Proc. Natl. Acad.

Sci. USA 88, 9006-9010 (1991)) under the control of TDH3 promoter, to prepare a cDNA library.

Example 2 Screening of cDNA library The cDNA library prepared in Example 1 was screened using Saccharomyces cerevisiae SY1984-P (his3A, Stel 1A, FUSlp:HIS3, STE7P368). In the signal transducing system of the mating pheromone for this yeast, Ste 11 is mutated causing loss of function, the serine at position 368 of Ste-7 is replaced with proline, and the FUS1 upstream activating sequence is linked to the HIS3 open reading frame to form a reporter gene.

This yeast line lacks the native his3, and therefore can only grow when exogenous histidine is present in the culture or when the Stell activity lost by the mutation is complemented.

S. cerevisiae SY1984-P was transformed with one of various different plasmids.

The plasmids used were YCplac22 (vector), pRS314PGKMEKKCT (expressing MEKKAN which lacks the N-terminal domain downstream of the PGK1 promoter J.

Blumer et al., Proc. Natl. Acad. Sci. USA, Vol.91, 4925 (1994)) and pADU-RafAN (expressing RafAN which lacks the N-terminal domain from the ADH1 promoter Irie et al, Science, Vol.265, 1716 5 These transformants were applied to SC-His plates without histidine, and incubated at 300 C. As a result, the yeast transformed with P \OPER\AJH\Spccs\124RI510 Mod 06-12-07.doc 7/12107 0-11- SYCplac22 vector failed to growth, whereas the yeasts transformed with O pRS314PGKMEKKCT and pADU-RafAN did reproduce. The validity of the screening system was thus confirmed.

The screening system yeast line YS1984-P was transformed with the cDNA library prepared in Example 1 and screened on an SC-His plate, yielding the positive S clone pNVll-HUll. The cDNA of this clone was named TAK1AN cDNA. The nucleotide sequence of this cDNA was determined by the dideoxynucleotide chain 0termination method. The nucleotide sequence corresponds to the sequence of nucleotides 223 to 1893 of Sequence No.l, and the amino acid sequence encoded thereby corresponds to amino acids 23 (Ser) to 579 (Ser) of the amino acid sequence of SEQ ID NO: 1.

The aforementioned TAK1AN cDNA was radioactively labelled and used as a probe for cloning of the full-length cDNA, and the cDNA library obtained in Example 1 was further screened. Positive clones were thus obtained. The cDNA of these clones were subcloned at the EcoRI site of vector pBS (product of Stratagene) to obtain pBS- TAK1-5'. This clone was the full-length clone containing the initiation codon ATG. The cDNA was named TAK1 cDNA. Its nucleotide sequence is represented by SEQ ID NO:1. The full-length amino acid sequence from Met at position 1 to Ser at position 579 is encoded by nucleotides 1 to 2443 of this sequence.

Example 3 Distribution of TAK1 gene in tissues Total RNA was extracted from different mouse tissues, and radioactively labelled TAKI cDNA was used as a probe for Northern blotting, which revealed expression of TAK1 cDNA-hybridizing RNA in all of the tissues and organs tested (spleen, thymus, lungs, heart, liver and brain). High levels were found in the spleen, thymus and brain, while low levels were found in the lungs, heart and liver.

Example 4 Properties of TAK1 kinase In order to investigate the function of TGF-P-activated kinase in mammalian cells, TAK1 cDNA and TAK1AN cDNA were inserted into the mammalian expression vector P \OPER\AJH\Spccs\l2481510 Mod 06-I2-07doc 7/12107 1-12- SpEF Shibuya et al., Nature Vol.357, 700 (1992)) under the control of human 0elongation factor (EF) promoter, to obtain expression plasmids pEF-TAK1 and pEF- TAK1AN. The expression plasmids pEF-TAKI and pEF-TAKAN contain the respective full-length TAK-1-coding and TAK1AN-coding sequences, respectively, under the control of EF promoter.

Specifically, the 2.3 kb XhoI fragment of pNVll-HUll was inserted into the SXhoI gap of pBS to obtain pBS-TAK1AN. pEF-MSSI Shibuya et al., Nature SVol.357, 700 (1992)) was cleaved with EcoRI and XbaI, and a synthetic EcoRI-XhoI linker (sense strand: 5'-AATTCGCCACCATGGC-3' (Sequence No.2), antisense strand: 5'-TCGAGCCATGGTGGCG-3' (Sequence No.3) (containing the initiation codon ATG) and XhoI-HindIII and HindIII-Xbal fragments from pBS-TAKlAN were inserted therein to prepare pEF-TAK1AN. pBS was cleaved with EcoRI and XhoI, and the EcoRI-SacI fragment from Pbs-TAK1-5' and the SacI-XhoI fragment from pBS-TAK1AN were inserted therein to obtain pBS-TAKI containing the full-length cDNA of TAKI (TAK1 cDNA). pEF-MSS1 was cleaved with EcoRI and Sail, and the EcoRI-SacI fragment from pBS-TAK1 was inserted therein to prepare pEF-TAK1.

E. coli containing the plasmid pEF-TAKI and E. coli containing the plasmid PEF- TAKlAN were internationally deposited as Escherichia coli MC1061/P3 (pEF-TAKI) and Escherichia coli MC1061/P3 (pEF-TAKIAN) at the National Institute of Bioscience and Human Technology Agency of Industrial Science and Technology 1-chome, Tsukuba City, Ibaraki, Japan) on Sep. 28, 1995 under the respective Assigned Nos.

FERM-BP-5246 and FERM-BP-5245, in accordance with the Budapest Treaty.

The TAKI gene included in plasmid pEF-TAKI may be cut out using suitable restriction endonucleases such as EcoRI and BamHI.

As a result of testing the effect of TAKI on induction of gene expression by various ligands, it was discovered that TAKI has an effect on induction of gene expression by TGF-P. The initial cellular response to TGF-P results in increased levels of mRNA for plasminogen activator inhibitor I(PAI-1) R. Keeton et al, J. Biol. Chem.

Vol. 266, 23048 (1991)). In order to investigate the effect of TAK1 on the TGF-P response, the TGF-P reporter plasmid p800neoLUC Abe et al, Analyt. Biochem., Vol.216, 276 (1994)) containing the luciferase gene controlled by the PAI-1 promoter P \OPER\AJH\Spcs\12481 510 Mod 06-12-07doc 7112/07 -13- Q induced by TGF-P was used for transient transfection of MvlLu lung epithelial cells by O the calcium phosphate method Shibuya et al., Nature Vol.357, 700 (1992)). This assay method allows measurement of luciferase activity induced by TGF-P through Stransfection of MvlLu lung epithelial cells with p800neoLUC. The MvlLu cells transiently transfected with p800neoLUC responded to TGF-P with 4- to 5-fold reinforced Sreporter gene activity. The results are shown in the vector column of FIG. 2.

The previously prepared TAK1 and TAK1AN expression plasmids were used to Stransiently transfect MvlLu cells together with p800neoLUC. TAK1 expression slightly reinforced TGF-P induced gene expression, and TAK1AN constitutively activated PAI-1 gene expression (TAKIAN column of FIG. The level of constitutive expression of the reporter gene by TAKIAN is comparable to that of the transfectants treated with TGF-P.

.Hence, activated TAK1 TAK1AN) can transfer signals in the absence of TGF-3.

Furthermore, expression of the PAI-1 gene increased when TGF-3 was added to the TAK 1 AN transfectant.

In FIG. 2, the white bars represent no induction with TGF-P and the shaded bars represent induction with TGF-P. After transfection in the experiment described above, the cells were cultured for 20 hours in the presence of and in the absence of human TGF-31 ng/ml), extracts were taken from the cells, and luciferase was measured according to H. Shibuya, et al., Mol. Cell. Biol. Vol.14, 5812 (1994). The graph of FIG. 2 shows relative activities, with 1 being the luciferase activity when cells transformed by the vector (without the TAKI gene) were not induced by TGF-P1. The results shown in this bar graph represent an average of the results from 3 consecutive experiments.

In order to confirm that these results were mediated by TAK1 kinase activity, catalytically inactive TAK1AN -K63W was prepared. This was accomplished using the PCR for site specific mutagenesis. In this vector, the 63rd amino acid lysine of the ATPbinding site is replaced with tryptophan. It is supposed that this mutation destroys TAK1AN kinase activity and signal transducing activity. When TAK1AN-K63W was simultaneously transfected with p800neoLUC, the ability to constitutively induce PAI-1 gene expression was lost (FIG. This result suggests that TAK1AN kinase activity is required for TGF-P-independent expression of the PAI-I gene. Furthermore, the kinasenegative TAK1AN provoked a partial reduction in TGF-P-induced expression. This result P\OPER.AH'Spcs\1 2481 510 Mod 06-I2-07doc 7112J07 -14- Ssupports the belief that TAK1 functions as a mediator in the TGF-P-signal transducing O pathway.

In order to obtain direct conclusive proof that TAK1 functions in the TGF-P signal transducing pathway, it was determined whether or not TAK1 kinase activity is activated by treating cells with TGF-P. A suitable foreign substrate was identified by in vitro Skinase reaction of TAKI immunoprecipitated from yeast cells expressing TAKI labelled with a hemagglutinin (HA) epitope (TAK1-HA) (prepared using the PCR to link the DNA Ssequence coding for the epitope labelled with anti-HA monoclonal antibody 12CA5 to the 3'-end of DNA coding for TAK1 in the reading frame). Judging from the immunocomplex kinase measurement, the active form of TAKI was able to phosphorylate and activate the XMEK2/SEK1 subfamily of MAPKK M. Yasher et al., Nature Vol. 372, 794 (1994)). However, no phosphorylation of the original MAPKK- MEK1 Nishida et al., Trends Biochem. Sci., 128 (1993); K. J. Blumer et al., ibid Vol.

19, 286 (1994); R. J. Davis, ibid Vol.19, 470 (1990); C. L. Marchall, Cell, Vol.80, 179 (1995)), histone and myelin basic protein was detected. It is therefore possible to measure TAK1 kinase activity based on its ability to activate XMEK2 in vitro.

The construct for expression of the HA epitope-tagged TAK1 (HA-TAK1) was prepared in the following manner. A synthetic oligonucleotide coding for the HA epitope Tyr-Pro-Tyr-Asp-Val-Pro-Asp-Tyr-Ala (Sequence Nc.4) labelled with monoclonal antibody 12CA5 was cloned at the Sail site position from the ATG codon) and EcoRI site ofpBS-TAK1 to construct pBS-HA-TAK1. pEF-MSSI was cleaved with EcoRI and Sall and the EcoRI-XhoI fragment from pBS-HA-TAKI was inserted therein to construct pEF-HA-TAKI. pBS-HA-TAKlAN was constructed by digesting pNVII-HUll with XhoI and HindIII. The fragment was isolated and inserted at the HinclI-HindIIl site of pBS-HA-TAK1. pEF-MSSI was cleaved with EcoRI and SalI and the PstI-XhoI fragment from pBS-HA- TAK1AN was inserted therein to construct pEF-HA-TAKIAN.

Both constructs have two copies of the N-terminal HA epitope expressed from the EF promoter.

The constructs pEF-HA-TAKI and pEF-HA-TAKIAN were used for transient transfection of MC3T3-E1 mouse osteoblasts Ohta et al., FEBS Lett. Vol.314, 356 (1992)). After stimulation with TGF-p, the expressed HA-TAKI was isolated by P:\OPER\AJH\Specs\l2481510 Mod 06-12-07.doc 7/12/07 Simmunoprecipitation, and its activity was determined by the coupled kinase assay (S.

O Matsuda et al., J. Biol. Chem. Vol.270, 12969 (1995)).

Specifically, the transfected cells were treated with TGF-11 (20 ng/ml) or BMP-4 (100 ng/ml) from 0 (untreated) to 30 minutes. The cells were scraped into a buffer solution Matsuda et al., J. Biol. Chem. Vol.270, 12781 (1995); T. Moriguchi et al., J.

0Biol. Chem. Vol.270, 12969 (1995)), and the cell extract solution was centrifuged at 15,000xg for 10 minutes. The resulting supernatant was subjected to Simmunoprecipitation with anti-HA antibody. That is, a 300 pl aliquot of the supernatant was mixed with 20 pl of antibody and 20 pl of protein A Sepharose, and the immunocomplex was washed twice with pBS and used for kinase assay Matsuda et al., J. Biol. Chem. Vol.270, 12781 (1995); T. Moriguchi et al., J. Biol. Chem. Vol.270, 12969 (1995)). The activity is shown as the multiple of increase with respect to the activity of HA-TAKI from non-stimulated cells. The activity of the immunoprecipitated TAKI was measured based on ability to activate recombinant XMEX2/SEK1. The activity of XMEX2/SEK was measured based on phosphorylation of the recombinant kinase-negative (KN) p38/MPK2 Matsuda et al., J. Biol. Chem. Vol.270, 12781 (1995); T. Moriguchi et al., J. Biol. Chem. Vol.270, 12781 (1995)). It was confirmed that HA-TAKl does not directly phosphorylate KN-p38/MPK2. The immunoblotting with each of the immunoprecipitating anti-HA antibodies recovered virtually equal amounts of HA-TAK1 at each stage of the immunoprecipitation.

The results of the above experiment show that TAK kinase activity began to increase within 5 minutes after stimulation by TGF-P, reached a peak at 10 minutes and returned almost to the baseline within 30 minutes (FIG. In addition, TGF-11 stimulated TAKI kinase activity in a dose-dependent manner (FIG. It was next determined whether TAK1 is activated by BMP, a member of the TGF-P superfamily (A.

H. Reddi et al., Carr. Opin. Genet. Dev. Vol.4, 737 (1994) or epithelial growth factor (EGF). Interestingly, BMP-4 also activated TAK1 kinase in a time-and dose-dependent manner (FIG. On the other hand, no TAK1 activation was observed in cells treated with EGF. It is believed that the reason EGF does not induce TAKI activation is not that MC3T3-ET cells do not respond to EGF, but rather that the EGF signal does not include TAKI. This is also evident from the fact that EGF induces expression of fos in MC3T3- P \OPER\AJH\Specs\12481510 Mod 06-12.07doc 7/12/07 S-16- El cells. These data collectively indicate that TAK1 is activated by the TGF-P 0superfamily.

That TAKIAN can activate expression of the PAI-1 gene independently of TGF- S(FIG. 2) suggests that TAKIAN protein has increased kinase activity even without TGF-P treatment of cells. To test this possibility, TAK1AN labelled with the HA epitope (HA- 0TAK1AN) (see above) was used to transiently transfect MC3T3-EI cells, and the STAK1AN activity was measured by immunocomplex kinase assay. Specifically, the 0 MC3T3-E1 cells were transfected with pEF-HA-TAK1AN, the HA-TAK1AN was immunoprecipitated from the transfected cells in the manner described earlier, and the activity was measured. All of the data are shown as multiples of increase with respect to activity of HA-TAK 1 from non-stimulated cells.

As shown in FIG. 4, the TAK1AN protein exhibited higher base kinase activity, supporting the hypothesis that TAK1AN which lacks 22 N-terminal amino acid residues is constitutively active.

Example Construction of cDNA library Poly RNA was prepared from a human cell line Jurkat, and cDNA was synthesized from the poly RNA according to a conventional procedure. The cDNA was inserted into a position downstream of TDH3 promoter in a yeast expression vector pNV7 (J.

Ninomiya-Tsuji et al., Proc. Natl. Acad. Sci. USA 88, 9006-9010 (1991)).

Example 6 Screening of cDNA library A Saccharomyces cerevisiae mutant lacking Ssk2/Ssk22 and Shol activities which work in a high osmotic pressure stress signal transducing system can grow in YEPD medium (yeast extract 10 g/l, tryptone 20 g/l, glucose 20 but cannot grow in the same medium supplemented with 1M sorbitol (T Maeda et al., Science 269, 554, 1995). Therefore, said yeast mutant can be used to screen the cDNA library. Namely, when said yeast is transformed with cDNA, and if the cDNA comprises a desired gene, the cDNA complements the lacked Sskl/Ssk2 activities resulting in the growth of the yeast mutant in P \OPER\AJHSpccs\l 2481510 Mod 06-12-07 doc 7/12/07 -17- (.2 the sorbitol-containing medium.

SFor confirmation, Saccharomyces cerevisiae (ssk2A, ssk22A, sholA) lacking Ssk2/Ssk22 and Shol activities was transformed with pNV1l-HUll (mouse TAK1AN) obtained in Example 2, and the transformed yeast cells were plated on a YEPD medium plate containing 1M sorbitol and incubated at 30° C. As a result, the yeast cells Stransformed with pNVII-HUll grew under high osmotic pressure stress. This experiment Sconfirmed that the screening system is effective.

0Accordingly, Saccharomyces cerevisiae (ssk2A, ssk22A, sholA) was transformed with the cDNA library constructed in Example 5, and the transformant was screened under a high osmotic pressure stress by incubating the transformant in a YEPD medium containing 1M sorbitol at 300 so as to obtain one positive clone, pNV7-hTAK1. A cDNA contained in this clone was amplified by PRISM Dye Terminator Cycle Sequencing kit (Perkin Elmer) and sequenced. The nucleotide sequence thus determined and a corresponding amino acid sequence is shown in SEQ ID NO:5. This nucleotide sequence of cDNA derived from human shows 92% homology with nucleotide sequence of mouse TAKI, and the human amino acid sequence shows 99% homology with the amino acid sequence of mouse TAKI. FIGS. 5A to 5E and FIGS. 6A to 6B compose nucleotide sequence and amino acid sequence, respectively, of mouse TAK1 and those of human TAK1. The human TAK1 cDNA was sub-cloned into pUC19 digested with Sall to obtain phTAK1 containing the full-length of cDNA of the human TAK1. E. coli containing the plasmid phTAKI was internationally deposited as Escherichia coli JM109 (phTAKI) at the National Institute of Bioscience and Human Technology Agency of Industrial Science and Technology 1-chome, Tsukuba City, Ibaraki, Japan) on Jul.

19, 1996 under the Assigned No. FERM-BP-5598 in accordance with the Budapest Treaty.

Depositing of microorganisms The following microorganisms were deposited at the Patent Microorganisms Depository Center of the National Institute of the Bioscience and Human Technology Agency of Industrial Science and Technology 1-chome, Tsukuba City, Ibaraki, Japan) on Sep. 28, 1995, under the Assigned Nos. listed below.

P \OPER' J-lpccs\1248l 510 Mod 06-12-07 doc 7/12107 -18- Name: Escherichia coli MC1O61/P3 (pEF-TAKI) Date of deposit: Sep. 28, 1995 Assigned No.: FERM-BP-5246 Name: Escherichia Ccli MClO6I/P3 (pEF-TAKIAN) Date of deposit: Sep. 28, 1995 Assigned No.: FERM-BP-5245 Name: Escherichia coli JM109 (phTAK I) Date of deposit: Jul. 19, 1996 Assigned No.: FERM BP-5 598 P:\OPER\AJH\Specs\l2481510 Mod 06-12-07 doc 7/1207 0 -19- SSEQUENCE LISTING 0 GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: S(A) LENGTH: 2443 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear C (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 157..1893 SEQUENCE DESCRIPTION: SEQ ID NO:1: 20 GAATTCGGGA CGAGGAGGAG CCGAAGCCGG GACTCGGCGG CCACGGAGCG GCGGGCGGCG GGCTGCGGGG CTCCGGGCTG AGGGCGGGCG CGGCCCCCCG GGCGCCGCGG GGGATC ATG Met TGGCCCGGGT CGGTCCCGCG AAGGGCGCTG CGCGAGCCGG TCG ACA GCC TCC GCC Ser Thr Ala Ser Ala GCC 'IGG TCC Ala Ser Ser TCC TCC TCG ICT TCI GCC AGI GAG Ser Ser Ser Ser Ser Ala Ser Glu ATC ATC Met Ile GAA GCG CCG Giu Ala Pro 15 ICG GAG GTC GIG AAC TIC GAA GAG ATC GAC TAC MAG GAG ATC GAG G'rG Ser Gin Val Leu Asn Phe Glu Glu Ile Asp Tyr Lys Glu Ile Glu Val 30 GMA GAG GTT GTC GGA AGA GGA GCT TTT OGA GTA GTT TGC AMA GOT AAG Giu Glu Val Val Gly Arg Gly Ala Phe Oly Val Val Cys Lys Ala Lys 45 TGG AGA GCA AAA GAT GTCGOCT ATT AMA GAG ATA GMA AGT GAG TCT GAG Trp Arg Ala Lys Asp Val Ala Ile Lys Gin Ile Glu Ser Olu Ser Glu 60 65 222 270 318 366 21 AGG AAG GCT TTC ATT GTG GAG CTC Arg Lys Ala Phe CCT AAC ATT GTC Pro Asn Ile Val GTG ATG GAA TAT Val Met Glu Tyr Ile Val AAG TTG Lys Leu Glu Leu

TAC

Tyr

GGA

Gly CGG CAG Arg Gin 80 GCC TGC Ala Cys 95 TCA TTG Ser Leu TTG TCG Leu Ser CGT GTG AAC CAT Arg Val Asn His CTG AAT CCA GTA TGT CTT Leu Asn Pro Val Cys Leu 100 TAT AAT GTG CTG CAT GGT Tyr Asn Val Leu His Gly GCA GAG GGG GGC Ala Glu Gly Gly 105 GCT GAA CCA Ala Glu Pro 120 TTA CAG TGT Leu Gin Cys TTG CCT TAC TAC Leu Pro Tyr Tyr 125 TCC CAA GGA GTG Ser Gin Gly Val 140 CAC AGG GAC CTC His Arg Asp Leu 110

ACT

Thr GCT GCT CAT Ala Ala His TAC CTG CAC Tyr Leu His

GCT

Ala 135

GCG

Ala 115 GCC ATG AGC TGG TGT Ala Met Ser Trp Cys 130 AGC ATG CAG CCC AAA Ser Met Gin Pro Lys .150 TTG CTG CTG GTT GCA Leu Leu Leu Val Ala 165 ACA GCT TGT GAC ATC Thr Ala Cys Asp ile CTG ATT Leu Ile 155

ATC

Ile AAG CCT CCA Lys Pro Pro 160 TGC GAT TTT Cvs Aso Phe 558 606 654 702 750 798 GGA GGG ACA GTT Gly Gly Thr Val 170 CAA ACA CAC ATG Gin Thr His Met 185 GAA GTG TTT GAA Glu Val Phe Glu CTA AAA Leu Lys

GGT

Gly v j 175 GGG AGT Gly Ser 180 GCT GCT TGG ATG GCG Ala Ala Tro Met Ala ACC AAT AAT AAA Thr Asn Asn Lys 190 GGT AGC AAT TAC Gly Ser Asn Tyr 205

CCT

Pro 195

GAT

Asp AGT GAA AAG TGT Ser Glu Lys Cys 210 GTC TTC AGC Val Phe Ser 200 22 TGG OGT Trp Gly 215 GAG ATC Olu Ile ACT CGA Thr Arg ATT ATC CTC TG Ile Ile Leu Trp 220 GGT GGC CCA OCT Glv Gly Pro Ala CMA OTO ATA ACA Ciii Val Ile Thr TTO AOA ATC ATO Phe Arg Ile Met 240

COO

Arg 225

TOO

T rp COO AAA CCC TTO OAT Arg Lys Pro Phe Asp 230 GOT OTT CAT MAT 000 Ala Val His Asn Gly 245 235 CCA OCA CTG Pro Pro Leu 250

ATC

Ile AAA MAT TTA Lys Asn Leu 255

OCA

Pro ATO ACA COO TOT Met Thr Arg Cys 265 GMA ATT OTO AAA Ohu Ile Val Lys 280 OAT GAG OCA TTA Asp Glu Pro Leu TOG TOT MAG CAC Trp Ser Lys Asp 270 ATA ATO ACT CAC Ile Met Thr His OCT MOG 000 ATT GAG AGO TTG Pro Lys Pro Ile Oiu Ser Leu 260 TOT CAG CCCOCT TOA ATC GAG Ser Gin Arg Pro Ser Met Olu 275 ATG OGG TAO TTO OCA GGA 000 Met Arg Tyr Phe Pro Gly Ala 846 894 942 990 1038 1086 1134 1182

TTG

Leu 285 OAO TAT COT Gin Tyr Pro CAG TAO TOT Gin Tyr Ser

TGT

Cys 295

MOC

Asn 300 305 TCA 000 ACC AGO ACA Ser Ala Thr S er Thr 315 AGT MAT AAA AGT GAG Ser Asn Lys Ser Asp GO TOG TTO ATG GAO Gly Ser Phe Met Asp 320 ACA MAT ATG GMA CAG Thr Asn Met Giu Gin 335 290 OGAT OMA 000 CAG AGO Asp Giu Gly Gin Ser 310 ATT GOT TOT ACA AAT Ile Ala Ser Thr Asn 325 OTT OCT 0CC ACA AAO Val Pro Ala Thr Asn

ACC

Thr 330 GAO ACT ATT AMA000 TTG Arg Leu GAO TOA AAA OTG TTG AAA AAO CAG GCA AAG Ohu Ser Lys Leu Leu Lys Asn Gin Ala Lys 1230 Asp.Thr Ile Lys 23 CAA CAG AGT GAA TOT GGA CGC Gin Gin 360 AGT GTG Ser Val Ser Glu Ser Gly GAG AGO TTG CCC Clu Ser Leu Pro 380 Arg 365

CCC

Pro CTG AGO TTG GGA GCC Leu Ser Leu Cly Ala 370 ACT TCO GAG GGC AAG Thr Ser Glu Gly Lys TCT CGT Ser Arg CGG AGG Cly Ser

AGG

Arg 375

GAG

Asp 385

GCC

Ala ATG ACT GCT Met Ser Ala 390

ATG

Met TCT GAA ATA Ser Glu Ile 395 CAA CCA AGG CGT Gin Pro Arg Arg 410 CCT GGT GAG GTG Pro Gly Gin Val 425

AGA

Arg GAA GCC AGG ATO Glu Ala Arg Ile TCC ATC CAA GAG Ser Ile Gin Asp 415 AGO CGG TOA TCC Ser Arg Ser Ser

GTG

Val 400 ACT CCA GGT AAC GGG Thr Ala Gly Asn Gly 405

AGO

Ser TTG ACT GTT ACT GGG ACA CAA Leu Thr Val Thr Gly Thr Glu, 420 AGO COT AGT GTC AGA ATG ATC Ser Pro Ser Val Arg Met Ile 435 OCA GOT CGC ACT CAC CCA TGG Pro Ala Arg Ser His Pro Trp 1278 1326 1374 1422 1470 1518 1566 1614 1662 430 ACT ACG Thr Thr 440 ACC CCT Thr Pro 45'5 ATG GCG Met Ala TCA GCA CCA ACC TCA Ser Gly Pro Thr Ser 445 CAT CAT TCC AGA GAG Asp Asp Ser Thr Asp 460 TAT OTT ACA CTG GAT Tyr Leu Thr Leu Asp

GAG

Glu

AAG

Lys 450 ACC AAT GGC TOA CAT AAO TCC Thr Asn Gly Ser Asp Asn Ser 465 CAC CAG OTA GAG OCT OTA GCG His Gin Leu Gin Pro Leu Ala ATC CCA Ile Pro 470 CCG TGC Pro Cys 475 GAA TCC Glu Ser CCA AAO TCC AAA Pro Asn Ser Lys 490 480 ATG GCA GTC TTC CAA Met Ala Val Phe GLu 495 485

AAA

Lys CAC CAC TGT Gin His Cys 500

ATG

Met 24 GCA CAG GAG TAT ATG AAA GTT CAA ACC GAA ATC GCA TTG TTA CTA CAG 1710 Ala Gin Glu Tyr Met Lys 505 AGA AAG CAA GAA CTA GTT Arg Lys Gin Giu Leu Val 520 Val Gin Thr Giu Ile Ala Leu Leu Leu Gin 510 515 GCA GAA TTG GAC GAG GAT GMA AAG GAG GAG Ala Giu Leu Asp Gin Asp Glu Lys Asp Gin 1758 CAA AT Gin Asn 535 AAC AAA Asn Lys ACA TC7 CGT Thr Ser Arg AGC CTT TCT Ser Leu Ser C TG Leu 540

ACT

Thr GAG GMA CAT Gin Giu His

A

Ly s 545

TGC

Gys TAT TAG GAG GMA Tyr Tyr Gin Gin MAG CT? Lys Leu AAA A Lys Lys ACT TCA Thr Ser TTA GAT GMA Leu Asp Giu 550 GMA CTA GAG Gin Leu Glu.

565

TGATTCTCTG

1806 1854 1903 555 GTG ATC AGA AGC CAA GAG GAG Val lie Arg Ser Gin Gin Gin 570 560 MAA CGA CAA GGG Lys Arg Gin Gly 575 G GAGC G TTAG C AG TT CATGA

TTTGTTATGT

GAGATGATGG

TGMACCTGGC

CTGGCTACTG

TTCTTCAAAG

GTAAAGACAG

GAG TTCAAAG GT TTTAAAAT

GTGTTAGGTT

GAG? TAGCCTT

CAGCTGTGGC

TGTATGTGCA

TGTGCAGGCA

TGCTCATCTC

AGTATTAAAA

ACAGGGTTTG

ATGGMAAGAC

TTTGGGGTGT

TGTTCTTATG

TTGGGMAGGC

TGCTCCTGGA

TCATCCTTTC

AAAAATCTGA

TAACTTGTGG

TGCCAACACA

CTTTTT TM

TCTGMATGCC

GTGGATATAA

GAG SGTGCTC

GTGAGCTACC

TCTGTAGTAA

TTTTTTTCCC

TACATTACAG

TCCTGGCTTT

GAG AAGA CAA

AAATGCCTCT

AATCCACTGT

AGCTTCAGGG

TAACAGGAGG

MAGGTGGGAC

ACTAGATOCT

AGGGACAGAA

AGAGCACMAT

ACCATTATAA

CTTTGC TGCA

CGTGTTGCAG

GCACATGAAG

GGGTAGCACA

CTCMAGAATT

ATGCTCCMAT

TGTTGAGCCT

GGATC TCCAG 1963 2023 2083 2143 2203 2263 2323 2383 2443 POPER\AJH\Spccs\12481510 Mod 06-12.07.doc 7/1112/07 0-25- S(2) INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 16 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: AATTCGCCAC CATGGC 16 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 16 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: TCGAGCCATG GTGGCG 16 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 9 amino acids TYPE: amino acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 2656 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (xi) FEATURE: NAME/KEY: CDS LOCATION: 183..1922 (xi) SEQUENCE DESCRIPTION: SEQ ID 26 G TC GAGATC C CGCCGGG TAG

GGGTCTCACC

TC ATG TCT Met Ser ATTGTGCTCT AAAGACGGCT GTGGCCGCTG CCTCTACCCC CGACGGAT TAGGACTGCG CGGCTCCAGG CTGAGGGTCG GTCCGGAGGC GGGTGGGCGC CGGATTGTCG GGGTGGGACC GTTCCCGGGG CCACCGGGCG CCGGGAGGGA ACA GCC TCT GCC GCG TGC TCC TCC TCC TCG TCT TCG GCC Thr Ala Ser Ala Ala Set Set Ser Ser Ser Ser Ser Ala 1 GGT GAG ATG ATC GAA Giy Giu Met Ile Giu GAC TAG AAG GAG ATG Asp Tyr Lys Giu Ile GGA GTT CTT TGC AAA Giy Val Val Cys Lys 5 GGC CCT TGC GAG GTG Ala Pro Ser Gin Vai 25 10 GTG MGC TTT GAA GAG ATG Leu Asn Phe Giu Giu Ile GTT GGA AGA GGA GCG TTT Val Giy Arg Gly Ala Phe GAG GTG GMA Giu Val Giu GGT MAG TGG Ala Lys Trp 55

GAG

Glu 40

GTT

ValI GMA ATA Gin Ilie AGA GGA AMA GAT GTT GCT AT? AAA Arg Ala Lys Asp Vai Ala Ile Lys 275 323 371 419 467 515 GMA AGT GMA TGT GAG Giu Set Giu Set Glu TGG GGT GTG MGC CAT Ser Arg Val Asn His

GAG

Gin

TG

Gys

TTA

Leu AGG MAA GGG TTT AT? Arg Lys Aia Phe Ile GGT MAT ATT GTA MAG Pro Asn Ile Val Lys 90 GTG, ATG GMA TAT GOT Val Met Giu Tyr Ala GTA GAG GTT CGO Vai Oiu Leu Arg GTT TAT GGA GOG Leu Tyr Gly Ala GAA GGG GO TOT Giu Gly Gly Ser 85 TTG MAT GGA GTG TOT Leu Asn Pro Val Gys

GTT

Leu 100 105 27 TTA TAT AAT OTO CTO CAT GGT GOT GAA CCA TTG OCA TAT TAT ACT GOT Leu Tyr Asn Val Leu 115 GOC CAC OCA ATO ACT Ala His Ala Met Ser His Gly Ala

TG

T rp TG T Cys 130 OTT CAC AGO ATO Leu His Ser Met TT A Leu 135 000 Ala Glu Pro Leu 120 CAG TOT TC Gin Cys Ser OTA ATT CAC Leu Ile His 000 ACA OTT Cly Thr Val Pro 125 Tyr Tyr Thr Ala 145 OGA AAO Pro Asn CAA COO AAA Gin Pro Lys 150 TTA OTG OTO Leu Leu Leu ACA GOC TOT Thr Ala Cys

GTT

Val 165

GOA

Ala 000 Gly 170 CAA OGA OTO GOT TAT Gin Gly Val Ala Tyr 140 AGO GAO OTO AAA OCA Arg Asp Leu Lys Pro 155 OTA AAA ATT TGr OAT Leu Lys Ile Cys ASP, 175 ACC AAT AAC AAG 000 Thr Asn Asn Lys Gly 190 GOT ACT AAT TAO ACT Gly Ser Asn Tyr Ser 205 OTT TOG GAA GTG ATA Leu Tro Glu Val Ile

GT

Gly 180 ACT GOT GOT TG Ser Ala Ala Trp 195 GAA AAA TOT GAO Glu Lys Cys Asp

ATO

Met GAO ATT CAG ACA CAO ATO Asp Ile Gin Thr His Met 185 OCA OCT OAA OTT TTT OAA Ala Pro Glu Val Phe Glu 200 TTC AGO TOG GOT ATT ATT Phe Ser Trp Gly Ile Ile 611 659 707 755 803 851 899 947

OTO

Val1 210 215 220

GOT

Ala ACO COT COG AAA COO Thr Arg Arg Lys Pro 225 ATO TOO GOT OTT OAT Met Trp Ala V al His TTT OAT Phe Asp 230 A.AT GOT Asn Gly

GAG

Glu ATT GOT Ile Oly 000 OCA Oly Pro 235 OGA OTO Pro Leu TTC OGA Phe Arg

ATO,

Ile ACT OGA OGA Thr Arg Pro ATA AAA AAT TTA Ile Lys Asn Leu 245 250 28 CCT AAG CCC ATT Pro Lys Pro Ile TCC CAG CGC CCT Ser Gin Arg Pro 275 ATG CGG TAC TTT Met Arg Tyr Phe GAG AGC Glu Ser 260 CTG ATG Leu Met ACT CGT Thr Arg 265 TGT TGG TCT AAA GAT CCT Cys Trp Ser Lys Asp Pro 270 AAA ATA ATG ACT CAC TTG Lvs Ile Met Thr His Leu

TCA

Ser ATG GAG GAA ATT Met Glu Glu Ile 280 GGA GCA GAT GAG Gly Ala Asp Glu

GTG

Val 285

CCA

Pro v 290

GAT

Asp 295

AAC

Asn CCA TTA CAG TAT CCT Pro Leu Gin Tyr Pro 300 GCC ACC AGT ACA GGC Ala Thr Ser Thr Gly

TGT

Cys

CAG

Gin

TAT

Tyr

ATG

Met 320

TCA

Ser 305

GAC

Asp GAA GGA CAG AGC Glu Gly Gin Ser 310

TCT

Ser TOA TTC Ser Phe- 315 ATT GCT TCT ACA Ile Ala Ser Thr 325 GAG CAA GTT CCT GCC Glu Gin Val Pro Ala 340 TTG TTG AAA AAT CAG Leu Leu Lys Asn Gin 355 TTG GGA GCC TCC CAT Leu Gly Ala Ser His 370 GAG GGC AAG AGG ATG Glu Gly Lys Arg Met

ACA

Thr AAT ACG AGT AAC AAA Asn Thr Ser Asn Lys 330 AAT GAT ACT ATT AAG Asn Asp Thr Ile Lys 345 AAG CAA CAG AGT GAA Lys Gin Gin Ser Glu 315 AGT GAC ACT AAT ATG Ser Asp Thr Asn Met 335 CGC TTA GAA TCA AAA Arg Leu Glu Ser Lys 350 TCT GGA CGT TTA AGC Ser Gly Arg Leu Ser 995 1043 1091 1139 1187 1235 1283 1331 1379

GCA

Ala 360 GGG AGC AGT GTG GAG AGC Gly Ser Ser Val Glu Ser 375 AGT GCT GAC ATG TCT GAA Ser Ala Asp Met Ser Glu 365 TTG CCC CCA Leu Pro Pro 380 ATA GAA GCT Ile Glu Ala ACC TCT Thr Ser AGG ATC Arg Ile 390 395 29

GCC

Ala 400

TTG

Leu GCA ACC ACA GGC AAC Ala Thr Thr Gly Asn 405 ACT GTA ACT GGA ACA Thr Val Thr Gly Thr 420 CCC AGT GTC AGA ATG Pro Ser Val Arg Met GGA CAG CCA AGA Gly Gin Pro Arg GAA CCT GGT CAG Glu Pro Gly Gin 425 CGT AGA TCC ATC CAA GAC Arg 410

GTG

Val Arg Ser Ile Gin AGC AGT AGG TCA Ser Ser Arg Ser 430 Asp 415

TCC

Ser

AGT

Ser ATT ACT ACC Ile Thr Thr 435 CCA ACT CGA AGT Pro Thr Arg Ser 450 GGA TCA GAT AAC Gly Ser Asp Asn 465 440 CAT CCA TGG ACC CCT His Pro Trp Thr Pro 455 TCA GGA CCA ACC TCA GAA AAG Ser Gly Pro Thr Ser Glu Lys 445 GAT GAT TCC ACA GAT ACC AAT Asp Asp Ser Thr Asp Thr Asn 460 1427 1475 1523 1571 1619 1667 1715 1763 TCC ATC CCA Ser Ile Pro 470

TGC

Cys

CTA

Leu 480

TTT

Phe CAG CCT CTA GCA CCG Gin Pro Leu Ala Pro 485 GAA CAG CAT TGT AAA Glu Gin His Cys Lys ATG GCT TAT CTT ACA CTG Met Ala Tyr Leu Thr Leu 475 CCA AAC TCC AAA GAA TCT Pro Asn Ser Lys Glu Ser 490 GCA CAA GAA TAT ATG AAA Ala Gin Glu Tyr Met Lys GAT CAC CAA Asp His Gin ATG GCA GTG Met Ala Val 495 GTT CAA ACA Val Gin Thr

ATG

Met 500 TTA TTA Leu Leu 505 CAG AGA AAG CAA G(n Arg Lvs Gin 510 GAA CTG GAA CTA GTT GCA GAA ATT Glu Ile GCA TTG Ala Leu 515 Glu Leu Val o 520 Ala Glu Leu 525 GAC CAG GAT GAA AAG GAC CAG CAA AAT ACA TCT CGC CTG GTA CAG GAA Asp Gin Asp Glu Lys Asp Gin Gin Asn Thr Ser Arg Leu Val Gin Glu 530 535 540 1811 P \OER\AJH\Sptcs\2481510 Mod 06.12.07.doc 7/12/07 30 CAT AAA AAG CTT TTA GAT GAA AAC AAA AGC CTT TCT ACT TAC TAC CAG His Lys Lys Leu Leu Asp Giu Asn Lys Ser Leu Ser Thr Tyr Tyr Gin 545 550 555 CAA TGC AAA AAA CAA CTA GAG GTC ATC AGA AGT CAG CAG CAG AAA CGA Gin Cys Lys Lys Gin Leu Giu Val Ile Arg Ser Gin Gin Gin Lye Arg 560 565 570 575 CAA GGC ACT TCA TGA TTCTCTGGGA CCGTTACATT TTGAAATATG CAAAGAAAGA Gin Giy Thr Ser 580 1859 1907 1962

CTTTTTTTTT

GTGTTCTGAA

CTCATGGTGG

AATGAGCAGC

TCATTGTGTG

CAGCGTCCAT

ATCTCAAAAT

TATTATAAAT

TCCGTGTAAG

AAAATATGTA

GTTAGACTAA

TAAAGATTTT

AAGGAAAGGA

TGCCAACTGC

ACATACAATT

ACTTTGCAAC

AAGGCTAGCC

TTTTTCATAT

ATTAATAATT

TTAGAGTGAT

GGCTTTGATG

AAGGTAAGTG

AATTTGATTG

CTTTAGAGCA

AAACCTTATA

CTATATTTGC

TTACTGTTTC

TTCAAAACAG

TAACAGAACA

TAGAGGTGGA

TTTTTCCCAA

TGGTGGTATA

CCAGCATCCT

GCAGCTGCTC

TGATACATTG

CAATGGATCT

ATGACGATTC

TGCATTTTTT

ATTGCATAAC

ATGCAGTGAA

GGAGGTATCA

ACCTCAAGAA

AAGATGGTAT

TTACGGAAAT

TGGATCAGTA

TATTTAATGA

AACAAAATGG

CGAC

ATGAGTGTTA

TCATTGTGTA

ATGGTAGCAT

CTGTGGCTGT

AACTAGCTGC

TGACTTTATT

ATACCAAGTT

ACGGAACCTT

CTGAACTCAG

AAGCAGTTTT

AACTCATTTT

GCTTTTTGGC

TTTTCCTTTT

CTGTGACTTG

ATATGCATGC

TATGTGCAAA

CTTGTATCTC

AAAGACAGGG

TAGGGATAGT

TTCCATCCGT

ACCGGATTTT

TTTTAAGGAG

2022 2082 2142 2202 2262 2322 2382 2442 2502 2562 2622 2656

Claims (29)

1. An isolated DNA coding for a polypeptide with kinase activity which is Vt activated by transforming growth factor (TGF)-P and comprises the amino acid sequence CN of SEQ ID NO:1 from Ser at position 23 to Ser at position 579. N 2. An isolated DNA according to claim 1 which has the nucleotide sequence of SEQ ID NO:1 from T at position 223 to A at position 1893.

3. A method of producing a polypeptide with kinase activity which is activated by TGF-P, characterized by culturing host cells transformed with a vector comprising DNA according to claim 1, and collecting the expressed product from the culture.

4. A polypeptide with kinase activity which is activated by TGF-p, and which is produced according to the method of claim 3. A vector comprising a DNA according to claim 1.

6. A host transformed with a vector according to claim

7. An isolated DNA coding for a polypeptide with kinase activity which is activated by TGF-P and comprises the amino acid sequence of SEQ ID NO:1 from Met at position 1 to Ser at position 579.

8. An isolated DNA according to claim 2 which has the nucleotide sequence of SEQ ID NO:1 from A at position 157 to A at position 1893.

9. A method of producing a polypeptide with kinase activity which is P \OPER\AH\Specs\l12481510 Mod 06-I2-07.doc 7/12/07 -32- Q activated by TGF-P, characterized by culturing host cells transformed with a vector O comprising DNA according to claim 7, and collecting the expressed product from the culture. A vector comprising a DNA according to claim 7.

11. A host transformed with a vector according to claim ("1

12. An isolated DNA coding for a polypeptide with kinase activity which is activated by TGF-P and is capable of hybridizing with the nucleotide sequence complementary to SEQ ID NO:1 under conditions of 600 0.1 xSSC and 0.1% SDS.

13. An isolated DNA according to claim 6 which has the nucleotide sequence of SEQ ID

14. A method of producing a polypeptide with kinase activity which is activated by TGF-P, characterized by culturing host cells transformed with a vector comprising DNA according to claim 12, and collecting the expressed product from the culture. An isolated DNA coding for a polypeptide with kinase activity which is activated by TGF-P and comprises the amino acid sequence of SEQ ID NO:5 from Ser at position 23 to Ser at position 579.

16. An isolated DNA according to claim 14 which has the nucleotide sequence of SEQ ID NO:5 from T at position 249 to A at position 1919.

17. A method of producing a polypeptide with kinase activity which is activated by TGF-P, characterized by culturing host cells transformed with a vector comprising DNA according to claim 15, and collecting the expressed product from the culture. P \OPER\AJH\Spes\I2481510 Mod 06-12-07.doc 7/12J07 S-33- U O 18. A vector comprising a DNA according to claim

19. A host transformed with a vector according to claim 18. An isolated DNA coding for a polypeptide with kinase activity which is 4 activated by TGF-P and comprises the amino acid sequence of SEQ ID NO:5 from Met at Sposition 1 to Ser at position 579.

21. An isolated DNA according to claim 20 which has the nucleotide sequence of SEQ ID NO: 5 from A at position 183 to A at position 1919.

22. A method of producing a polypeptide with kinase activity which is activated by TGF-P, characterized by culturing host cells transformed with a vector comprising DNA according to claim 20, and collecting the expressed product from the culture.

23. A vector comprising a DNA according to claim

24. A host transformed with a vector according to claim 23. An isolated DNA coding for a polypeptide with kinase activity which is activated by TGF-P and is capable of hybridizing with the nucleotide sequence complementary to SEQ ID NO:5 under conditions of 600 0.lxSSC and 0.1% SDS.

26. A method of producing a polypeptide with kinase activity which is activated by TGF-P, characterized by culturing host cells transformed with a vector comprising DNA according to claim 25, and collecting the expressed product from the culture.

27. An isolated kinase which is activated by TGF-B and has the amino acid P:\OPER\AJH\Specs\ 2481510 Mod 06-12-07doc 7/12/07 -34- Ssequence of SEQ ID NO: 1 from Ser at position 23 to Ser at position 579. O

28. An isolated kinase which is activated by TGF-P and has the amino acid sequence of SEQ ID NO:5 from Ser at position 23 to Ser at position 579.

29. An isolated DNA coding for a fusion protein which comprises: Sa first polypeptide having kinase activity which is activated by 0 transforming growth factor (TGF)-3 and comprises the amino acid sequence of SEQ ID NO: 1 from Ser at position 23 to Ser at position 579; and a second polypeptide. A vector comprising a DNA according to claim 29.

31. A host transformed with a vector according to claim

32. An isolated DNA coding for a fusion protein which comprises: a first polypeptide having kinase activity which is activated by TGF-P and comprises the amino acid sequence of SEQ ID NO:1 from Met at position 1 to Ser at position 579; and a second polypeptide.

33. A vector comprising a DNA according to claim 32.

34. A host transformed with a vector according to claim 33. An isolated DNA coding for a fusion protein comprising: a first polypeptide having kinase activity which is activated by TGF-P and comprises the amino acid sequence of SEQ ID NO:5 from Ser at position 23 to Ser at position 579; and a second polypeptide. P \OPER\AJH\Specs\I 2481 510 Mod 06-12-07doc 7/1207 O U O 36. A vector comprising a DNA according to claim

37. A host transformed with a vector according to claim 36.

38. An isolated DNA coding for a fusion protein comprising: a first polypeptide with kinase activity which is activated by TGF-P and Scomprises the amino acid sequence of SEQ ID NO:5 from Met at position 1 to Ser at position 579; and a second polypeptide.

39. A vector comprising a DNA according to claim 38. A host transformed with a vector according to claim 39.