KR20060062550A - Minimal fragment for catalytic activity of ptk6 and a method for detecting ptk6 inhibitors using the fragment - Google Patents

Abstract

The present invention identifies the site involved in self-inhibition and the site essential for catalytic activity in PTK6, to provide a minimally active fragment of protein tyrosine kinase-6 (PTK6) protein containing a site essential for catalytic activity without self-suppression And reacting the protein fragment with a polypeptide substrate having a tyrosine residue in the presence of an inhibitor, and comparing the phosphorylation of the substrate with phosphorylation in the absence of an inhibitory candidate.

PTK6, PTK6 linker region, PTK6 catalytic domain, inhibitor

Description

Minimal fragment for catalytic activity of PTK6 and a method for detecting PTK6 inhibitors using the fragment}             

1 is a schematic diagram showing domains constituting PTK6 and PTK6. PTK6 consists of an Src homology-3 (SH3) domain, an SH2 domain, a linker region and a catalytic domain. Proteins containing PTK6 SH3 domain and PTK6 catalytic domain were expressed by GST-PTK6-SH3 and GST-PTK6-Kinase fused to glutathione S-transferase (GST). The PTK6 linker region was expressed in the form of Trx-PTK6-Linker fused to thioredoxin (Trx; thioredoxin). In the detection system for measuring PTK6 catalytic activity, GST-PTK6-ΔNLinker-Kinase in which the linker region (ΔNLinker) without the N-terminal half region and the catalytic domain (Kinase) was fused to GST was used.

FIG. 2 is a diagram of GST-PTK6-ΔNLinker-Kinase expressed and purified in Escherichia coli and stained with Coomassie Brilliant Blue. The first lane shows the protein size marker and the size of the protein on the left side. The second lane is the cell lysate after breaking E. coli expressing GST-PTK6-ΔNLinker-Kinase by ultrasonic grinding method, and the third lane is the glutathione Sepharose 4B resin from the cell lysate. Purified GST-PTK6-ΔNLinker-Kinase.

FIG. 3 shows that the PTK6 SH3 domain and the PTK6 linker region have intramolecular interactions. GST or GST-fusion protein was reacted with Trx or Trx-fusion protein bound to Ni ²⁺ -NTA resin and the resin was pulled down. GST or GST-fusion protein subsided with Trx or Trx-fusion protein in pull-down samples was examined by Western-blot with GST antibody.

FIG. 4 shows that the PTK6 catalytic domain and the PTK6 linker region have intramolecular interactions. The figure also shows that the tryptophan 184 (W184) amino acid residue of the PTK6 linker region plays an important role in this intramolecular-interaction. Trx or Trx-fusion protein was reacted with GST or GST-fusion protein bound to glutathione Sepharose 4B resin and the resin was pulled down. The pull-down samples were examined by Western-blot with His-tag antibody to determine whether Trx or Trx-fusion protein had subsided with GST or GST-fusion protein.

5 is a diagram showing that the catalytic activity of PTK6 requires not only the PTK6 catalytic domain but also the C-terminal half of the PTK6 linker region, of which the tryptophan 184 (W184) amino acid residue plays an important role. In order to measure the catalytic activity of PTK6, the reaction was performed by adding PTK substrate and [γ ³² ] ATP to GST-PTK6-ΔNLinker-Kinase, GST-PTK6-ΔNLinkerW184A-Kinase or GST-PTK6-Kinase, and reacting the reaction with P81 chromatograph. After binding to the chromatography paper, the degree of phosphorylation of the substrate was examined by measuring the cpm value using a scintillation counter.

FIG. 6 is a graph illustrating the phosphorylation of PTK substrate according to GST-PTK6-ΔNLinker-Kinase concentration by a method that does not use radioactivity. GST-PTK6-ΔNLinker-Kinase was added to the PTK substrate attached to the 96-well dish to phosphorylate the substrate, and the degree of phosphorylation of the substrate was determined by binding of the mouse antiphosphate-tyrosine antibody to the HRP-conjugated anti mouse IgG antibody. The absorbance was measured at 485 nm through the color reaction.

Figure 7 is a picture of the phosphorylation of PTK substrate by GST-PTK6-ΔNLinker-Kinase according to the reaction time was measured by the method using no radiation.

FIG. 8 is a graph illustrating the degree of inhibition of the phosphorylation of PTK substrate by GST-PTK6-ΔNLinker-Kinase in the presence of various kinase inhibitors under optimal conditions obtained by analyzing the results of FIGS. 6 and 7. Statistical analysis by Student t test (marked with *** when the confidence is 0.999 or more).

The present invention relates to a minimally active fragment of protein tyrosine kinase-6 (PTK6) protein and a method for detecting a catalytic activity inhibitor of PTK6 using the protein fragment.

Protein tyrosine kinase (PTK) is found in all multicellular organisms and is an enzyme that carries important intercellular and intracellular signals that regulate cell growth, differentiation and cell death (Blume-Jensen). et al ., Nature , 411: 355-365, 2001). The catalytic activity of PTK is tightly regulated in such a way that PTK, which is usually in an inactive state, is temporarily activated upon receiving a specific signal. When such PTKs have hyperactive activity due to retrovirus or somatic mutation, or abnormally overexpressed by gene amplification or transcriptional activity, normal control fails to cause cancer (Yarden and Ullrich, Ann. Rev. Biochem). ., 57: 443-478, 1988; Druker et al, New Eng J. Med, 321: 1383-1391, 1989; Ullrich and Schlessinger, Cell, 61:... 203-212, 1990).

There are more than 100 PTKs present in humans, and they have receptor type PTK and non-receptor type PTK depending on whether they have receptor sites and membrane transmembrane sites that can receive extracellular signals. Divided into Receptor type PTK acts to receive extracellular signals such as growth factors and transmits them into cells, while non-receptor type PTKs play an important role in cell growth and differentiation by mediating signal transduction within cells. (Tsygankov, Front Biosci. , 8: s595-635, 2003; Neet and Hunter, Genes Cells, 1: 147-169, 1996). Non-receptor type PTKs found in vertebrates can be classified into 9 families of Abl, Hck, Fak, Fes / Fer, Jak, Src, Csk, Syk / Zap70 and Tec based on similarities in amino acid sequence and functional site composition. (Neet and Hunter, Genes Cells, 1: 147-169, 1996). Most of the PTK is regulated by a self-suppressing mechanism.

PTK6 (also known as breast tumor kinase (Brk), and mouse isologs are called Src-related intestinal kinase (Sik)) is expressed in these cells by reverse transcription PCR with degenerate primer pairs specific for PT. Is a non-receptor type PTK that was discovered in the course of mass irradiation of the resulting PTK mRNAs (Lee et al., Oncogene, 8: 3403-3410, 1993). PTK6 polypeptides inferred from full-length cDNA were observed to be similar to PTKs belonging to the Src family in terms of amino acid sequence similarity and the construction of functional sites with catalytic domains of SH3, SH2 and tyrosine kinases (Mitchell et al., Oncogene , 9: 2383-2390, 1994; Lee et al., Mol. Cells, 8: 401-407, 1998). However, amino acid sequence similarity (41-43%) between PTK6 and Src family is lower than amino acid sequence similarity (55-75%) between PTKs of Src family, and unlike the Src families, there is no site of myristoylation (Lee et al. , Mol. Cells, 8: 401-407, 1998; Mitchell et al., Oncogene , 9 (8): 2383-2390, 1994). In addition, unlike the Src family of PTK genes where the exon-intron structure was evolutionarily conserved (Sideras et al., J. Immunol., 153: 5607-5617, 1994), the PTK6 gene had a unique exon-intron structure. (Lee et al., Mol. Cells, 8: 401-407, 1998). PTK6 was therefore expected to constitute a unique non-receptor PTK family different from the Src family.

PTK6 has been observed to be expressed in epithelial cells that differentiate in the gastrointestinal tract or skin and in breast and colon cancer tissues and cell lines (Derry et al., Oncogene, 22: 4212-20, 2003). Overexpression of PTK6 in human breast epithelial cells enhanced EGF-induced mitogenic effects, resulting in increased cell division and anchorage-independent cell proliferation (Kamalati et al., J Biol.Chem ., 29: 30956-30963, 1996). These results indicate that when PTK6 is abnormally expressed, it is tumorigenic like many other PTKs. PTK6 is also regulated by a self-suppressing mechanism.

Since excessive expression and activity of PTK can cause tumors, PTK catalytic activity inhibitors can be used as an effective cancer treatment. Gleevec and Iressa are two examples of cancer drugs developed as PTK inhibitors. Gleevec (STI571) selectively acts on leukemia cancer cells with an abnormal chromosome called the Philadelphia chromosome. The Philadelphia chromosome produces the Bcr / Abl fusion gene and produces the BCR / ABL fusion protein. Normal ABL proteins are non-receptor type PTKs that promote cell proliferation under strict control, but BCR / ABL fusion proteins are abnormally expressed in leukocytes resulting in leukemia due to excessive cell proliferation. Gleevec inhibits the catalytic activity of ABL, inhibits the proliferation of cancer cells and induces apoptosis. Iresa is an effective anticancer agent for non-small cell lung cancer involving epidermal growth factor (EGF) and EGF receptor receptor type PTK. Iresa inhibits the catalytic activity of the EGF receptor, blocks cell proliferation signals transmitted into the cell from outside the cell, thereby inhibiting cell proliferation and inducing cell death.

In the same principle, the development of PTK6 catalytic activity inhibitors is important for treating diseases related to abnormal cell proliferation such as breast cancer and colon cancer caused by abnormal expression and activity of PTK6.

The type of PTK6 used to detect PTK6 catalytic activity inhibitors is more effective in the form in which self-inhibition does not occur. In order to obtain PTK6 fragments without self-inhibition, mechanisms based on the intramolecular interactions of PTK6 involved in inhibition and activity must be identified. However, the exact mechanism of the regulation of self-inhibition of PTK6 is not known, and therefore, an efficient method for detecting PTK6 inhibitors has not yet been established.

Thus, the present inventors have found that, in addition to the mechanism of self-inhibition regulation by specific binding between the SH2 domain of PTK6 and the phosphorylated tyrosine residue at the C-terminus of the catalytic domain, the N- The self-inhibitory mechanism of specific binding of the terminal proline residues and the C-terminal tryptophan residue of the linker region of the PTK6 domain were specifically regulated through the self-promoting mechanism. By determining the intramolecular-interaction of PTK6, we determined the smallest active fragment of PTK6 protein with substrate phosphorylation activity without self-inhibition, and maximized the substrate phosphorylation catalytic activity while using the least soluble fragment of PTK6 with high solubility in recombinant protein preparation. By confirming that PTK6 inhibitors can be effectively detected, the present invention was completed.

One object of the invention is an amino acid sequence of amino acids 184 to 190 and a catalytic domain of the linker region of PTK6, 0 to 13 amino acid sequences located adjacent to the N-terminal side of amino acid 184 of the linker region, and a catalytic domain To provide a minimally active fragment of the PTK6 protein containing 0 to 6 amino acid sequences located adjacent to the C-terminal side of amino acid 445 of the.

Another object of the present invention is to provide a nucleic acid molecule encoding the amino acid sequence of the least active fragment of the PTK6 protein.

Another object of the present invention comprises the step of reacting the least active fragment of the PTK6 protein with a polypeptide substrate having a tyrosine residue in the presence of a candidate and comparing the phosphorylation of the substrate with phosphorylation in the absence of the candidate. It is to provide a method for detecting a catalytic activity inhibitor of.

In one embodiment, the present invention relates to the least active fragment of PTK6 protein.

The minimally active fragment of the PTK6 protein of this type is characterized in that it does not cause self-inhibition and comprises an essential part for catalytic activity.

PTK6 is a protein having a structure including an SH3 domain, an SH2 domain, a linker region, and a catalytic domain, and is described by SEQ ID NO: 2. The nucleic acid sequence encoding the PTK6 protein is described as SEQ ID NO: 1, and the gene sequence information is described in GenBank NM_005975. In the case of human PTK6, it consists of a total of 451 amino acids, among which the SH3 domain is an 11-72 amino acid sequence, the SH2 domain is an 78-170 amino acid sequence, the linker region is an 171-190 amino acid sequence, and the catalytic domain is 191-445. Corresponds to the amino acid sequence.

In the present invention, “self-inhibition” is a mechanism by which the activity of many non-receptor type PTKs is regulated, and the enzyme is in an inactive state by taking a structure that prevents access of ATP and a substrate to an enzyme active site. It means a phenomenon to be maintained. Through this enzyme regulation mechanism, the cell reacts only to a specific signal. In particular, self-inhibition of non-receptor type PTKs of the Src family is well reported by the organic interaction of each domain (Xu et al., Nature , 385: 595-602, 1997; Sicheri et al., Nature , 385: 602-609, 1997; Sicheri and Kuriyan, Curr Opin Struct Biol ., 7: 777-785, 1997). It is known that the inactive structure of the enzyme is maintained by intramolecular interactions in which the SH2 domain binds to the phosphorylated tyrosine at the C-terminus of the catalytic domain and the SH3 domain binds to the proline of the linker region.

Even in the case of “self-inhibition of PTK6 (or mouse isotope Sik)” reports that SH2 domains bind to phosphorylated Tyr447 and inhibit catalytic activity by self-inhibition mechanisms (Derry et al., Mol. Cell. Biol., 20: 6114). -6126, 2000; Qiu and Miller, J. Biol. Chem., 277: 34634-34641, 2002; Hong et al., J. Biol. Chem., 279: 29700-29708, 2004). In addition, it is proposed that the SH3 domain and the linker region of PTK6 bind to inhibit the catalytic activity by a self-suppressing mechanism. However, the direct binding of these sites was not analyzed, the present inventors confirmed that the catalytic activity of PTK6 is inhibited by intramolecular-interaction of the linker region and SH3 domain of PTK6 directly, PTK6 The N-terminal half region (particularly three amino acid residues of Pro175, Pro177, and Pro179) of the linker region of is important for binding. In addition, in the non-receptor PTKs, the N-terminal region of the linker region binds to the catalytic domain and inhibits catalytic activity by a self-suppressing mechanism, whereas in PTK6, the C-terminal half region of the linker region, particularly the 184 tryptophan residue It was found that the binding of and the catalytic domain is essential for the catalytic activity of PTK6 phosphorylating the substrate.

In the present invention, "minimum active fragment of the PTK6 protein" refers to a form in which the site of self-inhibition occurs in PTK6 is removed and retained a site essential for substrate phosphorylation catalytic activity, and is a fragment that can be effectively used for the detection of PTK6 inhibitors. Therefore, the least active fragment of the PTK6 protein of the present invention, the SH2 domain is removed in order to avoid self-inhibition by the binding of the phosphorylated Tyr447 with the SH2 domain of PTK6, the binding of the SH3 domain and the N-terminal half of the linker region The SH3 domain is removed in order to avoid self-inhibition by and essentially includes the 184 tryptophan residue of the linker region for catalytic activity to phosphorylate the substrate.

Thus, one object of the present invention is to sequence amino acids 184 to 190 of the linker region of PTK6 and amino acid sequence of the catalytic domain, 0 to 13 amino acid sequences located adjacent to the N-terminal side of amino acid 184 of the linker region, and A minimally active fragment of PTK6 protein comprising 0-6 amino acid sequences located adjacent to the C-terminal side of amino acid 445 of the catalytic domain.

In the above description, the term “n-terminal side” and “c-terminal side” refers to the left side of the N-terminal and the right side of the C-terminal center around amino acids, regions, or sites.

In the above, the term “0 to 13 amino acid sequence located adjacent to the N-terminal side of amino acid 184 of the linker region” refers to 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. 11, 12, or 13 amino acid sequences. When having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. 11, 12, or 13 amino acid sequences, 183, 182 to 183, 181 to 183, 180 of the linker region, respectively 183, 179 to 183, 178 to 183, 177 to 183, 176 to 183, 175 to 183, 174 to 183, 173 to 183, 172 to 183, or 171 to 183.

In the above, the term “0 to 6 amino acid sequence located adjacent to the C-terminal side of amino acid 445 of the catalytic domain” refers to 0, 1, 2, 3, 4, 5, Or six amino acid sequences. In the case of having 1, 2, 3, 4, 5, or 6 amino acid sequences, SEQ ID NOs: 446, 446 to 447, 446 to 448, 446 to 449, 446 to 450, or Amino acid sequences 446 through 451.

In a preferred embodiment of the present invention, amino acid sequences 184 to 190 of the linker region of PTK6 and the amino acid sequence of the catalytic domain, four amino acid sequences located adjacent to the N-terminal side of amino acid 184 of the linker region, and 445 of the catalytic domain It provides the least active fragment of the PTK6 protein comprising six amino acid sequences located adjacent to the C-terminal side of the amino acid. The protein fragment set forth in SEQ ID NO: 4 was expressed as PTK6-ΔNLinker-Kinase in the present invention. PTK6-ΔNLinker-Kinase is preferably used because of its high catalytic activity for phosphorylating substrates and its high solubility in the production of recombinant proteins.

The least active fragment of the PTK6 protein of the present invention is the least active fragment of PTK6 protein from all eukaryotes with PTK6, including fish, amphibians, reptiles, birds, mammals and the like. Preferably it is derived from mammals such as cattle, goats, sheep, pigs, mice, rabbits, and more preferably, it is the least active fragment of PTK6 protein derived from humans.

The least active fragment of the PTK6 protein of the present invention includes the amino acid sequence variant as well as the amino acid sequence variant of the native type derived from the living organism within the scope of the present invention. Sequence variant of the least active fragment of PTK6 protein means a protein having a sequence in which the natural amino acid sequence differs from one or more amino acid residues, and any cleavage in the final structure of the protein as long as it retains enzymatic activity to phosphorylate tyrosine residues in the substrate. , Combinations of deletions, insertions, and substitutions are also possible. One example of a sequence variant is a form in which amino acid residues at sites not essential for catalytic activity are truncated or deleted, or amino acid residue substitutions at sites important for self-inhibition. In some cases, it may be modified by phosphorylation, glycosylation, methylation, farnesylation, or the like. Through such variations and modifications of the sequence, it is more desirable if the change in amino acid sequence of the least active fragment of PTK6 protein increases the function and / or stability (thermal stability, pH stability, structural stability, etc.) and / or solubility of the protein. Do.

A method of causing a mutation in the amino acid sequence of a native protein is by using a method of preparing to have a nucleic acid molecule comprising a nucleotide sequence corresponding to the amino acid sequence to be altered by mutating a nucleotide sequence encoding a native protein. The method of obtaining the gene encoding the least active site can be mutated in vivo or in vitro using any mutation technique well known in the art. Site-directed mutagenesis (Hutchinson et al., J. Biol. Chem ., 253: 6551, 1978; Zoller and Smith, DNA, 3: 479-488, 1984; Oliphant et al., Gene, 44: 177, 1986; Hutchinson et al ., Proc. Natl. Acad. Sci. USA ., 83: 710, 1986), TAB linker (Pharmacia), PCR techniques (Higuchi, 1989, “Using PCR to Engineer DNA” in PCR Technology: Principles and Applications for DNA Amplification , H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70).

The least active fragments of PTK6 protein and variants thereof may be isolated from a source of natural origin, chemically synthesized, or introduced into and expressed from a recombinant expression vector encoding the least active fragment of PTK6 protein into a suitable host. You can get it by

Preferably, a vector comprising a nucleic acid sequence encoding a minimally active fragment amino acid sequence of PTK6 protein is prepared and transformed into a host cell with the vector to recover the least active fragment of PTK6 protein from the cultured transformant. Through the steps, the least active fragment of the natural or variant PTK6 protein can be prepared.

In another aspect, the invention relates to a nucleic acid molecule encoding the amino acid sequence of the least active fragment of the PTK6 protein.

Preferably, a nucleic acid molecule encoding a protein as set forth in SEQ ID NO: 4 having an amino acid sequence of 180 to 190 in the linker region, an amino acid sequence of a catalytic domain, and an amino acid sequence of 446 to 451 under the catalytic domain is provided. It may be encoded by the nucleic acid sequence of SEQ ID NO: 3.

The nucleic acid sequence encoding the least active fragment of PTK6 protein as described above may be mutated by substitution, deletion, insertion or combination thereof, as long as it encodes a protein having equivalent activity.

Nucleic acid sequences encoding the least active fragments of the PTK6 protein of the invention and variants thereof may be isolated from nature or may be prepared by artificially synthetic or genetic recombination methods. Nucleic acid sequences encoding the least active fragments of PTK6 protein of the invention are provided by operably linked to an expression vector capable of expressing them.

In another aspect, the present invention relates to a recombinant expression vector comprising the nucleic acid molecule.

In the present invention, "expression vector" is a vector capable of expressing a target protein or RNA by introducing a nucleic acid sequence encoding a gene of interest into a suitable host cell, comprising an essential regulatory element operably linked to express the gene insert Refers to the genetic construct. Such expression vectors include all vectors such as plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors and the like.

Suitable expression vectors have expression control elements such as promoters, initiation codons, termination codons, polyadenylation signals and enhancers. Initiation and termination codons are generally considered to be part of a nucleic acid sequence encoding a protein, and the protein coding sequence is constructed to be in frame to be operable in a vector. The promoter may be constitutive or inducible. Conventional expression vectors also include a selection marker. Operational linkage with expression vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation uses enzymes commonly known in the art.

In a specific embodiment of the present invention, a vector expressing GST-fusion PTK6 ΔNLinker-Kinase was prepared using pGEX-4T3.

A vector comprising a nucleic acid sequence encoding the least active fragment of PTK6 protein can be transformed into an appropriate host cell.

Any method for introducing a "transformation" nucleic acid into a host cell in an organism, cell, tissue or organ is included in the present invention and may be carried out by selecting a suitable standard technique according to the host cell as is known in the art. These methods include electroporation, plasma fusion, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, agitation with silicon carbide fibers, agro bacterial mediated transformation, PEG, dextran sulfate, lipo Pectamine and dry / inhibited mediated transformation methods and the like.

Host cells include Escherichia coli, Bacillus subtilis, Streptomyces, Pseudomonas (e.g. Pseudomonas putida), Proteus mirabilis Prokaryotic host cells such as Proteus mirabilis or Staphylococcus (eg, Staphylocus carnosus) are but are not limited to these. Preferred host cells are E. coli. Coli, Lee. E. coli XL-1 blue E. coli BL21 (DE3), two. E. coli JM109, Lee. E. coli DH series, Lee. Coli TOP10, Lee. E. coli HB101 or Yi. E. coli ER2566. Also, fungi such as Aspergillus, Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, Neurospora crasa Yeast, other lower eukaryotic cells including crassa), or cells derived from higher eukaryotes, including insect cells, plant cells, mammals, and the like, can be used as host cells.

After expressing the least active fragments of PTK6 protein in the transformants, conventional biochemical separation techniques, such as treatment with protein precipitants (salting), centrifugation, sonication, ultrafiltration, dialysis, for isolation and purification , Molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, affinity chromatography, and the like can be used. In order to separate proteins of high purity, they are commonly used in combination (Sambrook et al., Molecular Cloning: A laborarory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press (1989); Deuscher, M., Guide to Protein Purification Methods Enzymology, Vol. 182. Academic Press. Inc., San Diego, CA (1990)).

Preferably, the least active fragment of PTK6 protein is purified by adding a tag to the protein sequence and using an antibody or a substance having a suitably high affinity for such a tag. Tags that may be used in the present invention include His-tag, T7-tag, S-tag, FLAG-tag, thioredoxin (Trx: thioredoxin) -tag, His-patch thioredoxin (Tag), lacZ (β-Galactosidase) -tag, chloroamphenicol acetyltransferase-tag, trpE-tag, avidin / streptavidin / Strep -tag, T7gene10-tag, staphylo Staphylococcal protein A-tag, staphylococcal protein G-tag, glutathione- S- transferase-tag, DHFR (dihydrofolate reductase) -tag, CBD's (cellulose binding domains)- Tags, MBP (maltose binding protein) -tag, galactose-binding protein (tag), tag, calmodulin binding protein (CBP) -tag, hemagglutinin influenza virus (HAI) -tag, HSV Tag, B- (VP7 protein region of bluetongue virus) -tag, polycysteine eine) -tag, polyphenyalanine-tag, (Ala-Trp-Trp-Pro) _n -tag, polyaspartic acid-tag, c-myc-tag, lac -pressor ( lac repressor ) -Tags, and the like, but are not limited thereto. The tag may be located at the N-terminus, C-terminus or the inside of the target protein according to the tag type. The tag not only facilitates the purification of the protein, but also enhances the expression of the protein, secretes the protein, and increases the solubility of the protein, depending on the tag used. Preferably, the least active fragment of PTK6 protein is fused to GST-tag. GST is a protein of about 26 kDa that can be fused to the N-terminus of the nucleic acid encoding PTK6-ΔNLinker-Kinase and is cleavable using glutathione affinity or GST antibodies. In addition to the hassle, when proteins are aggregated, the protein yields a complex additional process of dissolving and denaturing the protein in the appropriate solution to refold it with a refolding agent such as urea, guanidine, arginine, and the like. The disadvantage is that efficiency and activity are very poor. However, when the smallest active fragment of the GST-fusion PTK6 protein of the present invention was transformed into E. coli, it was found that the solubility was high and the purification was easy.

In a specific embodiment of the present invention, GST-PTK6-ΔNLinker-Kinase in which PTK6-ΔNLinker-Kinase is fused with GST was used. A recombinant expression vector was prepared to express GST-PTK6-ΔNLinker-Kinase having a GST label on the N-terminus of PTK6-ΔNLinker-Kinase, and GST-PTK6-ΔNLinker-Kinase was expressed in Escherichia coli, thereby producing a glutathione cell. Purification using a Rose 4B affinity column. GST-PTK6-ΔNLinker-Kinase not only has the advantage of easy purification, but also shows that it is expressed more as a soluble protein and has higher enzymatic activity than the total PTK6 protein fused to GST (GST-PTK6). there was.

Minimal active fragments of PTK6 protein having enzyme catalytic activity but no self-inhibition prepared by the above method can be used for detection of inhibitors that inhibit the substrate catalytic activity of PTK6.

In another aspect, the invention comprises reacting a least active fragment of the PTK6 protein with a polypeptide substrate having a tyrosine residue in the presence of an inhibitor and comparing the phosphorylation of the substrate with phosphorylation in the absence of the candidate. A method for detecting catalytic activity inhibitors of PTK6.

In the present invention, the "inhibitor" generically refers to agents or compounds that reduce or eliminate the biological activity of PTK6. By "removal of activity" is meant that the enzymatic catalytic activity is completely blocked by the inhibitor treatment, which means that the catalytic activity is reduced relative to the "reduction of activity" control. Inhibitors may be synthetic compounds or may be extracts from plants, animals or microorganisms.

The inhibitor blocks PTK6 signal transduction and can be used as a therapeutic agent for diseases caused by overexpression and overactivity of PTK6. The term "disease" refers to all pathological conditions induced by or related to PTK6, including but not limited to cancers such as breast cancer, colon cancer, and the like.

As used herein, "substrate" refers to any polypeptide comprising a tyrosine residue that can be recognized and phosphorylated by the least active fragment of PTK6 protein. Naturally existing in vivo substrates or synthetic polymers containing artificially made tyrosine may be used as the substrate. In the present invention, poly-Glu-Tyr (Cat. No. P0275, Sigma, USA) was used.

Since PTK6 is an enzyme that phosphorylates a substrate, the degree of phosphorylation of the substrate is a measure of enzyme activity. Determination of the degree of phosphorylation of a substrate can detect PTK6 inhibitors by forming a substrate-antibody complex using an antibody that specifically reacts with phosphorylated tyrosine residues and quantifying it using a detection label. The antibody may be an antibody directly labeled with a detection indicator or an unlabeled antibody. In the case of an unlabeled antibody, it is labeled indirectly through a secondary antibody labeled with a detection label.

In the present invention, the "label or detectable moiety" spectroscopic, photochemical, biochemical, immunochemical, chemical, other physical It refers to a composition that can be detected by means. Such detection markers include, but are not limited to, enzymes, fluorescent materials, ligands, luminescent materials, microparticles, redox molecules and radioisotopes.

Enzymes include β-glucuronidase, β-D-glucosidase, β-D-galactosidase, urease, peroxidase or alkaline phosphatase, acetylcholinesterase, glucose oxidase, hexokinase And GDPase, RNase, glucose oxidase and luciferase, phosphofructokinase, phosphoenolpyruvate carboxylase, aspartate aminotransferase, phosphphenolpyruvate decarboxylase or β-latamases Including but not limited to. Fluorescent materials include, but are not limited to, fluorescein, isothiocyanate, rhodamine, phycoerythrin, phycocyanin, allophycocyanin, o-phthalaldehyde or fluorescamine, and the like. Ligands include, but are not limited to, biotin derivatives, and the like. Luminescent materials include, but are not limited to, acridinium esters, luciferin, luciferase, and the like. Microparticles include, but are not limited to, colloidal gold, colored latex, and the like. Redox molecules include, but are not limited to, ferrocene, ruthenium complex, biologen, quinone, Ti ions, Cs ions, diimides, 1,4-benzoquinone, hydroquinone and the like. Radioisotopes include, but are not limited to, ³ H, ¹⁴ C, ³² P, ³⁵ S, ³⁶ Cl, ⁵¹ Cr, ⁵⁷ Co, ⁵⁸ Co, ⁵⁹ Fe, ⁹⁰ Y, ¹²⁵ I, ¹³¹ I, ¹⁸⁶ Re, and the like. .

In the present invention, the treatment of the "detection of inhibitor" inhibitor is possible by comparing the effect of the least active fragment of PTK6 protein on the phosphorylation of the substrate with the phosphorylation of the control group not treated with inhibitor. The degree of phosphorylation can be compared by absolute or relative (eg relative intensity of signal) values. For example, if the phosphorylation of the substrate is reduced by at least 2 fold compared to the control by treating the inhibitor candidate, it can be considered as a significant difference and can be considered an effective inhibitor.

As long as the phosphorylation of the substrate can be confirmed, any method is not limited, but ELISA using an antibody specific for phosphorylated tyrosine is preferable because it can simply examine a large number of samples at once.

ELISA is a direct ELISA using a labeled antibody that recognizes an antigen attached to a solid support, an indirect ELISA using a labeled secondary antibody that recognizes a capture antibody in a complex of antibodies that recognize an antigen attached to a solid support, and attaches to a solid support Direct sandwich ELISA using another labeled antibody that recognizes the antigen in a complex of antibodies and antigens, a label that recognizes the antibody after reacting with another antibody that recognizes the antigen in a complex of the antibody and antigen attached to a solid support Various ELISA methods, such as indirect sandwich ELISA using secondary antibodies.

In a specific embodiment of the present invention, in order to measure PTK6 catalytic activity, a reaction was performed by adding a minimally active fragment of PTK6 protein to a PTK substrate bound to 96 wells, followed by mouse antiphosphate-tyrosine antibody, HRP conjugated (horseradish peroxidase- conjugated) The anti-mouse IgG antibody and the HRP chromogenic substrate were reacted in order, and the degree of phosphorylation of the tyrosine residue of the substrate was measured by absorbance. After setting the concentration and reaction time of GST-PTK6-ΔNLinker-Kinase, the smallest active fragment of PTK6 protein to measure PTK6 catalytic activity, AG1478 (EGF receptor inhibitor), PP1 (Src family PTK inhibitor), LY294002 (PI3 kinase) Changes in PTK6 catalytic activity were analyzed in the presence of various kinase inhibitors of inhibitors), AG490 (JAK2 kinase inhibitor), Genistein (common PTK inhibitor), PD98059 (MEK inhibitor), and U0126 (MEK inhibitor). In this assay, GST-PTK6-ΔNLinker-Kinase was confirmed to be inhibited only by genistein, a general inhibitor of PTK. It can be confirmed that it can be used effectively.

In order to determine the optimal concentration and the reaction time of GST-PTK6-ΔNLinker-Kinase for the determination of PTK6 catalyst activity, the substrate phosphorylation at the concentration of 0 to 1000 ng of GST-PTK6-ΔNLinker-Kinase and the reaction time of 0 to 60 minutes was measured. As a result of the analysis, preferably, the amount of PTK6-ΔNLinker-Kinase is about 200 to 400 ng / well, the reaction time is 10 to 30 minutes, and more preferably the amount of PTK6-ΔNLinker-Kinase is about 300 ng / well, reaction time Is 20 minutes.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 E. coli Expression Vector Cloning to Express Functional Sites of PTK6

PTK6 consists of SH3, SH2, a linker region, and a catalyst domain (FIG. 1). To analyze intramolecular binding in PTK6, E. coli expression vectors were cloned to express the SH3 domain, linker region, and catalytic domain of PTK6 in the form of GST- or Trx-fusion proteins in E. coli (FIG. 1).

To construct a vector expressing the GST-fusion PTK6 SH3 domain (GST-PTK6-SH3), cDNA fragments encoding the PTK6 SH3 domain (amino acids 3 to 72) were prepared using pBS-PTK6-MR (Lee et al., Mol. Cells , 8: 401-407, 1998) were used as a template for PCR amplification. The primer pair is a primer of SEQ ID NO: 5 [5'-CG GGATCC CGGGACCAGGCTCACCTG- 3 'used; BamHI site (underlined) and PTK6 cDNA (GeneBank NM_005975) nt 47-67] and primers of SEQ ID NO: 6 [5'-G GAAT TC A CGTCTCCCTCTCGGCCAGGT-3 '; EcoRI site (underlined), termination codon (bold) and PTK6 cDNA nt 256-239]. PCR products were digested with BamHI and EcoRI and then ligated to pGEX-4T-3 (Pharmacia, USA) digested with the same restriction enzymes. The ligation mixture was transformed into E. coli XLI-Blue to obtain a pGEX-4T-3-PTK6-SH3 clone that was confirmed by restriction digestion and DNA sequencing.

To prepare a vector expressing the Trx-fusion PTK6 linker (Trx-PTK6-Linker), cDNA fragments encoding the linker region (amino acids 171 to 191) of PTK6 were PCR amplified using pBS-PTK6-MR as a template. The primer pair used at this time is the primer of SEQ ID NO: 7 [5'-CATG CCATGG GCCGGAAGCACGAGCCTGAG-3 '; NcoI portion (underlined) and PTK6 cDNA nt 551-568] and a primer of SEQ ID NO: 8 [5'-CGC GGATCC TCA GAACTCCTCCCAGGCCTC -3 '; BamHl site (underlined), stop codon (bold) and PTK6 cDNA nt 613-593]. PCR products were digested with NcoI and BamHI and then ligated to pET-32c (+) (Novagen, USA) digested with the same restriction enzymes. The ligation mixture was transformed into E. coli XLI-Blue to obtain a pET-32c (+)-PTK6-Linker clone, which was confirmed by DNA sequencing.

In order to construct a vector expressing the GST-fusion PTK6 catalytic domain (GST-PTK6-Kinase), cBS fragments encoding PTK6 catalytic domains (amino acids 189-451; PTK6 cDNA nt 605-1372) were identified by pBS-PTK6-MR. PCR amplification was carried out as a template. The primer pair used at this time is [5'-G GAATTCC GAGGAGTTCACGC-3 'of SEQ ID NO: 9; EcoRI site (underlined) and PTK6 cDNA nt 605-617] and the T7 promoter primer of SEQ ID NO: 10 [5'-CATTATGCTGAGTGATATCCCG-3 '; nt 668-689 (GeneBank X52327). Since the PCR product obtained had an EcoRI site introduced into Primer 5 and a NotI site upstream of the T7 promoter primer, the PCR product was digested with EcoRI and NotI and then ligated to pGEX-4T3 digested with the same enzyme. . The ligation mixture was transformed into E. coli XL1-Blue to obtain a pGEX-4T3-PTK6-Kinase clone, which was confirmed by restriction digestion and DNA sequencing.

To prepare a vector expressing GST-fused PTK6 ΔNLinker-Kinase, PCR was performed using pBS-PTK6-MR as a template for cDNA fragments encoding ΔNLinker-Kinase (aa 180-451; PTK6 cDNA nt 578-1396) of PTK6. Amplified. The primer pair used at this time is [5'-G GAATTCC CATTGGGATGACT-3 'of SEQ ID NO: 11; EcoRI site (underlined) and PTK6 cDNA nt 578-592] and T7 promoter primer of SEQ ID NO: 10 [5'-CATTATGCTGAGTGATATCCCG-3 '; nt 668-689 (GeneBank X52327)]. Since the PCR product obtained had an EcoRI site introduced into Primer 6 and a NotI site upstream of the T7 promoter primer, the PCR product was digested with EcoRI and NotI and then digested with pGEX-4T3. Lg. The ligation mixture was transformed into E. coli XL1-Blue to obtain a pGEX-4T3-PTK6-ΔNLinker-Kinase clone, which was confirmed by restriction digestion and DNA sequencing.

Trx-PTK6-LinkerW184A and GST-PTK6- in order to obtain a protein in which the 184th tryptophan at the linker C-terminal half region was mutated to alanine (W184A) in the proteins Trx-PTK6-Linker and GST-PTK6-ΔNLinker-Kinase. A vector expressing ΔNLinkerW184A-Kinase was constructed. The introduction of the W184A mutation used the QuickChange site-directed mutation method (Kirson et al., Nucleic Acids Res. , 26: 1848-1850, 1998). a primer of SEQ ID NO: 12 using pET-32c (+)-PTK6-Linker and pGEX-4T3-PTK6-ΔNLinker-Kinase as a template [5'-TTGGGATGAC GCG GAGAGGCCGAGGGAGGA-3 '; Mutation of Trp184 codon into Ala codon in PTK6 cDNA nt 580-609 (underlined) and primer of SEQ ID NO: 13 [5'-TCGGCCTCTC CGC GTCATCCCAATGGGGCA-3 '; Mutant transduction of Trp184 codon in PTK6 cDNA nt 602-573 into Ala codon (underlined)] was amplified by PCR. After PCR-amplified samples were treated with DpnI to remove template DNA, the remaining PCR product was transformed into E. coli XLI-Blue to obtain pET-32c (+)-PTK6-LinkerW184A and pGEX-4T3-PTK6-ΔNLinkerW184A-Kinase clones. Confirmed by DNA sequencing.

Example 2: Expression and Purification of Functional Sites of PTK6 in Escherichia Coli

In order to express and purify GST-PTK6-SH3 in E. coli, E. coli XL1-Blue transformed with pGEX-4T-3-PTK6-SH3 was inoculated into 100 ml of LB medium until absorbance was 0.5 at 600 nm. After incubation at 占 폚, 0.5 mM IPTG was added and further incubated at 23 占 폚 at 100 rpm for 5 hours. After E. coli was harvested by centrifugation, 10 ml of lysis buffer solution (PBS-A (137 mM NaCl, 2.7 mM KCl, 10 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ , pH 7.4), 1 mM EDTA, 1 mM PMSF] and then 100 μg / ml of lysozyme and 0.1% (v / v) Triton X-100 were added and reacted at 30 ° C. for 15 minutes. The reaction solution was pulverized ultrasonically to pulverize the DNA of E. coli, and centrifuged at 12,000 × g for 30 minutes at 4 ℃ to obtain a supernatant. Apply supernatant to a 300 μl bed volume of Glutathione Sepharose 4B affinity column (Sigma, USA) in lysis buffer and wash buffer solution for GS4B column at least 10 times the bed volume. After washing with A), GST-PTK6-SH3 was eluted with an elution buffer (20 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0) for a 1.5 ml GS4B column. Eluted GST-PTK6-SH3 protein was dialyzed in 20 mM Tris-HCl, pH 7.4.

To express and purify GST-PTK6-Kinase, GST-PTK6-ΔNLinker-Kinase, GST-PTK6-ΔNLinkerW184A-Kinase from E. coli, pGEX-4T3-PTK6-Kinase, pGEX-4T3-PTK6-ΔNLinker-Kinase, pGEX- E. coli XL1-Blue transformed with 4T3-PTK6-ΔNLinkerW184A-Kinase was inoculated into 500 ml of LB medium and incubated at 37 ° C until the absorbance was 0.5 at 600 nm, followed by addition of 0.1 mM IPTG at 23 ° C, Further incubation at 100 rpm for 23 hours. E. coli was harvested by centrifugation, suspended in 50 ml lysis buffer, and the supernatant was added to a 1 ml bed volume of glutathione Sepharose 4B affinity column through the same process as the purification of GST-PTK6-SH3. And wash with GS4B column wash buffer solution at least 10 times the bed volume, then use GST-PTK6-Kinase, GST-PTK6-ΔNLinker-Kinase, GST-PTK6-ΔNLinkerW184A-Kinase with elution buffer solution for 5 ml GS4B column. Eluted. Eluted GST-PTK6-Kinase, GST-PTK6-ΔNLinker-Kinase, GST-PTK6-ΔNLinkerW184A-Kinase proteins were dialyzed with active buffer solution (20 mM Tris-HCl, pH 7.4, 10 mM MgCl ₂ , 1 mM MnCl ₂ ) It was.

To express and purify Trx-PTK6-Linker and Trx-PTK6-LinkerW184A in E. coli, pET-32c (+)-PTK6-Linker and pET-32c (+)-PTK6-LinkerW184A were transformed into E. coli BL21 (DE3) (Novagen, USA). The transformed E. coli was inoculated in 100 ml of LB medium and incubated at 37 ° C. until the absorbance was 0.5 at 600 nm, followed by further incubation at 23 ° C. and 100 rpm for 5 hours by adding 0.1 mM of IPTG. E. coli was harvested by centrifugation, suspended in 10 ml of a buffer solution containing 5 mM imidazole, and then 100 μg / ml of lysozyme and 0.1% (v / v) Triton X-100 was added. The reaction was carried out at 30 ° C. for 15 minutes. The reaction solution was ultrasonically pulverized, and the DNA of E. coli was pulverized and centrifuged at 12,000 × g for 30 minutes at 4 ° C. to obtain a supernatant. Apply the supernatant to a 300 μl bed volume of Ni ²⁺ -NTA affinity column (Amersham, USA) in lysis buffer and wash buffer solution (20 mM imidazole, PBS-A for at least 10 times the bed volume). , pH 7.4), and then Trx-PTK6-Linker and Trx-PTK6-LinkerW184A were eluted with elution buffer (100 mM imidazole, PBS-A, pH 7.4) for 1.5 ml NTA column. The eluted Trx-PTK6-Linker and Trx-PTK6-LinkerW184A proteins were dialyzed in PBS-A.

Based on 1 liter of E. coli culture, GST-PTK6-SH3, Trx-PTK6-Linker, Trx-PTK6-LinkerW184A, GST-PTK6-Kinase, GST-PTK6-ΔNLinker-Kinase, GST-PTK6-ΔNLinkerW184A-Kinase 30 mg / l, 25 mg / l, 25 mg / l, 5 mg / l, 5 mg / l, 5 mg / l, respectively. Purification with an affinity column suitable for each protein showed greater than 95% purity. As an example of these proteins, the state of GST-PTK6-ΔNLinker-Kinase expressed in E. coli and purified for the catalytic activity measurement of PTK6 is shown in FIG. 2.

Example 3: Establishment of Expression Sites Suitable for Measuring Catalyst Activity of PTK6

SH2 Domain and Phosphorylated Tyr447 : PTK6 reports that SH2 domain binds to phosphorylated Tyr447 at the C-terminus of the catalytic domain and inhibits catalytic activity by a self-suppressing mechanism (Derry et al., Mol. Cell. Biol., 20: 6114-6126, 2000; Qiu and Miller, J. Biol. Chem., 277: 34634-34641, 2002), PTK6 via binding of SH2 domain and phosphorylated Tyr447 by removing the SH2 domain from the binding partner. Self-inhibition did not occur.

SH3 domain and linker region : There have been reports of mutations in PTK6's SH3 domain and linker region to promote the activity of PTK6 (Qiu and Miller, Oncogene , 22: 1-8, 2003), but the SH3 domain of PTK6 Direct linkage of and linker region was not analyzed. Therefore, the SH3 domain and the linker region of PTK6 bind and the sites important for the binding were analyzed by a pull-down assay.

2 ug of GST or GST-fusion protein was added to the precipitate containing Ni ²⁺ -NTA resin bound to the Trx or Trx-PTK6-SH2 protein described in Example 2, and then reacted at 4 ° C. for 8 hours at 1,2,500 rpm. The supernatant was carefully removed by centrifugation for 5 minutes, suspended in 1 ml of cold PBS-A, and centrifuged at 2,500 rpm for 1 minute to remove the supernatant three times. 2 x SDS sample buffer (100 mM Tris-HCl, pH 6.8, 4% SDS, 0.2% bromophenol blue, 20% glycerol) and β-mercaptoethanol (final concentration 5%) were added to the precipitate. After boiling for 1 minute, the mixture was centrifuged at 14,000 rpm to obtain a supernatant containing the denatured protein. The protein thus obtained was loaded on 10% SDS-PAG at 20 ul and then developed. Proteins deployed on SDS-PAG were coated with a rabbit anti-GST antibody (1: 1000, Cat no. PC53-100UG, Calbiochem) and an HRP-conjugated goat anti-rabbit IgG antibody (1: 5000, Western blot analysis was performed by sequentially reacting Cat no.NA934, Amersham).

As can be seen in Figure 3, Trx-PTK6-Linker did not bind with GST, Trx did not bind with GST-PTK6-SH3, but Trx-PTK6-Linker was found to bind with GST-PTK6-SH3. . Thus, it was found that the SH3 domain and the linker region of PTK6 are mutually bonded. In addition, similar pull-down assays revealed that each protein mutated to Ala by Pro175, Pro177, and Pro179, located in the N-terminal half region of the PTK6 linker region, was partially impaired with SH3. It was confirmed that the protein mutated to Prola, Pro177, Pro179, and Ala completely damaged the binding to SH3. From these results, it was confirmed that the SH3 domain of PTK6 and the N-terminal half of the linker region specifically bind to inhibit the catalytic activity of PTK6. Accordingly, both binding partners of the N-terminal half region of the SH3 domain and the linker region of PTK6 were removed to prevent self-inhibition of PTK6 through the coupling of the SH3 domain and the linker region.

Catalytic domain and linker domain: Intramolecular-inhibition of linker and catalytic domains in non-receptor PTKs belonging to the Src family has been reported (Ref: LaFevre-Bernt et al., J. Biol. Chem) , 273: 32129-32134, 1998; Xu et al., Nature , 385: 595-602, 1997; Sicheri et al., Nature , 385: 602-609, 1997). Therefore, it was analyzed whether the catalytic domain and the linker region of PTK6 are mutually bonded and whether the binding is involved in self-inhibition.

A pull-down assay was performed in the same manner as described above by adding 2 ug of Trx or Trx-fusion protein to the precipitate containing glutathione Sepharose 4B resin bound to the GST or GST-PTK6-Kinase protein described in Example 2, respectively. . Proteins deployed on SDS-PAG were transfected with mouse anti-His-tag antibody (1: 1000, Cat no. 34660, Qiagen) and HRP-conjugated sheep anti-mouse IgG antibody (1: 3000, Western blot analysis was performed by sequentially reacting Cat no.A6782, Sigma).

As can be seen in Figure 4, GST-PTK6-Kinase did not bind to Trx, GST did not bind to Trx-PTK6-Linker, but GST-PTK6-Kinase was confirmed to bind to Trx-PTK6-Linker. . Therefore, it was found that the catalytic domain and the linker region of PTK6 specifically interlinked. However, since GST-PTK6-Kinase hardly bound with Trx-PTK6-LinkerW184A, it was found that the Trp184 residue in the C-terminal half region is important for binding to the catalytic domain. In addition, through a similar pull-down assay, the protein mutated to Ala of Pro175, Pro177, and Pro179 in the N-terminal half of the PTK6 linker region had no effect on binding to the catalytic domain. From these results, it was confirmed that the three half of the catalytic domain and the linker region of PTK6 specifically bind.

The effect of intramolecular-interaction on the catalytic domain of PTK6 and the C-terminal half of the linker region was analyzed on the catalytic activity of PTK6. To measure the catalytic activity, poly-Glu-Tyr (Cat.No. P0275, Sigma, GT-PTK6-ΔNLinker-Kinase, GST-PTK6-ΔNLinkerW184A-Kinase or GST-PTK6-Kinase) is used as a PTK substrate. USA) and reacted in the presence of [γ ³² ] ATP, and then reacted with P81 Chromatography paper (Cat. No. 3698-915, Whatman) to measure the extent of substrate phosphorylation using a scintillation counter to analyze the degree of catalytic activity. It was.

For each 100 ng of GST-PTK6-ΔNLinker-Kinase, GST-PTK6-ΔNLinker W184A-Kinase or GST-PTK6-Kinase and 500 ug / ml poly-Glu-Tyr, 0.1 mM NaVO ₄ A total of 10 μl of the reactants, in which 0.5 μl [γ ³² ] ATP (3000 Ci / mmol, 10 μCi / μl) was added to the included 1X buffer solution, were reacted at 30 ° C. for 30 minutes. After 30 minutes the reaction was stopped by the addition of 5 μl of 7.5 M Guanidine-HCl. 10 μl of this was spotted on a P81 chromatography paper. Wash this paper 6 times with 10% solution containing 5% Trichloroacetic acid (Cat.No.T8657, Sigma) and 0.5% H ₃ PO ₄ (Cat.No. P6560, Sigma), and finally with 100% ethanol. Rinse and dry. The dry paper was placed in a tube containing 4 ml of scintillation cocktail (Cat. No. 882475, ICN) and the cpm value was measured with a scintillation counter.

As can be seen in Figure 5, it can be seen that GST-PTK6-Kinase having a catalytic domain of GST and PTK6 has little catalytic activity. However, GST-PTK6-ΔNLinker-Kinase, which includes the C-terminal half region and the catalytic domain of the linker region of PTK6, exhibited very high catalytic activity, and GST-PTK6- mutated Trp184 to Ala in the linker region of the same protein. It was found that ΔNLinkerW184A-Kinase loses catalytic activity. This result shows that, unlike the Src family non-receptor PTKs in which the intramolecular interaction of the catalytic domain with the linker region self-inhibits catalytic activity, in the case of PTK6 the C-terminal half region of the catalytic domain and the linker region (particularly the Trp184 residue) ) Is essential for catalytic activity.

To summarize these results, in order to avoid self-inhibition, the N-terminal half region of the SH2 domain, the SH3 domain and the linker region is removed, and PTK6- having the C-terminal half region and the catalytic domain of the linker region essential for catalytic activity ΔNLinker-Kinase (aa 180-451, PTK6 cDNA nt 587-1372) was determined as the least active site of PTK6. Therefore, in the present invention, GST-PTK6-ΔNLinker-Kinase was used for the catalytic activity measurement.

Example 4: Setting of PTK6 Catalyst Activity Measurement Conditions

In order to establish a method for measuring the catalytic activity of PTK6 in high through-put, GST-PTK6-ΔNLinker-Kinase was added to the PTK substrate bound to 96 wells, followed by reaction with a mouse antiphosphate-tyrosine antibody, HRP. An ELISA method was used to measure the degree of phosphorylation of the tyrosine residues on the PTK substrate by sequentially reacting the conjugated anti-mouse IgG antibody and the HRP chromogenic substrate.

Poly-Glu-Tyr, a synthetic polymer used as PTK substrate in 96-well ELISA microplates (Greiner, Germany), was added to PBS-A at a concentration of 250 ug / ml, and 100 ul was added to each well for 12 hours at 37 ° C. After the above coating, each well was washed five times with PBS-A. Each well was blocked for 2 hours at 37 ° C. with 200 ul of 1% BSA per well and each well was washed five times with PBS-A. If the plate was not used immediately, the plate was stored by removing PBS-A and drying at 37 ° C. for about 2 hours.

For the phosphorylation reaction, add 100 ul of 10X buffer buffer (final concentration 1X buffer), ATP (final concentration 0.3 mM), GST-PTK6-ΔNLinker-Kinase (various concentration or 300 ng) and water to each well. After incubation at room temperature for a certain time (various hours or 20 minutes), washed five times with PBS-A. Each washed well was reacted with a primary antibody, a mouse anti-phosphate-tyrosine antibody (1: 1000, 4G10, Upstate, USA) for 2 hours at room temperature, and washed with PBS-A containing 0.05% Tween-20. Washed once. HRP-conjugated goth anti-mouse IgG antibody (1: 1000, Sigma, USA) as a secondary antibody was reacted at room temperature for 2 hours and washed 5 times with PBS-A containing 0.05% Tween-20. Color reaction was performed using HRP color substrate (OPD, O -phenylenediamine dihydrochloride) (Sigma, USA). 100 ul of urea hydrogen peroxide phosphate-citrate buffer containing 0.014% H ₂ O ₂ and OPD (final concentration 0.5 mg / ml) in each well (Sigma, USA) After the addition, it was reacted for 7 minutes at room temperature in a light-blocked environment. The color reaction was stopped by adding 100 μl of 2.5NH ₂ SO ₄ to each well. The degree of color development was analyzed by measuring absorbance at 485 nm using SPECTRAFLUOR PLUS (Tecan, Austria).

To determine the appropriate amount of GST-PTK6-ΔNLinker-Kinase for this phosphorylation reaction, 0, 200, 400, 600, 800 and 1000 ng of GST-PTK6-ΔNLinker-Kinase were reacted for 20 minutes under the above conditions. The absorbance, indicating the activity of PTK6, was found to increase concentration-dependent and saturate at 800 ng or more (FIG. 6). Therefore, the optimal amount of GST-PTK6-ΔNLinker-Kinase protein per well was determined to be 300 ng / well, which is the GST-PTK6-ΔNLinker-Kinase protein concentration when the absorbance was half the maximum absorbance.

In order to determine an appropriate reaction time for this phosphorylation reaction, the reaction time was reacted at intervals of 10 minutes to 0 minutes to 60 minutes under conditions using 300 ng of GST-PTK6-ΔNLinker-Kinase. The absorbance was found to increase time-dependent and to saturate after 30 minutes or more (FIG. 7). Therefore, the optimal reaction time for screening the inhibitor was determined to be 20 minutes, the time when the absorbance was at the level of 80% of the maximum absorbance.

Through this phosphorylation reaction, 300 ng of GST-PTK6-ΔNLinker-Kinase per 96-well and reacted for 20 minutes were set as optimal reaction conditions for PTK6 activity analysis.

Example 5: Validation of PTK6 Catalytic Activity Detection System for Kinase Inhibitors

In order to investigate whether the PTK6 catalyst activity assay conditions set forth in Example 4 were suitable for the search for PTK6 inhibitors, changes in PTK6 catalyst activity were analyzed under various kinase inhibitor conditions. As set in the optimum condition, 300 ng of GST-PTK6-ΔNLinker-Kinase was used for each well. Prior to the reaction with ATP, 10X activated buffer solution (final concentration 1X activated buffer solution), 300 ng of GST-PTK6-ΔNLinker-Kinase, various inhibitors and water were added to make 20 ul and incubated in advance for 30 minutes at room temperature. To each well of 96-well plate, add 10X Buffer (final concentration 1X Buffer), ATP (final concentration 0.3 mM) and water to 80 ul, and then mix GST-PTK6-ΔNLinker-Kinase with inhibitor 20 ul was added to make the final 100 ul and incubated for 20 minutes at room temperature, and washed 5 times with PBS-A. The subsequent procedure followed the method of Example 4. This reaction includes DMSO, a solvent of kinase inhibitors, 100 nM AG1478 (EGF receptor inhibitor), 200 nM PP1 (Src family PTK inhibitor), 10 uM LY294002 (PI3 kinase inhibitor), 100 nM AG490 (JAK2 kinase inhibitor), 100 uM genistein (Typical PTK inhibitor), 5 uM PD98059 (MEK inhibitor) and 5 uM U0126 (MEK inhibitor) were used and the results were as in FIG. 8. As can be seen in FIG. 8, GST-PTK6-ΔNLinker-Kinase was not inhibited by an inhibitor specific to a specific protein, and was inhibited only by Genistein, a general PTK inhibitor. Thus, it was confirmed that the present PTK6 catalytic activity measurement method set at the 96-multiwell level can be efficiently used to search for PTK6 inhibitors in high through-put.

PTK6 protein minimally active fragments, more preferably PTK6-ΔNLinker-Kinase, prepared to include a site essential for catalytic activity without self-inhibition in the present invention, can specifically measure the catalytic activity of PTK6, The present invention provides a detection method for identifying inhibitors that can treat diseases such as cancer caused by overexpression and overactivity of PTK6.

<110> Yonsei University <120> Minimal fragment for catalytic activity of PTK6 and a method for detecting PTK6 inhibitors using the fragment <160> 13 <170> KopatentIn 1.71 <210> 1 <211> 1356 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (1353) <223> Minimal catalytic domain of PTK6 <400> 1 atg gtg tcc cgg gac cag gct cac ctg ggc ccc aag tat gtg ggc ctc 48 Met Val Ser Arg Asp Gln Ala His Leu Gly Pro Lys Tyr Val Gly Leu   1 5 10 15 tgg gac ttc aag tcc cgg acg gac gag gag ctg agc ttc cgc gcg ggg 96 Trp Asp Phe Lys Ser Arg Thr Asp Glu Glu Leu Ser Phe Arg Ala Gly              20 25 30 gac gtc ttc cac gtg gcc agg aag gag gag cag tgg tgg tgg gcc acg 144 Asp Val Phe His Val Ala Arg Lys Glu Glu Gln Trp Trp Trp Ala Thr          35 40 45 ctg ctg gac gag gcg ggt ggg gcc gtg gcc cag ggc tat gtg ccc cac 192 Leu Leu Asp Glu Ala Gly Gly Ala Val Ala Gln Gly Tyr Val Pro His      50 55 60 aac tac ctg gcc gag agg gag acg gtg gag tcg gaa ccg tgg ttc ttt 240 Asn Tyr Leu Ala Glu Arg Glu Thr Val Glu Ser Glu Pro Trp Phe Phe  65 70 75 80 ggc tgc atc tcc cgc tcg gaa gct gtg cgt cgg ctg cag gcc gag ggc 288 Gly Cys Ile Ser Arg Ser Glu Ala Val Arg Arg Leu Gln Ala Glu Gly                  85 90 95 aac gcc acg ggc gcc ttc ctg atc agg gtc agc gag aag ccg agt gcc 336 Asn Ala Thr Gly Ala Phe Leu Ile Arg Val Ser Glu Lys Pro Ser Ala             100 105 110 gac tac gtc ctg tcg gtg cgg gac acg cag gct gtg cgg cac tac aag 384 Asp Tyr Val Leu Ser Val Arg Asp Thr Gln Ala Val Arg His Tyr Lys         115 120 125 atc tgg cgg cgt gcc ggg ggc cgg ctg cac ctg aac gag gcg gtg tcc 432 Ile Trp Arg Arg Ala Gly Gly Arg Leu His Leu Asn Glu Ala Val Ser     130 135 140 ttc ctc agc ctg ccc gag ctt gtg aac tac cac agg gcc cag agc ctg 480 Phe Leu Ser Leu Pro Glu Leu Val Asn Tyr His Arg Ala Gln Ser Leu 145 150 155 160 tcc cac ggc ctg cgg ctg gcc gcg ccc tgc cgg aag cac gag cct gag 528 Ser His Gly Leu Arg Leu Ala Ala Pro Cys Arg Lys His Glu Pro Glu                 165 170 175 ccc ctg ccc cat tgg gat gac tgg gag agg ccg agg gag gag ttc acg 576 Pro Leu Pro His Trp Asp Asp Trp Glu Arg Pro Arg Glu Glu Phe Thr             180 185 190 ctc tgc agg aag ctg ggg tcc ggc tac ttt ggg gag gtc ttc gag ggg 624 Leu Cys Arg Lys Leu Gly Ser Gly Tyr Phe Gly Glu Val Phe Glu Gly         195 200 205 ctc tgg aaa gac cgg gtc cag gtg gcc att aag gtg att tct cga gac 672 Leu Trp Lys Asp Arg Val Gln Val Ala Ile Lys Val Ile Ser Arg Asp     210 215 220 aac ctc ctg cac cag cag atg ctg cag tcg gag atc cag gcc atg aag 720 Asn Leu Leu His Gln Gln Met Leu Gln Ser Glu Ile Gln Ala Met Lys 225 230 235 240 aag ctg cgg cac aaa cac atc ctg gcg ctg tac gcc gtg gtg tcc gtg 768 Lys Leu Arg His Lys His Ile Leu Ala Leu Tyr Ala Val Val Ser Val                 245 250 255 ggg gac ccc gtg tac atc atc acg gag ctc atg gcc aag ggc agc ctg 816 Gly Asp Pro Val Tyr Ile Ile Thr Glu Leu Met Ala Lys Gly Ser Leu             260 265 270 ctg gag ctg ctc cgc gac tct gat gag aaa gtc ctg ccc gtt tcg gag 864 Leu Glu Leu Leu Arg Asp Ser Asp Glu Lys Val Leu Pro Val Ser Glu         275 280 285 ctg ctg gac atc gcc tgg cag gtg gct gag ggc atg tgt tac ctg gag 912 Leu Leu Asp Ile Ala Trp Gln Val Ala Glu Gly Met Cys Tyr Leu Glu     290 295 300 tcg cag aat tac atc cac cgg gac ctg gcc gcc agg aac atc ctc gtc 960 Ser Gln Asn Tyr Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val 305 310 315 320 ggg gaa aac acc ctc tgc aaa gtt ggg gac ttc ggg tta gcc agg ctt 1008 Gly Glu Asn Thr Leu Cys Lys Val Gly Asp Phe Gly Leu Ala Arg Leu                 325 330 335 atc aag gag gac gtc tac ctc tcc cat gac cac aat atc ccc tac aag 1056 Ile Lys Glu Asp Val Tyr Leu Ser His Asp His Asn Ile Pro Tyr Lys             340 345 350 tgg acg gcc cct gaa gcg ctc tcc cga ggc cat tac tcc acc aaa tcc 1104 Trp Thr Ala Pro Glu Ala Leu Ser Arg Gly His Tyr Ser Thr Lys Ser         355 360 365 gac gtc tgg tcc ttt ggg att ctc ctg cat gag atg ttc agc agg ggt 1152 Asp Val Trp Ser Phe Gly Ile Leu Leu His Glu Met Phe Ser Arg Gly     370 375 380 cag gtg ccc tac cca ggc atg tcc aac cat gag gcc ttc ctg agg gtg 1200 Gln Val Pro Tyr Pro Gly Met Ser Asn His Glu Ala Phe Leu Arg Val 385 390 395 400 gac gcc ggc tac cgc atg ccc tgc cct ctg gag tgc ccg ccc agc gtg 1248 Asp Ala Gly Tyr Arg Met Pro Cys Pro Leu Glu Cys Pro Pro Ser Val                 405 410 415 cac aag ctg atg ctg aca tgc tgg tgc agg gac ccc gag cag aga ccc 1296 His Lys Leu Met Leu Thr Cys Trp Cys Arg Asp Pro Glu Gln Arg Pro             420 425 430 tgc ttc aag gcc ctg cgg gag agg ctc tcc agc ttc acc agc tac gag 1344 Cys Phe Lys Ala Leu Arg Glu Arg Leu Ser Ser Phe Thr Ser Tyr Glu         435 440 445 aac ccg acc tga 1356 Asn Pro Thr     450 <210> 2 <211> 451 <212> PRT <213> Homo sapiens <400> 2 Met Val Ser Arg Asp Gln Ala His Leu Gly Pro Lys Tyr Val Gly Leu   1 5 10 15 Trp Asp Phe Lys Ser Arg Thr Asp Glu Glu Leu Ser Phe Arg Ala Gly              20 25 30 Asp Val Phe His Val Ala Arg Lys Glu Glu Gln Trp Trp Trp Ala Thr          35 40 45 Leu Leu Asp Glu Ala Gly Gly Ala Val Ala Gln Gly Tyr Val Pro His      50 55 60 Asn Tyr Leu Ala Glu Arg Glu Thr Val Glu Ser Glu Pro Trp Phe Phe  65 70 75 80 Gly Cys Ile Ser Arg Ser Glu Ala Val Arg Arg Leu Gln Ala Glu Gly                  85 90 95 Asn Ala Thr Gly Ala Phe Leu Ile Arg Val Ser Glu Lys Pro Ser Ala             100 105 110 Asp Tyr Val Leu Ser Val Arg Asp Thr Gln Ala Val Arg His Tyr Lys         115 120 125 Ile Trp Arg Arg Ala Gly Gly Arg Leu His Leu Asn Glu Ala Val Ser     130 135 140 Phe Leu Ser Leu Pro Glu Leu Val Asn Tyr His Arg Ala Gln Ser Leu 145 150 155 160 Ser His Gly Leu Arg Leu Ala Ala Pro Cys Arg Lys His Glu Pro Glu                 165 170 175 Pro Leu Pro His Trp Asp Asp Trp Glu Arg Pro Arg Glu Glu Phe Thr             180 185 190 Leu Cys Arg Lys Leu Gly Ser Gly Tyr Phe Gly Glu Val Phe Glu Gly         195 200 205 Leu Trp Lys Asp Arg Val Gln Val Ala Ile Lys Val Ile Ser Arg Asp     210 215 220 Asn Leu Leu His Gln Gln Met Leu Gln Ser Glu Ile Gln Ala Met Lys 225 230 235 240 Lys Leu Arg His Lys His Ile Leu Ala Leu Tyr Ala Val Val Ser Val                 245 250 255 Gly Asp Pro Val Tyr Ile Ile Thr Glu Leu Met Ala Lys Gly Ser Leu             260 265 270 Leu Glu Leu Leu Arg Asp Ser Asp Glu Lys Val Leu Pro Val Ser Glu         275 280 285 Leu Leu Asp Ile Ala Trp Gln Val Ala Glu Gly Met Cys Tyr Leu Glu     290 295 300 Ser Gln Asn Tyr Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val 305 310 315 320 Gly Glu Asn Thr Leu Cys Lys Val Gly Asp Phe Gly Leu Ala Arg Leu                 325 330 335 Ile Lys Glu Asp Val Tyr Leu Ser His Asp His Asn Ile Pro Tyr Lys             340 345 350 Trp Thr Ala Pro Glu Ala Leu Ser Arg Gly His Tyr Ser Thr Lys Ser         355 360 365 Asp Val Trp Ser Phe Gly Ile Leu Leu His Glu Met Phe Ser Arg Gly     370 375 380 Gln Val Pro Tyr Pro Gly Met Ser Asn His Glu Ala Phe Leu Arg Val 385 390 395 400 Asp Ala Gly Tyr Arg Met Pro Cys Pro Leu Glu Cys Pro Pro Ser Val                 405 410 415 His Lys Leu Met Leu Thr Cys Trp Cys Arg Asp Pro Glu Gln Arg Pro             420 425 430 Cys Phe Lys Ala Leu Arg Glu Arg Leu Ser Ser Phe Thr Ser Tyr Glu         435 440 445 Asn Pro Thr     450 <210> 3 <211> 819 <212> DNA <213> Homo sapiens <220> <221> CDS (222) (1) .. (816) <400> 3 cat tgg gat gac tgg gag agg ccg agg gag gag ttc acg ctc tgc agg 48 His Trp Asp Asp Trp Glu Arg Pro Arg Glu Glu Phe Thr Leu Cys Arg   1 5 10 15 aag ctg ggg tcc ggc tac ttt ggg gag gtc ttc gag ggg ctc tgg aaa 96 Lys Leu Gly Ser Gly Tyr Phe Gly Glu Val Phe Glu Gly Leu Trp Lys              20 25 30 gac cgg gtc cag gtg gcc att aag gtg att tct cga gac aac ctc ctg 144 Asp Arg Val Gln Val Ala Ile Lys Val Ile Ser Arg Asp Asn Leu Leu          35 40 45 cac cag cag atg ctg cag tcg gag atc cag gcc atg aag aag ctg cgg 192 His Gln Gln Met Leu Gln Ser Glu Ile Gln Ala Met Lys Lys Leu Arg      50 55 60 cac aaa cac atc ctg gcg ctg tac gcc gtg gtg tcc gtg ggg gac ccc 240 His Lys His Ile Leu Ala Leu Tyr Ala Val Val Ser Val Gly Asp Pro  65 70 75 80 gtg tac atc atc acg gag ctc atg gcc aag ggc agc ctg ctg gag ctg 288 Val Tyr Ile Ile Thr Glu Leu Met Ala Lys Gly Ser Leu Leu Glu Leu                  85 90 95 ctc cgc gac tct gat gag aaa gtc ctg ccc gtt tcg gag ctg ctg gac 336 Leu Arg Asp Ser Asp Glu Lys Val Leu Pro Val Ser Glu Leu Leu Asp             100 105 110 atc gcc tgg cag gtg gct gag ggc atg tgt tac ctg gag tcg cag aat 384 Ile Ala Trp Gln Val Ala Glu Gly Met Cys Tyr Leu Glu Ser Gln Asn         115 120 125 tac atc cac cgg gac ctg gcc gcc agg aac atc ctc gtc ggg gaa aac 432 Tyr Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Gly Glu Asn     130 135 140 acc ctc tgc aaa gtt ggg gac ttc ggg tta gcc agg ctt atc aag gag 480 Thr Leu Cys Lys Val Gly Asp Phe Gly Leu Ala Arg Leu Ile Lys Glu 145 150 155 160 gac gtc tac ctc tcc cat gac cac aat atc ccc tac aag tgg acg gcc 528 Asp Val Tyr Leu Ser His Asp His Asn Ile Pro Tyr Lys Trp Thr Ala                 165 170 175 cct gaa gcg ctc tcc cga ggc cat tac tcc acc aaa tcc gac gtc tgg 576 Pro Glu Ala Leu Ser Arg Gly His Tyr Ser Thr Lys Ser Asp Val Trp             180 185 190 tcc ttt ggg att ctc ctg cat gag atg ttc agc agg ggt cag gtg ccc 624 Ser Phe Gly Ile Leu Leu His Glu Met Phe Ser Arg Gly Gln Val Pro         195 200 205 tac cca ggc atg tcc aac cat gag gcc ttc ctg agg gtg gac gcc ggc 672 Tyr Pro Gly Met Ser Asn His Glu Ala Phe Leu Arg Val Asp Ala Gly     210 215 220 tac cgc atg ccc tgc cct ctg gag tgc ccg ccc agc gtg cac aag ctg 720 Tyr Arg Met Pro Cys Pro Leu Glu Cys Pro Pro Ser Val His Lys Leu 225 230 235 240 atg ctg aca tgc tgg tgc agg gac ccc gag cag aga ccc tgc ttc aag 768 Met Leu Thr Cys Trp Cys Arg Asp Pro Glu Gln Arg Pro Cys Phe Lys                 245 250 255 gcc ctg cgg gag agg ctc tcc agc ttc acc agc tac gag aac ccg acc 816 Ala Leu Arg Glu Arg Leu Ser Ser Phe Thr Ser Tyr Glu Asn Pro Thr             260 265 270       tga 819 <210> 4 <211> 272 <212> PRT <213> Homo sapiens <400> 4 His Trp Asp Asp Trp Glu Arg Pro Arg Glu Glu Phe Thr Leu Cys Arg   1 5 10 15 Lys Leu Gly Ser Gly Tyr Phe Gly Glu Val Phe Glu Gly Leu Trp Lys              20 25 30 Asp Arg Val Gln Val Ala Ile Lys Val Ile Ser Arg Asp Asn Leu Leu          35 40 45 His Gln Gln Met Leu Gln Ser Glu Ile Gln Ala Met Lys Lys Leu Arg      50 55 60 His Lys His Ile Leu Ala Leu Tyr Ala Val Val Ser Val Gly Asp Pro  65 70 75 80 Val Tyr Ile Ile Thr Glu Leu Met Ala Lys Gly Ser Leu Leu Glu Leu                  85 90 95 Leu Arg Asp Ser Asp Glu Lys Val Leu Pro Val Ser Glu Leu Leu Asp             100 105 110 Ile Ala Trp Gln Val Ala Glu Gly Met Cys Tyr Leu Glu Ser Gln Asn         115 120 125 Tyr Ile His Arg Asp Leu Ala Ala Arg Asn Ile Leu Val Gly Glu Asn     130 135 140 Thr Leu Cys Lys Val Gly Asp Phe Gly Leu Ala Arg Leu Ile Lys Glu 145 150 155 160 Asp Val Tyr Leu Ser His Asp His Asn Ile Pro Tyr Lys Trp Thr Ala                 165 170 175 Pro Glu Ala Leu Ser Arg Gly His Tyr Ser Thr Lys Ser Asp Val Trp             180 185 190 Ser Phe Gly Ile Leu Leu His Glu Met Phe Ser Arg Gly Gln Val Pro         195 200 205 Tyr Pro Gly Met Ser Asn His Glu Ala Phe Leu Arg Val Asp Ala Gly     210 215 220 Tyr Arg Met Pro Cys Pro Leu Glu Cys Pro Pro Ser Val His Lys Leu 225 230 235 240 Met Leu Thr Cys Trp Cys Arg Asp Pro Glu Gln Arg Pro Cys Phe Lys                 245 250 255 Ala Leu Arg Glu Arg Leu Ser Ser Phe Thr Ser Tyr Glu Asn Pro Thr             260 265 270 <210> 5 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of PTK6 SH3 domain <400> 5 cgggatcccg ggaccaggct cacctg 26 <210> 6 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of PTK6 SH3 domain <400> 6 ggaattcacg tctccctctc ggccaggt 28 <210> 7 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of PTK6-Linker domain <400> 7 catgccatgg gccggaagca cgagcctgag 30 <210> 8 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of PTK6-Linker domain <400> 8 cgcggatcct cagaactcct cccaggcctc 30 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of GST-PTK6-Kinase domain <400> 9 ggaattccga ggagttcacg c 21 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of GST-PTK6-Kinase domain and          PTK6-NLinker-Kinase domain <400> 10 cattatgctg agtgatatcc cg 22 <210> 11 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> primer for amplification of PTK6-NLinker-Kinase domain <400> 11 ggaattccca ttgggatgac t 21 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer for site-directed mutagenesis of PTK6 linker region <400> 12 ttgggatgac gcggagaggc cgagggagga 30 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> primer for site-directed mutagenesis of PTK6 linker region <400> 13 tcggcctctc cgcgtcatcc caatggggca 30  

Claims (7)

Amino acid sequence 184 to 190 of the linker region of the protein tyrosine kinase-6 (PTK6) and amino acid sequence of the catalytic domain, 0 to 13 amino acid sequences located adjacent to the N-terminal side of amino acid 184 of the linker region, and the catalytic domain Least active fragment of PTK6 protein comprising 0-6 amino acid sequences located adjacent to the C-terminal side of amino acid 445; The minimally active fragment of PTK6 protein according to claim 1, as set forth in SEQ ID NO: 4. A nucleic acid molecule encoding the amino acid sequence of the least active fragment of the PTK6 protein of claim 1. A recombinant expression vector comprising the nucleic acid molecule of claim 3. A method of inhibiting catalytic activity of PTK6, comprising reacting a least active fragment of the PTK6 protein of claim 1 with a polypeptide substrate having a tyrosine residue in the presence of an inhibitor and comparing phosphorylation of the substrate to phosphorylation in the absence of the candidate. Detection method. The method of claim 5, wherein the method for confirming phosphorylation is ELISA using an antibody specific for phosphorylated tyrosine. 6. The method of claim 5, wherein the amount of protein is about 200 to 400 ng / well and the reaction time is 10 to 30 minutes.

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