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WO1998035016A1 - Protein tyrosine kinase 2 (pyk2), nucleic acids, and assay - Google Patents

Protein tyrosine kinase 2 (pyk2), nucleic acids, and assay Download PDF

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Publication number
WO1998035016A1
WO1998035016A1 PCT/US1998/002494 US9802494W WO9835016A1 WO 1998035016 A1 WO1998035016 A1 WO 1998035016A1 US 9802494 W US9802494 W US 9802494W WO 9835016 A1 WO9835016 A1 WO 9835016A1
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Prior art keywords
pyk2
leu
glu
lys
pro
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French (fr)
Inventor
Le T. Duong
Gideon A. Rodan
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Merck and Co Inc
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Merck and Co Inc
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Priority to CA002277028A priority Critical patent/CA2277028A1/en
Priority to EP98905026A priority patent/EP0998553A4/en
Priority to JP53498698A priority patent/JP2002513284A/en
Publication of WO1998035016A1 publication Critical patent/WO1998035016A1/en
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/91—Transferases (2.)
    • G01N2333/912—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases

Definitions

  • PROTEIN TYROSINE KINASE 2 PYK2
  • NUCLEIC ACIDS NUCLEIC ACIDS
  • This invention is directed to nucleic acids encoding protein tyrosine kinase 2 (PYK2), to murine PYK2, to methods of making this protein using the nucleic acids, and to assays for inhibitors of PYK2.
  • PYK2 protein tyrosine kinase 2
  • PYK2 Protein tyrosine kinase 2
  • CAK ⁇ Cell Adhesion Kinase ⁇
  • RAFTK Adhesion Focal Tyrosine Kinase
  • PYK2 was first cloned from human brain as a Grb-2 binding protein, and has also been cloned from rat and human brain libraries. There have been conflicting reports as to its cellular expression. In one study, abundant PYK2 transcripts were found in brain and lower levels were detected in the kidney. In another report, PYK2 expression was also found to be most abundant in rat brain, but its transcripts could also be detected in kidney, spleen, lung, intestine and epididymis. PYK2 transcripts were also detected in rat fibroblast 3Y1 and WFB cell lines, as well as in the human T cell leukemia Jurkat line.
  • nucleic acids substantially free from associated nucleic acids, which encode murine protein tryosine kinase 2 (PYK2).
  • PYK2 murine protein tryosine kinase 2
  • the nucleic acid which encodes PYK2 is a DNA.
  • Murine PYK2 cDNA is set forth in Figure 1 (SEQ ID NO:5).
  • FIG. 6 Yet another aspect of this invention is murine PYK2 which is free from associated murine proteins.
  • FIG. 6 One murine PYK2 is set forth in Figure 1 (SEQ ID NO:6).
  • Another aspect of this invention is a method of making PYK2 by introducing nucleic acids into a cell, the nucleic acids comprising nucleic acids which encode PYK2, under conditions which transcription and translation of PYK2 occur. It is preferred that the nucleic acids be present in a vector, such as a plasmid or baculovirus vector. It is also preferred that the nucleic acids be under the control of transcriptional control elements, such as promoters and optionally enhancers. Such control elements are well known in the art.
  • Host cells which express PYK2 are also part of this invention.
  • Preferred host cells include mammalian cells, insect cells, yeast and bacterial cells such as E. coli. Cell lines which permanently (rather than transiently) express murine PYK2 are also another aspect of this invention.
  • the recombinant PYK2 which is made using the cloning process of this invention may be used in assays in order to further characterize the biological function of PYK2 and to identify compounds such as agonists and antagonists which modulate its activity.
  • a further aspect of this invention is an assay for the identification of compounds which modulate the activitiy of PYK2, and particularly inhibitors of PYK2 activity.
  • This assay comprises the steps of: contacting recombinant PYK2 with a tyrosine substrate in the presence of radiolabeled ATP and a putative activity-modifying compound, and measuring the amount of radiolabeled tyrosine which is formed; and optionally comparing the amount of radiolabeled tyrosine formed in the presence of the putative activity-modifying compound with that formed in the absence of the putative activity-modifying compound.
  • Integrins are the major family of cell surface receptors that mediate adhesive interactions, either to adjacent cells or to the extracellular matrix. Integrin signalling is mediated through the focal adhesion kinase (FAK) family of proteins. PYK2 is a member of the FAK family, and is involved in integrin-mediated signal transduction pathways in megakaryocytes, brain tissue and hematopoietic cells. Modulators of PYK2 would therefore be potential therapeutic agents for modulating platelet levels.
  • FAK focal adhesion kinase
  • Figure 1 is the cDNA sequence of mouse PYK2 and the deduced protein sequence. Intron sequences are in lower case letters. The exon sequence is capitalized. The boxed sequence of the deduced protein indicates the kinase domain. The circled prolines of the deduced protein indicate the proline rich domain.
  • PYK2 means protein tyrosine kinase 2, allelic variations of protein tyrosine kinase 2, and mutations or fragments thereof which retain at least about 85%, and preferably at least about 90% of the biological activity of native PYK2.
  • Native PYK2 means the protein tyrosine kinase which is naturally occuring in an organism.
  • Substantially free from associated nucleic acids means that in a sample, there is less than about 5% (by weight) nucleic acids present which are other than nucleic acids encoding PYK2.
  • Substantially free from associated murine proteins means that in a sample, there is less than about 5% (by weight) protein which is other than murine PYK2.
  • FAM focal adhesion kinase.
  • Heterologous PYK2 nucleic acid means that the nucleic acid was introduced to the cell, without regard as to whether the nucleic acid is from the same species as the cell; alternatively it refers to nucleic acids encoding PYK2 in a cell whose ancestor had PYK2 introduced into the cell.
  • Heterologous PYK2 protein means that the PYK2 was encoded by a heterologous nucleic acid.
  • FAK proteins which are involved in cell adhesion processes, were not detected in a number of macrophage cell lines and it was therefore hypothesized that another cell adhesion-dependent kinase, homologous to FAK, may assume its function in these cells.
  • PYK2 was recently identified as another member of the FAK family and its expression was detected in spleen, thymus, lung and peripheral blood leukocytes (Avraham, et al., 1995 supra; Sasaki, et al., 1995 supra).
  • specific probes were generated for PYK2 and FAK which were used to examine the expression of PYK2 and FAK in mouse tissues. As previously reported, for other species, PYK2 is highly expressed in brain and spleen, and at lower levels in kidney, lung and liver and has a more restricted tissue distribution than FAK.
  • the full length cDNA was cloned from a mouse spleen cDNA library.
  • the deduced amino acid sequence of the full length clone was found to be identical to the recently published amino acid sequence of the mouse RAFTK (Avraham, et al., 1995, supra).
  • PYK2 and FAK cDNAs were subsequently transfected into human embryonic kidney (HEK) 293 cells.
  • HEK human embryonic kidney
  • Macrophages were induced by thioglycolate injection into the peritoneal cavities of adult BALB/c mice. After 4 days, cells were collected, washed and cultured in RPMI 1640 medium containing 10% FBS. After 3h at 37°C, the cultures were washed extensively to remove non-adherent cells and cultured overnight before samples were prepared for immunoprecipitation. Bone marrow derived macrophages were prepared as described by Li and Chen, 1995 J. Leuk. Biol. 57:484- 490, which is hereby incorporated by reference.
  • Non adherent cells were cultured in RPMI completed medium in the presence of human macrophage colony-stimulating factor (MCS-F, 250 units/ml, Genetics Institute, Cambridge, MA).
  • MCS-F human macrophage colony-stimulating factor
  • Differentiated macrophages were prepared for immunoprecipitation after 5 days in culture.
  • Bone marrow derived osteoclast-like cells were prepared as described by Wesolowski, et al., 1995 Exp. Cell Res. 219:679-686, which is hereby incorporated by reference. After collagenase-dispase treatment, mononucleated tartrate resistant phosphatase positive cells were released from the tissue culture plate using 30 nM echistatin (Merck Res. Labs., West Point, PA). Freshly isolated osteoclast-like cells were immediately solubilized in immunoprecipitation buffer.
  • a specific probe for FAK (700bp) was generated using the following primers: 5'- CAGCA CACAA TCCTG GAGGA G (SEQ. ID. NO:3) and 3'- GCTGA AGCTT GACAC CCTCA T (SEQ. ID. NO. 4) with cDNAs of mouse osteoblastic MB 1.8 cells as template (Wesolowski, et al., 1995, supra). These probes were confirmed by sequencing analysis. PYK2 cDNA fragments were cloned from a mouse spleen ⁇ -ZAP II cDNA library (Stratagene, La Jolla, CA) using the specific PYK2 probe.
  • Full length PYK2 cDNA were constructed by ligation of two overlapping clones at the Vspl site.
  • the amino acid sequence of the isolated PYK2 cDNA clone was identical to the previously published mouse RAFTK sequence (Avraham, et al., 1995 supra).
  • Full length FAK cDNA was generated by PCR according to the published sequence as described in Hanks, et al., 1992 Proc. Natl. Acad. Sci. USA. 89:8487-8491.
  • Both PYK2 and FAK cDNAs were subcloned into pCDNA3 plasmid (InVitrogen, San Diego, CA) and transfected into human embryonic kidney (HEK) 293 cells (ATCC, Rockland, MD) by electroporation at 200V, 960 ⁇ F using a GenePulser (Biorad Labs, Richmond, CA).
  • HEK 293 cells were subsequently subjected to G418 selection (800 ⁇ g/ml, Gibco BRL) and clones were picked after 3 weeks in selection medium.
  • Expression of PYK2 and FAK in HEK293 cells were confirmed by Northern analysis using the respective probes, and Western blots were performed using anti-PYK2 antibodies.
  • Mouse multiple tissue Northern blot was purchased from Clonetech (Palo Alto, CA) and hybridization of the Northern blot using probes specific for PYK2, FAK and glyceraldehde 3-phosphate dehydrogenase (GAPDH) were performed as described previously (Wesolowski, et al., 1995, supra).
  • the PYK2 C-terminal domain (from Methionine residue 685 to end) was amplified by PCR using the mouse PYK2 as template. Amplified product was cloned into plasmid pGEX-4T (Pharmacia Biotech., Piscataway, NJ) and transformed in E.coli XLl-Blue (Stratagene). Expression of GST-PYK2 C-terminal fragment was induced using 0.5 mM IPTG, purified and cleaved from GST with thrombin, essentially according to the instructions of the manufacturer (Pharmacia).
  • the purified C-terminal fragment of mouse PYK2 was used to immunize two rabbits (Research Genetics, Huntsville, AL) and the titers of both antisera were initially determined by ELISA using the recombinant C-terminal fragment of PYK2. Specificity of the immune sera was subsequently determined by Western blot by comparison to the preimmune sera. Polyclonal antibodies were then affinity purified by passing the combined fractions of both antisera through an affinity column, which was constructed using the same purified antigen cross linked to CNBr-activated Sepharose 4B according to the instructions of the manufacturer (Pharmacia).
  • the antibodies were eluted from the column using 0.2 M Glycine, pH 2.5 and lmM EGTA and the eluted fraction was then dialyzed against PBS containning 0.02% azide.
  • Anti-PYK2 antibodies were stored at -70°C at a concentration of 0.5mg/ml.
  • IC-21 cells were solubilized in TNE lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, lmM EDTA, 10% glycerol, 50 mM NaF, 1 mM sodium vanadate and protease inhibitors as described above.
  • PYK2 were immunoprecipitated from the clarified lysates, half of the sample was subjected to immunoblotting with anti PYK2 antibodies, as described above, and the other half was washed 2 times with the same lysis buffer, and with kinase assay buffer (IX) containing 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM MnCl2 and 1 mM dithiothreitol.
  • IX kinase assay buffer
  • kinase assay buffer containing 5 ⁇ Ci [ ⁇ -32p] ATP (3000 Ci/mmol, Amersham), 10 mM ATP, 0.1% BSA and 100 ⁇ g of poly (Glu,Tyr) (molar ratio 4:1; Sigma) was added to the beads and incubated for 10 min at 30°C (Howell and Cooper, 1995 Mol. Cell. Biol. 14:5402-5411).
  • the reaction mixtures (25 ⁇ l) were added to 25 ⁇ l of 30% trichloroacetic acid (TCA) and 0.1 M sodium pyrophosphate, followed by incubation at 4°C for 15 min.
  • TCA trichloroacetic acid
  • Multiscreen-FC filter plate (Millipore, Marlborough, MA), washed with ice cold 15% TCA (3X), allowed to dry and incorporation of 32p into the substrate was counted on a Packard top count microplate scintillation counter (Packard, Meriden, CT). Each assay were performed as triplicate. The specific activity was determined by comparing the radioactive counts with immunoblot signals.
  • MOLECULE TYPE cDNA
  • SEQUENCE DESCRIPTION SEQ ID NO : 5 :
  • AGTGTACCCC TAACGGCCAA GATGGCTTTC TGCATGGACA TTTGAGAGCC AGTATTCCTC 3540
  • CTTTTCTTAC GTCTCCTTTT TCTCCTCCCC
  • CTTTTCACAT CTGCTCCCCT CCTCTCTCAT 3900
  • Val Glu Lys Glu Asp Val Arg lie Leu Lys Val Cys Phe Tyr Ser Asn

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Abstract

This invention is directed to nucleic acids encoding protein tyrosine kinase 2 (PYK2), to murine PYK2, to methods of making this protein using the nucleic acids, and to assays for inhibitors of PYK2.

Description

TITLE OF THE INVENTION
PROTEIN TYROSINE KINASE 2 (PYK2), NUCLEIC ACIDS, AND
ASSAY
BRIEF DESCRIPTION OF THE INVENTION
This invention is directed to nucleic acids encoding protein tyrosine kinase 2 (PYK2), to murine PYK2, to methods of making this protein using the nucleic acids, and to assays for inhibitors of PYK2.
BACKGROUND OF THE INVENTION
Protein tyrosine kinase 2 (PYK2), also known as Cell Adhesion Kinase β (CAKβ) and Related Adhesion Focal Tyrosine Kinase (RAFTK) is a recently described member of the focal adhesion kinase family (Avraham et al., 1995 J. Biol. Chem. 270:27742-27751; Lev et al., 1995 Nature. 376:737-745; and Sasaki, et al., 1995 J. Biol. Chem.
270:21206-21219.). PYK2 was first cloned from human brain as a Grb-2 binding protein, and has also been cloned from rat and human brain libraries. There have been conflicting reports as to its cellular expression. In one study, abundant PYK2 transcripts were found in brain and lower levels were detected in the kidney. In another report, PYK2 expression was also found to be most abundant in rat brain, but its transcripts could also be detected in kidney, spleen, lung, intestine and epididymis. PYK2 transcripts were also detected in rat fibroblast 3Y1 and WFB cell lines, as well as in the human T cell leukemia Jurkat line. When cloned from the human megakaryocytic CMK cell line and from mouse brain, it was found to have wider tissue distribution beyond brain, notably spleen, lung, thymus and peripheral blood leukocytes. In addition, expression of PYK2 was reported in human CD34+ marrow cells, megakaryocytes and platelets. DETAILED DESCRIPTION OF THE INVENTION
One aspect of this invention is are nucleic acids, substantially free from associated nucleic acids, which encode murine protein tryosine kinase 2 (PYK2). In one embodiment, the nucleic acid which encodes PYK2 is a DNA.
Another aspect of this invention is murine PYK2 cDNA, Murine PYK2 DNA is set forth in Figure 1 (SEQ ID NO:5).
Yet another aspect of this invention is murine PYK2 which is free from associated murine proteins. One murine PYK2 is set forth in Figure 1 (SEQ ID NO:6).
Another aspect of this invention is a method of making PYK2 by introducing nucleic acids into a cell, the nucleic acids comprising nucleic acids which encode PYK2, under conditions which transcription and translation of PYK2 occur. It is preferred that the nucleic acids be present in a vector, such as a plasmid or baculovirus vector. It is also preferred that the nucleic acids be under the control of transcriptional control elements, such as promoters and optionally enhancers. Such control elements are well known in the art.
Host cells which express PYK2 are also part of this invention. Preferred host cells include mammalian cells, insect cells, yeast and bacterial cells such as E. coli. Cell lines which permanently (rather than transiently) express murine PYK2 are also another aspect of this invention.
The recombinant PYK2, which is made using the cloning process of this invention may be used in assays in order to further characterize the biological function of PYK2 and to identify compounds such as agonists and antagonists which modulate its activity. A further aspect of this invention is an assay for the identification of compounds which modulate the activitiy of PYK2, and particularly inhibitors of PYK2 activity. This assay comprises the steps of: contacting recombinant PYK2 with a tyrosine substrate in the presence of radiolabeled ATP and a putative activity-modifying compound, and measuring the amount of radiolabeled tyrosine which is formed; and optionally comparing the amount of radiolabeled tyrosine formed in the presence of the putative activity-modifying compound with that formed in the absence of the putative activity-modifying compound.
Integrins are the major family of cell surface receptors that mediate adhesive interactions, either to adjacent cells or to the extracellular matrix. Integrin signalling is mediated through the focal adhesion kinase (FAK) family of proteins. PYK2 is a member of the FAK family, and is involved in integrin-mediated signal transduction pathways in megakaryocytes, brain tissue and hematopoietic cells. Modulators of PYK2 would therefore be potential therapeutic agents for modulating platelet levels.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 is the cDNA sequence of mouse PYK2 and the deduced protein sequence. Intron sequences are in lower case letters. The exon sequence is capitalized. The boxed sequence of the deduced protein indicates the kinase domain. The circled prolines of the deduced protein indicate the proline rich domain.
As used throughout the specification and claims, the following definitions apply: "PYK2" means protein tyrosine kinase 2, allelic variations of protein tyrosine kinase 2, and mutations or fragments thereof which retain at least about 85%, and preferably at least about 90% of the biological activity of native PYK2.
"Native PYK2" means the protein tyrosine kinase which is naturally occuring in an organism.
"Substantially free from associated nucleic acids" means that in a sample, there is less than about 5% (by weight) nucleic acids present which are other than nucleic acids encoding PYK2.
"Substantially free from associated murine proteins" means that in a sample, there is less than about 5% (by weight) protein which is other than murine PYK2.
"FAK" means focal adhesion kinase. "Heterologous" PYK2 nucleic acid means that the nucleic acid was introduced to the cell, without regard as to whether the nucleic acid is from the same species as the cell; alternatively it refers to nucleic acids encoding PYK2 in a cell whose ancestor had PYK2 introduced into the cell.
"Heterologous" PYK2 protein means that the PYK2 was encoded by a heterologous nucleic acid.
FAK proteins, which are involved in cell adhesion processes, were not detected in a number of macrophage cell lines and it was therefore hypothesized that another cell adhesion-dependent kinase, homologous to FAK, may assume its function in these cells. PYK2 was recently identified as another member of the FAK family and its expression was detected in spleen, thymus, lung and peripheral blood leukocytes (Avraham, et al., 1995 supra; Sasaki, et al., 1995 supra). To evaluate PYK2 as a possible adhesion-dependent kinase in macrophages, specific probes were generated for PYK2 and FAK which were used to examine the expression of PYK2 and FAK in mouse tissues. As previously reported, for other species, PYK2 is highly expressed in brain and spleen, and at lower levels in kidney, lung and liver and has a more restricted tissue distribution than FAK.
Using a PYK2 probe, the full length cDNA was cloned from a mouse spleen cDNA library. The deduced amino acid sequence of the full length clone was found to be identical to the recently published amino acid sequence of the mouse RAFTK (Avraham, et al., 1995, supra).
In addition, the full length FAK was cloned from a mouse osteoblastic MB1.8 cell line (Wesolowski, et al, 1995, Exp. Cell Res., 219: 679-686.).
PYK2 and FAK cDNAs were subsequently transfected into human embryonic kidney (HEK) 293 cells. Cell lines which permanently express either PYK2 or FAK were established and the expression levels of the exogeneously expressed mouse kinases were assessed by northern analysis.
The following Examples are presented to better illustrate the invention. EXAMPLE 1 Cell Culture
Macrophages were induced by thioglycolate injection into the peritoneal cavities of adult BALB/c mice. After 4 days, cells were collected, washed and cultured in RPMI 1640 medium containing 10% FBS. After 3h at 37°C, the cultures were washed extensively to remove non-adherent cells and cultured overnight before samples were prepared for immunoprecipitation. Bone marrow derived macrophages were prepared as described by Li and Chen, 1995 J. Leuk. Biol. 57:484- 490, which is hereby incorporated by reference. Non adherent cells were cultured in RPMI completed medium in the presence of human macrophage colony-stimulating factor (MCS-F, 250 units/ml, Genetics Institute, Cambridge, MA). Differentiated macrophages were prepared for immunoprecipitation after 5 days in culture. Bone marrow derived osteoclast-like cells were prepared as described by Wesolowski, et al., 1995 Exp. Cell Res. 219:679-686, which is hereby incorporated by reference. After collagenase-dispase treatment, mononucleated tartrate resistant phosphatase positive cells were released from the tissue culture plate using 30 nM echistatin (Merck Res. Labs., West Point, PA). Freshly isolated osteoclast-like cells were immediately solubilized in immunoprecipitation buffer.
EXAMPLE 2
cDNA Cloning and Expression of mouse PYK2
Specific probes for mouse .PYK2 and FAK were initially generated based on the non-homologous region between the proteins, which is adjacent to the C-terminal of the kinase domain. Using polymerase chain reaction (PCR), a specific probe for PYK2 (570bp) was generated using the 5'-primer AGTGA CATTT ATCAG ATGGA G
(SEQ. ID. NO:l) and the 3'-primer GAATG GACTG TGCAC CGAGC C (SEQ. ID. NO:2) with cDNAs of mouse bone marrow derived osteoclast- like cells as template (Wesolowski, et al., 1995, supra).
Similarly, a specific probe for FAK (700bp) was generated using the following primers: 5'- CAGCA CACAA TCCTG GAGGA G (SEQ. ID. NO:3) and 3'- GCTGA AGCTT GACAC CCTCA T (SEQ. ID. NO. 4) with cDNAs of mouse osteoblastic MB 1.8 cells as template (Wesolowski, et al., 1995, supra). These probes were confirmed by sequencing analysis. PYK2 cDNA fragments were cloned from a mouse spleen λ-ZAP II cDNA library (Stratagene, La Jolla, CA) using the specific PYK2 probe. Full length PYK2 cDNA were constructed by ligation of two overlapping clones at the Vspl site. The amino acid sequence of the isolated PYK2 cDNA clone was identical to the previously published mouse RAFTK sequence (Avraham, et al., 1995 supra). Full length FAK cDNA was generated by PCR according to the published sequence as described in Hanks, et al., 1992 Proc. Natl. Acad. Sci. USA. 89:8487-8491.
Both PYK2 and FAK cDNAs were subcloned into pCDNA3 plasmid (InVitrogen, San Diego, CA) and transfected into human embryonic kidney (HEK) 293 cells (ATCC, Rockland, MD) by electroporation at 200V, 960 μF using a GenePulser (Biorad Labs, Richmond, CA). HEK 293 cells were subsequently subjected to G418 selection (800 μg/ml, Gibco BRL) and clones were picked after 3 weeks in selection medium. Expression of PYK2 and FAK in HEK293 cells were confirmed by Northern analysis using the respective probes, and Western blots were performed using anti-PYK2 antibodies. Mouse multiple tissue Northern blot was purchased from Clonetech (Palo Alto, CA) and hybridization of the Northern blot using probes specific for PYK2, FAK and glyceraldehde 3-phosphate dehydrogenase (GAPDH) were performed as described previously (Wesolowski, et al., 1995, supra).
EXAMPLE 3
Production and Affinity Purification of Polyclonal Antibodies to mouse PYK2 The PYK2 C-terminal domain (from Methionine residue 685 to end) was amplified by PCR using the mouse PYK2 as template. Amplified product was cloned into plasmid pGEX-4T (Pharmacia Biotech., Piscataway, NJ) and transformed in E.coli XLl-Blue (Stratagene). Expression of GST-PYK2 C-terminal fragment was induced using 0.5 mM IPTG, purified and cleaved from GST with thrombin, essentially according to the instructions of the manufacturer (Pharmacia). The purified C-terminal fragment of mouse PYK2 was used to immunize two rabbits (Research Genetics, Huntsville, AL) and the titers of both antisera were initially determined by ELISA using the recombinant C-terminal fragment of PYK2. Specificity of the immune sera was subsequently determined by Western blot by comparison to the preimmune sera. Polyclonal antibodies were then affinity purified by passing the combined fractions of both antisera through an affinity column, which was constructed using the same purified antigen cross linked to CNBr-activated Sepharose 4B according to the instructions of the manufacturer (Pharmacia).
The antibodies were eluted from the column using 0.2 M Glycine, pH 2.5 and lmM EGTA and the eluted fraction was then dialyzed against PBS containning 0.02% azide. Anti-PYK2 antibodies were stored at -70°C at a concentration of 0.5mg/ml.
EXAMPLE 4
In vitro Kinase Assay After cell attachment to ECM, IC-21 cells were solubilized in TNE lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, lmM EDTA, 10% glycerol, 50 mM NaF, 1 mM sodium vanadate and protease inhibitors as described above. PYK2 were immunoprecipitated from the clarified lysates, half of the sample was subjected to immunoblotting with anti PYK2 antibodies, as described above, and the other half was washed 2 times with the same lysis buffer, and with kinase assay buffer (IX) containing 20 mM Tris-HCl, pH 7.4, 100 mM NaCl, 10 mM MnCl2 and 1 mM dithiothreitol. After removal of the wash buffer, 50 μl of kinase assay buffer containing 5 μCi [γ-32p] ATP (3000 Ci/mmol, Amersham), 10 mM ATP, 0.1% BSA and 100 μg of poly (Glu,Tyr) (molar ratio 4:1; Sigma) was added to the beads and incubated for 10 min at 30°C (Howell and Cooper, 1995 Mol. Cell. Biol. 14:5402-5411). The reaction mixtures (25 μl) were added to 25 μl of 30% trichloroacetic acid (TCA) and 0.1 M sodium pyrophosphate, followed by incubation at 4°C for 15 min. The precipitated proteins were transferred to a
Multiscreen-FC filter plate (Millipore, Marlborough, MA), washed with ice cold 15% TCA (3X), allowed to dry and incorporation of 32p into the substrate was counted on a Packard top count microplate scintillation counter (Packard, Meriden, CT). Each assay were performed as triplicate. The specific activity was determined by comparing the radioactive counts with immunoblot signals.
SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: DUONG, LE T.
RODAN, GIDEON A.
(ii) TITLE OF THE INVENTION: PROTEIN TYROSINE KINASE 2 (PYK2), NUCLEIC ACIDS AND ASSAY
(iii) NUMBER OF SEQUENCES: 6
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Merck & Co . , Inc.
(B) STREET: P.O. Box 2000, 126 E. Lincoln Avenue
(C) CITY: Rahway
(D) STATE: NJ
(E) COUNTRY: USA
(F) ZIP: 07065-0900
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: DOS
(D) SOFTWARE: FastSEQ for Windows Version 2.0
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: 60/037,561
(B) FILING DATE: ll-FEB-1997
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Sabatelli, Anthony D
(B) REGISTRATION NUMBER: 34,714
(C) REFERENCE/DOCKET NUMBER: 19792
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 732-594-1935
(B) TELEFAX: 732-594-4720
(C) TELEX:
(2) INFORMATION FOR SEQ ID NO : 1 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :
AGTGACATTT ATCAGATGGA G 21
(2) INFORMATION FOR SEQ ID NO : 2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 2 : GAATGGACTG TGCACCGAGC C 21
(2) INFORMATION FOR SEQ ID NO : 3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 3 : CAGCACACAA TCCTGGAGGA G 21
(2) INFORMATION FOR SEQ ID NO : 4 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: Other
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 4 : GCTGAAGCTT GACACCCTCA T 21
(2) INFORMATION FOR SEQ ID NO : 5 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3981 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 5 :
ATTCGCGGCC GCTCGACCTC AGCCTCTGCA GGCAGAGCCG CGCGTCCTAC CTGCGGCGGC 60
TGCGCTCACC TGGCCCAGCC CGGAGCCCTG GCCCGAGTCC GCGCCTCGCC CGAGGGACTG 120
CAATGTGCCG GTCCTAGCTG CAGTCTGAGA GGATGTCCGG GGTGTCTGAG CCCTTGAGCC 180
GTGTAAAAGT GGGCACTTTA CGCCGGCCTG AGGGCCCCCC AGAGCCCATG GTGGTGGTAC 240
CAGTGGATGT GGAGAAGGAA GACGTGCGCA TCCTCAAGGT CTGCTTCTAC AGCAACAGCT 300
TCAACCCAGG GAAGAACTTC AAGCTTGTCA AATGCACAGT GCAGACAGAG ATCCAGGAGA 360
TCATCACCTC CATCCTCCTG AGTGGGCGAA TAGGGCCCAA CATCCAGCTG GCTGAATGCT 420
ATGGGCTGAG GCTGAAGCAC ATGAAGTCAG ACGAGATCCA CTGGCTGCAC CCACAGATGA 480
CCGTGGGCGA AGTGCAGGAC AAGTATGAAT GTCTACACGT GGAAGCTGAG TGGAGGTATG 540
ACCTTCAAAT CCGCTACTTG CCGGAAGACT TCATGGAGAG CCTGAAAGAA GACAGGACCA 600
CATTGCTGTA CTTTTATCAA CAGCTCCGGA ATGACTACAT GCAACGCTAC GCCAGCAAGG 660
TCAGTGAAGG CATGGCTCTG CAGCTGGGCT GTCTGGAGCT CAGGAGATTC TTCAAGGACA 720
TGCCCCACAA TGCACTGGAC AAAAAGTCCA ACTTTGAACT CCTGGAAAAA GAAGTCGGTC 780
TGGACCTGTT TTTCCCAAAG CAGATGCAGG AAAACTTAAA GCCCAAGCAG TTCCGGAAGA 840
TGATCCAGCA GACCTTCCAG CAGTATGCAT CACTCCGGGA GGAAGAGTGT GTCATGAAAT 900
TCTTCAATAC CCTAGCGGGC TTTGCCAACA TTGACCAGGA GACCTACCGC TGCGAACTCA 960
TTCAAGGATG GAACATTACT GTGGACCTGG TCATCGGCCC TAAAGGCATC CGTCAGCTGA 1020
CAAGTCAAGA TACAAAGCCC ACCTGCCTGG CCGAGTTTAA GCAGATCAGA TCCATCAGGT 1080
GCCTCCCATT GGAAGAGACC CAGGCAGTCC TGCAGCTGGG CATCGAGGGT GCCCCCCAGT 1140
CCTTGTCTAT CAAAACGTCG TCCCTGGCAG AGGCTGAGAA CATGGCTGAT CTCATAGATG 1200
GCTACTGCAG GCTGCAAGGA GAACATAAGG GCTCTCTCAT CATGCATGCC AAGAAAGATG 1260
GTGAGAAGAG GAACAGCCTG CCTCAGATCC CCACACTAAA CCTGGAGGCT CGGCGGTCGC 1320
ACCTCTCAGA AAGCTGCAGC ATAGAGTCAG ACATCTATGC GGAGATTCCC GATGAGACCC 1380
TGCGAAGACC AGGAGGTCCA CAGTACGGTG TTGCCCGTGA AGAAGTAGTT CTTAACCGC 1440
TTCTGGGTGA AGGCTTCTTT GGGGAGGTCT ATGAAGGTGT CTACACGAAC CACAAAGGGG 1500
AAAAAATTAA TGTGGCCGTC AAGACCTGTA AGAAAGACTG TACCCAGGAC AACAAGGAGA 1560
AGTTCATGAG TGAGGCAGTG ATCATGAAGA ATCTTGACCA CCCTCACATC GTGAAGCTGA 1620
TTGGCATCAT TGAAGAGGAA CCCACCTGGA TTATCATGGA ACTGTATCCT TATGGGGAGC 1680
TGGGACACTA CCTGGAACGA AATAAAAACT CCCTGAAGGT ACCCACTCTG GTCCTGTACA 1740
CCCTACAGAT ATGCAAAGCC ATGGCCTATC TGGAGAGCAT CAACTGTGTG CACAGGGATA 1800
TTGCTGTCCG GAACATCCTG GTGGCCTCTC CTGAGTGTGT GAAGCTGGGG GACTTTGGGC 1860
TCTCCCGGTA CATTGAGGAC GAAGACTATT ACAAAGCCTC TGTGACACGT CTACCCATCA 1920
AATGGATGTC CCCCGAGTCC ATCAACTTCC GCCGCTTCAC AACCGCCAGT GATGTCTGGA 1980
TGTTTGCTGT ATGCATGTGG GAGATCCTCA GCTTTGGGAA GCAGCCTTTC TTCTGGCTCG 2040
AAAATAAGGA TGTCATCGGA GTGCTGGAGA AAGGGGACAG GCTGCCCAAG CCCGAACTCT 2100
GTCCGCCTGT CCTTTACACA CTCATGACTC GCTGCTGGGA CTACGACCCC AGTGACCGGC 2160
CCCGCTTC C GGAGCTTGTG TGCAGCCTCA GTGACATTTA TCAGATGGAG AAGGACATTG 2220
CCATAGAGCA AGAAAGGAAT GCTCGCTACC GACCCCCTAA AATATTGGAG CCTACTACCT 2280
TTCAGGAACC CCCACCCAAG CCCAGCCGGC CCAAGTACAG ACCTCCTCCA CAGACCAACC 2340
TGCTGGCTCC TAAGCTGCAG TTCCAGGTCC CTGAGGGTC.T GTGTGCCAGC TCTCCTACGC 2400
TTACCAGCCC TATGGAGTAT CCATCTCCAG TTAACTCGCT GCACACCCCA CCTCTCCACC 2460
GGCACAATGT CTTCAAGCGC CACAGCATGC GGGAGGAGGA CTTCATCCGG CCCAGTAGCC 2520
GAGAAGAGGC CCAGCAGCTC TGGGAGGCAG AGAAGATCAA GATGAAGCAG GTCCTAGAAA 2580
GACAGCAGAA GCAGATGGTG GAAGATTCCC AGTGGCTGAG GCGAGAGGAA AGATGCTTGG 2640
ACCCTATGGT TTATATGAAT GACAAGTCCC CACTGACTCC AGAGAAGGAG GCCGGCTACA 2700
CGGAGTTCAC AGGGCCCCCA CAGAAACCAC CTCGGCTCGG TGCACAGTCC ATTCAGCCCA 2760
CAGCCAACCT GGACAGGACC GATGACCTCG TGTACCACAA TGTCATGACC CTGGTGGAGG 2820
CTGTGCTGGA ACTCAAGAAC AAGCTTGGCC AGTTGCCCCC TGAGGACTAT GTGGTGGTGG 2880
TGAAGAACGT GGGGCTGAAC CTGCGGAAGC TCATCGGCAG TGTGGACGAT CTCTTGCCCT 2940
CCTTGCCGGC ATCTTCGAGG ACAGAGATTG AAGGGACCCA GAAACTGCTC AACAAAGACC 3000
TGGCAGAGCT CATCAACAAG ATGAAGTTGG CTCAGCAGAA CGCCGTGACG TCCCTGAGTG 3060
AGGACTGCAA GCGGCAGATG CTCACAGCGT CCCATACCCT GGCTGTGGAT GCCAAGAACC 3120
TGCTGGATGC TGTGGACCAA GCCAAGGTTG TGGCTAATCT GGCCCACCCG CCTGCAGAGT 3180
GATCAAGAGA GGGGCCACCT GCCTGCATCT TCTGCCCCCA CCTGTCTTGG CATACCTTTC 3240 CTGCCTTGCC TTTGGTTATT GGTCTTCCAG GGAAAGCTGA GAAGAGTCCA TCCCCCTTGC 3300
CACTTTGCAC GACACCCCCT CTTCCCCCAA CCCACCCCAG ACTGTGCTAC TCAGGCTGCA 3360
TCTGGACAGA AAGGACTCTG GGCACAGACA CGGGGTGGGG TGACATAGTT CATAGGGGTA 3420
CTACTGCCAG CCACTCCCTC TTACCCCAGC CTGGGTTGCT GGAGCATCAT TGGGGTCATG 3480
AGTGTACCCC TAACGGCCAA GATGGCTTTC TGCATGGACA TTTGAGAGCC AGTATTCCTC 3540
CTTCCTCTTC AGCCCTCAGG GACCCCTGAT ACAGAGGGGA CAGAGAGGGG TTTTATTTGT 3600
AGAGAAGCTG GTGAGATGAG GGCTGGACCT GGCTCTCTTG TACAGTGTAC ATTGGAATTT 3660
ATTTAATGTG AGTTTGACCT GGATGGACAG CCAAGGGCCA TAGTCCAGGA GCAAACCAAT 3720
CCAGTCACAG GACTCTGTGT TTTATGGAAC TGAGTGCCAC AGGAAGAAGC AGAGAGTCGG 3780
AGGTCAGAAT GGACTTTGTG CCCTTCCTGC GTTTCTCTTC TCCCTCTTTC CTTCTCCCCT 3840
CTTTTCTTAC GTCTCCTTTT TCTCCTCCCC CTTTTCACAT CTGCTCCCCT CCTCTCTCAT 3900
GTCTGTGGAG AACATTTACC TTCCTTCTTT TTGATCGGTG GTTGAATTAA AATTATTACC 3960
ATTTGCTTTG TGGCTCGTGC C 3981
(2) INFORMATION FOR SEQ ID NO : 6 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1009 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 6 :
Met Ser Gly Val Ser Glu Pro Leu Ser Arg Val Lys Val Gly Thr Leu
1 5 10 15
Arg Arg Pro Glu Gly Pro Pro Glu Pro Met Val Val Val Pro Val Asp
20 25 30
Val Glu Lys Glu Asp Val Arg lie Leu Lys Val Cys Phe Tyr Ser Asn
35 40 45
Ser Phe Asn Pro Gly Lys Asn Phe Lys Leu Val Lys Cys Thr Val Gin
50 55 60
Thr Glu He Gin Glu He He Thr Ser He Leu Leu Ser Gly Arg He 65 70 75 80
Gly Pro Asn He Gin Leu Ala Glu Cys Tyr Gly Leu Arg Leu Lys His
85 90 95
Met Lys Ser Asp Glu He His Trp Leu His Pro Gin Met Thr Val Gly
100 105 110
Glu Val Gin Asp Lys Tyr Glu Cys Leu His Val Glu Ala Glu Trp Arg
115 120 . 125
Tyr Asp Leu Gin He Arg Tyr Leu Pro Glu Asp Phe Met Glu Ser Leu
130 135 140
Lys Glu Asp Arg Thr Thr Leu Leu Tyr Phe Tyr Gin Gin Leu Arg Asn 145 150 155 160
Asp Tyr Met Gin Arg Tyr Ala Ser Lys Val Ser Glu Gly Met Ala Leu
165 170 175
Gin Leu Gly Cys Leu Glu Leu Arg Arg Phe Phe Lys Asp Met Pro His
180 185 190
Asn Ala Leu Asp Lys Lys Ser Asn Phe Glu Leu Leu Glu Lys Glu Val
195 200 205
Gly Leu Asp Leu Phe Phe Pro Lys Gin Met Gin Glu Asn Leu Lys Pro
210 215 220
Lys Gin Phe Arg Lys Met He Gin Gin Thr Phe Gin Gin Tyr Ala Ser 225 230 235 240 Leu Arg Glu Glu Glu Cys Val Met Lys Phe Phe Asn Thr Leu Ala Gly
245 250 255
Phe Ala Asn He Asp Gin Glu Thr Tyr Arg Cys Glu Leu He Gin Gly
260 265 270
Trp Asn He Thr Val Asp Leu Val He Gly Pro Lys Gly He Arg Gin
275 280 285
Leu Thr Ser Gin Asp Thr Lys Pro Thr Cys Leu Ala Glu Phe Lys Gin
290 295 300
He Arg Ser He Arg Cys Leu Pro Leu Glu Glu Thr Gin Ala Val Leu 305 310 315 320
Gin Leu Gly He Glu Gly Ala Pro Gin Ser Leu Ser He Lys Thr Ser
325 330 335
Ser Leu Ala Glu Ala Glu Asn Met Ala Asp Leu He Asp Gly Tyr Cys
340 345 350
Arg Leu Gin Gly Glu His Lys Gly Ser Leu He Met His Ala Lys Lys
355 360 365
Asp Gly Glu Lys Arg Asn Ser Leu Pro Gin He Pro Thr Leu Asn Leu
370 375 380
Glu Ala Arg Arg Ser His Leu Ser Glu Ser Cys Ser He Glu Ser Asp 385 390 395 400
He Tyr Ala Glu He Pro Asp Glu Thr Leu Arg Arg Pro Gly Gly Pro
405 410 415
Gin Tyr Gly Val Ala Arg Glu Glu Val Val Leu Asn Arg He Leu Gly
420 425 430
Glu Gly Phe Phe Gly Glu Val Tyr Glu Gly Val Tyr Thr Asn His Lys
435 440 445
Gly Glu Lys He Asn Val Ala Val Lys Thr Cys Lys Lys Asp Cys Thr
450 455 460
Gin Asp Asn Lys Glu Lys Phe Met Ser Glu Ala Val He Met Lys Asn 465 470 475 480
Leu Asp His Pro His He Val Lys Leu He Gly He He Glu Glu Glu
485 490 495
Pro Thr Trp He He Met Glu Leu Tyr Pro Tyr Gly Glu Leu Gly His
500 505 510
Tyr Leu Glu Arg Asn Lys Asn Ser Leu Lys Val Pro Thr Leu Val Leu
515 520 525
Tyr Thr Leu Gin He Cys Lys Ala Met Ala Tyr Leu Glu Ser He Asn
530 535 540
Cys Val His Arg Asp He Ala Val Arg Asn He Leu Val Ala Ser Pro 545 550 555 560
Glu Cys Val Lys Leu Gly Asp Phe Gly Leu Ser Arg Tyr He Glu Asp
565 570 . 575
Glu Asp Tyr Tyr Lys Ala Ser Val Thr Arg Leu Pro He Lys Trp Met
580 585 590
Ser Pro Glu Ser He Asn Phe Arg Arg Phe Thr Thr Ala Ser Asp Val
595 600 605
Trp Met Phe Ala Val Cys Met Trp Glu He Leu Ser Phe Gly Lys Gin
610 615 620
Pro Phe Phe Trp Leu Glu Asn Lys Asp Val He Gly Val Leu Glu Lys 625 630 635 640
Gly Asp Arg Leu Pro Lys Pro Glu Leu Cys Pro Pro Val Leu Tyr Thr
645 650 655
Leu Met Thr Arg Cys Trp Asp Tyr Asp Pro Ser Asp Arg Pro Arg Phe
660 665 670
Thr Glu Leu Val Cys Ser Leu Ser Asp He Tyr Gin Met Glu Lys Asp 675 680 685 He Ala He Glu Gin Glu Arg Asn Ala Arg Tyr Arg Pro Pro Lys He
690 695 700
Leu Glu Pro Thr Thr Phe Gin Glu Pro Pro Pro Lys Pro Ser Arg Pro 705 710 715 720
Lys Tyr Arg Pro Pro Pro Gin Thr Asn Leu Leu Ala Pro Lys Leu Gin
725 730 735
Phe Gin Val Pro Glu Gly Leu Cys Ala Ser Ser Pro Thr Leu Thr Ser
740 745 750
Pro Met Glu Tyr Pro Ser Pro Val Asn Ser Leu His Thr Pro Pro Leu
755 760 765
His Arg His Asn Val Phe Lys Arg His Ser Met Arg Glu Glu Asp Phe
770 775 780
He Arg Pro Ser Ser Arg Glu Glu Ala Gin Gin Leu Trp Glu Ala Glu 785 790 795 800
Lys He Lys Met Lys Gin Val Leu Glu Arg Gin Gin Lys Gin Met Val
805 810 815
Glu Asp Ser Gin Trp Leu Arg Arg Glu Glu Arg Cys Leu Asp Pro Met
820 825 830
Val Tyr Met Asn Asp Lys Ser Pro Leu Thr Pro Glu Lys Glu Ala Gly
835 840 845
Tyr Thr Glu Phe Thr Gly Pro Pro Gin Lys Pro Pro Arg Leu Gly Ala
850 855 860
Gin Ser He Gin Pro Thr Ala Asn Leu Asp Arg Thr Asp Asp Leu Val 865 870 875 880
Tyr His Asn Val Met Thr Leu Val Glu Ala Val Leu Glu Leu Lys Asn
885 890 895
Lys Leu Gly Gin Leu Pro Pro Glu Asp Tyr Val Val Val Val Lys Asn
900 905 910
Val Gly Leu Asn Leu Arg Lys Leu He Gly Ser Val Asp Asp Leu Leu
915 920 925
Pro Ser Leu Pro Ala Ser Ser Arg Thr Glu He Glu Gly Thr Gin Lys
930 935 940
Leu Leu Asn Lys Asp Leu Ala Glu Leu He Asn Lys Met Lys Leu Ala 945 950 955 960
Gin Gin Asn Ala Val Thr Ser Leu Ser Glu Asp Cys Lys Arg Gin Met
965 970 975
Leu Thr Ala Ser His Thr Leu Ala Val Asp Ala Lys Asn Leu Leu Asp
980 985 990
Ala Val Asp Gin Ala Lys Val Val Ala Asn Leu Ala His Pro Pro Ala
995 1000 1005
Glu 1

Claims

WHAT IS CLAIMED IS:
1. A nucleic acid, free from associated nucleic acids which encodes murine protein tyrosine kinase 2 (PYK2).
2. A nucleic acid according to Claim 1 which is DNA.
3. Murine PYK2 cDNA.
4. Murine PYK2 cDNA which is set forth in Figure 1.
5. A cell line comprising heterologous PYK2, and which expresses PYK2.
6. An assay to identify compounds which alter the activity of PYK2 comprising: a) contacting recombinant PYK2 with a tyrosine substrate in the presence of radiolabeled ATP and a putative activity-modifying compound; b) measuring the amount of radiolabeled tyrosine which is formed; and c) optionally comparing the amount of radiolabeled tyrosine formed in the presence of the putative activity-modifying compound with that formed in the absence of the putative activity-modifying compound.
TITLE OF THE INVENTION
PROTEIN TYROSINE KINASE 2 (PYK2), NUCLEIC ACIDS, AND
ASSAY
ABSTRACT OF THE INVENTION
This invention is directed to nucleic acids encoding protein tyrosine kinase 2 (PYK2), to murine PYK2, to methods of making this protein using the nucleic acids, and to assays for inhibitors of PYK2.
PCT/US1998/002494 1997-02-11 1998-02-09 Protein tyrosine kinase 2 (pyk2), nucleic acids, and assay Ceased WO1998035016A1 (en)

Priority Applications (3)

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CA002277028A CA2277028A1 (en) 1997-02-11 1998-02-09 Protein tyrosine kinase 2 (pyk2), nucleic acids, and assay
EP98905026A EP0998553A4 (en) 1997-02-11 1998-02-09 PROTEIN TYROSINE-KINASE 2 (PYK2), NUCLEIC ACIDS AND DETECTION METHOD
JP53498698A JP2002513284A (en) 1997-02-11 1998-02-09 Protein tyrosine kinase 2 (PYK2), nucleic acids and assays

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US3756197P 1997-02-11 1997-02-11
US60/037,561 1997-02-11

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1009395A4 (en) * 1997-02-18 2000-11-08 Lxr Biotechnology Inc Bak promotor expression system

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5538858A (en) * 1994-04-08 1996-07-23 Pierce Chemical Company Rapid assay for radioactive determination of protein kinase activity
US5573944A (en) * 1994-07-22 1996-11-12 President And Fellows Of Harvard College Yeast cell expressing heterologous receptor kinase

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5538858A (en) * 1994-04-08 1996-07-23 Pierce Chemical Company Rapid assay for radioactive determination of protein kinase activity
US5573944A (en) * 1994-07-22 1996-11-12 President And Fellows Of Harvard College Yeast cell expressing heterologous receptor kinase

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
AVRAHAM S., ET AL.: "IDENTIFICATION AND CHARACTERIZATION OF A NOVEL RELATED ADHESION FOCAL TYROSINE KINASE (RAFTK) FROM MEGAKARYOCYTES AND BRAIN.", JOURNAL OF BIOLOGICAL CHEMISTRY, AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGY, US, vol. 270., no. 46., 17 November 1995 (1995-11-17), US, pages 27742 - 27751., XP002912212, ISSN: 0021-9258, DOI: 10.1074/jbc.270.46.27742 *
See also references of EP0998553A4 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1009395A4 (en) * 1997-02-18 2000-11-08 Lxr Biotechnology Inc Bak promotor expression system
US6436639B1 (en) 1997-02-18 2002-08-20 Tanox, Inc. Bak promoter expression system

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EP0998553A1 (en) 2000-05-10
CA2277028A1 (en) 1998-08-13
JP2002513284A (en) 2002-05-08

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