JP2002513284A - Protein tyrosine kinase 2 (PYK2), nucleic acids and assays - Google Patents

Abstract

  (57) [Summary] The present invention relates to a nucleic acid encoding protein tyrosine kinase 2 (PYK2), mouse PYK2, a method for producing the protein using the nucleic acid, and an assay for inhibition of PYK2.

Description

DETAILED DESCRIPTION OF THE INVENTION Protein tyrosine kinase 2 (PYK2), BRIEF DESCRIPTION OF THE INVENTION The nucleic and assay invention, a nucleic acid encoding a protein tyrosine kinase 2 (PYK2), mouse PYK2, production of the protein using the nucleic acid Methods and assays for PYK2 inhibitors. 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 announced member of the focal adhesion kinase family ( Avraham et al., J. Biol. Chem., 270: 27742-27751 (1995); Lev et al., Nature, 376: 737-745 (1995); and Sasaki et al., J. Biol. Chem., 270: 21 206-21219. (1995)). PYK2 was first cloned from human brain as a Grb-2 binding protein, and also from rat and human brain libraries. There are also conflicting reports on its cellular expression. In one study, large amounts of PYK2 transcripts were found in the brain and small amounts were detected in the kidney. In another study, PYK2 expression was found to be most abundant in rat brain, but its transcript was also detected in kidney, spleen, lung, small intestine and epididymis. PYK2 transcript was also detected in rat fibroblast 3T1 and WFB cell lines, and in human T-cell leukemia Jurkat cells. When cloned from the human megakaryocyte CMK cell line and mouse brain, it was found to be distributed over the brain in tissues that are broad, especially in the spleen, lung, thymus and peripheral blood leukocytes. In addition, expression of PYK2 in human CD34 + bone marrow cells, megakaryocytes and platelets has been reported. DETAILED DESCRIPTION OF THE INVENTION A first aspect of the invention is a nucleic acid encoding mouse protein tyrosine kinase 2 (PYK2) substantially free of related nucleic acids. In one embodiment, the nucleic acid encoding PYK2 is DNA. Another aspect of the invention is the mouse PYK2 cDNA. Mouse PYK2 DNA is set forth in FIG. 1 (SEQ ID NO: 5). Another aspect of the invention is mouse PYK2 that does not contain a related mouse protein. One mouse PYK2 is described in FIG. 1 (SEQ ID NO: 6). Another aspect of the present invention is a method for producing PYK2 by introducing a nucleic acid comprising a nucleic acid encoding PYK2 into a cell under conditions that cause transcription and translation of PYK2. Preferably, the nucleic acid is in a plasmid or a vector such as a baculovirus vector. It is also preferred that the nucleic acid be under the control of a promoter and optionally a transcriptional regulator such as an enhancer. Said modulators are known in the art. Host cells expressing PYK2 also form part of the invention. Preferred host cells include mammalian cells, insect cells, yeast, and bacterial cells such as E. coli. Cell lines expressing mouse PYK2 permanently (rather than transiently) also form part of the invention. Recombinant PYK2 produced using the cloning method of the invention can be used in assays to further characterize the biological function of PYK2 and identify compounds such as agonists and antagonists that modulate its activity. . A further aspect of the invention is an assay for identifying compounds that modulate the activity of PYK2, and in particular, inhibitors of PYK2 activity. This assay involves contacting recombinant PYK2 with a tyrosine substrate in the presence of radiolabeled ATP and a putative activity-modulating compound, measuring the amount of radiolabeled tyrosine formed, and optionally presuming Comparing the amount of radiolabeled tyrosine formed in the presence of the activity modulating compound with the estimated amount of radiolabeled tyrosine formed in the absence of the activity modulating compound. Integrins are a major family of cell surface receptors that mediate adhesive interactions with either neighboring cells or the extracellular matrix. Integrin signaling 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 tissues and hematopoietic cells. Thus, modulators of PYK2 would be potential therapeutics for regulating platelet levels. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the cDNA sequence and predicted protein sequence of mouse PYK2. Intron sequences are shown in lower case, exon sequences in upper case. The boxed sequence of the putative protein indicates the kinase domain. Proline circled for putative protein indicates high proline domain. The terms used in the description and the claims are defined as follows. "PYK2" refers to protein tyrosine kinase 2, allelic variants of protein tyrosine kinase 2, and mutants or fragments thereof that retain at least about 85%, preferably about 90%, of the biological activity of native PYK2. Point. "Native PYK2" refers to a protein tyrosine kinase that is naturally present in an organism. "Substantially free of related nucleic acids" means that less than about 5% by weight of nucleic acids other than the nucleic acid encoding PYK2 are present in the sample. “Substantially free of related mouse proteins” means that there is less than about 5% by weight of proteins other than mouse PYK2 in the sample. “FAK” refers to focal adhesion kinase. A "heterologous" PYK2 nucleic acid means that the nucleic acid has been introduced into the cell regardless of whether the nucleic acid is from the same species as the cell. Alternatively, it refers to a nucleic acid encoding PYK2 in a cell whose ancestor has PYK2 introduced into the cell. “Heterologous” PYK2 protein means that PYK2 is encoded by a heterologous nucleic acid. FAK proteins involved in the cell adhesion process were not detected in many macrophage cell lines. Therefore, it was hypothesized that another cell adhesion-dependent kinase homologous to FAK could have its function in these cells. PYK2 was recently identified as another member of the FAK family and its expression has been detected in spleen, thymus, lung and peripheral blood leukocytes (Avraham (1995) supra; Sasaki (1995) supra). To evaluate PYK2 as a potential adhesion-dependent kinase in macrophages, specific probes for PYK2 and FAK were generated and used to test PYK2 and FAK expression in mouse tissues. As previously reported for other species, PYK2 is highly expressed in brain and spleen, with lower levels in kidney, lung and liver, distributed in smaller tissues compared to FAK. I have. The full-length cDNA was cloned from a mouse spleen cDNA library using the PYK2 probe. The deduced amino acid sequence of the full-length clone was found to be identical to the recently published amino acid sequence of mouse RAFTK (Avraham et al. (1995) supra). In addition, full-length FAK was cloned from a mouse osteoblast MB1.8 cell line (Weslowski et al., Exp. Cell Res., 219: 679-686 (1995)). PYK2 cDNA and FAK cDNA were subsequently transfected into human embryonic kidney (HEK) 293 cells. Permanent cell lines expressing PYK2 or FAK were established and expression levels of exogenously expressed mouse kinase were assessed by Northern analysis. The following examples are provided to further illustrate the present invention. Example 1 Macrophages were induced by injecting cell culture thioglycolate into the peritoneal cavity of adult BALB / c mice. Four days later, cells were collected, washed, and cultured in RPMI 1640 medium containing 10% FBS. After 3 hours at 37 ° C., the culture was sufficiently washed to remove non-adherent cells, and after overnight culture, a sample for immunoprecipitation was prepared. Bone marrow-derived macrophages are described in Li and Chen, J. et al. Leuk. Biol. , 57: 484-490 (1995). Non-adherent cells were cultured in RPMI-supplemented medium in the presence of human macrophage colony stimulating factor (MCS-F, 250 units / ml, Genetics Institute, Cambridge, Mass.). After culturing for 5 days, differentiated macrophages for immunoprecipitation were prepared. Bone marrow-derived osteoblast-like cells were prepared as described in Wesolowski et al., Exp. Cell Res. , 219: 679-686 (1995). After collagenase-dispase treatment, mononuclear tartrate-resistant phosphatase-positive cells were released from the tissue culture plates using 30 nM echistatin (Merck Res. Labs., West Point, PA). Freshly isolated osteoblast-like cells were immediately lysed with immunoprecipitation buffer. Example 2 cDNA cloning and expression of mouse PYK2 Specific probes for mouse PYK2 and FAK were first generated based on the heterologous region between proteins adjacent to the C-terminus of the kinase domain. By polymerase chain reaction (PCR), specific probes for PYK2 (570 bp) were converted to 5′-primer-AGTGA CATTT ATCAG ATGGA G (SEQ ID NO: 1) and 3′-primer-GAATG GACTG TGCAC CG AGC C (SEQ ID NO: 2). A cDNA of mouse bone marrow-derived osteoblast-like cells was used as a template (Weslowski et al. (1995), supra). Similarly, a specific probe for FAK (700 bp) was used as a mouse osteoblast cell MB1 using primers 5′-CAGCA CACAA TCCTG GAGGA G (SEQ ID NO: 3) and 3′-GCTGA AGCTT GACAC CCTCAT (SEQ ID NO: 4). .8 cells as a template (Weslowski et al. (1995) supra). These probes were confirmed by sequencing analysis. The PYK2 cDNA fragment was cloned from a mouse spleen λ-ZAP II cDNA library (Stratagene, La Jolla, Calif.) Using a specific PYK2 probe. A full-length PYK2 cDNA was constructed by ligating two overlapping clones at the VspI site. The amino acid sequence of the isolated PYK2 cDNA clone was identical to the mouse RAFTK sequence reported previously (Avraham et al. (1995) supra). Full-length FAK cDNA was obtained from Hanks et al., Proc. Natl. Acad. S ci. USA, 89: 8487-8491 (1992). Both PYK2 and FAK cDNAs were subcloned into the pCDNA3 plasmid (InVitrogen, San Diego, Calif.) And human embryos were electroporated at 200V, 960 μF using a GenePulser (Biora d Labs, Richmond, Calif.). Kidney (HEK) 293 cells (ATCC, Rockland, MD) were transfected. Thereafter, HEK293 cells were subjected to G418 selection (800 μg / ml, Gibco BRL), and clones were selected after 3 weeks in a selection medium. Expression of PYK2 and FAK in HEK293 cells was confirmed by Northern analysis using the respective probes, and Western blots were performed using anti-PYK2 antibodies. Mouse multi-tissue Northern blots were purchased from Clonetech (Palo Alto, Calif.) And hybridization of Northern blots using probes specific for PYK2, FAK and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was described above (supra). Weslowski et al. (1995)). Example 3 Production of Polyclonal Antibody Against Mouse PYK2 and Affinity Purification The C-terminal domain of PYK2 (from methionine residue 685 to the end) was amplified by PCR using mouse PYK2 as a template. The amplification product was cloned into plasmid pGEX-4T (Pharmacia Biotech, Piscataway, NJ) and transformed into E. coli XL1-Blue (Stratage). Expression of the GST-PYK2 C-terminal fragment was induced with 0.5 mM IPTG, purified and cleaved from GST with thrombin essentially according to the manufacturer's instructions (Pharmacia). Purified C-terminal fragments of mouse PYK2 were used to immunize two rabbits (Research Genetics, Huntsville, Ala.), And the titers of both antisera were first determined by recombinant C-terminal PYK2. It was measured by ELISA using terminal fragments. Thereafter, the specificity of the immune sera was determined by Western blot by comparing with the pre-immune sera. The polyclonal antibody was then affinity-passed by passing the combined fractions of both antisera through an affinity column constructed with the same purified antigen cross-linked to CNBr-activated Sepharose 4B according to the manufacturer's instructions (Pharmacia). Purified. After the antibody was eluted from the column with 0.2 M glycine (pH 2.5) and 1 mM EGTA, the eluted fraction was dialyzed against PBS containing 0.02% azide. Anti-PYK2 antibody was stored at -70 ° C at a concentration of 0.5 mg / ml. Example 4 In Vitro Kinase Activity After cell adhesion to ECM, IC-21 cells were treated with 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 1 mM EDTA, 10% glycerol, 50 mM NaF, 1 mM sodium vanadate And dissolved in a TNE lysis buffer containing the above protease inhibitor. PYK2 was immunoprecipitated from the clarified lysate, half of the sample was immunoblotted using the anti-PYK2 antibody described above, and the other half was the same lysis buffer and 20 mM Tris-HCl (pH 7.4), 10 mM NaCl, 10 mM MnCl. Washed twice with kinase assay buffer (1 ×) containing ₂ and 1 mM dithiothreitol. After removal of the wash buffer, 5 μCi [γ- ³² P] ATP (3000 Ci / mmol, Amersham), 10 mM ATP, 0.1% BSA and 100 μg poly (Glu, Tyr) (molar ratio 4: 1, Sigma) 50 μl of the kinase assay buffer was added to the beads and incubated at 30 ° C. for 10 minutes (Howell and Cooper, Mol. Cell. Biol., 14: 5402-5411 (1995)). The reaction mixture (25 μl) was added to 30% trichloroacetic acid (TCA) and 25 μl of 0.1 M sodium pyrophosphate and then incubated at 4 ° C. for 15 minutes. The precipitated proteins were transferred to Multiscreen-FC filter plate (MA Marlborough the whereabouts Millipore), and washed with ice cold 15% TCA (3 ×), dried, and ³² P incorporation into a substrate Packard TopCount Microplate Scintillation Counting was performed on a counter (Packard, Meriden, CT). Each assay was performed three times. Specific activity was determined by comparing radioactivity counts to immunoblot signals.

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Claims (1)

[Claims] 1. Related nuclei encoding mouse protein tyrosine kinase 2 (PYK2) Acid free nucleic acids. 2. The nucleic acid according to claim 1, which is DNA. 3. Mouse PYK2 cDNA. 4. The mouse PYK2 cDNA shown in FIG. 5. A cell line that contains heterologous PYK2 and expresses PYK2. 6. An assay for identifying a compound that alters the activity of PYK2, comprising: a) Recombinant PYK2 in the presence of radiolabeled ATP and putative activity modulating compounds Is contacted with a tyrosine substrate, b) measuring the amount of radiolabeled tyrosine formed, c) radioactively labeled fir tree optionally formed in the presence of the putative activity modulating compound The amount of radiolabeled tyrosine formed in the absence of the putative activity-modulating compound. Said method comprising comparing to the amount of