Differential Regulation of Pyk2 and Focal Adhesion Kinase (FAK)

Abstract

Pyk2 is a recently described cytoplasmic tyrosine kinase that is related to focal adhesion kinase (FAK) and can be activated by a variety of stimuli that elevate intracellular calcium. In this report, we showed that Pyk2 and FAK tyrosine phosphorylation are regulated differentially by integrin-mediated cell adhesion and soluble factors both in rat aortic smooth muscle cells, which express endogenous Pyk2 and FAK, and in transfected Chinese hamster ovary cells. We also found that Pyk2 is diffusely present throughout the cytoplasm, while FAK is localized in focal contacts as expected, suggesting that the different localization may account for their differential regulation. By analyzing a chimeric protein contain N-terminal and kinase domains of Pyk2 and C-terminal domain of FAK, we provided evidence that the distinctive C-terminal domains of Pyk2 and FAK were responsible for their differential regulation by integrins and soluble stimuli as well as their subcellular localization. Finally, we correlated FAK, Pyk2, and the chimeric protein binding to talin, but not paxillin, with their regulation by integrins and focal contact localization. These results demonstrate that the distinctive C-terminal domain of Pyk2 and FAK confer their differential regulation by different subcellular localization and association with the cytoskeletal protein talin. Pyk2 is a recently described cytoplasmic tyrosine kinase that is related to focal adhesion kinase (FAK) and can be activated by a variety of stimuli that elevate intracellular calcium. In this report, we showed that Pyk2 and FAK tyrosine phosphorylation are regulated differentially by integrin-mediated cell adhesion and soluble factors both in rat aortic smooth muscle cells, which express endogenous Pyk2 and FAK, and in transfected Chinese hamster ovary cells. We also found that Pyk2 is diffusely present throughout the cytoplasm, while FAK is localized in focal contacts as expected, suggesting that the different localization may account for their differential regulation. By analyzing a chimeric protein contain N-terminal and kinase domains of Pyk2 and C-terminal domain of FAK, we provided evidence that the distinctive C-terminal domains of Pyk2 and FAK were responsible for their differential regulation by integrins and soluble stimuli as well as their subcellular localization. Finally, we correlated FAK, Pyk2, and the chimeric protein binding to talin, but not paxillin, with their regulation by integrins and focal contact localization. These results demonstrate that the distinctive C-terminal domain of Pyk2 and FAK confer their differential regulation by different subcellular localization and association with the cytoskeletal protein talin. Proline-rich tyrosine kinase 2 (Pyk2 1The abbreviations used are: Pyk2, proline-rich tyrosine kinase 2; FAK, focal adhesion kinase; FN, fibronectin; RASM, rat aortic smooth muscle; HA, hemagglutinin; PLL, poly-l-lysine; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; CHO, Chinese hamster ovary; GST, glutathioneS-transferase; PDGF, platelet-derived growth factor; PAGE, polyacrylamide gel electrophoresis; phosphate-buffered saline.1The abbreviations used are: Pyk2, proline-rich tyrosine kinase 2; FAK, focal adhesion kinase; FN, fibronectin; RASM, rat aortic smooth muscle; HA, hemagglutinin; PLL, poly-l-lysine; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; CHO, Chinese hamster ovary; GST, glutathioneS-transferase; PDGF, platelet-derived growth factor; PAGE, polyacrylamide gel electrophoresis; phosphate-buffered saline.; also known as CAKβ, RAFTK, and CADTK) is a recently described cytoplasmic tyrosine kinase that is related to the focal adhesion kinase (FAK) (1Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1248) Google Scholar, 2Sasaki H. Nagura K. Ishino M. Tobioka H. Kotani K. Sasaki T. J. Biol. Chem. 1995; 270: 21206-21219Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 3Avraham S. London R. Fu Y. Ota S. Hiregowdara D. Li J. Jiang S. Pasztor L.M. White R.A. Groopman J.E. Avraham H. J. Biol. Chem. 1995; 270: 27742-27751Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 4Yu H. Li X. Marchetto G.S. Dy R. Hunter D. Calvo B. Dawson T.L. Wilm M. Anderegg R.J. Graves L.M. Earp H.S. J. Biol. Chem. 1996; 271: 29993-29998Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). Recent studies have shown that Pyk2 is involved in calcium-induced regulation of ion channel and mitogen-activated protein kinase activation (1Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1248) Google Scholar), stress-induced c-Jun N-terminal kinase activation (5Tokiwa G. Dikic I. Lev S. Schlessinger J. Science. 1996; 273: 792-794Crossref PubMed Scopus (285) Google Scholar), and Src-mediated activation of the mitogen-activated protein kinase signaling pathway in PC12 cells (6Dikic I. Tokiwa G. Lev S. Courtneidge S. Schlessinger J. Nature. 1996; 383: 547-549Crossref PubMed Scopus (877) Google Scholar). Although these studies indicate that Pyk2 is involved in several signal transduction pathways, many questions concerning the regulation and function of Pyk2, especially the role of Pyk2 in cell adhesion, remain unanswered. Pyk2 and FAK share a similar structural organization with a tyrosine kinase domain flanked by non-catalytic domains at both the N and C termini. These two kinases are approximately 60% identical in the central catalytic domain and share approximately 40% identity in both the N- and C-terminal domains (1Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1248) Google Scholar, 2Sasaki H. Nagura K. Ishino M. Tobioka H. Kotani K. Sasaki T. J. Biol. Chem. 1995; 270: 21206-21219Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, 3Avraham S. London R. Fu Y. Ota S. Hiregowdara D. Li J. Jiang S. Pasztor L.M. White R.A. Groopman J.E. Avraham H. J. Biol. Chem. 1995; 270: 27742-27751Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar). Because of the high sequence homology and similar overall organization between Pyk2 and FAK, it is especially interesting to compare the regulation of Pyk2 with that of FAK, in particular their responses to integrin-mediated cell adhesion. Several recent reports have presented somewhat conflicting data regarding regulation of Pyk2 by integrin-mediated cell adhesion in different cell types. It has been reported that Pyk2 displays an integrin-dependent phosphorylation and is localized in focal contacts in B lymphocytes, megakaryocytes, and transfected COS cells (7Li J. Avraham H. Rogers R.A. Raja S. Avraham S. Blood. 1996; 88: 417-428Crossref PubMed Google Scholar, 8Astier A. Avraham H. Manie S.N. Groopman J. Canty T. Avraham S. Freedman A.S. J. Biol. Chem. 1997; 272: 228-232Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar); however, in transfected 3Y1 cells, Pyk2 phosphorylation is not increased upon plating on fibronectin (FN) and Pyk2 has been shown to localize in intercellular junctions (2Sasaki H. Nagura K. Ishino M. Tobioka H. Kotani K. Sasaki T. J. Biol. Chem. 1995; 270: 21206-21219Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar). In addition, Siciliano et al. (9Siciliano J.C. Toutant M. Derkinderen P. Sasaki T. Girault J.-A. J. Biol. Chem. 1996; 271: 28942-28946Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar) reported that, in rat hippocampal slices and cortical synaptosomes, Pyk2 and FAK are regulated differentially by pathways involving calcium and protein kinase C. To determine whether induction of Pyk2 phosphorylation by integrins is as effective as that by soluble factors and how it compares with FAK phosphorylation in cell adhesion in the same cells, we investigated regulation of Pyk2 and FAK in rat aortic smooth muscle (RASM) cells, which express both kinases. Using a CHO cell transfection system, we also analyzed regulation of Pyk2, FAK, or a chimeric protein containing the N-terminal and kinase domains of Pyk2 fused to the C-terminal domain of FAK. Our results demonstrated that Pyk2 and FAK were regulated differentially in both RASM cells and transfected CHO cells and that the distinctive C-terminal domains are responsible for the differential regulation by directing different subcellular localization and association with the cytoskeletal protein talin. Antiserum against human Pyk2 was prepared in rabbits using a GST fusion protein containing the C-terminal 250 residues (GST-Pyk2.C). Anti-Pyk2 antibodies were then affinity-purified from the antiserum using GST-Pyk2.C immobilized on glutathione-Sepharose as an affinity matrix, as described previously (10Devor B.B. Zhang X. Patel S.K. Polte T. Hanks S.K. Biochem. Biophys. Res. Commun. 1993; 190: 1084-1089Crossref PubMed Scopus (14) Google Scholar). Rabbit polyclonal anti-FAK serum and mouse monoclonal antibody 12CA5 against an epitope of the hemagglutinin (HA) protein were as described (11Xing Z. Chen H.-C. Nowlen J.K. Taylor S.J. Shalloway D. Guan J.-L. Mol. Biol. Cell. 1994; 5: 413-421Crossref PubMed Scopus (284) Google Scholar, 12Chen H.-C. Appeddu P.A. Parsons J.T. Hildebrand J.D. Schaller M.D. Guan J.-L. J. Biol. Chem. 1995; 270: 16995-16999Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). Mouse monoclonal anti-phosphotyrosine antibody PY20 and mouse monoclonal anti-paxillin antibody were obtained from Transduction Laboratories (Lexington, KY). Mouse monoclonal anti-vinculin and anti-talin antibodies, rhodamine-conjugated goat anti-rabbit IgG, fluorescein-conjugated goat anti-mouse IgG, normal goat serum, Protein A-Sepharose 4B, calcium ionophore A23187, angiotensin II, sorbitol, and poly-l-lysine (PLL) were purchased from Sigma. Human plasma FN was from Life Technologies, Inc. Recombinant PDGF-BB was obtained from UBI (Lake Placid, RASM cells were obtained from and in Dulbecco's modified Eagle's with fetal bovine serum Life Technologies, and cells were in with and then to different as In to addition, cells were that been with FN or and with 2 bovine serum as described previously J.-L. J.E. PubMed Scopus Google Scholar). of CHO cells were using Technologies, to the has been described previously H.-C. Appeddu P.A. H. Guan J.-L. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). human Pyk2 was from a by on an provided by Y. Li of Inc. C. Z. C. and J. showed that is identical with the Pyk2 for the of at It was then the of the with the N fused in to the to chimeric was by with the by as described previously S.N. J.K. PubMed Scopus Google Scholar). fusion was by and this was then the of were with and 2 as described previously H.-C. Appeddu P.A. Parsons J.T. Hildebrand J.D. Schaller M.D. Guan J.-L. J. Biol. Chem. 1995; 270: 16995-16999Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, H.-C. Appeddu P.A. H. Guan J.-L. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). was at by cell for 2 with antibodies by for with Protein A-Sepharose or Protein the were then in for and by was with antibodies as using the Life as described previously H.-C. Appeddu P.A. Parsons J.T. Hildebrand J.D. Schaller M.D. Guan J.-L. J. Biol. Chem. 1995; 270: 16995-16999Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar, H.-C. Appeddu P.A. H. Guan J.-L. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar). In cell were analyzed by on were in with and in in for at and then in and in in for at were in and with antibody in normal goat serum in for at two in were with or rhodamine-conjugated antibody in normal goat serum in for at in were on and and using a Because express both endogenous Pyk2 and FAK, RASM cells an to compare regulation of tyrosine of Pyk2 and FAK by cell adhesion and by soluble factors in the same cells, which may the somewhat results on Pyk2 regulation by integrins in reports (2Sasaki H. Nagura K. Ishino M. Tobioka H. Kotani K. Sasaki T. J. Biol. Chem. 1995; 270: 21206-21219Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, J. Avraham H. Rogers R.A. Raja S. Avraham S. Blood. 1996; 88: 417-428Crossref PubMed Google Scholar, 8Astier A. Avraham H. Manie S.N. Groopman J. Canty T. Avraham S. Freedman A.S. J. Biol. Chem. 1997; 272: 228-232Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). described were prepared from cells or cells that been with soluble factors shown to Pyk2 previously (1Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1248) Google Scholar, 4Yu H. Li X. Marchetto G.S. Dy R. Hunter D. Calvo B. Dawson T.L. Wilm M. Anderegg R.J. Graves L.M. Earp H.S. J. Biol. Chem. 1996; 271: 29993-29998Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, G. Dikic I. Lev S. Schlessinger J. Science. 1996; 273: 792-794Crossref PubMed Scopus (285) Google Scholar, I. Tokiwa G. Lev S. Courtneidge S. Schlessinger J. Nature. 1996; 383: 547-549Crossref PubMed Scopus (877) Google Scholar). were with or anti-FAK antibodies, by and then analyzed by using a monoclonal antibody against to tyrosine phosphorylation or or anti-FAK antibodies to similar shown in is a of Pyk2 phosphorylation in RASM cells with Pyk2 phosphorylation in cells. of the cells by angiotensin II, PDGF, sorbitol, calcium ionophore A23187, and in Pyk2 phosphorylation as by In FAK phosphorylation in RASM cells was the and showed with or and a as by upon with angiotensin II, PDGF, and These results a differential regulation of Pyk2 and FAK in the same while FAK phosphorylation is by cell adhesion, Pyk2 phosphorylation is by soluble factors that elevate intracellular calcium. To determine the of the cell adhesion we Pyk2 and FAK phosphorylation in RASM cells on FN or tyrosine phosphorylation was for Pyk2 or FAK in cells on adhesion to FN increased Pyk2 phosphorylation while a as shown in A. In cell adhesion to FN FAK phosphorylation to a and a in FAK phosphorylation 2 These results the differential responses of FAK and Pyk2 to integrin-mediated cell adhesion and to soluble factors in RASM cells. To of the differential of Pyk2 and FAK to cell adhesion, we localization of these two kinases in RASM cells upon their adhesion to on were by using polyclonal anti-FAK or antibodies and mouse monoclonal anti-vinculin as described the of FAK in focal contacts of cells on FN which with a of focal contacts In Pyk2 was as throughout the with the of the same cell These results that differential localization of Pyk2 and FAK be responsible for their differential regulation by C-terminal domain of FAK the focal adhesion sequence that has been shown to be both and for FAK localization to focal contacts J.D. Schaller M.D. Parsons J.T. J. Biol. 1993; PubMed Scopus Google Scholar). the C-terminal domain of Pyk2 is identical to that of FAK in a of homology the focal adhesion the of focal contact localization of Pyk2 in RASM cells the that the in the C-terminal domains of these two kinases are responsible for their differential regulation and subcellular localization. To this we a chimeric which of the N-terminal and kinase domains of Pyk2 fused in to the C-terminal domain of FAK as described of fusion was at a of high homology between Pyk2 and FAK to structural of the chimeric protein To regulation of the chimeric protein FAK, and Pyk2 were transfected CHO cells. the cells were with calcium ionophore or with were prepared from these cells and by monoclonal antibody 12CA5 that the present in these of the using that similar of FAK, Pyk2, and were in the transfected CHO cells with showed a high of tyrosine phosphorylation of transfected FAK in the cells as well as the cells In a of tyrosine phosphorylation was in the transfected Pyk2 in the cells and a was in cells with or These results are similar to that for endogenous FAK and Pyk2 in RASM cells suggesting that transfection in CHO cells a for analyzing these two kinases. the tyrosine phosphorylation of the chimeric protein that of high phosphorylation in the cells and a with or These results that the C-terminal domain of FAK was responsible for high of phosphorylation in cells. To the regulation of by we analyzed the phosphorylation of the transfected FAK, Pyk2, and in CHO cells that been on or FN adhesion to FN to a of Pyk2 phosphorylation with Pyk2 phosphorylation in cells on PLL, and of cells on FN in a in Pyk2 phosphorylation In plating cells on FN increased tyrosine phosphorylation of FAK and to the and was by and In addition, a of FAK and phosphorylation was in cells on PLL, which was the of to the high by transfection in CHO cells. or was for Pyk2 a high the regulation of that of FAK, but not Pyk2 in transfected CHO cells. To determine the C-terminal domain of FAK of by it to focal we subcellular localization of with that of Pyk2 and FAK in transfected CHO cells the transfected FAK and were with in focal contacts as by using polyclonal anti-FAK and and mouse monoclonal anti-vinculin antibody In Pyk2 was present in the transfected cells in a throughout the as by antibody which is from the in focal contacts To the that different of FAK and Pyk2 were to the of different antibodies Pyk2 antibody was against the C-terminal of Pyk2 and not the chimeric protein we the transfected cells with the antibody which the epitope on transfected the focal contact localization of FAK and as well as the cytoplasmic of Pyk2 these data demonstrate that the C-terminal domain of Pyk2 with that of FAK results in a chimeric protein which FAK in both regulation of tyrosine phosphorylation and subcellular localization. that the C-terminal domains of Pyk2 and FAK are share 40% C-terminal domain of FAK has been shown to with several localized in focal contacts paxillin, and H.-C. Appeddu P.A. Parsons J.T. Hildebrand J.D. Schaller M.D. Guan J.-L. J. Biol. Chem. 1995; 270: 16995-16999Abstract Full Text Full Text PDF PubMed Scopus (327) Google Hanks S.K. S. A. 1995; PubMed Scopus Google Scholar, Hildebrand J.D. Parsons J.T. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. 1994; PubMed Google Scholar, M.D. Parsons J.T. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar), which be responsible for the of FAK to focal To the the different localization of Pyk2 and FAK, we the in of Pyk2 and FAK with and in RASM cells were prepared from RASM cells, by antibodies as and then with anti-talin or anti-paxillin was in with anti-FAK as reported previously in cells H.-C. Appeddu P.A. Parsons J.T. Hildebrand J.D. Schaller M.D. Guan J.-L. J. Biol. Chem. 1995; 270: 16995-16999Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar). it was not present in with antibody or serum with results J. J. 1994; PubMed Google Scholar, M.D. Parsons J.T. Mol. Cell. Biol. 1995; PubMed Scopus Google Scholar, D. Avraham H. Fu Y. London R. Avraham S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, R. Avraham S. E. Li J.-L. Raja S. M. Avraham H. J.D. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, X. Earp H.S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), was by both anti-FAK and antibodies were with CHO cells transfected with an Pyk2, FAK, or using antibody to the transfected was with FAK and but not with Pyk2 or from the cells In was with with antibody similar of Pyk2, FAK, and in the transfected cells These results correlated FAK, Pyk2 and binding to talin, but not paxillin, with their regulation by integrins and focal contact localization. studies have that regulation of Pyk2 tyrosine phosphorylation by cell adhesion is cell and Pyk2 has been shown to an integrin-dependent phosphorylation in B lymphocytes, cells, and transfected COS cells (7Li J. Avraham H. Rogers R.A. Raja S. Avraham S. Blood. 1996; 88: 417-428Crossref PubMed Google Scholar, 8Astier A. Avraham H. Manie S.N. Groopman J. Canty T. Avraham S. Freedman A.S. J. Biol. Chem. 1997; 272: 228-232Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). In Pyk2 phosphorylation has been found to be of cell adhesion in transfected 3Y1 cells or (2Sasaki H. Nagura K. Ishino M. Tobioka H. Kotani K. Sasaki T. J. Biol. Chem. 1995; 270: 21206-21219Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, S. Avraham S. Avraham H. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). It is not however, whether induction of Pyk2 phosphorylation by integrins is as effective as that by soluble factors or how it compares with FAK phosphorylation in cell adhesion in the same cells. In this report, we investigated these questions using both RASM cells endogenous Pyk2 and FAK, and transfected CHO cells. We showed that tyrosine phosphorylation of FAK is by integrin-mediated cell adhesion in both cell as Schaller M.D. Biol. 1995; PubMed Scopus Google Scholar). In a in Pyk2 tyrosine phosphorylation is upon cell adhesion in these cells. in Pyk2 tyrosine phosphorylation is obtained the cells are with soluble factors as angiotensin II, PDGF, sorbitol, and calcium ionophore A23187, these in FAK These results demonstrate that Pyk2 and FAK are differentially regulated by cell adhesion and soluble factors in these cell types. by which cell adhesion differentially Pyk2 and FAK is at it has been that of FAK with integrins and cytoskeletal in focal contacts may be responsible for FAK activation and by integrins in cell adhesion Schaller M.D. Biol. 1995; PubMed Scopus Google Scholar, J.-L. Biol. 1997; PubMed Scopus Google Scholar). we have that, in to the localization of FAK in focal Pyk2 is diffusely in the in both RASM cells and the transfected CHO cells. the of of Pyk2 by cell adhesion be by the of focal contact localization in these cells. studies in several cell have also demonstrated a between integrin-dependent Pyk2 phosphorylation and localization in focal (2Sasaki H. Nagura K. Ishino M. Tobioka H. Kotani K. Sasaki T. J. Biol. Chem. 1995; 270: 21206-21219Abstract Full Text Full Text PDF PubMed Scopus (364) Google Scholar, J. Avraham H. Rogers R.A. Raja S. Avraham S. Blood. 1996; 88: 417-428Crossref PubMed Google Scholar). Finally, this is by of a chimeric protein containing the N-terminal and kinase domains of Pyk2 and C of FAK, which is localized in the focal contacts and by cell adhesion to the same as FAK. of FAK with the cytoplasmic domain or cytoskeletal as and have been to focal contact localization and activation of FAK Schaller M.D. Biol. 1995; PubMed Scopus Google Scholar, J.-L. Biol. 1997; PubMed Scopus Google Scholar). Recent studies have an of Pyk2 with D. Avraham H. Fu Y. London R. Avraham S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar, R. Avraham S. E. Li J.-L. Raja S. M. Avraham H. J.D. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, X. Earp H.S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar), suggesting that binding to is to account for the differential regulation of FAK and Pyk2 with we have found that was with FAK, Pyk2, and the chimeric protein in both RASM cells and transfected CHO cells. In we have found an association of with FAK, but not Pyk2, in these cells. In addition, the chimeric protein which FAK in of regulation and was also found to with as as FAK in transfected CHO cells. association with may be responsible for the differential regulation and localization of Pyk2 and FAK in these cells, we the that binding to the cytoplasmic domain or to as are also In to activation by Pyk2 has been shown to be activated by a variety of soluble factors that elevate intracellular calcium (1Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1248) Google Scholar, 4Yu H. Li X. Marchetto G.S. Dy R. Hunter D. Calvo B. Dawson T.L. Wilm M. Anderegg R.J. Graves L.M. Earp H.S. J. Biol. Chem. 1996; 271: 29993-29998Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar, G. Dikic I. Lev S. Schlessinger J. Science. 1996; 273: 792-794Crossref PubMed Scopus (285) Google Scholar, I. Tokiwa G. Lev S. Courtneidge S. Schlessinger J. Nature. 1996; 383: 547-549Crossref PubMed Scopus (877) Google Scholar). results presented indicate that in with these soluble cell adhesion by integrins is a of Pyk2 in both RASM cells and transfected CHO cells. in Pyk2 phosphorylation upon cell adhesion be to of focal contact localization of Pyk2 that are the of the used in these We have localization of Pyk2 in focal contacts in a of cells not which to the in Pyk2 we not in Pyk2 localization in focal with or angiotensin not Pyk2 may be regulated by intracellular calcium and protein kinase as in studies (1Lev S. Moreno H. Martinez R. Canoll P. Peles E. Musacchio J.M. Plowman G.D. Rudy B. Schlessinger J. Nature. 1995; 376: 737-745Crossref PubMed Scopus (1248) Google Scholar, I. Tokiwa G. Lev S. Courtneidge S. Schlessinger J. Nature. 1996; 383: 547-549Crossref PubMed Scopus (877) Google Scholar, X. H. Graves L.M. Earp H.S. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). has been shown to calcium in cell Schaller M.D. Biol. 1995; PubMed Scopus Google Scholar, S. J. Biol. 1993; PubMed Scopus Google Scholar). It is that integrin-mediated cell adhesion intracellular calcium may be with by the soluble factors to Pyk2 to in these cells. It be interesting to compare the of calcium in to cell adhesion and to the soluble factors used in these cells. from these studies of Pyk2 and FAK may also have for of FAK regulation by the calcium upon cell adhesion, to a in FAK phosphorylation which may in focal contacts in an in FAK activation is with reports a role for calcium and protein kinase C in FAK activation Mol. Biol. Cell. PubMed Scopus Google Scholar, S. B. M. Parsons J.T. J. Biol. Chem. 1994; Full Text PDF PubMed Google Scholar, K. E. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar), as well as with the of focal contact localization in the regulation as of FAK by protein kinase C and by kinases has been A. Parsons J.T. 1995; PubMed Scopus Google Scholar, A. J.D. Schaller M.D. Parsons J.T. Biochem. J. 1997; PubMed Scopus Google Scholar), their role in FAK regulation the high of sequence homology between Pyk2 and FAK, it is somewhat to that these kinases are differentially of a chimeric protein that the C-terminal domains of the kinases are responsible for these regulation their 40% Although we a role for the N-terminal and kinase domains in the regulation of Pyk2 and FAK, it is that of the in the C-terminal domains are for the differential focal contact and regulation as described We have previously reported that of the focal adhesion sequence of FAK binding to H.-C. Appeddu P.A. Parsons J.T. Hildebrand J.D. Schaller M.D. Guan J.-L. J. Biol. Chem. 1995; 270: 16995-16999Abstract Full Text Full Text PDF PubMed Scopus (327) Google Scholar), suggesting that the different residues this may be studies are to residues that are responsible for differential regulation of Pyk2 and FAK. We Li of Inc. for the used in this We are to and for their of the and