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Protein kinase C-mediated tyrosine phosphorylation of paxillin and focal adhesion kinase requires cytoskeletal integrity and is uncoupled to mitogen-activated protein kinase activation in human hepatoma cells

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Journal of Biomedical Science

Abstract

Treatment of cultured human hepatoma HepG2 cells with the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), results in an increase in tyrosine phosphorylation of several proteins, including the focal adhesion kinase (FAK) and paxillin using anti-phosphotyrosine Western blotting and immunoprecipitation. However, when cells are in suspension or in the presence of cytochalasin D which disrupts the intracellular network of actin microfilaments, TPA loses its ability to stimulate tyrosine phosphorylation of FAK and paxillin but it still activates mitogen-activated protein kinase (MAPK) and induces PKC translocation from cytosol to the membrane in HepG2 cells. On the other hand, PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, blocks TPA-induced MAPK activation but has no effect on TPA-induced tyrosine phosphorylation. Our findings suggest that TPA-induced tyrosine phosphorylation of FAK and paxillin in human hepatoma cells is PKC dependent and requires the integrity of the cell cytoskeleton but is uncoupled to the signal transduction pathway of PKC leading to the translocation of PKC and MAPK activation.

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References

  1. Ahn NG, Seger R. The mitogen-activated protein kinase activator. Curr Opin Cell Biol 4:992–999;1992.

    Google Scholar 

  2. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254;1976.

    Google Scholar 

  3. Burridge K, Turner CE, Romer LH. Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: A role in cytoskeletal assembly. J Cell Biol 119:893–903;1992.

    Google Scholar 

  4. Calb MB, Polte TR, Hanks SK. Tyrosine phosphorylation of focal adhesion kinase at sites in the catalytic domain regulates kinase activity: A role for src family kinases. Mol Cell Biol 15:954–963;1995.

    Google Scholar 

  5. Chin JE, Dickens M, Tavare JM, Roth RA. Overexpression of protein kinase C isoenzymes alpha, beta I, gamma, and epsilon in cells over-expressing the insulin receptor. Effects on receptor phosphorylation and signaling. J Biol Chem 268:6338–6347;1993.

    Google Scholar 

  6. Clark EA, Brugge JS. Integrins and signal transduction pathways: The road taken. Science 268:233–239;1995.

    Google Scholar 

  7. Cooper DR, Konda TS, Standaert ML, Davis JS, Pollet RJ, Farese RV. Insulin increases membrane and cytosolic protein kinase C activity in BC3H-1 myocytes. J Biol Chem 262:3633–3639;1987.

    Google Scholar 

  8. Emkey R, Kahn CR. Cross-talk between phorbol ester-mediated signaling and tyrosine kinase proto-oncogenes. II. Comparison of phorbol ester and sphingomyelinase-induced phosphorylation of ErbB2 and ErbB3. J Biol Chem 272:31182–31189;1997.

    Google Scholar 

  9. Emkey R, Kahn CR. Cross-talk between phorbol ester-mediated signaling and tyrosine kinase proto-oncogenes. I. Activation of protein kinase C stimulates tyrosine phosphorylation and activation of ErbB2 and ErbB3. J Biol Chem 272:31172–31181;1997.

    Google Scholar 

  10. Guan JL, Shalloway D. Regulation of focal adhesion-associated protein tyrosine kinase by both cellular adhesion and oncogenic transformation. Nature 358:690–692;1992.

    Google Scholar 

  11. Guan JL. Role of focal adhesion kinase in integrin signaling. Int J Biochem Cell Biol 29:1085–1096;1997.

    Google Scholar 

  12. Hartwig JH, Kung S, Kovacsovics T, Janmey PA, Cantley LC, Stossel TP, Toker A. D3 phosphoinositides and outside-in integrin signaling by glycoprotein IIb-IIIa mediate platelet actin assembly and filopodial extension induced by phorbol 12-myristate 13-acetate. J Biol Chem 271:32986–32993;1996.

    Google Scholar 

  13. Hildebrand JD, Schaller MD, Parsons JT. Identification of sequences required for the efficient localization of the focal adhesion kinase, p125FAK, to cellular focal adhesions. J Cell Biol 123:993–1005;1993.

    Google Scholar 

  14. Jaken S. Increased diacylglycerol content with phospholipase C or hormone treatment: Inhibition of phorbol ester binding and induction of phorbol ester-like biological responses. Endocrinology 117:2301–2306;1985.

    Google Scholar 

  15. Kanner SB, Reynolds AB, Vines RR, Parsons JT. Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases. Proc Natl Acad Sci USA 87:3328–3332;1990.

    Google Scholar 

  16. Kiley SC, Parker PJ, Fabbro D, Jaken S. Differential regulation of protein kinase C isozymes by thyrotropin-releasing hormone in GH4C1 cells. J Biol Chem 266:23761–2376;1991.

    Google Scholar 

  17. Kim B, Feldman EL. Differential regulation of focal adhesion kinase and mitogen-activated protein kinase tyrosine phosphorylation during insulin-like growth factor-I-mediated cytoskeletal reorganization. J Neurochem 71:1333–1336;1998.

    Google Scholar 

  18. Knight JB, Yamauchi K, Pessin JE. Divergent insulin and platelet-derived growth factor regulation of focal adhesion kinase (pp125FAK) tyrosine phosphorylation, and rearrangement of actin stress fibers. J Biol Chem 270:10199–10203;1995.

    Google Scholar 

  19. Kolch W, Heidecker G, Kochs G, Humml R, Vahidi H, Mischak H, Finkenzeller G, Marme D, Rapp UR. Protein kinase C alpha activates RAF-1 by direct phosphorylation. Nature 364:249–252;1993.

    Google Scholar 

  20. Kornberg L, Earp HS, Parsons JT, Schaller M, Juliano RL. Cell adhesion or integrin clustering increases phosphorylation of a focal adhesion-associated tyrosine kinase. J Biol Chem 267:23439–23442;1992.

    Google Scholar 

  21. Lacerda HM, Lax AJ, Rozengurt E. Pasteurella nultocida toxin, a potent intracellularly acting mitogen, induces p125FAK and paxillin tyrosine phosphorylation, actin stress fiber formation, and focal contact assembly in Swiss 3T3 cells. J Biol Chem 271:439–445;1996.

    Google Scholar 

  22. Leeb Lundberg LM, Song XH, Mathis SA. Focal adhesion-associated protein's p125FAK and paxillin are substrates for bradykinin-stimulated tyrosine phosphorylation in Swiss 3T3 cells. J Biol Chem 269:24328–24334;1994.

    Google Scholar 

  23. Leventhal PS, Feldman EL. Tyrosine phosphorylation and enhanced expression of paxillin during neuronal differentiation in vitro. J Biol Chem 271:5957–5960;1996.

    Google Scholar 

  24. Lewis JM, Cherish DA, Schwartz MA. Protein kinase C regulates alpha beta 5-dependent cytoskeletal associations and focal adhesion kinase phosphorylation. J Cell Biol 134:1323–1332;1996.

    Google Scholar 

  25. Li DM, Li H. TEP1, encoded by a candidate tumor suppressor locus, is a novel protein tyrosine phosphatase regulated by transforming growth factor beta. Cancer Res 57:2124–2129;1997.

    Google Scholar 

  26. Li L, Ernsting BR, Wishart MJ, Lohse DL, Dixon JE. A family of putative tumor suppressors is structurally and functionally conserved in humans and yeast. J Biol Chem 272:29403–29406;1997.

    Google Scholar 

  27. Lipfert L, Haimovich B, Schaller MD, Cobb BS, Parsons JT, Brugge JS. Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets. J Cell Biol 119:905–912;1992.

    Google Scholar 

  28. Myers MP, Stolarov JP, Eng C, Li J, Wang SI, Wigler MH, Parsons R, Tonks NK. PTEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatase. Proc Natl Acad Sci USA 94:9302–9057;1997.

    Google Scholar 

  29. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 258:607–614;1992.

    Google Scholar 

  30. Nojima Y, Morino N, Mimura T, Hamasaki K, Furuya H, Sakai R, Sato T, Tachibana K, Morimoto C, Yazaki Y, Hirai H. Integrin-mediated cell adhesion promotes tyrosine phosphorylation of p130Cas, a src homology 3-containing molecule having multiple src homology 2-binding motifs. J Biol Chem 270:15398–15402;1995.

    Google Scholar 

  31. Owens LV, Xu LH, Craven RJ, Dent GA, Weiner TM, Kornberg L, Liu ET, Cance WG. Overexpression of the focal adhesion kinase (p125FAK) in invasive human tumors. Cancer Res 55:2752–2755;1995.

    Google Scholar 

  32. Petricoin E III, David M, Igarashi K, Benjamin C, Ling L, Goelz S, Finbloom DS, Larner AC. Inhibition of alpha interferon but not gamma interferon signal transduction by phorbol esters is mediated by a tyrosine phosphatase. Mol Cell Biol 16:1419–1424;1995.

    Google Scholar 

  33. Rankin S, Rozengurt E. Platelet-derived growth factors modulation of focal adhesion kinase (p125FAK) and paxillin tyrosine phosphorylation in Swiss 3T3 cells. Bell-shaped dose response and cross talk with bombesin. J Biol Chem 269:704–710;1994.

    Google Scholar 

  34. Ridley A, Hall JA. The small GTP-binding protein rho regulates the assembly of focal adhesions and actin stress fibers in response to growth factors. Cell 70:389–399;1992.

    Google Scholar 

  35. Rodriguez-Fernandez JL, Rodriguez-Fernandez E. Bombesin, vasopressin, lysophosphatidic acid, and sphingosylphosphorylcholine induce focal adhesion kinase activation in intact Swiss 3T3 cells. J Biol Chem 273:19321–19328;1998.

    Google Scholar 

  36. Schaller MD, Hilddebrand JD, Shannon JD, Fox JW, Vines RR, Parsons JT. Autophosphorylation of the focal adhesion kinase, ppFAK, directs SH2-dependent binding of pp60src. Mol Cell Biol 14:1680–1688;1994.

    Google Scholar 

  37. Seufferlein T, Rozengurt E. Lysophosphatidic acid stimulates tyrosine phosphorylation of focal adhesion kinase, paxillin, and p130. Signaling pathways and cross-talk with platelet-derived growth factor. J Biol Chem 269:9345–9351;1994.

    Google Scholar 

  38. Sinnett-Smith J, Zachary I, Valverde A, Rozengurt ME. Bombesin stimulation of p125 focal adhesion kinase tyrosine phosphorylation. J Biol Chem 268:14261–14268;1993.

    Google Scholar 

  39. Slack B. Tyrosine phosphorylation of paxillin and focal adhesion kinase by activation of muscarinic m3 receptors is dependent on integrin engagement by the extracellular matrix. Proc Natl Acad Sci USA 95:7281–7286;1998.

    Google Scholar 

  40. Tamura MT, Gu J, Matsumoto K, Aota S, Parsons R, Yamada KM. Inhibition of cell migration, spreading, and focal adhesions by tumor suppressor PTEN. Science 280:1614–1617;1998.

    Google Scholar 

  41. Turner CE, Glenny JR Jr, Burridge K. Paxillin: A new vinculin-binding protein present in focal adhesions. J Cell Biol 111:1059–1068;1990.

    Google Scholar 

  42. Turner CE, Miller JT. Primary sequence of paxillin contains putative SH2 and SH3 domain binding motifs and multiple LIM domains: Identification of vinculin and pp125Fak-binding region. J Cell Sci 107:1583–1591;1994.

    Google Scholar 

  43. Turner CE, Schaller MD, Parsons JT. Tyrosine phosphorylation of the focal adhesion kinase pp125FAK during development: Relation to paxillin. J Cell Sci 105:637–645;1993.

    Google Scholar 

  44. Vuori K, Ruoslahti E. Activation of protein kinase C precedes alpha 5 beta 1 integrinmediated cell spreading on fibronectin. J Biol Chem 268:21459–21462;1993.

    Google Scholar 

  45. Zachary I, Sinnett-Smith J, Turner CE, Rozengurt E. Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation of the focal adhesion-associated protein paxillin in Swiss 3T3 cells. J Biol Chem 268:22060–22065;1993.

    Google Scholar 

  46. Zhao Z, Shen SH, Fischer EH. Phorbol ester-induced expression, phosphorylation, and translocation of protein-tyrosine-phosphatase 1C in HL-60 cells. Proc Natl Acad Sci USA 91:5007–5011;1994.

    Google Scholar 

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Authors and Affiliations

  1. Institute of Biochemistry, National Yang-Ming University, Nan-Kan, Taipei, Taiwan, ROC

    Lan Chun Tu, Hua-Chien Chen & Sheau-Farn Yeh

  2. Department of Medical Research, Veterans General Hospital, Nan-Kan, Taipei, Taiwan, ROC

    Chen-Kung Chou

  3. Division of Molecular and Genomic Medicine, National Health Research Institutes, Nan-Kan, Taipei, Taiwan, ROC

    Chen-Kung Chou

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Tu, L.C., Chou, CK., Chen, HC. et al. Protein kinase C-mediated tyrosine phosphorylation of paxillin and focal adhesion kinase requires cytoskeletal integrity and is uncoupled to mitogen-activated protein kinase activation in human hepatoma cells. J Biomed Sci 8, 184–190 (2001). https://doi.org/10.1007/BF02256411

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