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. 1996 Dec;7(12):1865–1875. doi: 10.1091/mbc.7.12.1865

Dissociation of mitogen-activated protein kinase activation from p125 focal adhesion kinase tyrosine phosphorylation in Swiss 3T3 cells stimulated by bombesin, lysophosphatidic acid, and platelet-derived growth factor.

T Seufferlein 1, D J Withers 1, D Mann 1, E Rozengurt 1
PMCID: PMC276036  PMID: 8970151

Abstract

The experiments presented here were designed to examine the contribution of p125 focal adhesion kinase (p125FAK) tyrosine phosphorylation to the activation of the mitogen-activated protein kinase cascade induced by bombesin, lysophosphatidic acid (LPA), and platelet-derived growth factor (PDGF) in Swiss 3T3 cells. We found that tyrosine phosphorylation of p125FAK in response to these growth factors is completely abolished in cells treated with cytochalasin D or in cells that were suspended in serum-free medium for 30 min. In marked contrast, the activation of p42mapk by these factors was independent of the integrity of the actin cytoskeleton and of the interaction of the cells with the extracellular matrix. The protein kinase C inhibitor GF 109203X and down-regulation of protein kinase C by prolonged pretreatment of cells with phorbol esters blocked bombesin-stimulated activation of p42mapk, p90rsk, and MAPK kinase-1 but did not prevent bombesin-induced tyrosine phosphorylation of p125FAK. Furthermore, LPA-induced p42mapk activation involved a pertussis toxin-sensitive guanylate nucleotide-binding protein, whereas tyrosine phosphorylation of p125FAK in response to LPA was not prevented by pretreatment with pertussis toxin. Finally, PDGF induced maximum p42mapk activation at concentrations (30 ng/ml) that failed to induce tyrosine phosphorylation of p125FAK. Thus, our results demonstrate that p42mapk activation in response to bombesin, LPA, and PDGF can be dissociated from p125FAK tyrosine phosphorylation in Swiss 3T3 cells.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Alessi D. R., Saito Y., Campbell D. G., Cohen P., Sithanandam G., Rapp U., Ashworth A., Marshall C. J., Cowley S. Identification of the sites in MAP kinase kinase-1 phosphorylated by p74raf-1. EMBO J. 1994 Apr 1;13(7):1610–1619. doi: 10.1002/j.1460-2075.1994.tb06424.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Blenis J. Signal transduction via the MAP kinases: proceed at your own RSK. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):5889–5892. doi: 10.1073/pnas.90.13.5889. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Burridge K., Turner C. E., Romer L. H. Tyrosine phosphorylation of paxillin and pp125FAK accompanies cell adhesion to extracellular matrix: a role in cytoskeletal assembly. J Cell Biol. 1992 Nov;119(4):893–903. doi: 10.1083/jcb.119.4.893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Böhmer R. M., Scharf E., Assoian R. K. Cytoskeletal integrity is required throughout the mitogen stimulation phase of the cell cycle and mediates the anchorage-dependent expression of cyclin D1. Mol Biol Cell. 1996 Jan;7(1):101–111. doi: 10.1091/mbc.7.1.101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chen Q., Kinch M. S., Lin T. H., Burridge K., Juliano R. L. Integrin-mediated cell adhesion activates mitogen-activated protein kinases. J Biol Chem. 1994 Oct 28;269(43):26602–26605. [PubMed] [Google Scholar]
  6. Claesson-Welsh L. Platelet-derived growth factor receptor signals. J Biol Chem. 1994 Dec 23;269(51):32023–32026. [PubMed] [Google Scholar]
  7. Crespo P., Xu N., Simonds W. F., Gutkind J. S. Ras-dependent activation of MAP kinase pathway mediated by G-protein beta gamma subunits. Nature. 1994 Jun 2;369(6479):418–420. doi: 10.1038/369418a0. [DOI] [PubMed] [Google Scholar]
  8. Crews C. M., Alessandrini A., Erikson R. L. The primary structure of MEK, a protein kinase that phosphorylates the ERK gene product. Science. 1992 Oct 16;258(5081):478–480. doi: 10.1126/science.1411546. [DOI] [PubMed] [Google Scholar]
  9. Davis R. J. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem. 1993 Jul 15;268(20):14553–14556. [PubMed] [Google Scholar]
  10. Fang F., Orend G., Watanabe N., Hunter T., Ruoslahti E. Dependence of cyclin E-CDK2 kinase activity on cell anchorage. Science. 1996 Jan 26;271(5248):499–502. doi: 10.1126/science.271.5248.499. [DOI] [PubMed] [Google Scholar]
  11. Guadagno T. M., Assoian R. K. G1/S control of anchorage-independent growth in the fibroblast cell cycle. J Cell Biol. 1991 Dec;115(5):1419–1425. doi: 10.1083/jcb.115.5.1419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guadagno T. M., Ohtsubo M., Roberts J. M., Assoian R. K. A link between cyclin A expression and adhesion-dependent cell cycle progression. Science. 1993 Dec 3;262(5139):1572–1575. doi: 10.1126/science.8248807. [DOI] [PubMed] [Google Scholar]
  13. Guan J. L., Shalloway D. Regulation of focal adhesion-associated protein tyrosine kinase by both cellular adhesion and oncogenic transformation. Nature. 1992 Aug 20;358(6388):690–692. doi: 10.1038/358690a0. [DOI] [PubMed] [Google Scholar]
  14. Hanks S. K., Calalb M. B., Harper M. C., Patel S. K. Focal adhesion protein-tyrosine kinase phosphorylated in response to cell attachment to fibronectin. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8487–8491. doi: 10.1073/pnas.89.18.8487. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hawes B. E., van Biesen T., Koch W. J., Luttrell L. M., Lefkowitz R. J. Distinct pathways of Gi- and Gq-mediated mitogen-activated protein kinase activation. J Biol Chem. 1995 Jul 21;270(29):17148–17153. doi: 10.1074/jbc.270.29.17148. [DOI] [PubMed] [Google Scholar]
  16. Heldin C. H. Structural and functional studies on platelet-derived growth factor. EMBO J. 1992 Dec;11(12):4251–4259. doi: 10.1002/j.1460-2075.1992.tb05523.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hordijk P. L., Verlaan I., van Corven E. J., Moolenaar W. H. Protein tyrosine phosphorylation induced by lysophosphatidic acid in Rat-1 fibroblasts. Evidence that phosphorylation of map kinase is mediated by the Gi-p21ras pathway. J Biol Chem. 1994 Jan 7;269(1):645–651. [PubMed] [Google Scholar]
  18. Hotchin N. A., Hall A. The assembly of integrin adhesion complexes requires both extracellular matrix and intracellular rho/rac GTPases. J Cell Biol. 1995 Dec;131(6 Pt 2):1857–1865. doi: 10.1083/jcb.131.6.1857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Howe L. R., Marshall C. J. Lysophosphatidic acid stimulates mitogen-activated protein kinase activation via a G-protein-coupled pathway requiring p21ras and p74raf-1. J Biol Chem. 1993 Oct 5;268(28):20717–20720. [PubMed] [Google Scholar]
  20. Kornberg L., Earp H. S., Parsons J. T., Schaller M., Juliano R. L. Cell adhesion or integrin clustering increases phosphorylation of a focal adhesion-associated tyrosine kinase. J Biol Chem. 1992 Nov 25;267(33):23439–23442. [PubMed] [Google Scholar]
  21. Kumagai N., Morii N., Fujisawa K., Yoshimasa T., Nakao K., Narumiya S. Lysophosphatidic acid induces tyrosine phosphorylation and activation of MAP-kinase and focal adhesion kinase in cultured Swiss 3T3 cells. FEBS Lett. 1993 Aug 30;329(3):273–276. doi: 10.1016/0014-5793(93)80236-n. [DOI] [PubMed] [Google Scholar]
  22. Leevers S. J., Marshall C. J. Activation of extracellular signal-regulated kinase, ERK2, by p21ras oncoprotein. EMBO J. 1992 Feb;11(2):569–574. doi: 10.1002/j.1460-2075.1992.tb05088.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Marshall C. J. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell. 1995 Jan 27;80(2):179–185. doi: 10.1016/0092-8674(95)90401-8. [DOI] [PubMed] [Google Scholar]
  24. Meloche S., Seuwen K., Pagès G., Pouysségur J. Biphasic and synergistic activation of p44mapk (ERK1) by growth factors: correlation between late phase activation and mitogenicity. Mol Endocrinol. 1992 May;6(5):845–854. doi: 10.1210/mend.6.5.1603090. [DOI] [PubMed] [Google Scholar]
  25. Mitchell F. M., Heasley L. E., Qian N. X., Zamarripa J., Johnson G. L. Differential modulation of bombesin-stimulated phospholipase C beta and mitogen-activated protein kinase activity by [D-Arg1,D-Phe5,D-Trp7,9,Leu11]substance P. J Biol Chem. 1995 Apr 14;270(15):8623–8628. doi: 10.1074/jbc.270.15.8623. [DOI] [PubMed] [Google Scholar]
  26. Morino N., Mimura T., Hamasaki K., Tobe K., Ueki K., Kikuchi K., Takehara K., Kadowaki T., Yazaki Y., Nojima Y. Matrix/integrin interaction activates the mitogen-activated protein kinase, p44erk-1 and p42erk-2. J Biol Chem. 1995 Jan 6;270(1):269–273. doi: 10.1074/jbc.270.1.269. [DOI] [PubMed] [Google Scholar]
  27. Pagès G., Lenormand P., L'Allemain G., Chambard J. C., Meloche S., Pouysségur J. Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8319–8323. doi: 10.1073/pnas.90.18.8319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Payne D. M., Rossomando A. J., Martino P., Erickson A. K., Her J. H., Shabanowitz J., Hunt D. F., Weber M. J., Sturgill T. W. Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). EMBO J. 1991 Apr;10(4):885–892. doi: 10.1002/j.1460-2075.1991.tb08021.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rankin S., Rozengurt E. Platelet-derived growth factor 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. 1994 Jan 7;269(1):704–710. [PubMed] [Google Scholar]
  30. Schaller M. D., Borgman C. A., Cobb B. S., Vines R. R., Reynolds A. B., Parsons J. T. pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5192–5196. doi: 10.1073/pnas.89.11.5192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schlaepfer D. D., Hanks S. K., Hunter T., van der Geer P. Integrin-mediated signal transduction linked to Ras pathway by GRB2 binding to focal adhesion kinase. Nature. 1994 Dec 22;372(6508):786–791. doi: 10.1038/372786a0. [DOI] [PubMed] [Google Scholar]
  32. 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. 1994 Mar 25;269(12):9345–9351. [PubMed] [Google Scholar]
  33. Seufferlein T., Rozengurt E. Sphingosine induces p125FAK and paxillin tyrosine phosphorylation, actin stress fiber formation, and focal contact assembly in Swiss 3T3 cells. J Biol Chem. 1994 Nov 4;269(44):27610–27617. [PubMed] [Google Scholar]
  34. Seufferlein T., Rozengurt E. Sphingosylphosphorylcholine activation of mitogen-activated protein kinase in Swiss 3T3 cells requires protein kinase C and a pertussis toxin-sensitive G protein. J Biol Chem. 1995 Oct 13;270(41):24334–24342. doi: 10.1074/jbc.270.41.24334. [DOI] [PubMed] [Google Scholar]
  35. Seufferlein T., Rozengurt E. Sphingosylphosphorylcholine rapidly induces tyrosine phosphorylation of p125FAK and paxillin, rearrangement of the actin cytoskeleton and focal contact assembly. Requirement of p21rho in the signaling pathway. J Biol Chem. 1995 Oct 13;270(41):24343–24351. doi: 10.1074/jbc.270.41.24343. [DOI] [PubMed] [Google Scholar]
  36. Seufferlein T., Withers D. J., Broad S., Herget T., Walsh J. H., Rozengurt E. The human CCKB/gastrin receptor transfected into rat1 fibroblasts mediates activation of MAP kinase, p74raf-1 kinase, and mitogenesis. Cell Growth Differ. 1995 Apr;6(4):383–393. [PubMed] [Google Scholar]
  37. Seufferlein T., Withers D. J., Rozengurt E. Reduced requirement of mitogen-activated protein kinase (MAPK) activity for entry into the S phase of the cell cycle in Swiss 3T3 fibroblasts stimulated by bombesin and insulin. J Biol Chem. 1996 Aug 30;271(35):21471–21477. doi: 10.1074/jbc.271.35.21471. [DOI] [PubMed] [Google Scholar]
  38. Sherr C. J. Mammalian G1 cyclins. Cell. 1993 Jun 18;73(6):1059–1065. doi: 10.1016/0092-8674(93)90636-5. [DOI] [PubMed] [Google Scholar]
  39. Sherr C. J., Roberts J. M. Inhibitors of mammalian G1 cyclin-dependent kinases. Genes Dev. 1995 May 15;9(10):1149–1163. doi: 10.1101/gad.9.10.1149. [DOI] [PubMed] [Google Scholar]
  40. Sinnett-Smith J., Zachary I., Valverde A. M., Rozengurt E. Bombesin stimulation of p125 focal adhesion kinase tyrosine phosphorylation. Role of protein kinase C, Ca2+ mobilization, and the actin cytoskeleton. J Biol Chem. 1993 Jul 5;268(19):14261–14268. [PubMed] [Google Scholar]
  41. Sturgill T. W., Wu J. Recent progress in characterization of protein kinase cascades for phosphorylation of ribosomal protein S6. Biochim Biophys Acta. 1991 May 17;1092(3):350–357. doi: 10.1016/s0167-4889(97)90012-4. [DOI] [PubMed] [Google Scholar]
  42. Touhara K., Hawes B. E., van Biesen T., Lefkowitz R. J. G protein beta gamma subunits stimulate phosphorylation of Shc adapter protein. Proc Natl Acad Sci U S A. 1995 Sep 26;92(20):9284–9287. doi: 10.1073/pnas.92.20.9284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Withers D. J., Bloom S. R., Rozengurt E. Dissociation of cAMP-stimulated mitogenesis from activation of the mitogen-activated protein kinase cascade in Swiss 3T3 cells. J Biol Chem. 1995 Sep 8;270(36):21411–21419. doi: 10.1074/jbc.270.36.21411. [DOI] [PubMed] [Google Scholar]
  44. Wu J., Harrison J. K., Vincent L. A., Haystead C., Haystead T. A., Michel H., Hunt D. F., Lynch K. R., Sturgill T. W. Molecular structure of a protein-tyrosine/threonine kinase activating p42 mitogen-activated protein (MAP) kinase: MAP kinase kinase. Proc Natl Acad Sci U S A. 1993 Jan 1;90(1):173–177. doi: 10.1073/pnas.90.1.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zachary I., Rozengurt E. Focal adhesion kinase (p125FAK): a point of convergence in the action of neuropeptides, integrins, and oncogenes. Cell. 1992 Dec 11;71(6):891–894. doi: 10.1016/0092-8674(92)90385-p. [DOI] [PubMed] [Google Scholar]
  46. Zachary I., Sinnett-Smith J., Rozengurt E. Bombesin, vasopressin, and endothelin stimulation of tyrosine phosphorylation in Swiss 3T3 cells. Identification of a novel tyrosine kinase as a major substrate. J Biol Chem. 1992 Sep 25;267(27):19031–19034. [PubMed] [Google Scholar]
  47. Zachary I., Sinnett-Smith J., Turner C. E., Rozengurt E. Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation of the focal adhesion-associated protein paxillin in Swiss 3T3 cells. J Biol Chem. 1993 Oct 15;268(29):22060–22065. [PubMed] [Google Scholar]
  48. Zhu X., Assoian R. K. Integrin-dependent activation of MAP kinase: a link to shape-dependent cell proliferation. Mol Biol Cell. 1995 Mar;6(3):273–282. doi: 10.1091/mbc.6.3.273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. van Biesen T., Hawes B. E., Raymond J. R., Luttrell L. M., Koch W. J., Lefkowitz R. J. G(o)-protein alpha-subunits activate mitogen-activated protein kinase via a novel protein kinase C-dependent mechanism. J Biol Chem. 1996 Jan 19;271(3):1266–1269. doi: 10.1074/jbc.271.3.1266. [DOI] [PubMed] [Google Scholar]
  50. van Corven E. J., Hordijk P. L., Medema R. H., Bos J. L., Moolenaar W. H. Pertussis toxin-sensitive activation of p21ras by G protein-coupled receptor agonists in fibroblasts. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1257–1261. doi: 10.1073/pnas.90.4.1257. [DOI] [PMC free article] [PubMed] [Google Scholar]

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