Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1994 Jan;14(1):147–155. doi: 10.1128/mcb.14.1.147

Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK.

B S Cobb 1, M D Schaller 1, T H Leu 1, J T Parsons 1
PMCID: PMC358365  PMID: 7505391

Abstract

Changes in cellular growth and dramatic alterations in cell morphology and adhesion are common features of cells transformed by oncogenic protein tyrosine kinases, such as pp60src and other members of the Src family. In this report, we present evidence for the stable association of two Src family kinases (pp60src and pp59fyn) with tyrosine-phosphorylated forms of a focal adhesion-associated protein tyrosine kinase, pp125FAK. In Src-transformed chicken embryo cells, most of the pp125FAK was stably complexed with activated pp60src (e.g., pp60(527F). The stable association of pp125FAK with pp60(527F) in vivo required the structural integrity of the Src SH2 domain. The association of pp60(527F) and pp125FAK could be reconstituted in vitro by incubation of normal cell extracts with glutathione S-transferase fusion proteins containing SH2 or SH3/SH2 domains of pp60src. Furthermore, the association of isolated SH2 or SH3/SH2 domains with in vitro 32P-labeled pp125FAK protected the major site of pp125FAK autophosphorylation from digestion with a tyrosine phosphatase, indicating that the autophosphorylation site of pp125FAK participates in binding with Src. Immunoprecipitation of Src family kinases from extracts of normal chicken embryo cells revealed stable complexes of pp59fyn and tyrosine-phosphorylated pp125FAK. These data provide evidence for a direct interaction between two cytoplasmic nonreceptor protein tyrosine kinases and suggest that Src may contribute to changes in pp125FAK regulation in transformed cells. Furthermore, pp125FAK may directly participate in the targeting of pp59fyn or possibly other Src family kinases to focal adhesions in normal cells.

Full text

PDF
147

Images in this article

149Image
on p.149
150Image
on p.150
151Image
on p.151
151Image
on p.151
152Image
on p.152
152Image
on p.152
153Image
on p.153
153Image
on p.153

Selected References

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

  1. Bryant D. L., Parsons J. T. Amino acid alterations within a highly conserved region of the Rous sarcoma virus src gene product pp60src inactivate tyrosine protein kinase activity. Mol Cell Biol. 1984 May;4(5):862–866. doi: 10.1128/mcb.4.5.862. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Cartwright C. A., Eckhart W., Simon S., Kaplan P. L. Cell transformation by pp60c-src mutated in the carboxy-terminal regulatory domain. Cell. 1987 Apr 10;49(1):83–91. doi: 10.1016/0092-8674(87)90758-6. [DOI] [PubMed] [Google Scholar]
  4. Cobb B. S., Payne D. M., Reynolds A. B., Parsons J. T. Regulation of the oncogenic activity of the cellular src protein requires the correct spacing between the kinase domain and the C-terminal phosphorylated tyrosine (Tyr-527). Mol Cell Biol. 1991 Dec;11(12):5832–5838. doi: 10.1128/mcb.11.12.5832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cooper J. A., Gould K. L., Cartwright C. A., Hunter T. Tyr527 is phosphorylated in pp60c-src: implications for regulation. Science. 1986 Mar 21;231(4744):1431–1434. doi: 10.1126/science.2420005. [DOI] [PubMed] [Google Scholar]
  6. Cooper J. A., King C. S. Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src. Mol Cell Biol. 1986 Dec;6(12):4467–4477. doi: 10.1128/mcb.6.12.4467. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Courtneidge S. A. Activation of the pp60c-src kinase by middle T antigen binding or by dephosphorylation. EMBO J. 1985 Jun;4(6):1471–1477. doi: 10.1002/j.1460-2075.1985.tb03805.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Damsky C. H., Werb Z. Signal transduction by integrin receptors for extracellular matrix: cooperative processing of extracellular information. Curr Opin Cell Biol. 1992 Oct;4(5):772–781. doi: 10.1016/0955-0674(92)90100-q. [DOI] [PubMed] [Google Scholar]
  9. Davis S., Lu M. L., Lo S. H., Lin S., Butler J. A., Druker B. J., Roberts T. M., An Q., Chen L. B. Presence of an SH2 domain in the actin-binding protein tensin. Science. 1991 May 3;252(5006):712–715. doi: 10.1126/science.1708917. [DOI] [PubMed] [Google Scholar]
  10. DeClue J. E., Martin G. S. Phosphorylation of talin at tyrosine in Rous sarcoma virus-transformed cells. Mol Cell Biol. 1987 Jan;7(1):371–378. doi: 10.1128/mcb.7.1.371. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Glenney J. R., Jr, Zokas L. Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton. J Cell Biol. 1989 Jun;108(6):2401–2408. doi: 10.1083/jcb.108.6.2401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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]
  13. Guan J. L., Trevithick J. E., Hynes R. O. Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein. Cell Regul. 1991 Nov;2(11):951–964. doi: 10.1091/mbc.2.11.951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hildebrand J. D., Schaller M. D., Parsons J. T. Identification of sequences required for the efficient localization of the focal adhesion kinase, pp125FAK, to cellular focal adhesions. J Cell Biol. 1993 Nov;123(4):993–1005. doi: 10.1083/jcb.123.4.993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hirst R., Horwitz A., Buck C., Rohrschneider L. Phosphorylation of the fibronectin receptor complex in cells transformed by oncogenes that encode tyrosine kinases. Proc Natl Acad Sci U S A. 1986 Sep;83(17):6470–6474. doi: 10.1073/pnas.83.17.6470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hughes S. H., Greenhouse J. J., Petropoulos C. J., Sutrave P. Adaptor plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors. J Virol. 1987 Oct;61(10):3004–3012. doi: 10.1128/jvi.61.10.3004-3012.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jove R., Hanafusa H. Cell transformation by the viral src oncogene. Annu Rev Cell Biol. 1987;3:31–56. doi: 10.1146/annurev.cb.03.110187.000335. [DOI] [PubMed] [Google Scholar]
  18. Juliano R. L., Haskill S. Signal transduction from the extracellular matrix. J Cell Biol. 1993 Feb;120(3):577–585. doi: 10.1083/jcb.120.3.577. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kanner S. B., Reynolds A. B., Vines R. R., Parsons J. T. Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases. Proc Natl Acad Sci U S A. 1990 May;87(9):3328–3332. doi: 10.1073/pnas.87.9.3328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kanner S. B., Reynolds A. B., Wang H. C., Vines R. R., Parsons J. T. The SH2 and SH3 domains of pp60src direct stable association with tyrosine phosphorylated proteins p130 and p110. EMBO J. 1991 Jul;10(7):1689–1698. doi: 10.1002/j.1460-2075.1991.tb07693.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kmiecik T. E., Shalloway D. Activation and suppression of pp60c-src transforming ability by mutation of its primary sites of tyrosine phosphorylation. Cell. 1987 Apr 10;49(1):65–73. doi: 10.1016/0092-8674(87)90756-2. [DOI] [PubMed] [Google Scholar]
  22. Koch C. A., Anderson D., Moran M. F., Ellis C., Pawson T. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science. 1991 May 3;252(5006):668–674. doi: 10.1126/science.1708916. [DOI] [PubMed] [Google Scholar]
  23. Kornberg L. J., Earp H. S., Turner C. E., Prockop C., Juliano R. L. Signal transduction by integrins: increased protein tyrosine phosphorylation caused by clustering of beta 1 integrins. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8392–8396. doi: 10.1073/pnas.88.19.8392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Lipfert L., Haimovich B., Schaller M. D., Cobb B. S., Parsons J. T., Brugge J. S. Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets. J Cell Biol. 1992 Nov;119(4):905–912. doi: 10.1083/jcb.119.4.905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Parsons J. T., Weber M. J. Genetics of src: structure and functional organization of a protein tyrosine kinase. Curr Top Microbiol Immunol. 1989;147:79–127. doi: 10.1007/978-3-642-74697-0_3. [DOI] [PubMed] [Google Scholar]
  27. Parsons S. J., McCarley D. J., Ely C. M., Benjamin D. C., Parsons J. T. Monoclonal antibodies to Rous sarcoma virus pp60src react with enzymatically active cellular pp60src of avian and mammalian origin. J Virol. 1984 Aug;51(2):272–282. doi: 10.1128/jvi.51.2.272-282.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Parsons S. J., McCarley D. J., Raymond V. W., Parsons J. T. Localization of conserved and nonconserved epitopes within the Rous sarcoma virus-encoded src protein. J Virol. 1986 Sep;59(3):755–758. doi: 10.1128/jvi.59.3.755-758.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pasquale E. B., Maher P. A., Singer S. J. Talin is phosphorylated on tyrosine in chicken embryo fibroblasts transformed by Rous sarcoma virus. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5507–5511. doi: 10.1073/pnas.83.15.5507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Pawson T., Gish G. D. SH2 and SH3 domains: from structure to function. Cell. 1992 Oct 30;71(3):359–362. doi: 10.1016/0092-8674(92)90504-6. [DOI] [PubMed] [Google Scholar]
  31. Piwnica-Worms H., Saunders K. B., Roberts T. M., Smith A. E., Cheng S. H. Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-src. Cell. 1987 Apr 10;49(1):75–82. doi: 10.1016/0092-8674(87)90757-4. [DOI] [PubMed] [Google Scholar]
  32. Reynolds A. B., Herbert L., Cleveland J. L., Berg S. T., Gaut J. R. p120, a novel substrate of protein tyrosine kinase receptors and of p60v-src, is related to cadherin-binding factors beta-catenin, plakoglobin and armadillo. Oncogene. 1992 Dec;7(12):2439–2445. [PubMed] [Google Scholar]
  33. Reynolds A. B., Kanner S. B., Wang H. C., Parsons J. T. Stable association of activated pp60src with two tyrosine-phosphorylated cellular proteins. Mol Cell Biol. 1989 Sep;9(9):3951–3958. doi: 10.1128/mcb.9.9.3951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Reynolds A. B., Roesel D. J., Kanner S. B., Parsons J. T. Transformation-specific tyrosine phosphorylation of a novel cellular protein in chicken cells expressing oncogenic variants of the avian cellular src gene. Mol Cell Biol. 1989 Feb;9(2):629–638. doi: 10.1128/mcb.9.2.629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Reynolds A. B., Vila J., Lansing T. J., Potts W. M., Weber M. J., Parsons J. T. Activation of the oncogenic potential of the avian cellular src protein by specific structural alteration of the carboxy terminus. EMBO J. 1987 Aug;6(8):2359–2364. doi: 10.1002/j.1460-2075.1987.tb02512.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Roussel R. R., Brodeur S. R., Shalloway D., Laudano A. P. Selective binding of activated pp60c-src by an immobilized synthetic phosphopeptide modeled on the carboxyl terminus of pp60c-src. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10696–10700. doi: 10.1073/pnas.88.23.10696. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. Schaller M. D., Borgman C. A., Parsons J. T. Autonomous expression of a noncatalytic domain of the focal adhesion-associated protein tyrosine kinase pp125FAK. Mol Cell Biol. 1993 Feb;13(2):785–791. doi: 10.1128/mcb.13.2.785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schaller M. D., Parsons J. T. Focal adhesion kinase: an integrin-linked protein tyrosine kinase. Trends Cell Biol. 1993 Aug;3(8):258–262. doi: 10.1016/0962-8924(93)90053-4. [DOI] [PubMed] [Google Scholar]
  40. Sefton B. M., Hunter T., Ball E. H., Singer S. J. Vinculin: a cytoskeletal target of the transforming protein of Rous sarcoma virus. Cell. 1981 Apr;24(1):165–174. doi: 10.1016/0092-8674(81)90512-2. [DOI] [PubMed] [Google Scholar]
  41. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  42. Songyang Z., Shoelson S. E., Chaudhuri M., Gish G., Pawson T., Haser W. G., King F., Roberts T., Ratnofsky S., Lechleider R. J. SH2 domains recognize specific phosphopeptide sequences. Cell. 1993 Mar 12;72(5):767–778. doi: 10.1016/0092-8674(93)90404-e. [DOI] [PubMed] [Google Scholar]
  43. Turner C. E. Paxillin is a major phosphotyrosine-containing protein during embryonic development. J Cell Biol. 1991 Oct;115(1):201–207. doi: 10.1083/jcb.115.1.201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Wu H., Reynolds A. B., Kanner S. B., Vines R. R., Parsons J. T. Identification and characterization of a novel cytoskeleton-associated pp60src substrate. Mol Cell Biol. 1991 Oct;11(10):5113–5124. doi: 10.1128/mcb.11.10.5113. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES