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. 1995 Sep 1;130(5):1181–1187. doi: 10.1083/jcb.130.5.1181

Focal adhesion kinase and paxillin bind to peptides mimicking beta integrin cytoplasmic domains

PMCID: PMC2120552  PMID: 7657702

Abstract

The integrins have recently been implicated in signal transduction. A likely mediator of integrin signaling is focal adhesion kinase (pp125FAK or FAK), a structurally distinct protein tyrosine kinase that becomes enzymatically activated upon engagement of integrins with their ligands. A second candidate signaling molecule is paxillin, a focal adhesion associated, cytoskeletal protein that coordinately becomes phosphorylated on tyrosine upon activation of pp125FAK. Paxillin physically complexes with two protein tyrosine kinases, pp60src and Csk (COOH-terminal src kinase), and the oncoprotein p47gag-crk, each of which could function as part of a paxillin signaling complex. Using an in vitro assay we have established that the cytoplasmic domain of the beta 1 integrin can bind to paxillin and pp125FAK from chicken embryo cell lysates. The NH2-terminal, noncatalytic domain of pp125FAK can bind directly to the cytoplasmic tail of beta 1 and recognizes integrin sequences distinct from those involved in binding to alpha-actinin. Paxillin binding is independent of pp125FAK binding despite the fact that both bind to the same region of beta 1. These results demonstrate that the cytoplasmic domain of the beta subunits of integrins contain binding sites for both signaling molecules and structural proteins suggesting that integrins can coordinate the generation of cytoplasmic signals in addition to their role in anchoring components of the cytoskeleton.

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Selected References

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  1. Akiyama S. K., Yamada S. S., Yamada K. M., LaFlamme S. E. Transmembrane signal transduction by integrin cytoplasmic domains expressed in single-subunit chimeras. J Biol Chem. 1994 Jun 10;269(23):15961–15964. [PubMed] [Google Scholar]
  2. Albelda S. M., Buck C. A. Integrins and other cell adhesion molecules. FASEB J. 1990 Aug;4(11):2868–2880. [PubMed] [Google Scholar]
  3. Balzac F., Retta S. F., Albini A., Melchiorri A., Koteliansky V. E., Geuna M., Silengo L., Tarone G. Expression of beta 1B integrin isoform in CHO cells results in a dominant negative effect on cell adhesion and motility. J Cell Biol. 1994 Oct;127(2):557–565. doi: 10.1083/jcb.127.2.557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Belkin A. M., Koteliansky V. E. Interaction of iodinated vinculin, metavinculin and alpha-actinin with cytoskeletal proteins. FEBS Lett. 1987 Aug 17;220(2):291–294. doi: 10.1016/0014-5793(87)80832-3. [DOI] [PubMed] [Google Scholar]
  5. Bennett J. P., Zaner K. S., Stossel T. P. Isolation and some properties of macrophage alpha-actinin: evidence that it is not an actin gelling protein. Biochemistry. 1984 Oct 9;23(21):5081–5086. doi: 10.1021/bi00316a039. [DOI] [PubMed] [Google Scholar]
  6. Birge R. B., Fajardo J. E., Reichman C., Shoelson S. E., Songyang Z., Cantley L. C., Hanafusa H. Identification and characterization of a high-affinity interaction between v-Crk and tyrosine-phosphorylated paxillin in CT10-transformed fibroblasts. Mol Cell Biol. 1993 Aug;13(8):4648–4656. doi: 10.1128/mcb.13.8.4648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Burridge K., Fath K., Kelly T., Nuckolls G., Turner C. Focal adhesions: transmembrane junctions between the extracellular matrix and the cytoskeleton. Annu Rev Cell Biol. 1988;4:487–525. doi: 10.1146/annurev.cb.04.110188.002415. [DOI] [PubMed] [Google Scholar]
  8. Burridge K., Mangeat P. An interaction between vinculin and talin. Nature. 1984 Apr 19;308(5961):744–746. doi: 10.1038/308744a0. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Cobb B. S., Schaller M. D., Leu T. H., Parsons J. T. Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK. Mol Cell Biol. 1994 Jan;14(1):147–155. doi: 10.1128/mcb.14.1.147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Crawford A. W., Michelsen J. W., Beckerle M. C. An interaction between zyxin and alpha-actinin. J Cell Biol. 1992 Mar;116(6):1381–1393. doi: 10.1083/jcb.116.6.1381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Eck M. J., Atwell S. K., Shoelson S. E., Harrison S. C. Structure of the regulatory domains of the Src-family tyrosine kinase Lck. Nature. 1994 Apr 21;368(6473):764–769. doi: 10.1038/368764a0. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Grinblat Y., Zusman S., Yee G., Hynes R. O., Kafatos F. C. Functions of the cytoplasmic domain of the beta PS integrin subunit during Drosophila development. Development. 1994 Jan;120(1):91–102. doi: 10.1242/dev.120.1.91. [DOI] [PubMed] [Google Scholar]
  15. 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]
  16. 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]
  17. 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]
  18. 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]
  19. Hildebrand J. D., Schaller M. D., Parsons J. T. Paxillin, a tyrosine phosphorylated focal adhesion-associated protein binds to the carboxyl terminal domain of focal adhesion kinase. Mol Biol Cell. 1995 Jun;6(6):637–647. doi: 10.1091/mbc.6.6.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Horwitz A., Duggan K., Buck C., Beckerle M. C., Burridge K. Interaction of plasma membrane fibronectin receptor with talin--a transmembrane linkage. Nature. 1986 Apr 10;320(6062):531–533. doi: 10.1038/320531a0. [DOI] [PubMed] [Google Scholar]
  21. Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. doi: 10.1016/0092-8674(92)90115-s. [DOI] [PubMed] [Google Scholar]
  22. Ihle J. N., Witthuhn B. A., Quelle F. W., Yamamoto K., Thierfelder W. E., Kreider B., Silvennoinen O. Signaling by the cytokine receptor superfamily: JAKs and STATs. Trends Biochem Sci. 1994 May;19(5):222–227. doi: 10.1016/0968-0004(94)90026-4. [DOI] [PubMed] [Google Scholar]
  23. Jaconi M. E., Theler J. M., Schlegel W., Appel R. D., Wright S. D., Lew P. D. Multiple elevations of cytosolic-free Ca2+ in human neutrophils: initiation by adherence receptors of the integrin family. J Cell Biol. 1991 Mar;112(6):1249–1257. doi: 10.1083/jcb.112.6.1249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. Kaelin W. G., Jr, Krek W., Sellers W. R., DeCaprio J. A., Ajchenbaum F., Fuchs C. S., Chittenden T., Li Y., Farnham P. J., Blanar M. A. Expression cloning of a cDNA encoding a retinoblastoma-binding protein with E2F-like properties. Cell. 1992 Jul 24;70(2):351–364. doi: 10.1016/0092-8674(92)90108-o. [DOI] [PubMed] [Google Scholar]
  26. Kanner S. B., Reynolds A. B., Parsons J. T. Immunoaffinity purification of tyrosine-phosphorylated cellular proteins. J Immunol Methods. 1989 Jun 2;120(1):115–124. doi: 10.1016/0022-1759(89)90296-2. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. 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]
  29. 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]
  30. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  31. 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]
  32. Lo S. H., Janmey P. A., Hartwig J. H., Chen L. B. Interactions of tensin with actin and identification of its three distinct actin-binding domains. J Cell Biol. 1994 Jun;125(5):1067–1075. doi: 10.1083/jcb.125.5.1067. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Miyamoto S., Akiyama S. K., Yamada K. M. Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function. Science. 1995 Feb 10;267(5199):883–885. doi: 10.1126/science.7846531. [DOI] [PubMed] [Google Scholar]
  34. Otey C. A., Pavalko F. M., Burridge K. An interaction between alpha-actinin and the beta 1 integrin subunit in vitro. J Cell Biol. 1990 Aug;111(2):721–729. doi: 10.1083/jcb.111.2.721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Otey C. A., Vasquez G. B., Burridge K., Erickson B. W. Mapping of the alpha-actinin binding site within the beta 1 integrin cytoplasmic domain. J Biol Chem. 1993 Oct 5;268(28):21193–21197. [PubMed] [Google Scholar]
  36. Pelletier A. J., Bodary S. C., Levinson A. D. Signal transduction by the platelet integrin alpha IIb beta 3: induction of calcium oscillations required for protein-tyrosine phosphorylation and ligand-induced spreading of stably transfected cells. Mol Biol Cell. 1992 Sep;3(9):989–998. doi: 10.1091/mbc.3.9.989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Reszka A. A., Hayashi Y., Horwitz A. F. Identification of amino acid sequences in the integrin beta 1 cytoplasmic domain implicated in cytoskeletal association. J Cell Biol. 1992 Jun;117(6):1321–1330. doi: 10.1083/jcb.117.6.1321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Richter J., Ng-Sikorski J., Olsson I., Andersson T. Tumor necrosis factor-induced degranulation in adherent human neutrophils is dependent on CD11b/CD18-integrin-triggered oscillations of cytosolic free Ca2+. Proc Natl Acad Sci U S A. 1990 Dec;87(23):9472–9476. doi: 10.1073/pnas.87.23.9472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Sabe H., Hata A., Okada M., Nakagawa H., Hanafusa H. Analysis of the binding of the Src homology 2 domain of Csk to tyrosine-phosphorylated proteins in the suppression and mitotic activation of c-Src. Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3984–3988. doi: 10.1073/pnas.91.9.3984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Sastry S. K., Horwitz A. F. Integrin cytoplasmic domains: mediators of cytoskeletal linkages and extra- and intracellular initiated transmembrane signaling. Curr Opin Cell Biol. 1993 Oct;5(5):819–831. doi: 10.1016/0955-0674(93)90031-k. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. 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]
  43. Schaller M. D., Hildebrand J. D., Shannon J. D., Fox J. W., Vines R. R., Parsons J. T. Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src. Mol Cell Biol. 1994 Mar;14(3):1680–1688. doi: 10.1128/mcb.14.3.1680. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Schaller M. D., Parsons J. T. pp125FAK-dependent tyrosine phosphorylation of paxillin creates a high-affinity binding site for Crk. Mol Cell Biol. 1995 May;15(5):2635–2645. doi: 10.1128/mcb.15.5.2635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Schwartz M. A., Ingber D. E., Lawrence M., Springer T. A., Lechene C. Multiple integrins share the ability to induce elevation of intracellular pH. Exp Cell Res. 1991 Aug;195(2):533–535. doi: 10.1016/0014-4827(91)90407-l. [DOI] [PubMed] [Google Scholar]
  46. Schwartz M. A., Lechene C., Ingber D. E. Insoluble fibronectin activates the Na/H antiporter by clustering and immobilizing integrin alpha 5 beta 1, independent of cell shape. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7849–7853. doi: 10.1073/pnas.88.17.7849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Schwartz M. A. Spreading of human endothelial cells on fibronectin or vitronectin triggers elevation of intracellular free calcium. J Cell Biol. 1993 Feb;120(4):1003–1010. doi: 10.1083/jcb.120.4.1003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Schwartz M. A. Transmembrane signalling by integrins. Trends Cell Biol. 1992 Oct;2(10):304–308. doi: 10.1016/0962-8924(92)90120-c. [DOI] [PubMed] [Google Scholar]
  49. Sefton B. M. The lck tyrosine protein kinase. Oncogene. 1991 May;6(5):683–686. [PubMed] [Google Scholar]
  50. 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]
  51. Stuchbury T., Shipton M., Norris R., Malthouse J. P., Brocklehurst K., Herbert J. A., Suschitzky H. A reporter group delivery system with both absolute and selective specificity for thiol groups and an improved fluorescent probe containing the 7-nitrobenzo-2-oxa-1,3-diazole moiety. Biochem J. 1975 Nov;151(2):417–432. doi: 10.1042/bj1510417. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Tapley P., Horwitz A., Buck C., Duggan K., Rohrschneider L. Integrins isolated from Rous sarcoma virus-transformed chicken embryo fibroblasts. Oncogene. 1989 Mar;4(3):325–333. [PubMed] [Google Scholar]
  53. Turner C. E., Burridge K. Transmembrane molecular assemblies in cell-extracellular matrix interactions. Curr Opin Cell Biol. 1991 Oct;3(5):849–853. doi: 10.1016/0955-0674(91)90059-8. [DOI] [PubMed] [Google Scholar]
  54. Turner C. E., Glenney J. R., Jr, Burridge K. Paxillin: a new vinculin-binding protein present in focal adhesions. J Cell Biol. 1990 Sep;111(3):1059–1068. doi: 10.1083/jcb.111.3.1059. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Ullrich A., Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990 Apr 20;61(2):203–212. doi: 10.1016/0092-8674(90)90801-k. [DOI] [PubMed] [Google Scholar]
  56. Wachsstock D. H., Wilkins J. A., Lin S. Specific interaction of vinculin with alpha-actinin. Biochem Biophys Res Commun. 1987 Jul 31;146(2):554–560. doi: 10.1016/0006-291x(87)90564-x. [DOI] [PubMed] [Google Scholar]
  57. Weng Z., Taylor J. A., Turner C. E., Brugge J. S., Seidel-Dugan C. Detection of Src homology 3-binding proteins, including paxillin, in normal and v-Src-transformed Balb/c 3T3 cells. J Biol Chem. 1993 Jul 15;268(20):14956–14963. [PubMed] [Google Scholar]
  58. Ziemiecki A., Harpur A. G., Wilks A. F. JAK protein tyrosine kinases: their role in cytokine signalling. Trends Cell Biol. 1994 Jun;4(6):207–212. doi: 10.1016/0962-8924(94)90143-0. [DOI] [PubMed] [Google Scholar]

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