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. 2001 Nov 15;360(Pt 1):57–66. doi: 10.1042/0264-6021:3600057

Multiple stimuli induce tyrosine phosphorylation of the Crk-binding sites of paxillin.

M D Schaller 1, E M Schaefer 1
PMCID: PMC1222202  PMID: 11695992

Abstract

Paxillin is a focal-adhesion-associated, tyrosine-phosphorylated protein. In cells transformed by the src, crk or BCR-Abl oncogenes, the phosphotyrosine content of paxillin is elevated. In normal cells paxillin functions in signalling following integrin-dependent cell adhesion or exposure to a number of stimuli, including growth factors and neuropeptides. These stimuli induce tyrosine phosphorylation of paxillin, regulating the association of Src homology 2 domain-containing signalling molecules with paxillin. There are multiple sites of tyrosine phosphorylation on paxillin. To elucidate the role of paxillin in transducing signals in response to various stimuli, it is essential to identify all of the sites of phosphorylation on paxillin and to define which residues are phosphorylated in response to distinct stimuli. We describe two new sites of tyrosine phosphorylation on paxillin, residues at positions 40 and 88. Using paxillin variants with phenylalanine substitutions at phosphorylation sites and phospho-specific paxillin antibodies, tyrosine phosphorylation of paxillin in response to distinct stimuli was examined. The results demonstrate that Tyr(31) and Tyr(118), which are binding sites for Crk, are major sites of tyrosine phosphorylation following cell adhesion or stimulation with platelet-derived growth factor or angiotensin II. Thus multiple stimuli may elicit similar signalling events downstream of paxillin.

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Selected References

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  1. Abedi H., Dawes K. E., Zachary I. Differential effects of platelet-derived growth factor BB on p125 focal adhesion kinase and paxillin tyrosine phosphorylation and on cell migration in rabbit aortic vascular smooth muscle cells and Swiss 3T3 fibroblasts. J Biol Chem. 1995 May 12;270(19):11367–11376. doi: 10.1074/jbc.270.19.11367. [DOI] [PubMed] [Google Scholar]
  2. Bellis S. L., Miller J. T., Turner C. E. Characterization of tyrosine phosphorylation of paxillin in vitro by focal adhesion kinase. J Biol Chem. 1995 Jul 21;270(29):17437–17441. doi: 10.1074/jbc.270.29.17437. [DOI] [PubMed] [Google Scholar]
  3. Bellis S. L., Perrotta J. A., Curtis M. S., Turner C. E. Adhesion of fibroblasts to fibronectin stimulates both serine and tyrosine phosphorylation of paxillin. Biochem J. 1997 Jul 15;325(Pt 2):375–381. doi: 10.1042/bj3250375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bergman M., Joukov V., Virtanen I., Alitalo K. Overexpressed Csk tyrosine kinase is localized in focal adhesions, causes reorganization of alpha v beta 5 integrin, and interferes with HeLa cell spreading. Mol Cell Biol. 1995 Feb;15(2):711–722. doi: 10.1128/mcb.15.2.711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Brinson A. E., Harding T., Diliberto P. A., He Y., Li X., Hunter D., Herman B., Earp H. S., Graves L. M. Regulation of a calcium-dependent tyrosine kinase in vascular smooth muscle cells by angiotensin II and platelet-derived growth factor. Dependence on calcium and the actin cytoskeleton. J Biol Chem. 1998 Jan 16;273(3):1711–1718. doi: 10.1074/jbc.273.3.1711. [DOI] [PubMed] [Google Scholar]
  7. Brown M. C., Curtis M. S., Turner C. E. Paxillin LD motifs may define a new family of protein recognition domains. Nat Struct Biol. 1998 Aug;5(8):677–678. doi: 10.1038/1370. [DOI] [PubMed] [Google Scholar]
  8. Brown M. C., Perrotta J. A., Turner C. E. Identification of LIM3 as the principal determinant of paxillin focal adhesion localization and characterization of a novel motif on paxillin directing vinculin and focal adhesion kinase binding. J Cell Biol. 1996 Nov;135(4):1109–1123. doi: 10.1083/jcb.135.4.1109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Brown M. C., Perrotta J. A., Turner C. E. Serine and threonine phosphorylation of the paxillin LIM domains regulates paxillin focal adhesion localization and cell adhesion to fibronectin. Mol Biol Cell. 1998 Jul;9(7):1803–1816. doi: 10.1091/mbc.9.7.1803. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dawid I. B., Breen J. J., Toyama R. LIM domains: multiple roles as adapters and functional modifiers in protein interactions. Trends Genet. 1998 Apr;14(4):156–162. doi: 10.1016/s0168-9525(98)01424-3. [DOI] [PubMed] [Google Scholar]
  11. De Nichilo M. O., Yamada K. M. Integrin alpha v beta 5-dependent serine phosphorylation of paxillin in cultured human macrophages adherent to vitronectin. J Biol Chem. 1996 May 3;271(18):11016–11022. doi: 10.1074/jbc.271.18.11016. [DOI] [PubMed] [Google Scholar]
  12. Feller S. M., Knudsen B., Hanafusa H. c-Abl kinase regulates the protein binding activity of c-Crk. EMBO J. 1994 May 15;13(10):2341–2351. doi: 10.1002/j.1460-2075.1994.tb06518.x. [DOI] [PMC free article] [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. 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]
  15. Li X., Earp H. S. Paxillin is tyrosine-phosphorylated by and preferentially associates with the calcium-dependent tyrosine kinase in rat liver epithelial cells. J Biol Chem. 1997 May 30;272(22):14341–14348. doi: 10.1074/jbc.272.22.14341. [DOI] [PubMed] [Google Scholar]
  16. Liu S., Thomas S. M., Woodside D. G., Rose D. M., Kiosses W. B., Pfaff M., Ginsberg M. H. Binding of paxillin to alpha4 integrins modifies integrin-dependent biological responses. Nature. 1999 Dec 9;402(6762):676–681. doi: 10.1038/45264. [DOI] [PubMed] [Google Scholar]
  17. Nakamura K., Yano H., Uchida H., Hashimoto S., Schaefer E., Sabe H. Tyrosine phosphorylation of paxillin alpha is involved in temporospatial regulation of paxillin-containing focal adhesion formation and F-actin organization in motile cells. J Biol Chem. 2000 Sep 1;275(35):27155–27164. doi: 10.1074/jbc.M000679200. [DOI] [PubMed] [Google Scholar]
  18. Petit V., Boyer B., Lentz D., Turner C. E., Thiery J. P., Vallés A. M. Phosphorylation of tyrosine residues 31 and 118 on paxillin regulates cell migration through an association with CRK in NBT-II cells. J Cell Biol. 2000 Mar 6;148(5):957–970. doi: 10.1083/jcb.148.5.957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Richardson A., Malik R. K., Hildebrand J. D., Parsons J. T. Inhibition of cell spreading by expression of the C-terminal domain of focal adhesion kinase (FAK) is rescued by coexpression of Src or catalytically inactive FAK: a role for paxillin tyrosine phosphorylation. Mol Cell Biol. 1997 Dec;17(12):6906–6914. doi: 10.1128/mcb.17.12.6906. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Richardson A., Parsons T. A mechanism for regulation of the adhesion-associated proteintyrosine kinase pp125FAK. Nature. 1996 Apr 11;380(6574):538–540. doi: 10.1038/380538a0. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Salgia R., Li J. L., Lo S. H., Brunkhorst B., Kansas G. S., Sobhany E. S., Sun Y., Pisick E., Hallek M., Ernst T. Molecular cloning of human paxillin, a focal adhesion protein phosphorylated by P210BCR/ABL. J Biol Chem. 1995 Mar 10;270(10):5039–5047. doi: 10.1074/jbc.270.10.5039. [DOI] [PubMed] [Google Scholar]
  24. Salgia R., Uemura N., Okuda K., Li J. L., Pisick E., Sattler M., de Jong R., Druker B., Heisterkamp N., Chen L. B. CRKL links p210BCR/ABL with paxillin in chronic myelogenous leukemia cells. J Biol Chem. 1995 Dec 8;270(49):29145–29150. doi: 10.1074/jbc.270.49.29145. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Schaller M. D., Sasaki T. Differential signaling by the focal adhesion kinase and cell adhesion kinase beta. J Biol Chem. 1997 Oct 3;272(40):25319–25325. doi: 10.1074/jbc.272.40.25319. [DOI] [PubMed] [Google Scholar]
  27. Schlaepfer D. D., Hauck C. R., Sieg D. J. Signaling through focal adhesion kinase. Prog Biophys Mol Biol. 1999;71(3-4):435–478. doi: 10.1016/s0079-6107(98)00052-2. [DOI] [PubMed] [Google Scholar]
  28. Shen Y., Schaller M. D. Focal adhesion targeting: the critical determinant of FAK regulation and substrate phosphorylation. Mol Biol Cell. 1999 Aug;10(8):2507–2518. doi: 10.1091/mbc.10.8.2507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Thomas J. W., Cooley M. A., Broome J. M., Salgia R., Griffin J. D., Lombardo C. R., Schaller M. D. The role of focal adhesion kinase binding in the regulation of tyrosine phosphorylation of paxillin. J Biol Chem. 1999 Dec 17;274(51):36684–36692. doi: 10.1074/jbc.274.51.36684. [DOI] [PubMed] [Google Scholar]
  31. Turner C. E., Brown M. C., Perrotta J. A., Riedy M. C., Nikolopoulos S. N., McDonald A. R., Bagrodia S., Thomas S., Leventhal P. S. Paxillin LD4 motif binds PAK and PIX through a novel 95-kD ankyrin repeat, ARF-GAP protein: A role in cytoskeletal remodeling. J Cell Biol. 1999 May 17;145(4):851–863. doi: 10.1083/jcb.145.4.851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Turner C. E., Miller J. T. Primary sequence of paxillin contains putative SH2 and SH3 domain binding motifs and multiple LIM domains: identification of a vinculin and pp125Fak-binding region. J Cell Sci. 1994 Jun;107(Pt 6):1583–1591. doi: 10.1242/jcs.107.6.1583. [DOI] [PubMed] [Google Scholar]
  33. Turner C. E. Paxillin. Int J Biochem Cell Biol. 1998 Sep;30(9):955–959. doi: 10.1016/s1357-2725(98)00062-4. [DOI] [PubMed] [Google Scholar]
  34. Turner C. E. Paxillin: a cytoskeletal target for tyrosine kinases. Bioessays. 1994 Jan;16(1):47–52. doi: 10.1002/bies.950160107. [DOI] [PubMed] [Google Scholar]
  35. 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]
  36. Yano H., Uchida H., Iwasaki T., Mukai M., Akedo H., Nakamura K., Hashimoto S., Sabe H. Paxillin alpha and Crk-associated substrate exert opposing effects on cell migration and contact inhibition of growth through tyrosine phosphorylation. Proc Natl Acad Sci U S A. 2000 Aug 1;97(16):9076–9081. doi: 10.1073/pnas.97.16.9076. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]

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