Search tips
Search criteria 


Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
Mol Cell Biol. 1995 May; 15(5): 2635–2645.
PMCID: PMC230493

pp125FAK-dependent tyrosine phosphorylation of paxillin creates a high-affinity binding site for Crk.


Paxillin, a focal-adhesion-associated protein, becomes phosphorylated in response to a number of stimuli which also induce the tyrosine phosphorylation of the focal-adhesion-associated protein tyrosine kinase pp125FAK. On the basis of their colocalization and coordinate phosphorylation, paxillin is a candidate for a substrate of pp125FAK. We describe here conditions under which the phosphorylation of paxillin on tyrosine is pp125FAK dependent, supporting the hypothesis that paxillin phosphorylation is regulated by pp125FAK. pp125FAK must localize to focal adhesions and become autophosphorylated to induce paxillin phosphorylation. Phosphorylation of paxillin on tyrosine creates binding sites for the SH2 domains of Crk, Csk, and Src. We identify two sites of phosphorylation as tyrosine residues 31 and 118, each of which conforms to the Crk SH2 domain binding motif, (P)YXXP. These observations suggest that paxillin serves as an adapter protein, similar to insulin receptor substrate 1, and that pp125FAK may regulate the formation of signaling complexes by directing the phosphorylation of paxillin on tyrosine.

Full Text

The Full Text of this article is available as a PDF (668K).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Albelda SM, Buck CA. Integrins and other cell adhesion molecules. FASEB J. 1990 Aug;4(11):2868–2880. [PubMed]
  • Batzer AG, Rotin D, Ureña JM, Skolnik EY, Schlessinger J. Hierarchy of binding sites for Grb2 and Shc on the epidermal growth factor receptor. Mol Cell Biol. 1994 Aug;14(8):5192–5201. [PMC free article] [PubMed]
  • Birge RB, Fajardo JE, Reichman C, Shoelson SE, Songyang Z, Cantley LC, 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. [PMC free article] [PubMed]
  • Boyle WJ, van der Geer P, Hunter T. Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol. 1991;201:110–149. [PubMed]
  • 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. [PubMed]
  • 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. 1992 Nov;119(4):893–903. [PMC free article] [PubMed]
  • Cobb BS, Schaller MD, Leu TH, Parsons JT. Stable association of pp60src and pp59fyn with the focal adhesion-associated protein tyrosine kinase, pp125FAK. Mol Cell Biol. 1994 Jan;14(1):147–155. [PMC free article] [PubMed]
  • Damsky CH, 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. [PubMed]
  • Davis S, Lu ML, Lo SH, Lin S, Butler JA, Druker BJ, Roberts TM, An Q, Chen LB. Presence of an SH2 domain in the actin-binding protein tensin. Science. 1991 May 3;252(5006):712–715. [PubMed]
  • De Bondt HL, Rosenblatt J, Jancarik J, Jones HD, Morgan DO, Kim SH. Crystal structure of cyclin-dependent kinase 2. Nature. 1993 Jun 17;363(6430):595–602. [PubMed]
  • Glenney JR, 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. [PMC free article] [PubMed]
  • Guan JL, 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. [PubMed]
  • Guan JL, Trevithick JE, Hynes RO. Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein. Cell Regul. 1991 Nov;2(11):951–964. [PMC free article] [PubMed]
  • Hanks SK, Calalb MB, Harper MC, Patel SK. 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. [PubMed]
  • Hildebrand JD, Schaller MD, Parsons JT. 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. [PMC free article] [PubMed]
  • Hughes SH, Greenhouse JJ, Petropoulos CJ, Sutrave P. Adaptor plasmids simplify the insertion of foreign DNA into helper-independent retroviral vectors. J Virol. 1987 Oct;61(10):3004–3012. [PMC free article] [PubMed]
  • Hynes RO. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. [PubMed]
  • Jaconi ME, Theler JM, Schlegel W, Appel RD, Wright SD, Lew PD. 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. [PMC free article] [PubMed]
  • Jelinek T, Weber MJ. Optimization of the resolution of phosphoamino acids by one-dimensional thin-layer electrophoresis. Biotechniques. 1993 Oct;15(4):628–630. [PubMed]
  • Juliano RL, Haskill S. Signal transduction from the extracellular matrix. J Cell Biol. 1993 Feb;120(3):577–585. [PMC free article] [PubMed]
  • Kanner SB, Reynolds AB, Parsons JT. Immunoaffinity purification of tyrosine-phosphorylated cellular proteins. J Immunol Methods. 1989 Jun 2;120(1):115–124. [PubMed]
  • 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 U S A. 1990 May;87(9):3328–3332. [PubMed]
  • Knighton DR, Zheng JH, Ten Eyck LF, Ashford VA, Xuong NH, Taylor SS, Sowadski JM. Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science. 1991 Jul 26;253(5018):407–414. [PubMed]
  • 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. 1992 Nov 25;267(33):23439–23442. [PubMed]
  • Kornberg LJ, Earp HS, Turner CE, Prockop C, Juliano RL. 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. [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • 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. 1992 Nov;119(4):905–912. [PMC free article] [PubMed]
  • Luo KX, Hurley TR, Sefton BM. Cyanogen bromide cleavage and proteolytic peptide mapping of proteins immobilized to membranes. Methods Enzymol. 1991;201:149–152. [PubMed]
  • Matsuda M, Hashimoto Y, Muroya K, Hasegawa H, Kurata T, Tanaka S, Nakamura S, Hattori S. CRK protein binds to two guanine nucleotide-releasing proteins for the Ras family and modulates nerve growth factor-induced activation of Ras in PC12 cells. Mol Cell Biol. 1994 Aug;14(8):5495–5500. [PMC free article] [PubMed]
  • Naldini L, Vigna E, Ferracini R, Longati P, Gandino L, Prat M, Comoglio PM. The tyrosine kinase encoded by the MET proto-oncogene is activated by autophosphorylation. Mol Cell Biol. 1991 Apr;11(4):1793–1803. [PMC free article] [PubMed]
  • Parsons SJ, McCarley DJ, Ely CM, Benjamin DC, Parsons JT. 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. [PMC free article] [PubMed]
  • Pawson T, Gish GD. SH2 and SH3 domains: from structure to function. Cell. 1992 Oct 30;71(3):359–362. [PubMed]
  • Pawson T, Schlessingert J. SH2 and SH3 domains. Curr Biol. 1993 Jul 1;3(7):434–442. [PubMed]
  • Pelletier AJ, Bodary SC, Levinson AD. 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. [PMC free article] [PubMed]
  • 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]
  • Reynolds AB, Roesel DJ, Kanner SB, Parsons JT. 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. [PMC free article] [PubMed]
  • 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. [PubMed]
  • Rosen OM, Herrera R, Olowe Y, Petruzzelli LM, Cobb MH. Phosphorylation activates the insulin receptor tyrosine protein kinase. Proc Natl Acad Sci U S A. 1983 Jun;80(11):3237–3240. [PubMed]
  • 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. [PubMed]
  • Salgia R, Li JL, Lo SH, Brunkhorst B, Kansas GS, Sobhany ES, Sun Y, Pisick E, Hallek M, Ernst T, et al. Molecular cloning of human paxillin, a focal adhesion protein phosphorylated by P210BCR/ABL. J Biol Chem. 1995 Mar 10;270(10):5039–5047. [PubMed]
  • Schaller MD, Borgman CA, Cobb BS, Vines RR, Reynolds AB, Parsons JT. 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. [PubMed]
  • Schaller MD, Borgman CA, Parsons JT. Autonomous expression of a noncatalytic domain of the focal adhesion-associated protein tyrosine kinase pp125FAK. Mol Cell Biol. 1993 Feb;13(2):785–791. [PMC free article] [PubMed]
  • Schaller MD, Hildebrand JD, Shannon JD, Fox JW, Vines RR, Parsons JT. Autophosphorylation of the focal adhesion kinase, pp125FAK, directs SH2-dependent binding of pp60src. Mol Cell Biol. 1994 Mar;14(3):1680–1688. [PMC free article] [PubMed]
  • Schaller MD, Parsons JT. Focal adhesion kinase and associated proteins. Curr Opin Cell Biol. 1994 Oct;6(5):705–710. [PubMed]
  • Schlessinger J, Ullrich A. Growth factor signaling by receptor tyrosine kinases. Neuron. 1992 Sep;9(3):383–391. [PubMed]
  • Schmeichel KL, Beckerle MC. The LIM domain is a modular protein-binding interface. Cell. 1994 Oct 21;79(2):211–219. [PubMed]
  • Schwartz MA. Transmembrane signalling by integrins. Trends Cell Biol. 1992 Oct;2(10):304–308. [PubMed]
  • Schwartz MA. Spreading of human endothelial cells on fibronectin or vitronectin triggers elevation of intracellular free calcium. J Cell Biol. 1993 Feb;120(4):1003–1010. [PMC free article] [PubMed]
  • Schwartz MA, Ingber DE, Lawrence M, Springer TA, Lechene C. Multiple integrins share the ability to induce elevation of intracellular pH. Exp Cell Res. 1991 Aug;195(2):533–535. [PubMed]
  • Schwartz MA, Lechene C, Ingber DE. 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. [PubMed]
  • 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]
  • Shattil SJ, Brugge JS. Protein tyrosine phosphorylation and the adhesive functions of platelets. Curr Opin Cell Biol. 1991 Oct;3(5):869–879. [PubMed]
  • Sinnett-Smith J, Zachary I, Valverde AM, 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]
  • Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. [PubMed]
  • Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG, King F, Roberts T, Ratnofsky S, Lechleider RJ, et al. SH2 domains recognize specific phosphopeptide sequences. Cell. 1993 Mar 12;72(5):767–778. [PubMed]
  • Songyang Z, Shoelson SE, McGlade J, Olivier P, Pawson T, Bustelo XR, Barbacid M, Sabe H, Hanafusa H, Yi T, et al. Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav. Mol Cell Biol. 1994 Apr;14(4):2777–2785. [PMC free article] [PubMed]
  • Tanaka S, Morishita T, Hashimoto Y, Hattori S, Nakamura S, Shibuya M, Matuoka K, Takenawa T, Kurata T, Nagashima K, et al. C3G, a guanine nucleotide-releasing protein expressed ubiquitously, binds to the Src homology 3 domains of CRK and GRB2/ASH proteins. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3443–3447. [PubMed]
  • ten Hoeve J, Morris C, Heisterkamp N, Groffen J. Isolation and chromosomal localization of CRKL, a human crk-like gene. Oncogene. 1993 Sep;8(9):2469–2474. [PubMed]
  • Turner CE. Paxillin is a major phosphotyrosine-containing protein during embryonic development. J Cell Biol. 1991 Oct;115(1):201–207. [PMC free article] [PubMed]
  • Turner CE, Burridge K. Transmembrane molecular assemblies in cell-extracellular matrix interactions. Curr Opin Cell Biol. 1991 Oct;3(5):849–853. [PubMed]
  • Turner CE, Glenney JR, Jr, Burridge K. Paxillin: a new vinculin-binding protein present in focal adhesions. J Cell Biol. 1990 Sep;111(3):1059–1068. [PMC free article] [PubMed]
  • Turner CE, Miller JT. 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. [PubMed]
  • Turner CE, Schaller MD, Parsons JT. Tyrosine phosphorylation of the focal adhesion kinase pp125FAK during development: relation to paxillin. J Cell Sci. 1993 Jul;105(Pt 3):637–645. [PubMed]
  • Ullrich A, Schlessinger J. Signal transduction by receptors with tyrosine kinase activity. Cell. 1990 Apr 20;61(2):203–212. [PubMed]
  • Weng Z, Taylor JA, Turner CE, Brugge JS, 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]
  • Yu KT, Czech MP. Tyrosine phosphorylation of the insulin receptor beta subunit activates the receptor-associated tyrosine kinase activity. J Biol Chem. 1984 Apr 25;259(8):5277–5286. [PubMed]
  • 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. 1993 Oct 15;268(29):22060–22065. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)