Evidence for in vivo phosphorylation of the Grb2 SH2-domain binding site on focal adhesion kinase by Src-family protein-tyrosine kinases

Mol Cell Biol. 1996 Oct;16(10):5623-33. doi: 10.1128/MCB.16.10.5623.

Abstract

Focal adhesion kinase (FAK) is a nonreceptor protein-tyrosine kinase (PTK) that associates with integrin receptors and participates in extracellular matrix-mediated signal transduction events. We showed previously that the c-Src nonreceptor PTK and the Grb2 SH2/SH3 adaptor protein bound directly to FAK after fibronectin stimulation (D. D. Schlaepfer, S.K. Hanks, T. Hunter, and P. van der Geer, Nature [London] 372:786-791, 1994). Here, we present evidence that c-Src association with FAK is required for Grb2 binding to FAK. Using a tryptic phosphopeptide mapping approach, the in vivo phosphorylation of the Grb2 binding site on FAK (Tyr-925) was detected after fibronectin stimulation of NIH 3T3 cells and was constitutively phosphorylated in v-Src-transformed NIH 3T3 cells. In vitro, c-Src phosphorylated FAK Tyr-925 in a glutathione S-transferase-FAK C-terminal domain fusion protein, whereas FAK did not. Using epitope-tagged FAK constructs, transiently expressed in human 293 cells, we determined the effect of site-directed mutations on c-Src and Grb2 binding to FAK. Mutation of FAK Tyr-925 disrupted Grb2 binding, whereas mutation of the c-Src binding site on FAK (Tyr-397) disrupted both c-Src and Grb2 binding to FAK in vivo. These results support a model whereby Src-family PTKs are recruited to FAK and focal adhesions following integrin-induced autophosphorylation and exposure of FAK Tyr-397. Src-family binding and phosphorylation of FAK at Tyr-925 creates a Grb2 SH2-domain binding site and provides a link to the activation of the Ras signal transduction pathway. In Src-transformed cells, this pathway may be constitutively activated as a result of FAK Tyr-925 phosphorylation in the absence of integrin stimulation.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3T3 Cells
  • Adaptor Proteins, Signal Transducing*
  • Amino Acid Sequence
  • Animals
  • Binding Sites
  • Cell Adhesion
  • Cell Adhesion Molecules / metabolism*
  • Cell Line
  • ErbB Receptors / metabolism
  • Focal Adhesion Kinase 1
  • Focal Adhesion Protein-Tyrosine Kinases
  • GRB2 Adaptor Protein
  • Glutathione Transferase
  • Humans
  • Integrins / physiology
  • Mice
  • Models, Biological
  • Mutagenesis, Site-Directed
  • Phosphopeptides / chemistry
  • Phosphopeptides / isolation & purification
  • Phosphorylation
  • Polylysine
  • Polymerase Chain Reaction
  • Protein-Tyrosine Kinases / metabolism*
  • Proteins / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Tyrosine
  • src Homology Domains*
  • src-Family Kinases / metabolism*

Substances

  • Adaptor Proteins, Signal Transducing
  • Cell Adhesion Molecules
  • GRB2 Adaptor Protein
  • GRB2 protein, human
  • Grb2 protein, mouse
  • Integrins
  • Phosphopeptides
  • Proteins
  • Recombinant Fusion Proteins
  • Polylysine
  • Tyrosine
  • Glutathione Transferase
  • ErbB Receptors
  • Protein-Tyrosine Kinases
  • Focal Adhesion Kinase 1
  • Focal Adhesion Protein-Tyrosine Kinases
  • PTK2 protein, human
  • Ptk2 protein, mouse
  • src-Family Kinases