Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins

Mol Cell Biol. 1997 Mar;17(3):1702-13. doi: 10.1128/MCB.17.3.1702.

Abstract

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.

Publication types

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

MeSH terms

  • Adaptor Proteins, Signal Transducing*
  • Animals
  • Calcium-Calmodulin-Dependent Protein Kinases / metabolism
  • Cell Adhesion Molecules / metabolism*
  • Cells, Cultured
  • Crk-Associated Substrate Protein
  • Enzyme Activation
  • Fibroblasts
  • Fibronectins / pharmacology*
  • Focal Adhesion Kinase 1
  • Focal Adhesion Protein-Tyrosine Kinases
  • GRB2 Adaptor Protein
  • Integrins / physiology
  • Mice
  • Mice, Knockout
  • Mitogen-Activated Protein Kinase 1
  • Oncogene Proteins / metabolism
  • Peptide Mapping
  • Phosphoproteins / metabolism*
  • Phosphorylation
  • Protein-Tyrosine Kinases / metabolism*
  • Proteins / metabolism
  • Proto-Oncogene Proteins pp60(c-src) / metabolism*
  • Retinoblastoma-Like Protein p130
  • Signal Transduction / physiology*
  • src Homology Domains

Substances

  • Adaptor Proteins, Signal Transducing
  • Bcar1 protein, mouse
  • Cell Adhesion Molecules
  • Crk-Associated Substrate Protein
  • Fibronectins
  • GRB2 Adaptor Protein
  • Grb2 protein, mouse
  • Integrins
  • Nck protein
  • Oncogene Proteins
  • Phosphoproteins
  • Proteins
  • Retinoblastoma-Like Protein p130
  • Protein-Tyrosine Kinases
  • Focal Adhesion Kinase 1
  • Focal Adhesion Protein-Tyrosine Kinases
  • Proto-Oncogene Proteins pp60(c-src)
  • Ptk2 protein, mouse
  • Calcium-Calmodulin-Dependent Protein Kinases
  • Mitogen-Activated Protein Kinase 1