Direct interaction of v-Src with the focal adhesion kinase mediated by the Src SH2 domain.

Abstract

The recently described focal adhesion kinase (FAK) has been implicated in signal transduction pathways initiated by cell adhesion receptor integrins and by neuropeptide growth factors. To examine the mechanisms by which FAK relays signals from the membrane to the cell interior, we carried out a series of experiments to detect potential FAK interactions with proteins containing Src homology 2 (SH2) domains that are important intracellular signaling molecules. Using v-Src-transformed NIH3T3 cells, we showed that FAK was present in the immune-complex precipitated by anti-Src antibody, suggesting potential interaction of FAK with v-Src in vivo. We also showed potentially direct interaction of FAK with v-Src in vivo using the yeast two-hybrid system. Using recombinant FAK expressed in insect cells and bacterial fusion proteins containing Src SH2 domains, we showed direct binding of FAK to the Src SH2 domain but not to the SH3 domain in vitro. A kinase-defective mutant of FAK, which is not autophosphorylated, did not interact with the Src SH2 domain under the same conditions, suggesting the involvement of the FAK autophosphorylation sites. Treatment of FAK with a protein-tyrosine phosphatase decreased its binding to the Src SH2 domain, whereas autophosphorylation in vitro increased its binding. These results confirm the importance of FAK autophosphorylation sites in its interaction with SH2 domain-containing proteins. Taken together, these results suggest that FAK may mediate signal transduction events initiated on the cell surface by kinase activation and autophosphorylation that result in its binding to other key intracellular signaling molecules.

Contribution of Src and Ras pathways in FGF-2 induced endothelial cell differentiation

“…It is thus possible that integrin activation contributes to modulation of Src signaling, in FGF-2 induced tube formation. Fibronectin induced activation of FAK and subsequent binding of Src to phosphorylated Tyr397 in FAK is a well established integrinmediated signaling event (Schaller et al, 1994;Xing et al, 1994). Since Src activity is indispensable for IBEC di erentiation, it is interesting to note that FGF-2 has a potentiating e ect on Src ± FAK complex-formation in the collagen-cultured IBEC, which occurs in spite of the modest level of tyrosine phosphorylated FAK in these cells.…”

“…The nonreceptor tyrosine kinase FAK contains a central kinase domain which is¯anked by N-and C-terminal domains lacking SH2 and SH3 domains. Several studies have shown direct binding of Src SH2 domain to the major autophosphorylation site of FAK (Schaller et al, 1994;Xing et al, 1994). This interaction is thought to occur in subcellular compartments called focal adhesions, where the cell contacts and binds to the extracellular matrix.…”

The Src SH2 domain interacts dynamically with the focal adhesion kinase binding site as demonstrated by paramagnetic nmr spectroscopy

“…The amino acid sequence of this site, YAEI, is close to the Src family SH2 consensus binding motif, and phosphorylation of the tyrosine creates a high-affinity binding site for the Src SH2 domain (8)(9)(10)(11).…”

Phosphorylation of focal adhesion kinase on tyrosine 194 by Met leads to its activation through relief of autoinhibition

“…It is generally believed that autophosphorylation of FAK on a particular tyrosine residue, Y397, is an early, essential step for the full activation of FAK in response to many extracellular stimuli. The Y397 is the major site of FAK autophosphorylation, which creates a high affinity binding site for the Src-homology 2 domain of several proteins including the Src family kinases (Cobb et al, 1994;Schaller et al, 1994;Xing et al, 1994;Eide et al, 1995). Activated Src phosphorylates FAK on multiple sites, including Y576 and Y577, both of which are located in the activation loop within the kinase domain (Calalb et al, 1995;Lietha et al, 2007).…”