Bombesin and Platelet-derived Growth Factor Induce Association of Endogenous Focal Adhesion Kinase with Src in Intact Swiss 3T3 Cells

Abstract

Stimulation of quiescent Swiss 3T3 cells with bombesin induces a rapid increase in the formation of complexes between focal adhesion kinase (FAK) and Src family members, which can be extracted with a buffer containing Triton, deoxycholate, and SDS but not with a buffer containing Triton alone. An increase in complex formation between FAK and Src in response to bombesin could be detected within 1 min, reached a maximum after 10 min, and declined toward base-line levels after 60 min of bombesin treatment. Bradykinin, endothelin, and lysophosphatidic acid also stimulated FAKSrc complex formation. Bombesin stimulated FAK/Src association through a Ca 2؉ and phosphatidylinositol 3-kinase-independent pathway that requires the integrity of the actin filament network and is partly dependent on functional protein kinase C. Treatment with the selective Src kinase inhibitor PP-2 inhibited both FAK activation and phosphorylation of FAK at Tyr 577 induced by bombesin in intact cells. Platelet-derived growth factor at low concentrations (1-10 ng/ml) also induced FAK-Src complex formation via a pathway that depended on the integrity of the actin cytoskeleton and phosphatidylinositol 3-kinase. Thus, G protein-coupled receptor agonists and platelet-derived growth factor promote complex formation between endogenous FAK and Src in attached cells through different signal transduction pathways.

Phosphospecific Site Tyrosine Phosphorylation of p125FAK and Proline-rich Kinase 2 Is Differentially Regulated by Cholecystokinin Receptor Type A Activation in Pancreatic Acini

“…Our results also differ with PYK2 from the effect of transforming growth factor-␤ in mammary cells where only two sites were tyrosine-phosphorylated (pY402 and pY881) (39). However, our results showing tyrosine phosphorylation in all three sites in FAK by CCK A receptor activation are similar to the effects of cannabinoids in hippocampal cells (48), bombesin in 3T3 cells (53), thrombin in platelets (6), and fibronectin in fibroblasts (42). Similarly, stimulation of tyrosine phosphorylation of all three sites of PYK2 is seen with endothelin-1 in mesangial cells (45), thrombin or histamine in HUVEC cells (43,46), as well as the effect of transforming growth factor-␤ in mammary epithelial cells (39).…”

Focal Adhesion Kinase Facilitates Platelet-derived Growth Factor-BB-stimulated ERK2 Activation Required for Chemotaxis Migration of Vascular Smooth Muscle Cells

“…8D). Previous studies in fibroblasts have shown that motility-promoting concentrations of PDGF-BB promote FAK phosphorylation at Tyr-397 and the stimulated SH2-mediated binding of Src family PTKs to this site on FAK (12,36). Using a phosphospecific antibody directed to the FAK Tyr-397 site, stable FRNK expression but not FRNK L1034S resulted in the reduced phosphorylation of FAK Tyr-397 after PDGF-BB stimulation of SMCs (Fig.…”

Re-expression of the tumor suppressor NF2/merlin inhibits invasiveness in mesothelioma cells and negatively regulates FAK

“…Samples were analysed by immunoblotting. For the FAK-Src and FAK-p85 co-immunoprecipotation experiments, cells were lysed in RIPA lysis buffer containing 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium dehoxycolate and protease inhibitors as reported previously (Salazar and Rozengurt, 1999). Cell lysates were incubated overnight at 41C with the appropriate primary antibodies and subsequently for 2 h with protein G-agarose or protein A-agarose (both from Invitrogen).…”