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

Eduardo Perez Salazar,Enrique Rozengurt

doi:10.1074/jbc.274.40.28371

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 FAK-Src 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.

Highlights

Neuropeptides stimulate DNA synthesis and cell proliferation in cultured cells and are implicated as growth factors in a variety of fundamental processes including development, inflammation, tissue regeneration, and tumorigenesis [1,2,3]
The extracts were immunoprecipitated with anti-Src antibody SRC-2, which recognizes the C-terminal sequence of the family members Src, Yes, and Fyn, and the immune complexes were analyzed by SDS-PAGE followed by Western blotting with anti-focal adhesion kinase (FAK) antibody
We did not detect significant FAK immunoreactivity in Src immunocomplexes when the cells were lysed in a buffer containing 1% Triton (Fig. 1A), a substantial amount of Src was recovered in these lysates (Fig. 1A, lower panel), in agreement with our previous results [23, 24] and with results obtained using cells replated onto fibronectin [52]

Summary

Introduction

Neuropeptides stimulate DNA synthesis and cell proliferation in cultured cells and are implicated as growth factors in a variety of fundamental processes including development, inflammation, tissue regeneration, and tumorigenesis [1,2,3]. Src Contributes to Maximal FAK Activation in Response to Bombesin—Recently, we demonstrated that GPCR agonists including bombesin induce FAK activation [23], and the results presented above indicate that bombesin stimulates the formation of a complex between FAK and Src in intact cells.

Results

Conclusion