Src family kinases are required for integrin-mediated but not for G protein-coupled receptor stimulation of focal adhesion kinase autophosphorylation at Tyr-397.
Plating suspended Swiss 3T3 cells onto fibronectin-coated dishes promoted phosphorylation of endogenous focal adhesion kinase (FAK) at Tyr-397, the major autophosphorylation site, and at Tyr-577, located in the activation loop, as revealed by site-specific antibodies that recognize the phosphorylated form of these residues. Treatment with the selective Src family kinase inhibitor pyrazolopyrimidine 2 (PP-2) markedly reduced the phosphorylation of both Tyr-397 and Tyr-577 induced by fibronectin. Furthermore, fibronectin-mediated FAK phosphorylation at Tyr-397 was dramatically reduced in SYF cells (deficient in Src, Yes, and Fyn expression). Stimulation of Swiss 3T3 cells with bombesin also induced a rapid increase in the phosphorylation of endogenous FAK at Tyr-397. In contrast to the results obtained with fibronectin, PP-2 did not prevent FAK Tyr-397 phosphorylation stimulated by bombesin at a concentration (10 micrometer) that suppressed bombesin-induced FAK Tyr-577 phosphorylation. Similarly, PP-2 did not prevent Tyr-397 phosphorylation in Swiss 3T3 cells stimulated with other G protein-coupled receptor agonists including vasopressin, bradykinin, endothelin, and lysophosphatidic acid. Lysophosphatidic acid also induced FAK phosphorylation at Tyr-397 in SYF cells. Our results identify, for first time, the existence of Src-dependent and Src-independent pathways leading to FAK autophosphorylation at Tyr-397 stimulated by adhesion-dependent signals and G protein-coupled receptor agonists in the same cell.
Salazar
E P
EP
Department of Medicine, School of Medicine and Molecular Biology Institute, UCLA, Los Angeles, California 90095, USA.
Rozengurt
E
E
D016475:3T3 Cells; D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins / Q000502:physiology; D016023:Integrins / Q000502:physiology; D051379:Mice; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D011956:Receptors, Cell Surface / Q000819:agonists / Q000502:physiology*; D015398:Signal Transduction; D014443:Tyrosine; D019061:src-Family Kinases / Q000502:physiology*
D016023:Integrins; D011956:Receptors, Cell Surface; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; D019061:src-Family Kinases; D019204:GTP-Binding Proteins