Bombesin, vasopressin, lysophosphatidic acid, and sphingosylphosphorylcholine induce focal adhesion kinase activation in intact Swiss 3T3 cells.
Treatment of quiescent Swiss 3T3 cells with bombesin rapidly increased focal adhesion kinase (FAK)-associated tyrosine kinase activity in immune complexes. The effect was rapid (maximum at 2.5 min) and dose dependent (half-maximum response at 0.05 nM). Addition of vasopressin, lysophosphatidic acid, and sphingosylphosphorylcholine also elicited a rapid increase in FAK-associated tyrosine kinase activity. Addition of the selective Src inhibitor pyrazolopyrimidine directly to the in vitro kinase assay potently inhibited Src kinase activity induced by bombesin but did not affect the kinase activity of FAK measured by autophosphorylation or by synthetic substrate phosphorylation in paralell assays. In addition, Src activity was not detected in FAK immunoprecipitates using an optimal Src peptide substrate. Thus, agonist-induced tyrosine kinase activity measured in FAK immunoprecipitates is mediated by FAK activation rather than by co-immunoprecipitating Src. Bombesin-induced FAK activation is not dependent either on protein kinase C or Ca2+ mobilization but was completely blocked by treatment with cytochalasin D or by placing the cells in suspension. These findings indicate that FAK activation requires an intact actin cytoskeleton. Our results demonstrate that agonists that act via 7-transmembrane domain receptors stimulate FAK kinase activation.
Imperial Cancer Research Fund, P. O. Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX, United Kingdom.
J L
JL
Rodríguez-Fernández
E
E
Rozengurt
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001839:Bombesin / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D015638:Cytochalasin D / Q000378:metabolism; D004789:Enzyme Activation; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D008246:Lysophospholipids / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D010767:Phosphorylcholine / Q000031:analogs & derivatives* / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D013110:Sphingosine / Q000031:analogs & derivatives* / Q000378:metabolism; D014667:Vasopressins / Q000378:metabolism*
D015815:Cell Adhesion Molecules; D008246:Lysophospholipids; C005356:sphingosine phosphorylcholine; D010767:Phosphorylcholine; D014667:Vasopressins; D015638:Cytochalasin D; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; D011493:Protein Kinase C; D013110:Sphingosine; D001839:Bombesin; D002118:Calcium