Bombesin, vasopressin, and endothelin rapidly stimulate tyrosine phosphorylation of the focal adhesion-associated protein paxillin in Swiss 3T3 cells.
Treatment of Swiss 3T3 cells with bombesin caused a striking increase (21-fold) in the tyrosine phosphorylation of the cytoskeleton-associated protein paxillin, as judged by anti-phosphotyrosine Western blots of anti-paxillin immunoprecipitates. Vasopressin and endothelin also stimulated paxillin tyrosine phosphorylation. Bombesin-stimulated tyrosine phosphorylation of paxillin was detectable within 1 min and was concentration-dependent (half-maximum effect at 0.09 nM). Bombesin stimulation of paxillin tyrosine phosphorylation could be dissociated from both protein kinase C (PKC) activation and the mobilization of Ca2+ from intracellular stores. Activation of PKC in quiescent Swiss 3T3 cells using the tumor promoter phorbol 12,13-dibutyrate (PDB) increased the tyrosine phosphorylation of paxillin in a time-dependent manner but was less effective than bombesin and stimulated detectable phosphorylation only within 5 min, considerably slower than bombesin-induced tyrosine phosphorylation of paxillin. Furthermore, the selective PKC inhibitor, GF109203X, or down-regulation of PKC using prolonged treatment with PDB markedly inhibited the stimulation of paxillin tyrosine phosphorylation by PDB but had little effect on the response to bombesin. In contrast, cytochalasin D, an agent that selectively disrupts the network of actin microfilaments, completely inhibited bombesin- and PDB-induced paxillin tyrosine phosphorylation. This is the first report to identify paxillin as a substrate for neuropeptide-stimulated tyrosine phosphorylation.
Zachary
I
I
Imperial Cancer Research Fund, London, United Kingdom.
Sinnett-Smith
J
J
Turner
C E
CE
Rozengurt
E
E
D016475:3T3 Cells; D000818:Animals; D001839:Bombesin / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D002118:Calcium / Q000378:metabolism; D015638:Cytochalasin D / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D016232:Endothelins / Q000494:pharmacology*; D004789:Enzyme Activation; D051379:Mice; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism; D015398:Signal Transduction; D014443:Tyrosine / Q000378:metabolism*; D014667:Vasopressins / Q000494:pharmacology*
D003598:Cytoskeletal Proteins; D016232:Endothelins; D051419:Paxillin; D010750:Phosphoproteins; C496062:Pxn protein, mouse; D014667:Vasopressins; D015638:Cytochalasin D; D014443:Tyrosine; D011493:Protein Kinase C; D001839:Bombesin; D002118:Calcium