Contortrostatin, a snake venom disintegrin, induces alpha nu beta 3-mediated tyrosine phosphorylation of CAS and FAK in tumor cells

Contortrostatin is a homodimeric disintegrin that inhibits platelet aggrega tion and cell adhesion to extracellular matrix proteins by blocking integri ns. The effect of contortrostatin on integrin-mediated signaling in tumor c ells was investigated by studying tyrosine phosphorylation events and activ ation of specific signaling molecules. We found that at concentrations as l ow as 1 nM, soluble contortrostatin activates integrin signals leading to i ncreased tyrosine phosphorylation of FAK and GAS, and that these signals ar e abolished by inhibiting Src family kinases. Using transfected 293 cells e xpressing specific integrins, it was determined that contortrostatin-genera ted signals are mediated exclusively by the alpha nu beta 3 integrin. This observation was extended by showing that cells lacking alpha nu beta 3, but expressing alpha nu beta 5 and alpha 5 beta 1, do not respond in this way to contortrostatin treatment. In cells expressing alpha nu beta 3, blocking contortrostatin binding with antibodies against alpha nu beta 3 completely abrogates contortrostatin signals. Monovalent disintegrins echistatin and favoridin were incapable of affecting tyrosine phosphorylation alone, but w hen added simultaneously with contortrostatin, completely inhibited contort rostatin-initiated signals. We propose that the homodimeric nature of conto rtrostatin imparts the ability to crosslink alpha nu beta 3 integrins, caus ing Src activation and hyperphosphorylation of FAK and GAS. This activity m ay represent a novel mechanism by which tumor cell motility can be inhibite d, (C) 2000 Wiley-Liss, Inc.