Tyrosine phosphorylation of paxillin and focal adhesion kinase during insulin-like growth factor-I-stimulated lamellipodial advance

In the current studies, we examined whether focal adhesion kinase (FAK) and paxillin play a role in insulin-like growth factor-I (IGF-I)-stimulated morphological changes in neuronal cells, In SH-SY5Y human neuroblastoma cells, 10 nM IGF-I enhanced the extension of lamellipodia within 30 min, Scanning electron microscopy and staining with rhodamine-phalloidin showed that these lamellipodia displayed ruffles, filopodia, and a distinct meshwork of actin filaments, Immunofluorescent staining identified focal concentrations of FAK, paxillin, and phosphotyrosine within the lamellipodia, Immunoprecipitation experiments revealed that FAK and paxillin are tyrosine phosphorylated during IGF-I-stimulated lamellipodial extension, Maximal phosphorylation of FAK and paxillin was observed 15-30 min after the addition of 10 nM IGF-I, whereas maximal IGF-I receptor phosphorylation occurred within 5 min, FAK, paxillin, and IGF-I receptor tyrosine phosphorylation had similar concentration-response curves and were inhibited by the receptor blocking antibody alpha IR-3. These results indicate that FAK and paxillin are tyrosine-phosphorylated during IGF-I-stimulated lamellipodial advance and suggest that the tyrosine phosphorylation of these two proteins helps mediate IGF-I stimulated cell and growth cone motility, These responses contrast directly with recent reports showing insulin-stimulated dephosphorylation of FAK and paxillin.