Focal adhesion kinase (p125(FAK)) and paxillin are substrates for sphingomyelinase-induced tyrosine phosphorylation in Swiss 3T3 fibroblasts

We examined the effect of sphingomyelinase on tyrosine phosphorylation of intracellular proteins in mouse Swiss 3T3 fibroblasts. Incubation of the cells with bacterial sphingomyelinase resulted in the elevation of tyrosine phosphorylation of multiple cellular proteins of 190, 130, 120, 97 and 70 kDa within minutes. The 120 and 70 kDa tyrosine-phosphorylated peptides were identified as p125 focal adhesion kinase (p125(FAK)) and paxillin respectively by the use of specific antibodies against the proteins. Tyrosine kinase activity associated with anti-p125(FAK) immunoprecipitate was stimulated by incubation of cells with sphingomyelinase. Cytochalasin D, which selectively disrupts the network of actin filaments, inhibited sphingomyelinase-induced tyrosine phosphorylation of p125(FAK) and elevation of tyrosine kinase activity in the anti-p125(FAK) immunoprecipitates. Sphingomyelinase-induced phosphorylation of p125(FAK) was not inhibited by wortmannin, an inhibitor of phosphatidylinositol 3-kinase. This was in sharp contrast with a wortmannin-sensitive phosphorylation of p125(FAK) observed in platelet-derived growth factor (PGDF)-stimulated cells, Thus hydrolysis of sphingomyelin is the tyrosine kinase cascade including p125(FAK) and paxillin by a mechanism distinct from PDGF.