C-SRC REGULATES THE SIMULTANEOUS REARRANGEMENT OF ACTIN CYTOSKELETON,P190RHOGAP, AND P120RASGAP FOLLOWING EPIDERMAL GROWTH-FACTOR STIMULATION

Analysis of C3H10T1/2 murine fibroblasts overexpressing wild type and dominant negative variants of c-Src has demonstrated a requirement for c-Src in EGF-induced mitogenesis. Correlating with the ability of c-S rc variants to potentiate or inhibit EGF-dependent DNA synthesis is th e phosphotyrosine content of multiple cellular proteins, including p19 0-RhoGAP, a protein thought to regulate growth factor-induced actin cy toskeleton remodeling by modulating the activity of the small GTP bind ing protein, Rho. Because the in vivo phosphotyrosine content of p190 varies with the level of active c-Src and not with EGF treatment, p190 is considered to be a preferred substrate of c-Src. To determine whet her tyrosyl phosphorylation of p190 (by c-Src) could influence EGF-dep endent actin remodeling, we used conventional and confocal immunofluor escence microscopy to examine the intracellular distribution of p190, actin, and p120RasGAP in EGF-stimulated or unstimulated 10T1/2 Neo con trol cells and cells that stably overexpress wild-type (K+) or kinase- defective (K-) c-Src. We found that in all cell lines, EGF induced a r apid and transient condensation of p190 and RasGAP into cytoplasmic, a rclike structures. However, in K+ cells the rate of appearance and num ber of cells exhibiting arcs increased when compared with control cell s, Conversely, K- cells exhibited delayed are formation and a reductio n in number of cells forming arcs. EGF-induced actin stress fiber disa ssembly and reassembly occurred with the same kinetics and frequency a s did p190 and RasGAP rearrangements in all three cell lines. These re sults, together with the documented Rho-GAP activity intrinsic to p190 and the ability of Rho to modulate actin stress fiber formation, sugg est that c-Src regulates EGF-dependent actin cytoskeleton reorganizati on through phosphorylation of p190.