ACTIN-FILAMENTS FACILITATE INSULIN ACTIVATION OF THE SRC AND COLLAGENHOMOLOGOUS MITOGEN-ACTIVATED PROTEIN-KINASE PATHWAY LEADING TO DNA-SYNTHESIS AND C-FOS EXPRESSION/

The exact mechanism of the spatial organization of the insulin signali ng pathway leading to nuclear events remains unknown, Here, we investi gated the involvement of the actin cytoskeleton in propagation of insu lin signaling events leading to DNA synthesis and expression of the im mediate early genes c-fos and c-jun in L6 muscle cells. Insulin reorga nized the cellular actin network and increased the rate of DNA synthes is and the levels of c-fos mRNA, but not those of c-jun mRNA, in undif ferentiated L6 myoblasts. Similarly, insulin markedly elevated the lev els of c-fos mRNA but not of c-jun mRNA in differentiated L6 myotubes. Disassembly of the actin filaments by cytochalasin D, latrunculin B, or botulinum Ca toxin significantly inhibited insulin-mediated DNA syn thesis in myoblasts and abolished stimulation of c-fos expression by t he hormone in myoblasts and myotubes, Actin disassembly abolished insu lin-induced phosphorylation and activation of extracellulor signal-reg ulated kinases, activation of a 65-kda member of the pal-activated kin ases, and phosphorylation of p38 mitogen-activated protein kinases but did not prevent activation of phosphatidylinositol 3-kinase and p70(S 6k). Under these conditions, insulin-induced Ras activation was also a bolished, and Grb2 association with the Src and collogen homologous (S hc) molecule was inhibited without inhibition of the tyrosine phosphor ylation of Shc, We conclude that the actin filament network plays an e ssential role in insulin regulation of She-dependent signaling events governing gene expression by facilitating the interaction of Shc with Grb2.