PHOSPHATIDYLINOSITOL 3-KINASE IS NECESSARY AND SUFFICIENT FOR INSULIN-STIMULATED STRESS FIBER BREAKDOWN

Rat-1 fibroblasts overexpressing the human insulin receptor undergo ra pid actin rearrangement in response to insulin. Breakdown of stress fi bers present in quiescent cells is followed by transient membrane ruff ling and a return of stress fibers. We investigated the signaling path ways that mediate this insulin-stimulated reorganization of the actin cytoskeleton, which was visualized with rhodamine-phalloidin. Treatmen t of cells with the phosphatidylinositol 3-kinase (PI3-kinase) inhibit or wortmannin prevented insulin action at the preliminary step of stre ss fiber breakdown. Cellular microinjection of a polyclonal antibody d irected against the p85 subunit of PI3-kinase as well as a purified re combinant p85-SH2 domain protein also inhibited actin reorganization. Transient expression of a constitutively active form of PI3-kinase (p1 10) was sufficient to cause both stress fiber breakdown and membrane ruffling in the absence of insulin. Microinjection of a polyclonal ant i-She antibody or dominant negative N17-Ras protein did not affect act in dynamics, and although constitutively active Vla-Ras caused modest cytoskeletal reorganization, this effect was blocked by pretreatment w ith wortmannin. In summary, activation of PI3-kinase is necessary and sufficient to stimulate actin rearrangement, indicating that PI3-kinas e may initiate the only signaling cascade required for insulin to indu ce cytoskeletal restructuring.