Phosphatidylinositol 3-kinase is necessary and sufficient for insulin-stimulated stress fiber breakdown.
Rat-1 fibroblasts overexpressing the human insulin receptor undergo rapid actin rearrangement in response to insulin. Breakdown of stress fibers present in quiescent cells is followed by transient membrane ruffling and a return of stress fibers. We investigated the signaling pathways that mediate this insulin-stimulated reorganization of the actin cytoskeleton, which was visualized with rhodamine-phalloidin. Treatment of cells with the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor wortmannin prevented insulin action at the preliminary step of stress fiber breakdown. Cellular microinjection of a polyclonal antibody directed against the p85 subunit of PI3-kinase as well as a purified recombinant p85-SH2 domain protein also inhibited actin reorganization. Transient expression of a constitutively active form of PI3-kinase (p110*) was sufficient to cause both stress fiber breakdown and membrane ruffling in the absence of insulin. Microinjection of a polyclonal anti-Shc antibody or dominant negative N17-Ras protein did not affect actin dynamics, and although constitutively active V12-Ras caused modest cytoskeletal reorganization, this effect was blocked by pretreatment with wortmannin. In summary, activation of PI3-kinase is necessary and sufficient to stimulate actin rearrangement, indicating that PI3-kinase may initiate the only signaling cascade required for insulin to induce cytoskeletal restructuring.
Martin
S S
SS
Department of Medicine, Veterans Administration Medical Center, University of California-San Diego, La Jolla 92093, USA.
Rose
D W
DW
Saltiel
A R
AR
Klippel
A
A
Williams
L T
LT
Olefsky
J M
JM
D000199:Actins / Q000187:drug effects / Q000648:ultrastructure; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005347:Fibroblasts; D006801:Humans; D007328:Insulin / Q000494:pharmacology*; D007700:Kinetics; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D019000:Phosphotyrosine / Q000032:analysis; D051381:Rats; D011972:Receptor, Insulin / Q000096:biosynthesis / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D013314:Stress, Mechanical; D014162:Transfection; D000077191:Wortmannin
D000199:Actins; D000730:Androstadienes; D004791:Enzyme Inhibitors; D007328:Insulin; D010750:Phosphoproteins; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D004815:Epidermal Growth Factor; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D011972:Receptor, Insulin; D000077191:Wortmannin