Platelet-derived growth factor-induced phosphatidylinositol 3-kinase activation mediates actin rearrangements in fibroblasts

Various agonist-induced cell responses in neutrophils and fibroblasts, such as chemotaxis and cytoskeletal rearrangements, have been shown to correlate with the synthesis of PtdIns(3,4,5)P3; however, the significance of this rise in second messenger levels is not clear. We show here that wortmannin inhibits platelet-derived growth factor (PDGF)-mediated production of PtdIns(3,4,5)P3 in human foreskin fibroblasts with an IC50 of about 5 nM. A similar inhibition was observed in in vitro assays (IC50 approximately 1 nM) with phosphatidylinositol 3-kinase immunoprecipitated by antibodies directed against the 85 kDa subunit (p85). On the other hand, wortmannin did not affect PDGF-mediated phosphorylation of p85 as detected by immunoprecipitation with anti-phosphotyrosine antibodies, and did not dissociate the complex of p85 and the catalytic subunit (p110) of phosphatidylinositol 3-kinase. These results are consistent with a direct, specific inhibition of the enzyme by wortmannin at concentrations relevant for its previously reported effects on cellular responses. When stimulated with PDGF, human foreskin fibroblasts form circular structures of filamentous actin. Preincubation of these cells with wortmannin inhibits PDGF-mediated actin rearrangements, suggesting a need for PtdIns(3,4,5)P3 formation as a signal for this cell response.