Regulation of the Actin Cytoskeleton, and Mitogenesis

We have examined the roles of the p85/ p110 a and hVPS34 phosphatidylinositol (PI) 3 9 -kinases in cellular signaling using inhibitory isoform-specific antibodies. We raised anti-hVPS34 and anti-p110 a antibodies that specifically inhibit recombinant hVPS34 and p110 a , respectively, in vitro. We used the antibodies to study cellular processes that are sensitive to lowdose wortmannin. The antibodies had distinct effects on the actin cytoskeleton; microinjection of anti-p110 a antibodies blocked insulin-stimulated ruffling, whereas anti-hVPS34 antibodies had no effect. The antibodies also had different effects on vesicular trafficking. Microinjection of inhibitory anti-hVPS34 antibodies, but not anti-p110 a antibodies, blocked the transit of internalized PDGF receptors to a perinuclear compartment, and disrupted the localization of the early endosomal protein EEA1. Microinjection of anti-p110 a antibodies, and to a lesser extent anti-hVPS34 antibodies, reduced the rate of transferrin recycling in CHO cells. Surprisingly, both antibodies inhibited insulin-stimulated DNA synthesis by 80%. Injection of cells with antisense oligonucleotides derived from the hVPS34 sequence also blocked insulin-stimulated DNA synthesis, whereas scrambled oligonucleotides had no effect. Interestingly, the requirement for p110 a and hVPS34 occurred at different times during the G1–S transition. Our data suggest that different PI 3 9 -kinases play distinct regulatory roles in the cell, and document an unexpected role for hVPS34 during insulin-stimulated mito-