J. Cell Biol., Volume 143, Number 6, December 14, 1998 1647-1659

Distinct Roles for the p110 and hVPS34 Phosphatidylinositol 3'-Kinases in Vesicular Trafficking, Regulation of the Actin Cytoskeleton, and Mitogenesis

Uma Siddhanta, James McIlroy, Amishi Shah, Yitao Zhang, and Jonathan M. Backer

Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461

We have examined the roles of the p85/ p110 and hVPS34 phosphatidylinositol (PI) 3'-kinases in cellular signaling using inhibitory isoform-specific antibodies. We raised anti-hVPS34 and anti-p110 antibodies that specifically inhibit recombinant hVPS34 and p110, respectively, in vitro. We used the antibodies to study cellular processes that are sensitive to low-dose wortmannin. The antibodies had distinct effects on the actin cytoskeleton; microinjection of anti-p110 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 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 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 and hVPS34 occurred at different times during the G1-S transition. Our data suggest that different PI 3'-kinases play distinct regulatory roles in the cell, and document an unexpected role for hVPS34 during insulin-stimulated mitogenesis.

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