Distinct roles for the p110 alpha and hVPS34 phosphatidylinositol 3 '-kinases in vesicular trafficking, regulation of the actin cytoskeleton, and mitogenesis

We have examined the roles of the p85/p110 alpha and hVPS34 phosphatidylino sitol (PI) 3'-kinases in cellular signaling using inhibitory isoform-specif ic antibodies. We raised anti-hVPS34 and anti-p110 alpha antibodies that sp ecifically inhibit recombinant hVPS34 and p110 alpha, respectively, in vitr o. We used the antibodies to study cellular processes that are sensitive to low-dose wortmannin. The antibodies had distinct effects on the actin cyto skeleton; microinjection of anti-p110 alpha antibodies blocked insulin-stim ulated ruffling, whereas anti-hVPS34 antibodies had no effect. The antibodi es also had different effects on vesicular trafficking. Microinjection of i nhibitory anti-hVPS34 antibodies, but not anti-p110 alpha antibodies, block ed the transit of internalized PDGF receptors to a perinuclear compartment, and disrupted the localization of the early endosomal protein EEA1. Microi njection of anti-p110 alpha antibodies, and to a lesser extent anti-hVPS34 antibodies, reduced the rate of transferrin recycling in CHO cells. Surpris ingly, both antibodies inhibited insulin-stimulated DNA synthesis by 80%. I njection of cells with antisense oligonucleotides derived from the hVPS34 s equence also blocked insulin-stimulated DNA synthesis, whereas scrambled ol igonucleotides had no effect. Interestingly, the requirement for p110 alpha and hVPS34 occurred at different times during the G1-S transition. Our dat a suggest that different PI 3'-kinases play distinct regulatory roles in th e cell, and document an unexpected role for hVPS34 during insulin-stimulate d mitogenesis.