Distinct roles for the p110alpha and hVPS34 phosphatidylinositol 3'-kinases in vesicular trafficking, regulation of the actin cytoskeleton, and mitogenesis.
We have examined the roles of the p85/ p110alpha and hVPS34 phosphatidylinositol (PI) 3'-kinases in cellular signaling using inhibitory isoform-specific antibodies. We raised anti-hVPS34 and anti-p110alpha antibodies that specifically inhibit recombinant hVPS34 and p110alpha, 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-p110alpha 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-p110alpha 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-p110alpha 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 p110alpha 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.
Siddhanta
U
U
Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
McIlroy
J
J
Shah
A
A
Zhang
Y
Y
Backer
J M
JM
D000199:Actins / Q000502:physiology*; D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D000918:Antibody Specificity; D016466:CHO Cells; D002455:Cell Division; D006224:Cricetinae; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D006801:Humans; D007328:Insulin / Q000494:pharmacology / Q000502:physiology; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D017479:Receptors, Platelet-Derived Growth Factor / Q000187:drug effects / Q000502:physiology; D011990:Receptors, Transferrin / Q000235:genetics / Q000502:physiology; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D013873:Thionucleotides; D014162:Transfection; D014407:Tumor Cells, Cultured
D000199:Actins; D000906:Antibodies; D007328:Insulin; D007527:Isoenzymes; D016376:Oligonucleotides, Antisense; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D010982:Platelet-Derived Growth Factor; D017479:Receptors, Platelet-Derived Growth Factor; D011990:Receptors, Transferrin; D011994:Recombinant Proteins; D013873:Thionucleotides