TY - JOUR
T1 - Distinct roles for the p110α and hVPS34 phosphatidylinositol 3'- kinases in vesicular trafficking, regulation of the actin cytoskeleton, and mitogenesis
AU - Siddhanta, Uma
AU - McIlroy, James
AU - Shah, Amishi
AU - Zhang, Yitao
AU - Backer, Jonathan M.
PY - 1998/12/14
Y1 - 1998/12/14
N2 - We have examined the roles of the p85/p110α and hVPS34 phosphatidylinositol (PI) 3'-kinases in cellular signaling using inhibitory isoform-specific antibodies. We raised anti-hVPS34 and anti-p110α antibodies that specifically inhibit recombinant hVPS34 and p110α, 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-p110α 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-p110α 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-p110α 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 p110α 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.
AB - We have examined the roles of the p85/p110α and hVPS34 phosphatidylinositol (PI) 3'-kinases in cellular signaling using inhibitory isoform-specific antibodies. We raised anti-hVPS34 and anti-p110α antibodies that specifically inhibit recombinant hVPS34 and p110α, 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-p110α 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-p110α 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-p110α 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 p110α 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.
KW - Endocytosis
KW - Phosphatidylinositol 3'-kinase
KW - Phosphoinositides
KW - Signal transduction
KW - Vesicular trafficking
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U2 - 10.1083/jcb.143.6.1647
DO - 10.1083/jcb.143.6.1647
M3 - Article
C2 - 9852157
AN - SCOPUS:0032517624
SN - 0021-9525
VL - 143
SP - 1647
EP - 1659
JO - Journal of Cell Biology
JF - Journal of Cell Biology
IS - 6
ER -