Distinct roles for the p110α and hVPS34 phosphatidylinositol 3'- kinases in vesicular trafficking, regulation of the actin cytoskeleton, and mitogenesis

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

Research output: Contribution to journalArticle

133 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)1647-1659
Number of pages13
JournalJournal of Cell Biology
Volume143
Issue number6
DOIs
StatePublished - Dec 14 1998

Fingerprint

Actin Cytoskeleton
Phosphatidylinositol 3-Kinases
Anti-Idiotypic Antibodies
Microinjections
Antibodies
Insulin
Insulin Antibodies
Platelet-Derived Growth Factor Receptors
CHO Cells
Antisense Oligonucleotides
DNA
Transferrin
Oligonucleotides
Protein Isoforms
Injections
Proteins

Keywords

  • Endocytosis
  • Phosphatidylinositol 3'-kinase
  • Phosphoinositides
  • Signal transduction
  • Vesicular trafficking

ASJC Scopus subject areas

  • Cell Biology

Cite this

Distinct roles for the p110α and hVPS34 phosphatidylinositol 3'- kinases in vesicular trafficking, regulation of the actin cytoskeleton, and mitogenesis. / Siddhanta, Uma; McIlroy, James; Shah, Amishi; Zhang, Yitao; Backer, Jonathan M.

In: Journal of Cell Biology, Vol. 143, No. 6, 14.12.1998, p. 1647-1659.

Research output: Contribution to journalArticle

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abstract = "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.",
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