Ca2+-regulated pool of phosphatidylinositol-3-phosphate produced by phosphatidylinositol 3-kinase C2alpha on neurosecretory vesicles.
Phosphatidylinositol-3-phosphate [PtdIns(3)P] is a key player in early endosomal trafficking and is mainly produced by class III phosphatidylinositol 3-kinase (PI3K). In neurosecretory cells, class II PI3K-C2alpha and its lipid product PtdIns(3)P have recently been shown to play a critical role during neuroexocytosis, suggesting that two distinct pools of PtdIns(3)P might coexist in these cells. However, the precise characterization of this additional pool of PtdIns(3)P remains to be established. Using a selective PtdIns(3)P probe, we have identified a novel PtdIns(3)P-positive pool localized on secretory vesicles, sensitive to PI3K-C2alpha knockdown and relatively resistant to wortmannin treatment. In neurosecretory cells, stimulation of exocytosis promoted a transient albeit large increase in PtdIns(3)P production localized on secretory vesicles sensitive to PI3K-C2alpha knockdown and expression of PI3K-C2alpha catalytically inactive mutant. Using purified chromaffin granules, we found that PtdIns(3)P production is controlled by Ca(2+). We confirmed that PtdIns(3)P production from recombinantly expressed PI3K-C2alpha is indeed regulated by Ca(2+). We provide evidence that a dynamic pool of PtdIns(3)P synthesized by PI3K-C2alpha occurs on secretory vesicles in neurosecretory cells, demonstrating that the activity of a member of the PI3K family is regulated by Ca(2+) in vitro and in living neurosecretory cells.
Molecular Dynamics of Synaptic Function Laboratory, Queensland Brain Institute and School of Biomedical Sciences, The University of Queensland, St. Lucia, 4072 Queensland, Australia.
Peter J
PJ
Wen
Shona L
SL
Osborne
Isabel C
IC
Morrow
Robert G
RG
Parton
Jan
J
Domin
Frederic A
FA
Meunier
D000730:Androstadienes / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002417:Cattle; D002460:Cell Line; D019439:Chromaffin Cells / Q000166:cytology / Q000378:metabolism; D058541:Class II Phosphatidylinositol 3-Kinases; D005089:Exocytosis / Q000502:physiology; D006801:Humans; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D047428:Protein Kinase Inhibitors / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D022142:Secretory Vesicles / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D000077191:Wortmannin
D000730:Androstadienes; D018129:Phosphatidylinositol Phosphates; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; C055525:phosphatidylinositol 3-phosphate; D019869:Phosphatidylinositol 3-Kinases; D058541:Class II Phosphatidylinositol 3-Kinases; D002118:Calcium; D000077191:Wortmannin