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MBC in Press, published online ahead of print October 8, 2008
Mol. Biol. Cell 10.1091/mbc.E08-06-0595

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Submitted on June 12, 2008
Revised on September 15, 2008
Accepted on September 30, 2008

Ca2+-regulated Pool of PtdIns(3)P Produced by PI3 kinase-C2{alpha} on Neurosecretory Vesicles

Peter J. Wen,* Shona L. Osborne,* Isabel C. Morrow,{dagger} Robert G. Parton,{dagger} Jan Domin,{ddagger} and Frederic A. Meunier*

*Molecular Dynamics of Synaptic Function Laboratory, Queensland Brain Institute and School of Biomedical Sciences, The University of Queensland, St Lucia, 4072 Queensland, Australia; {dagger}Institute for Molecular Bioscience, and Centre for Microscopy and Microanalysis, University of Queensland, Brisbane, QLD 4072, Australia, {ddagger}Renal Section, Faculty of Medicine, Imperial College, London W12 0NN, United Kingdom

Monitoring Editor: Sean Munro

Phosphatidylinositol-3-phosphate (PtdIns(3)P) is a key player in early endosomal trafficking and is mainly produced by class III PI3-kinase. In neurosecretory cells, class II PI3-kinase C2{alpha} 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 PI3-kinase C2{alpha} knock down 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 PI3-kinase C2{alpha} knock down and expression of PI3-kinase C2{alpha} catalytically-inactive mutant. Using purified chromaffin granules, we found that PtdIns(3)P production is controlled by Ca2+. We confirmed that PtdIns(3)P production from recombinantly-expressed PI3-kinase C2{alpha} is indeed regulated by Ca2+. We provide evidence that a dynamic pool of PtdIns(3)P synthesized by PI3-kinase C2{alpha} occurs on secretory vesicles in neurosecretory cells, demonstrating that the activity of a member of the PI3 kinase family is regulated by Ca2+ in vitro and in living neurosecretory cells.

Address correspondence to: Frederic A. Meunier (f.meunier{at}uq.edu.au)




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