PIKfyve negatively regulates exocytosis in neurosecretory cells.
Regulated secretion depends upon a highly coordinated series of protein-protein and protein-lipid interactions. Two phosphoinositides, phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3-phosphate, are important for the ATP-dependent priming of the secretory apparatus prior to Ca(2+)-dependent exocytosis. Mechanisms that control phosphoinositide levels are likely to play an important role in priming fine tuning. Here we have investigated the involvement of PIKfyve, a phosphoinositide 5-kinase that can phosphorylate phosphatidylinositol 3-phosphate to produce phosphatidylinositol 3,5-bisphosphate on large dense core vesicle exocytosis from neuroendocrine cells. PIKfyve localizes to a subpopulation of secretory granules in chromaffin and PC12 cells. Nicotine stimulation promoted recruitment of PIKfyve-EGFP onto secretory vesicles in PC12 cells. YM-201636, a selective inhibitor of PIKfyve activity, and PIKfyve knockdown by small interfering RNA potentiated secretory granule exocytosis. Overexpression of PIKfyve or its yeast orthologue Fab1p inhibited regulated secretion in PC12 cells, whereas a catalytically inactive PIKfyve mutant had no effect. These results demonstrate a novel inhibitory role for PIKfyve catalytic activity in regulated secretion and provide further evidence for a fine tuning of exocytosis by 3-phosphorylated phosphoinositides.
Molecular Dynamics of Synaptic Function Laboratory, Queensland Brain Institute and School of Biomedical Sciences, University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia.
Shona L
SL
Osborne
Peter J
PJ
Wen
Christine
C
Boucheron
Hao N
HN
Nguyen
Masahiko
M
Hayakawa
Hiroyuki
H
Kaizawa
Peter J
PJ
Parker
Nicolas
N
Vitale
Frederic A
FA
Meunier
D000818:Animals; D002417:Cattle; D019439:Chromaffin Cells / Q000502:physiology*; D005089:Exocytosis / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D051379:Mice; D009490:Neurosecretory Systems / Q000187:drug effects / Q000502:physiology; D016716:PC12 Cells; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000494:pharmacology; D014162:Transfection
D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D034741:RNA, Small Interfering; D011994:Recombinant Proteins