PIKfyve Negatively Regulates Exocytosis in Neurosecretory Cells

Regulated secretion depends upon a highly co-ordinated series of protein-protein and protein-lipid interactions. Two phosphoinositides, PtdIns(4,5)P2 and PtdIns(3)P, are important for the ATP-dependent priming of the secretory apparatus prior to Ca2+-dependent exocytosis. Mechanisms that control phosphoinositides 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 PtdIns(3)P to produce PtdIns(3,5)P2, on large dense core vesicle exocytosis from neuroendocrine cells. PIKfyve localises to a sub-population 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 knock-down by siRNA potentiated secretory granule exocytosis. Over-expression of PIKfyve or its yeast orthologue Fab1p inhibited regulated secretion in PC12 cells while 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 phospho notinositides.