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 Ca2+-depe...

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Veröffentlicht in:The Journal of biological chemistry 2008-02, Vol.283 (5), p.2804-2813
Hauptverfasser: Osborne, Shona L., Wen, Peter J., Boucheron, Christine, Nguyen, Hao N., Hayakawa, Masahiko, Kaizawa, Hiroyuki, Parker, Peter J., Vitale, Nicolas, Meunier, Frederic A.
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container_end_page 2813
container_issue 5
container_start_page 2804
container_title The Journal of biological chemistry
container_volume 283
creator Osborne, Shona L.
Wen, Peter J.
Boucheron, Christine
Nguyen, Hao N.
Hayakawa, Masahiko
Kaizawa, Hiroyuki
Parker, Peter J.
Vitale, Nicolas
Meunier, Frederic A.
description 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 Ca2+-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.
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Two phosphoinositides, phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3-phosphate, are important for the ATP-dependent priming of the secretory apparatus prior to Ca2+-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. 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subjects Animals
Cattle
Chromaffin Cells - physiology
Exocytosis - physiology
Humans
In Vitro Techniques
Mice
Neurosecretory Systems - drug effects
Neurosecretory Systems - physiology
PC12 Cells
Phosphatidylinositol 3-Kinases - genetics
Phosphatidylinositol 3-Kinases - physiology
Phosphoinositide-3 Kinase Inhibitors
Rats
Recombinant Proteins - genetics
Recombinant Proteins - pharmacology
RNA Interference
RNA, Small Interfering - genetics
Transfection
title PIKfyve Negatively Regulates Exocytosis in Neurosecretory Cells
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