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J. Biol. Chem., Vol. 283, Issue 5, 2804-2813, February 1, 2008

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PIKfyve Negatively Regulates Exocytosis in Neurosecretory Cells^*

Shona L. Osborne, Peter J. Wen, Christine Boucheron, Hao N. Nguyen, Masahiko Hayakawa^¶, Hiroyuki Kaizawa^¶, Peter J. Parker, Nicolas Vitale^||, and Frederic A. Meunier¹

From the Molecular Dynamics of Synaptic Function Laboratory, Queensland Brain Institute and School of Biomedical Sciences, University of Queensland, St. Lucia, Brisbane, Queensland 4072, Australia, Cancer Research UK, London Research Institute, Lincoln's Inn Fields Laboratories, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom, ^¶Astellas Pharma Inc., 21 Niyukigaoka, Tsukuba, Ibaraki 305-8585, Japan, and ^||Département Neurotransmission and Sécrétion Neuroendocrine, Institut des Neurosciences Cellulaires et Intégratives (UMR 7168/LC2), Centre National de la Recherche Scientifique and Université Louis Pasteur, 5 Rue Blaise Pascal, Strasbourg 67084, France

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.

Received for publication, June 13, 2007 , and in revised form, November 25, 2007.

^* This work was supported by Australian Research Council Grant DP0449683 (to F. A. M. and S. L. O.) and Cancer Research UK (to C. B. and P. J. P.), by Agence Nationale de la Recherche Grant ANR-05-BLAN-0326-01 (to N. V.), and by Association pour la Recherche sur le Cancer Grant 4051 (to N. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1–S4.

¹ To whom correspondence should be addressed. Tel.: 61-7-3346-6373; Fax: 61-7-3365-8836; E-mail: f.meunier{at}uq.edu.au.

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