PIKfyve Negatively Regulates Exocytosis in Neurosecretory Cells

Abstract

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.Neurotransmitters and hormones are released from their storage vesicles into the extracellular space via calciumdependent exocytosis. Much work has been directed toward identifying the proteins involved in and regulating the exocytic process. These include members of the SNARE family of proteins, synaptotagmins (1, 2, 4, 5, 13), and a host of regulatory proteins, such as Munc18a (6, 7). In addition to the critical role played by membrane proteins, there is an increasing amount of literature pointing to the importance of lipids, in particular phosphoinositides, in the regulation of membrane trafficking events, including regulated exocytosis (8 -13).Phosphoinositides are generated by the reversible phosphorylation of the inositol head group of phosphatidylinositol (PtdIns)2 by an array of kinases and phosphatases at one or more of the 3-, 4-, and 5-OH positions (10). Previous studies have highlighted the importance of a plasma membrane pool of phosphatidylinositol 4,5-bisphosphate for regulated exocytosis of large dense core vesicles (LDCV) (14 -17).We recently demonstrated an involvement of the type II PI3K, PI3K-C2␣, and its product PtdIns(3)P in priming, an ATPdependent step during which LDCV acquire the ability to fuse with the plasma membrane (12). This novel role played by PtdIns(3)P in promoting the priming stage of exocytosis has opened a number of new research avenues regarding the mechanism(s) regulating the concentration of PtdIns(3)P and the potential role played by other 3-phosphorylated phosphoinositides in neurosecretory cells.Priming is a reversible step and must be tightly controlled to prevent too many vesicles from reaching the primed state. Pt...

The molecular basis of insulin-stimulated glucose uptake: signalling, trafficking and potential drug targets

“…We showed that insulin stimulates the phosphorylation of Ser318 in cells and that this phosphorylation plays a role in GLUT4 trafficking because over-expression of the non-activatable PIKfyve[S318A] mutant in 3T3-L1 adipocytes enhanced insulin-stimulated GLUT4 vesicle translocation to the plasma membrane (Berwick et al 2004). This observation is consistent with recent studies showing that pharmacologic inhibition (using the selective PIKfyve inhibitor YM201636 ( Jefferies et al 2008)) and siRNA-mediated knockdown of PIKfyve both potentiate secretory granule exocytosis in chromaffin and PC12 cells (Osborne et al 2007). Together, however, these observations are not consistent with Shisheva's studies (Ikonomov et al 2002(Ikonomov et al , 2007 although care should be taken in interpreting experiments where PIKfyve activity has been disrupted by siRNA-mediated knockdown, a kinase-inactive mutant or the PIKfyve inhibitor YM201636 because these manipulations all cause vacuolation of intracellular membranes (Shisheva et al 2001, Rutherford et al 2006, Jefferies et al 2008.…”

The myotubularin phosphatase MTMR4 regulates sorting from early endosomes

“…Expression of dominant-negative PIKfyve K1831E , or PIKfyve protein knockdown in mammalian cells, does not affect receptor internalization, Tfn recycling or receptor trafficking to lysosomes (Ikonomov et al, 2003;Rutherford et al, 2006). In mammalian cells, PIKfyve RNAi-mediated knockdown impairs early endosome-to-TGN traffic and potentiates exocytosis of neurosecretory vesicles (Rutherford et al, 2006;Osborne et al, 2008). However, we found that MTMR4 expression did not alter retrograde trafficking from early endosomes to the Golgi, as shown by the normal retrograde trafficking of cholera toxin from early endosomes to the Golgi (see supplementary material Fig.…”

Targeting the glial-derived neurotrophic factor and related molecules for controlling normal and pathologic pain

“…In nociceptive pathways, an ideal drug to target the release of endogenous GFLs from LGVs could act e.g. onto the phosphatidylinositol-3-phosphate [PtdIns(3)P] pool, which is sensitive to Ca 2+ modulation [90].…”