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.
Highlights
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)
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]
Summary
Antibodies—PIKfyve antiserum was raised by inoculating a rabbit with a synthetic peptide corresponding to the last 18 amino acids of mouse PIKfyve (NP_035216). Aliquots of the supernatant were taken at the end of each experiment, and cells were lysed with 1% (v/v) Triton X-100 (Sigma) Both sets of samples were assayed fluorometrically for catecholamines, and the amount released was expressed as a percentage of the total catecholamine content of the cells [12, 28, 29]. Cells were incubated for 20 min in Buffer A at 37 °C, 5% CO2 with the indicated concentrations of YM-201636 or YM-211387 prior to fixation. Quantification of the area and size of the early endosomes was carried out by image analysis on EEA1 staining using Laserpix software (Bio-Rad). Analysis of Time Lapse Confocal Images—Labeled organelles that remained in the same optical section throughout the duration of imaging were selected for intensity analysis. Values are expressed as mean Ϯ S.E., and data were considered significant at p Ͻ 0.05 (*) and p Ͻ 0.01 (**) unless otherwise stated (Student’s t test)
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