Abstract
Exocytosis is one of the most fundamental cellular events. The basic mechanism of the final step, membrane fusion, is mediated by the formation of the SNARE complex, which is modulated by the phosphorylation of proteins controlled by the concerted actions of protein kinases and phosphatases. We have previously shown that a protein phosphatase-1 (PP1) anchoring protein, phospholipase C-related but catalytically inactive protein (PRIP), has an inhibitory role in regulated exocytosis. The current study investigated the involvement of PRIP in the phospho-dependent modulation of exocytosis. Dephosphorylation of synaptosome-associated protein of 25 kDa (SNAP-25) was mainly catalyzed by PP1, and the process was modulated by wild-type PRIP but not by the mutant (F97A) lacking PP1 binding ability in in vitro studies. We then examined the role of PRIP in phospho-dependent regulation of exocytosis in cell-based studies using pheochromocytoma cell line PC12 cells, which secrete noradrenalin. Exogenous expression of PRIP accelerated the dephosphorylation process of phosphorylated SNAP-25 after forskolin or phorbol ester treatment of the cells. The phospho-states of SNAP-25 were correlated with noradrenalin secretion, which was enhanced by forskolin or phorbol ester treatment and modulated by PRIP expression in PC12 cells. Both SNAP-25 and PP1 were co-precipitated in anti-PRIP immunocomplex isolated from PC12 cells expressing PRIP. Collectively, together with our previous observation regarding the roles of PRIP in PP1 regulation, these results suggest that PRIP is involved in the regulation of the phospho-states of SNAP-25 by modulating the activity of PP1, thus regulating exocytosis.
Highlights
Phospho-modulation of SNARE function has not yet been shown
We found that dephosphorylation of synaptosome-associated protein of 25 kDa (SNAP-25) phosphorylated by protein kinase (PKA) and protein kinase C (PKC) in vitro was mainly catalyzed by PP1, the process of which was modulated by wild-type phospholipase C-related but catalytically inactive protein (PRIP)-1 but not by mutant PRIP-1 (F97A) lacking the ability to bind to PP1
The phospho-pattern of SNAP-25 was similar to that in cells expressing GFP alone or mutant PRIP (F97A). These results indicate that the dephosphorylation process of SNAP-25 catalyzed mainly by PP1 was accelerated by the presence of PRIP-1 in PC12 cells, and PP1 binding ability is required for PRIP to execute the role in regulating the dephosphorylation of SNAP-25
Summary
Phospho-modulation of SNARE function has not yet been shown. Results: Phospho-SNAP-25 was dephosphorylated by protein phosphatase-1, whose activity was regulated by PRIP, regulating exocytosis. In the present study, we investigated the possible involvement of PRIP in the phospho-regulation of exocytosis through modulation of the dynamics of protein phosphorylation For this purpose, we employed rat pheochromocytoma cell line PC12 cells, which express no intrinsic PRIP-1 and -2, as a cell-based experimental model, because they have many characteristics of adrenal chromaffin cells, including the ability to secrete catecholamines [39, 40], and we have recently optimized the experimental procedures to prepare permeabilized cells with a preserved mechanism for exocytosis for manipulation from outside of cells [41]. Together with our previous observation of the roles of PRIP in PP1 regulation, the results suggest that PRIP is involved in the phospho-state of SNAP-25 through modulating the activity of protein phosphatase, regulating exocytosis
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