Phosphatidylinositol 3-Kinase C2α Is Essential for ATP-dependent Priming of Neurosecretory Granule Exocytosis

Abstract

Neurotransmitter release and hormonal secretion are highly regulated processes culminating in the calcium-dependent fusion of secretory vesicles with the plasma membrane. Here, we have identified a role for phosphatidylinositol 3-kinase C2␣ (PI3K-C2␣) and its main catalytic product, PtdIns3P, in regulated exocytosis. In neuroendocrine cells, PI3K-C2␣ is present on a subpopulation of mature secretory granules. Impairment of PI3K-C2␣ function specifically inhibits the ATP-dependent priming phase of exocytosis. Overexpression of wild-type PI3K-C2␣ enhanced secretion, whereas transfection of PC12 cells with a catalytically inactive PI3K-C2␣ mutant or a 2xFYVE domain sequestering PtdIns3P abolished secretion. Based on these results, we propose that production of PtdIns3P by PI3K-C2␣ is required for acquisition of fusion competence in neurosecretion. INTRODUCTIONThe analysis of the molecular mechanism controlling neuroexocytosis has been greatly helped by studies on neurosecretory cells (Dunn and Holz, 1983;Sarafian et al., 1987;Martin and Walent, 1989;Monck and Fernandez, 1994;Rettig and Neher, 2002). Early work has shed light on two major steps consisting of the reversible ATP-dependent priming of docked granules followed by their Ca 2ϩ -driven fusion with the plasma membrane and release of the granule content (Holz et al., 1989;Hay and Martin, 1992). The spatiotemporal control of intracellular Ca 2ϩ concentration together with capacitance measurements has allowed the identification of distinct pools of chromaffin granules involved in these distinct steps (Rettig and Neher, 2002). Further electrophysiological and biochemical approaches, such as the reconstitution of secretion in permeabilized neurosecretory cells (Martin and Walent, 1989), have highlighted the role of key factors involved in these two processes. Cytosolic and membrane proteins have been linked to priming and fusion, including the mammalian orthologues of the Caenorhabditis elegans UNC13 and UNC18 gene products, synaptotagmins, and members of the SNARE family and associated proteins (Jahn and Sudhof, 1999;Burgoyne et al., 2001;Brose and Rosenmund, 2002;Rettig and Neher, 2002).In contrast, less is known about the contribution of lipid dynamics during these processes with the exception of phosphatidic acid and phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P 2 ]. The former is produced by phospholipase D, which is essential for secretion in neurons (Humeau et al., 2001) and in neuroendocrine cells . The inhibition of Ca 2ϩ -dependent catecholamine release after depletion of phosphatidylinositol highlighted the role of phosphoinositides during the secretory events (Eberhard et al., 1990). Moreover, phosphatidylinositol transfer protein and phosphatidylinositol-4-phosphate 5 kinase (PI4P5K) were shown to be required for the ATP-dependent priming of secretory granules in PC12 cells (Hay and Martin, 1993;Hay et al., 1995). In addition, phosphatidylinositol 4-kinase (PI4K), an integral membrane protein of chromaffin granules and synaptic vesicles, is r...

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

“…It was shown that synapsin1-associated PI-3K regulates the size of the ready-releasable pool of synaptic vesicles [32]. Considering the essential role of PI-3K in ATP-dependent priming [59] and that PI-3K inhibitors can compete with PIP for the kinase ATP-binding site [81,95], inhibition of fusion extent seen here (Figs. 8a, b and 9a, b) is likely caused by CV depriming in the fully docked state.…”

“…Considering the suggested roles of PIP species in exocytosis [5,36,59, 63], we studied their possible functions in Ca 2+ -triggered fusion. Eggs were treated with Wrt and LY29 to down-regulate PI-3K activity.…”

“…Thus, PI-3K inhibition can dramatically affect protein localization (i.e., PFM). Some PIP-binding proteins help regulate the secretory pathway [46,59]. A type IV P-type ATPase, required for flippase activity, is a PI(4)P effector [62], suggesting that increased PI(4)P levels (Fig.…”

A phosphoinositide conversion mechanism for exit from endosomes

“…PI(3)P, synthesized by PI3KC2α on chromaffin granules, positively regulated secretion (Meunier, Osborne et al 2005;Wen, Osborne et al 2008). However, PI(3)P turnover and/ or PI conversion was not addressed by this study.…”

Activity-driven relaxation of the cortical actomyosin II network synchronizes Munc18-1-dependent neurosecretory vesicle docking

“…Chromaffin cells were isolated from bovine adrenal glands 48,49 and maintained in Dulbecco's modified Eagle's medium supplemented with 10% serum supreme, 2.5 mg ml À 1 fungizone, 50 mg ml À 1 gentamycin and 10 mM HEPES on 0.1 mg ml À 1 poly-D-lysine-coated culture dishes (MatTek Corporation). Cells were transfected using electroporation (Amaxa Basic Nucleofector Kit for primary neurons (Lonza), Program X-001) and cultured at 37°C/5% CO 2 for at least 24 h before experimentation.…”