Functional Implication of Neuronal Calcium Sensor-1 and Phosphoinositol 4-Kinase-β Interaction in Regulated Exocytosis of PC12 Cells

Jean De Barry,Agnes Janoshazi,Jean Luc Dupont,Odile Procksch,Sylvette Chasserot-Golaz,Andreas Jeromin,Nicolas Vitale

doi:10.1074/jbc.m509842200

Abstract

Several studies have shown that the neuronal calcium sensor (NCS-1) and phosphoinositol 4-kinase-beta (PI4K-beta) regulate the exocytotic process of nerve and neuroendocrine cells. The aim of our study was to investigate their possible interaction at rest and during stimulation in living cells and to decipher the role of this interaction in the secretory process. In PC12 cells, we observed a stimulation-induced recruitment of NCS-1 and PI4K-beta from the intracellular compartment toward the plasma membrane. This recruitment was highly correlated to the intracellular Ca(2+) rise induced by secretagogues. Using fluorescence resonance energy transfer between PI4K-beta-ECFP and NCS-1-EYFP, we show that both proteins are interacting in resting cells and that this interaction increases with stimulation. It appears that the membrane insertion of NCS-1 is necessary for the interaction with PI4K-beta, since a mutation that prevented the membrane insertion of NCS-1 abolished NCS-1-PI4K-beta interaction, as revealed by fluorescence resonance energy transfer analysis. Additionally, the overexpression of mutated NCS-1 prevents the stimulatory effect on secretion induced by PI4K-beta, suggesting that the interaction of the two proteins on a membrane compartment is necessary for the secretory function. Moreover, extinction of endogenous PI4K-beta by small interfering RNA inhibits secretion and completely prevents the stimulatory effect of NCS-1 on calcium-evoked exocytosis from permeabilized PC12 cells, showing directly for the first time the functional implication of a NCS-1.PI4K-beta complex in regulated exocytosis.

Highlights

Exocytosis in nerve cells and in neuroendocrine cells is mediated by the exocytotic fusion of synaptic vesicles and secretory granules with the plasma membrane in a process that is largely governed by Ca2ϩ
Localization of PI4K-␤ and NCS-1—In resting PC12 cells, endogenous PI4K-␤ immunoreactivity appeared as small patches inside the cytoplasm, and the density of the fluorescence was higher around the nucleus (Fig. 1), probably in the Golgi apparatus, consistent with earlier observations [1, 3]
No fluorescence staining of the cells was observed when the primary antibody was omitted, and the distribution pattern of PI4K-␤-ECFP was similar to endogenous PI4K-␤-immunoreactivity, albeit slightly more fuzzy

Summary

Introduction

Exocytosis in nerve cells and in neuroendocrine cells is mediated by the exocytotic fusion of synaptic vesicles and secretory granules (dense core vesicles) with the plasma membrane in a process that is largely governed by Ca2ϩ. The neuronal calcium sensor (NCS-1) [1], orthologue of frequenin in invertebrates, and phosphoinositol 4-kinase-␤ (PI4K-␤) [2] have been shown to play an important role in regulating synaptic vesicle and secretory granule exocytosis, the underlying molecular mechanisms are still not fully understood. These proteins are mostly found in the cytosol but are found on the membrane of secretory vesicles [1, 3]. Extinction experiments demonstrated the functional implication of the NCS-1-PI4K-␤ interaction in dense core secretory granule exocytosis

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Results

Conclusion