Functional implication of neuronal calcium sensor-1 and phosphoinositol 4-kinase-beta interaction in regulated exocytosis of PC12 cells

Overview & Synopsis

Several studies have shown that the neuronal calcium sensor (NCS-1) and phosphoinositol 4-kinase-β (PI4K-β) 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-β from the intracellular compartment toward the plasma membrane. This recruitment was highly correlated to the intracellular Ca2+ rise induced by secretagogues. Using fluorescence resonance energy transfer between PI4K-β-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-β, since a mutation that prevented the membrane insertion of NCS-1 abolished NCS-1-PI4K-β 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-β, suggesting that the interaction of the two proteins on a membrane compartment is necessary for the secretory function. Moreover, extinction of endogenous PI4K-β 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-β complex in regulated exocytosis. 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 Ca2+. Formation of the SNARE3 complex between the vesicular synaptobrevin and the plasma membrane syntaxin and SNAP-25 is generally accepted to play a major role in Ca2+-triggered exocytosis. Although the neuronal calcium sensor (NCS-1) (1), orthologue of frequenin in invertebrates, and phosphoinositol 4-kinase-β (PI4K-β) (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 also found on the membrane of secretory vesicles (1, 3). NCS-1 has been shown to modulate Ca2+ membrane conductance through a direct effect on Ca2+ channels (2, 4, 5). Indeed, the C-terminal part of NCS-1 interacts directly with the β subunit of P/Q type voltage-gated calcium channels (5), and a C-terminal peptide of NCS-1 inhibited the facilitation current through P/Q calcium channels after NCS-1 injection (4). On the other hand, PI4K-β together with phosphoinositide 5-kinase produces phosphoinositol 4,5-diphosphate (PIP2), which can form phospholipid microdomains (6–8). Besides its effect on calcium channels, NCS-1 has also been shown to regulate PI4K-β activity (2, 9). Indeed, PI4K-β and NCS-1 have been shown to interact in vitro (10), in Madin-Darby canine kidney (11), COS-7 (12), and PC12 cells (13). It was first believed that NCS-1 undergoes a Ca2+-induced conformational change, which allows its membrane insertion in secretory granules through its myristoyl residue and the subsequent interaction with PI4K-β (14, 15). However, this model has been recently challenged (16, 17), since the myristoyl group of NCS-1 was reported to be freely accessible, and the membrane association of NCS-1 was independent of intracellular Ca2+ concentration. The aim of our study was to determine the conditions of NCS-1 and PI4K-β type III interaction in living cells and to correlate this interaction with the secretory function. For this purpose, we used living transfected PC12 cells overexpressing PI4K-β-ECFP and/or NCS-1-EYFP as fusion proteins. A stimulation-induced recruitment of NCS-1 and PI4K-β was observed by biochemical and immunofluorescence approaches as well as by time lapse fluorescence imaging and correlated with the intracellular Ca2+ concentration changes. The interaction between NCS-1 and PI4K-β in PC12 cells was demonstrated through fluorescence resonance energy transfer (FRET) between two chromophores and by co-immunoprecipitation. Finally, extinction experiments demonstrated the functional implication of the NCS-1-PI4K-β interaction in dense core secretory granule exocytosis.