Functional implication of neuronal calcium sensor-1 and phosphoinositol 4-kinase-β interaction in regulated exocytosis of PC12 cells
Functional implication of neuronal calcium sensor-1 and phosphoinositol 4-kinase-β interaction in regulated exocytosis of PC12 cells
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DOI：
10.1074/jbc.m509842200
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发表时间：
2006-06-30
影响因子：
4.8
通讯作者：
Vitale, Nicolas
中科院分区：
文献类型：
--
作者：
de Barry, Jean;Janoshazi, Agnes;Vitale, Nicolas
一些研究表明，神经元钙感受器(NCS-1)和磷酸肌醇4-激酶-β(PI4K-β)调节神经和神经内分泌细胞的胞外过程。我们研究的目的是研究它们在静息和刺激时在活细胞中可能的相互作用，并破译这种相互作用在分泌过程中的作用。在PC12细胞中，我们观察到了刺激诱导的NCS-1和PI4K-β从细胞内室向质膜的募集。这种募集与促分泌剂诱导的细胞内钙升高高度相关。利用PI4K-β-ECFP和NCS-1-EYFP之间的荧光共振能量转移，我们证明了这两种蛋白质在静息细胞中相互作用，并且这种相互作用随着刺激的增加而增强。荧光共振能量转移分析显示，NCS-1的膜插入似乎是与PI4K-β相互作用所必需的，因为阻止NCS-1膜插入的突变取消了NCS-1-PI4K-β的相互作用。此外，突变的NCS-1的过表达阻止了PI4K-β对分泌的刺激作用，这表明这两种蛋白在膜间隔上的相互作用对于分泌功能是必要的。此外，内源性PI4K-β被小干扰RNA抑制分泌，并完全阻止NCS-1对通透性PC12细胞钙诱导的胞吐的刺激作用，首次直接显示了NCS-1中心点PI4K-β复合体在调节胞吐中的功能意义。
Several studies have shown that the neuronal calcium sensor (NCS-1) and phosphoinositol 4-kinase-beta (PI4K-beta) regulate the exo-cytotic 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 Ca2+ 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 center dot PI4K-beta complex in regulated exocytosis.