Functional implication of neuronal calcium sensor-1 and phosphoinositol 4-kinase-beta interaction in regulated exocytosis of PC12 cells.
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.
de Barry
Jean
J
Institut des Neurosciences Cellulaires et Intégratives, UMR 7168 LC2 CNRS/ULP, 5 Rue B. Pascal, F-67084 Strasbourg Cedex, France. barry@neurochem.u-strasbg.fr
Janoshazi
Agnes
A
Dupont
Jean Luc
JL
Procksch
Odile
O
Chasserot-Golaz
Sylvette
S
Jeromin
Andreas
A
Vitale
Nicolas
N
D019870:1-Phosphatidylinositol 4-Kinase / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004558:Electric Stimulation; D005089:Exocytosis / Q000502:physiology*; D015870:Gene Expression; D051580:Neuronal Calcium-Sensor Proteins; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D009479:Neuropeptides / Q000235:genetics / Q000378:metabolism*; D016716:PC12 Cells; D051381:Rats; D022142:Secretory Vesicles / Q000502:physiology
D002135:Calcium-Binding Proteins; D051580:Neuronal Calcium-Sensor Proteins; D009479:Neuropeptides; C101464:frequenin calcium sensor proteins; D019870:1-Phosphatidylinositol 4-Kinase; D002118:Calcium