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.

F-actin does not modulate the initial steps of the protein kinase C activation process in living nerve cells.

Actin is a major substrate for protein kinase C (PKC) and PKC is considered a modulator of the actin network. In addition in vitro studies (Biochemistry 39 (2000) 271) have suggested that all PKC isoforms bind to actin during the process of activation of the enzyme. To test the physiological significance of such a coupling we used living PC12 cells and primary cultures of cerebellar granule cells. When PC12 cells were treated with either latrunculin B, which impairs actin polymerization, or phalloidin, which stabilizes actin filaments, we observed a significant reduction of the [Ca2+]i response revealed by Fura-2 fluorescence, while the PKC conformational changes followed by Fim-1 fluorescence were unaffected. The responses induced either by cell depolarization or muscarinic receptor activation were similarly affected by the toxin treatment of PC12 cells. In cerebellar granule cells the [Ca2+]i response induced by KCl depolarization was increased by latrunculin treatment, whereas no effect was observed on the PKC response. Latrunculin had no effect on the NMDA-induced responses in these cells. Finally we also show that the response induced by a long-lasting depolarization, which mimics stimulation leading to neuronal plasticity, was not significantly altered by latrunculin or phalloidin treatment of the cells. These results suggest that the actin network is not involved in the initial steps of the PKC activation process in living nerve cells.