The ATP-dependent membrane localization of protein kinase Calpha is regulated by Ca2+ influx and phosphatidylinositol 4,5-bisphosphate in differentiated PC12 cells.
Signal transduction through protein kinase Cs (PKCs) strongly depends on their subcellular localization. Here, we investigate the molecular determinants of PKCalpha localization by using a model system of neural growth factor (NGF)-differentiated pheochromocytoma (PC12) cells and extracellular stimulation with ATP. Strikingly, the Ca2+ influx, initiated by the ATP stimulation of P2X receptors, rather than the Ca2+ released from the intracellular stores, was the driving force behind the translocation of PKCalpha to the plasma membrane. Furthermore, the localization process depended on two regions of the C2 domain: the Ca2+-binding region and the lysine-rich cluster, which bind Ca2+ and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2], respectively. It was demonstrated that diacylglycerol was not involved in the localization of PKCalpha through its C1 domain, and in lieu, the presence of PtdIns(4,5)P2 increased the permanence of PKCalpha in the plasma membrane. Finally, it also was shown that ATP cooperated with NGF during the differentiation process of PC12 cells by increasing the length of the neurites, an effect that was inhibited when the cells were incubated in the presence of a specific inhibitor of PKCalpha, suggesting a possible role for this isoenzyme in the neural differentiation process. Overall, these results show a novel mechanism of PKCalpha activation in differentiated PC12 cells, where Ca2+ influx, together with the endogenous PtdIns(4,5)P2, anchor PKCalpha to the plasma membrane through two distinct motifs of its C2 domain, leading to enzyme activation.
Marín-Vicente
Consuelo
C
Departamento de Bioquímica y Biología Molecular (A), Facultad de Veterinaria, Universidad de Murcia, E-30100 Murcia, Spain.
Gómez-Fernández
Juan C
JC
Corbalán-García
Senena
S
D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000032:analysis / Q000378:metabolism*; D002454:Cell Differentiation; D002462:Cell Membrane / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D016257:Fura-2 / Q000494:pharmacology; D007700:Kinetics; D008239:Lysine / Q000378:metabolism; D018613:Microscopy, Confocal; D008954:Models, Biological; D009414:Nerve Growth Factors / Q000494:pharmacology; D016501:Neurites / Q000187:drug effects / Q000378:metabolism; D016716:PC12 Cells; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats
D009414:Nerve Growth Factors; D019269:Phosphatidylinositol 4,5-Diphosphate; D000255:Adenosine Triphosphate; D008239:Lysine; D002118:Calcium; D016257:Fura-2