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Originally published as MBC in Press, 10.1091/mbc.E05-01-0067 on April 6, 2005

Vol. 16, Issue 6, 2848-2861, June 2005

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The ATP-dependent Membrane Localization of Protein Kinase C{alpha} Is Regulated by Ca2+ Influx and Phosphatidylinositol 4,5-Bisphosphate in Differentiated PC12 Cells

Consuelo Marín-Vicente, Juan C. Gómez-Fernández, and Senena Corbalán-García

Departamento de Bioquímica y Biología Molecular (A), Facultad de Veterinaria, Universidad de Murcia, E-30100 Murcia, Spain

Submitted January 25, 2005; Revised March 22, 2005; Accepted March 29, 2005
Monitoring Editor: John York

Signal transduction through protein kinase Cs (PKCs) strongly depends on their subcellular localization. Here, we investigate the molecular determinants of PKC{alpha} 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 PKC{alpha} 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 PKC{alpha} through its C1 domain, and in lieu, the presence of PtdIns(4,5)P2 increased the permanence of PKC{alpha} 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 PKC{alpha}, suggesting a possible role for this isoenzyme in the neural differentiation process. Overall, these results show a novel mechanism of PKC{alpha} activation in differentiated PC12 cells, where Ca2+ influx, together with the endogenous PtdIns(4,5)P2, anchor PKC{alpha} to the plasma membrane through two distinct motifs of its C2 domain, leading to enzyme activation.

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E05–01–0067) on April 6, 2005.

Abbreviations used: CFP, cyan fluorescent protein; DAG, diacylglycerol; DiC8, 1,2-sn-dioctanoylglycerol; GFP, green fluorescent protein; HBS, HEPES buffer saline; NGF, neural growth factor; PC12, pheochromocytoma cells; PH, pleckstrin homology; PI-PLC, phosphatidylinositol-specific-phospholipase C; PLD, phospholipase D; PtdCho, phosphatidylcholine; PtdOH, phosphatidic acid; PtdSer, phosphatidylserine; PtdIns(4,5)P2, phosphatidylinositol (4,5)-bisphosphate; YFP, yellow fluorescent protein.

Address correspondence to: Senena Corbalán-García (senena{at}um.es).




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