The Ptdins(4,5)P2 ligand itself influences the localization of PKCα in the plasma membrane of intact living cells

Rapamycin-triggered heterodimerization strategy is becoming an excellent tool for rapidly modifying phosphatidylinositol(4,5)-bisphosphate [PtdIns (4,5)P-2] levels at the plasma membrane and for studying their influence in different processes. In this work, we studied the effect of modulation of the Ptdlns(4,5)P-2 concentration on protein kinase C (PKC) alpha membrane localization in intact living cells. We showed that an increase in the Ptdlns(4,5)P2 concentration enlarges the permanence of PKC(x in the plasma membrane when PC12 cells are stimulated with ATP, independently of the diacylglycerol generated. The depletion of this phosphoinositide decreases both the percentage of protein able to translocate to the plasma membrane and its permanence there. Our results demonstrate that the polybasic cluster located in the C2 domain of PKC alpha is responsible for this phosphoinositide-protein interaction. Furthermore, the C2 domain acts as a dominant interfering module in the neural differentiation process of PC12 cells, a fact that was also supported by the inhibitory effect obtained by knocking down PKC alpha with small interfering RNA duplexes. Taken together, these data demonstrate that Ptdlns(4,5)P2 itself targets PKCa. to the plasma membrane through the polybasic cluster located in the C2 domain, with this interaction being critical in the signaling network involved in neural differentiation. (C) 2007 Elsevier Ltd. All rights reserved.