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)P2] levels at the plasma membrane and for studying their influence in different processes. In this work, we studied the effect of modulation of the PtdIns(4,5)P2 concentration on protein kinase C (PKC) α membrane localization in intact living cells. We showed that an increase in the PtdIns(4,5)P2 concentration enlarges the permanence of PKCα 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 PKCα 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 PKCα with small interfering RNA duplexes. Taken together, these data demonstrate that PtdIns(4,5)P2 itself targets PKCα 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.