Protein kinase Cα modulates depolarizaton-evoked changes of intracellular Ca2+ concentration in a rat pheochromocytoma cell

Conventional protein kinase C (cPKC) isoforms are activated by a coincident rise in cytosolic Ca2+ and membrane-bound diacylglycerol. In excitable cells, cPKC may be activated by Ca2+ influx through voltage-gated Ca2+ channels (VGCC). cPKCs, in turn, are known to modulate the activity of VGCC. We examined whether PKC alpha, a cPKC, could be activated by depolarization in a neuroendocrine cell line and whether activation occurred on a time scale that modulated the depolarization-evoked intracellular Ca2+ concentration ([Ca2+](i)) signal. Pheochromocytoma cells (PC12 cells) were transfected with wild-type and mutant forms of PKCa. labeled with yellow fluorescent protein to monitor kinase translocation. Simultaneously, [Ca2+](i) changes were monitored with fura-2. Two point mutations that render PKCa inactive, D187A in the Ca2+ binding site and K368R in the ATP binding site, significantly prolonged the time-to-peak of the depolarization-evoked [Ca2+](i) signal. A mutation that modulates membrane insertion (W58G) and two mutations of an autophosphorylation site (S657A, S657E) had no effect on the kinetics of the [Ca2+](i) signal. We conclude that in PC12 cells, Ca2+ entry through VGCC rapidly activates PKCa, and that PKC alpha can modulate the Ca2+ signal on a physiologically relevant time scale. Point mutations of PKC alpha can be used as specific and potent modulators of the PKC signaling pathway. (c) 2005 Published by Elsevier Ltd on behalf of IBRO.