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 PKCα, 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 PKCα labeled with yellow fluorescent protein to monitor kinase translocation. Simultaneously, [Ca2+]i changes were monitored with fura-2. Two point mutations that render PKCα inactive, D187A in the Ca2+ binding site and K368R in the ATP binding site, significantly prolonged the time-to-peak of the depolarization-evoked [Ca 2+]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 PKCα, and that PKCα can modulate the Ca2+ signal on a physiologically relevant time scale. Point mutations of PKCα can be used as specific and potent modulators of the PKC signaling pathway.
|Original language||English (US)|
|Number of pages||11|
|State||Published - Jun 7 2005|
- PC12 cells
- Voltage-gated Ca channels
ASJC Scopus subject areas