Protein kinase Calpha modulates depolarizaton-evoked changes of intracellular Ca2+ concentration in a rat pheochromocytoma cell line.
Conventional protein kinase C (cPKC) isoforms are activated by a coincident rise in cytosolic Ca(2+) and membrane-bound diacylglycerol. In excitable cells, cPKC may be activated by Ca(2+) influx through voltage-gated Ca(2+) channels (VGCC). cPKCs, in turn, are known to modulate the activity of VGCC. We examined whether PKCalpha, 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 Ca(2+) concentration ([Ca(2+)](i)) signal. Pheochromocytoma cells (PC12 cells) were transfected with wild-type and mutant forms of PKCalpha labeled with yellow fluorescent protein to monitor kinase translocation. Simultaneously, [Ca(2+)](i) changes were monitored with fura-2. Two point mutations that render PKCalpha inactive, D187A in the Ca(2+) 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 [Ca(2+)](i) signal. We conclude that in PC12 cells, Ca(2+) entry through VGCC rapidly activates PKCalpha, and that PKCalpha can modulate the Ca(2+) signal on a physiologically relevant time scale. Point mutations of PKCalpha can be used as specific and potent modulators of the PKC signaling pathway.
Fontainhas
A M
AM
Department of Pharmacology and Physiology, University of Medicine and Dentistry New Jersey-New Jersey Medical School, Newark, NJ 07101-1709, USA.
Obukhov
A G
AG
Nowycky
M C
MC
D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism*; D003952:Diagnostic Imaging / Q000379:methods; D005110:Extracellular Space / Q000187:drug effects* / Q000378:metabolism; D016257:Fura-2 / Q000378:metabolism; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D016296:Mutagenesis / Q000502:physiology; D009154:Mutation; D016716:PC12 Cells; D010673:Pheochromocytoma / Q000473:pathology; D010766:Phosphorylation; D011189:Potassium Chloride / Q000494:pharmacology*; D011493:Protein Kinase C / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D051571:Protein Kinase C-alpha; D021381:Protein Transport / Q000187:drug effects / Q000502:physiology; D051381:Rats; D014162:Transfection / Q000379:methods
D001426:Bacterial Proteins; D008164:Luminescent Proteins; C054562:yellow fluorescent protein, Bacteria; D011189:Potassium Chloride; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D002118:Calcium; D016257:Fura-2