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

Neuroscience. 2005;133(2):393-403. doi: 10.1016/j.neuroscience.2005.02.026.

Abstract

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.

Publication types

  • Comparative Study
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Bacterial Proteins / metabolism
  • Calcium / metabolism*
  • Diagnostic Imaging / methods
  • Extracellular Space / drug effects*
  • Extracellular Space / metabolism
  • Fura-2 / metabolism
  • Humans
  • Luminescent Proteins / metabolism
  • Mutagenesis / physiology
  • Mutation
  • PC12 Cells
  • Pheochromocytoma / pathology
  • Phosphorylation
  • Potassium Chloride / pharmacology*
  • Protein Kinase C / chemistry
  • Protein Kinase C / genetics
  • Protein Kinase C / physiology*
  • Protein Kinase C-alpha
  • Protein Transport / drug effects
  • Protein Transport / physiology
  • Rats
  • Transfection / methods

Substances

  • Bacterial Proteins
  • Luminescent Proteins
  • yellow fluorescent protein, Bacteria
  • Potassium Chloride
  • PRKCA protein, human
  • Protein Kinase C
  • Protein Kinase C-alpha
  • Calcium
  • Fura-2