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Oxidative activation of protein kinase Cgamma through the C1 domain. Effects on gap junctions

Dingbo Lin1, Dolores J Takemoto

  • 1Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.

The Journal of Biological Chemistry
|January 12, 2005
PubMed

Insights

Reactive oxygen species (ROS) activate Protein Kinase C (PKC) gamma through direct oxidation, leading to gap junction inhibition. This mechanism may protect cells from oxidative stress.

Area of Science:

  • Biochemistry
  • Cell Biology
  • Neuroscience

Background:

  • Reactive oxygen species (ROS) accumulation is linked to chronic diseases.
  • Protein Kinase C (PKC) gamma, found in neurons and eyes, is activated by ROS.
  • Mutations in PKCgamma are associated with cerebellar ataxia, potentially due to impaired ROS activation and gap junction inhibition.

Purpose of the Study:

  • To elucidate the cellular mechanism of PKCgamma activation by hydrogen peroxide (H2O2).
  • To investigate the effects of H2O2-induced PKCgamma activation on gap junction activity.

Main Methods:

  • Studied H2O2 effects on PKCgamma activity and cellular diacylglycerol levels.
  • Utilized phosphatase inhibitor (Okadaic acid) and reductant (dithiothreitol) to assess activation pathways.
  • Examined PKCgamma C1 and C2 domain translocation using cell imaging.
  • Analyzed H2O2-induced modifications of PKCgamma via two-dimensional SDS-PAGE.
  • Investigated PKCgamma localization in lipid rafts and phosphorylation of connexin 43.

Main Results:

  • H2O2 activated PKCgamma independently of diacylglycerol and dephosphorylation.
  • Dithiothreitol abolished H2O2 effects, suggesting direct oxidation of the Cys-rich C1 domain.
  • H2O2 induced C1 domain translocation to plasma membranes.
  • PKCgamma formed disulfide bonds in response to H2O2.
  • H2O2-activated PKCgamma localized to lipid rafts and phosphorylated connexin 43 at Ser-368.
  • This phosphorylation led to gap junction plaque disassembly and reduced activity.

Conclusions:

  • H2O2 directly oxidizes the PKCgamma C1 domain, leading to enzyme activation.
  • Activated PKCgamma inhibits gap junction activity by phosphorylating connexin 43.
  • This inhibition of gap junctions may serve as a protective mechanism against oxidative stress.

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