Oxidant-induced activation of protein kinase C in UC11MG cells.
Free radical formation and subsequent lipid peroxidation may participate in the pathogenesis of tissue injury, including the brain injury induced by hypoxia or trauma and cardiac injury arising from ischemia and reperfusion. However, the exact cellular mechanisms by which the initial oxidative insult leads to the ultimate tissue damage are not known. A number of reports have indicated that protein kinase C (PKC) may be activated following oxidative stress and that this enzyme may play an important role in the steps leading to cellular damage. In this work, we have examined in a cell model whether PKC is activated following oxidative exposure. UC11MG cells, a human astrocytoma cell line, were treated with H2O2. Incubation with 0.5 mM H2O2 increased malondialdehyde levels by as early as 15 minutes. To assess the effects of H2O2 treatment on PKC activation, we measured phosphorylation of an endogenous PKC substrate, the MARCKS (myristoylated alanine-rich C kinase substrate) protein. Treatment of cells with 0.2-1.0 mM H2O2 resulted in a rapid increase in MARCKS phosphorylation. Phosphorylation was stimulated approximately 2.5-fold following treatment with 0.5 mM H2O2 for ten minutes. Treatment with phorbol 12-myristate 13-acetate, a PKC activator, increased MARCKS phosphorylation approximately 4-fold. The H2O2-induced MARCKS phosphorylation was inhibited by the addition of the kinase inhibitors H-7 and staurosporine. Furthermore, specific down-regulation of PKC by phorbol ester also inhibited H2O2-induced MARCKS phosphorylation. These results indicate that PKC is rapidly activated in cells following an oxidative exposure and that this cell system may be a good model to further investigate the role of PKC in regulating oxidative damage in the cell.
Brawn
M K
MK
Upjohn Company, Department of Cell Biology, Kalamazoo, Michigan 49007.
Chiou
W J
WJ
Leach
K L
KL
D015536:Down-Regulation / Q000187:drug effects; D004789:Enzyme Activation; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D016877:Oxidants / Q000494:pharmacology*; D018384:Oxidative Stress; D010766:Phosphorylation; D011493:Protein Kinase C / Q000187:drug effects*; D011506:Proteins / Q000378:metabolism; D013268:Stimulation, Chemical; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D016877:Oxidants; D011506:Proteins; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D006861:Hydrogen Peroxide; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate