title: Methods for studying oxidative regulation of protein kinase C.
abstract: The protein kinase C (PKC) family of isoenzymes may be a crucial player in transducing H2O2-induced signaling in a wide variety of physiological and pathophysiological processes. PKCs contain unique structural features that make them highly susceptible to oxidative modification. Depending on the site of oxidation and the extent to which it is modified, PKC can be either activated or inactivated by H2O2. The N-terminal regulatory domain contains zinc-binding, cysteine-rich motifs that are readily oxidized by H2O2. When oxidized, the autoinhibitory function of the regulatory domain is compromised, and as a result, PKC is activated in a lipid cofactor-independent manner. The C-terminal catalytic domain contains several reactive cysteine residues, which when oxidized with a higher concentration of H2O2 leads to an inactivation of PKC. Here, we describe the methods used to induce oxidative modification of purified PKC isoenzymes by H2O2 and the methods to assess the extent of this modification. Protocols are given for isolating oxidatively activated PKC isoenzymes from cells treated with H2O2. Furthermore, we describe the methods used to assess indirect regulation of PKC isoenzymes by determining their cytosol to membrane or mitochondrial translocation and tyrosine phosphorylation of PKCδ in response to sublethal levels of H2O2. Finally, as an example, we describe the methods used to demonstrate the role of H2O2-mediated cell signaling of PKCɛ in green tea polyphenol-induced preconditioning against neuronal cell death caused by oxygen-glucose deprivation and reoxygenation, an in vitro model for cerebral ischemic/reperfusion injury.
authors.affiliation: Department of Cell and Neurobiology, Keck School of Medicine, University of Southern California, Los Angeles, California, USA. rgopalak@usc.edu
authors.forename: Rayudu
authors.initials: R
authors.lastname: Gopalakrishna
authors.forename: Thomas H
authors.initials: TH
authors.lastname: McNeill
authors.forename: Albert A
authors.initials: AA
authors.lastname: Elhiani
authors.forename: Usha
authors.initials: U
authors.lastname: Gundimeda
mesh_terms: D000818:Animals; D001681:Biological Assay; D028241:Camellia sinensis / Q000737:chemistry; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000187:drug effects* / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology*; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000187:drug effects; D015240:Phorbol 12,13-Dibutyrate / Q000032:analysis; D010766:Phosphorylation; D010936:Plant Extracts / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D051381:Rats; D015398:Signal Transduction
chemical_list: D007527:Isoenzymes; D010936:Plant Extracts; D015240:Phorbol 12,13-Dibutyrate; D006861:Hydrogen Peroxide; D011493:Protein Kinase C
keywords: Cerebral ischemia; Hydrogen peroxide; Oxidative activation; Oxidative inactivation; Oxygen–glucose deprivation; Phorbol ester; Protein kinase C; Reoxygenation