title: Detecting disulfide-bound complexes and the oxidative regulation of cyclic nucleotide-dependent protein kinases by H2O2.
abstract: Hydrogen peroxide regulates intracellular signaling by oxidatively converting susceptible cysteine thiols to a modified state, which includes the formation of intermolecular disulfides. This type of oxidative modification can occur within the cAMP- and cGMP-dependent protein kinases often referred to as PKA and PKG, which have important roles in regulating cardiac contractility and systemic blood pressure. Both kinases are stimulated through conical pathways that elevate their respective cyclic nucleotides leading to direct kinase stimulation. However, PKA and PKG can also be functionally modulated independently of cyclic nucleotide stimulation through direct cysteine thiol oxidation leading to intermolecular disulfide formation. In the case of PKG, the formation of an intermolecular disulfide between two parallel dimeric subunits leads to enhanced kinase affinity for substrate. For PKA, the formation of two intermolecular disulfides between antiparallel dimeric regulatory RI subunits increases the affinity of this kinase for its binding partners, the A-kinase anchoring proteins, leading to increased PKA localization to its substrates. In this chapter, we describe the methods for detecting intermolecular disulfide-bound proteins and monitoring PKA and PKG oxidation within biological samples.
authors.affiliation: King's College London, Cardiovascular Division, The Rayne Institute, London, United Kingdom.
authors.forename: Joseph R
authors.initials: JR
authors.lastname: Burgoyne
authors.forename: Philip
authors.initials: P
authors.lastname: Eaton
mesh_terms: D002478:Cells, Cultured; D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit / Q000737:chemistry / Q000378:metabolism*; D062965:Cyclic GMP-Dependent Protein Kinase Type I / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D004220:Disulfides / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000378:metabolism / Q000494:pharmacology*; D007150:Immunohistochemistry; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D054756:Cyclic AMP-Dependent Protein Kinase RIalpha Subunit; D004220:Disulfides; C517834:PRKAR1A protein, human; D006861:Hydrogen Peroxide; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D003545:Cysteine
keywords: Disulfide; Kinase; Oxidative stress; Redox; Signaling; Thiol