title: Purification of reversibly oxidized proteins (PROP) reveals a redox switch controlling p38 MAP kinase activity.
abstract: Oxidation of cysteine residues of proteins is emerging as an important means of regulation of signal transduction, particularly of protein kinase function. Tools to detect and quantify cysteine oxidation of proteins have been a limiting factor in understanding the role of cysteine oxidation in signal transduction. As an example, the p38 MAP kinase is activated by several stress-related stimuli that are often accompanied by in vitro generation of hydrogen peroxide. We noted that hydrogen peroxide inhibited p38 activity despite paradoxically increasing the activating phosphorylation of p38. To address the possibility that cysteine oxidation may provide a negative regulatory effect on p38 activity, we developed a biochemical assay to detect reversible cysteine oxidation in intact cells. This procedure, PROP, demonstrated in vivo oxidation of p38 in response to hydrogen peroxide and also to the natural inflammatory lipid prostaglandin J2. Mutagenesis of the potential target cysteines showed that oxidation occurred preferentially on residues near the surface of the p38 molecule. Cysteine oxidation thus controls a functional redox switch regulating the intensity or duration of p38 activity that would not be revealed by immunodetection of phosphoprotein commonly interpreted as reflective of p38 activity.
authors.affiliation: Department of Pathology, University of Virginia School of Medicine, Charlottesville, Virginia, United States of America.
authors.forename: Dennis J
authors.initials: DJ
authors.lastname: Templeton
authors.forename: Myo-Sabai
authors.initials: MS
authors.lastname: Aye
authors.forename: Joshua
authors.initials: J
authors.lastname: Rady
authors.forename: Fang
authors.initials: F
authors.lastname: Xu
authors.forename: Janet V
authors.initials: JV
authors.lastname: Cross
mesh_terms: D002623:Chemistry Techniques, Analytical / Q000379:methods*; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015151:Immunoblotting; D048308:Mitogen-Activated Protein Kinase 14; D008958:Models, Molecular; D009154:Mutation; D016877:Oxidants / Q000494:pharmacology; D010084:Oxidation-Reduction; D015230:Prostaglandin D2 / Q000031:analogs & derivatives / Q000494:pharmacology; D011487:Protein Conformation; D011506:Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000187:drug effects; D013438:Sulfhydryl Compounds / Q000378:metabolism; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D016877:Oxidants; D011506:Proteins; D013438:Sulfhydryl Compounds; C037112:9-deoxy-delta-9-prostaglandin D2; D006861:Hydrogen Peroxide; D048308:Mitogen-Activated Protein Kinase 14; D048051:p38 Mitogen-Activated Protein Kinases; D003545:Cysteine; D015230:Prostaglandin D2