Regulation of cAMP-dependent protein kinase activity by glutathionylation.
The catalytic subunit of cAMP-dependent protein kinase (cAPK) is susceptible to inactivation by a number of thiol-modifying reagents. Inactivation occurs through modification of cysteine 199, which is located near the active site. Because cysteine 199 is reactive at physiological pH, and modification of this site inhibits activity, we hypothesized that cAPK is a likely target for regulation by an oxidative mechanism, specifically glutathionylation. In vitro studies demonstrated the susceptibility of kinase activity to the sulfhydryl oxidant diamide, which inhibited by promoting an intramolecular disulfide bond between cysteines 199 and 343. In the presence of a low concentration of diamide and reduced glutathione, the kinase was rapidly and reversibly inhibited by glutathionylation. Mutant kinase containing an alanine to cysteine mutation at position 199 was resistant to inhibition by both diamide and glutathionylation, thus implicating this as the oxidation-sensitive site. Mouse fibroblast cells treated with diamide showed a reversible decrease in cAPK activity. Inhibition was dramatically enhanced when cells were first treated with cAPK activators. Using biotin-cysteine as means for detecting and purifying thiolated cAPK from cells, we were able to show that, under conditions in which cAPK is inactivated by diamide, it is also readily thiolated.
Humphries
Kenneth M
KM
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry, The University of California, San Diego, La Jolla, California 92093-0654, USA.
Juliano
Celina
C
Taylor
Susan S
SS
D000409:Alanine; D000595:Amino Acid Sequence; D001665:Binding Sites; D019921:Biotinylation; D017124:Conserved Sequence; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine; D003958:Diamide / Q000494:pharmacology; D005978:Glutathione / Q000494:pharmacology*; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011499:Protein Processing, Post-Translational; D021122:Protein Subunits / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013438:Sulfhydryl Compounds / Q000378:metabolism
D007527:Isoenzymes; D021122:Protein Subunits; D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D003958:Diamide; D017868:Cyclic AMP-Dependent Protein Kinases; D005978:Glutathione; D003545:Cysteine; D000409:Alanine