Enhanced dephosphorylation of cAMP-dependent protein kinase by oxidation and thiol modification.
The catalytic subunit of cAMP-dependent protein kinase (PKA) is phosphorylated at threonine 197 and serine 338. Phosphorylation of threonine 197, located in the activation loop, is required for coordinating the active site conformation and optimal enzymatic activity. However, this phosphorylation has not been widely appreciated as a regulatory site because of the apparent constitutive nature of the phosphorylation and the general resistance of the kinase to phosphatase treatment. We demonstrate here that the observed resistance of the catalytic subunit to dephosphorylation is due, in part, to the presence of the highly nucleophilic cysteine 199 located proximal to the phosphate on threonine 197. Experiments performed in vitro demonstrated that mutation (cysteine 199 to alanine), oxidation, such as by glutathionylation or internal disulfide bond formation, or alkylation of the C-subunit enhanced its ability to be dephosphorylated. Furthermore, rephosphorylation of reduced C-subunit by PDK1 created a cycle whereby the inactive kinase could be reactivated. To demonstrate that thiol modification of PKA can lead to enhanced dephosphorylation in vivo, PC12 cells were treated with N-ethylmaleimide (NEM). Such treatment resulted in complete PKA inactivation and dephosphorylation of threonine 197. This effect of NEM was contingent upon prior treatment of the cells with PKA activators, demonstrating the resistance of the holoenzyme to thiol alkylation-mediated dephosphorylation. Our results also demonstrated that NEM treatment of PC12 cells enhanced the dephosphorylation of the protein kinase Calpha activation loop, suggesting a common mechanism of regulation among members of the AGC family of kinases.
Humphries
Kenneth M
KM
Howard Hughes Medical Institute, Department of Chemistry and Biochemistry and Department of Pharmacology, The University of California, San Diego, La Jolla, California 92093-0654, USA.
Deal
Michael S
MS
Taylor
Susan S
SS
D000818:Animals; D001665:Binding Sites; D015153:Blotting, Western; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D005033:Ethylmaleimide / Q000494:pharmacology; D005978:Glutathione / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D009154:Mutation; D018384:Oxidative Stress; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism*; D016716:PC12 Cells; D010766:Phosphorylation; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D051571:Protein Kinase C-alpha; D017434:Protein Structure, Tertiary; D051381:Rats; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D013912:Threonine / Q000737:chemistry; D013997:Time Factors
D004220:Disulfides; D013438:Sulfhydryl Compounds; D013912:Threonine; D017868:Cyclic AMP-Dependent Protein Kinases; C496630:Prkca protein, mouse; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D005978:Glutathione; D003545:Cysteine; D005033:Ethylmaleimide; D010100:Oxygen