title: The activity of cGMP-dependent protein kinase Iα is not directly regulated by oxidation-induced disulfide formation at cysteine 43.
abstract: The type I cGMP-dependent protein kinases (PKGs) are key regulators of smooth muscle tone, cardiac hypertrophy, and other physiological processes. The two isoforms PKGIα and PKGIβ are thought to have unique functions because of their tissue-specific expression, different cGMP affinities, and isoform-specific protein-protein interactions. Recently, a non-canonical pathway of PKGIα activation has been proposed, in which PKGIα is activated in a cGMP-independent fashion via oxidation of Cys43, resulting in disulfide formation within the PKGIα N-terminal dimerization domain. A "redox-dead" knock-in mouse containing a C43S mutation exhibits phenotypes consistent with decreased PKGIα signaling, but the detailed mechanism of oxidation-induced PKGIα activation is unknown. Therefore, we examined oxidation-induced activation of PKGIα, and in contrast to previous findings, we observed that disulfide formation at Cys43does not directly activate PKGIαin vitroor in intact cells. In transfected cells, phosphorylation of Ras homolog gene family member A (RhoA) and vasodilator-stimulated phosphoprotein was increased in response to 8-CPT-cGMP treatment, but not when disulfide formation in PKGIα was induced by H2O2Using purified enzymes, we found that the Cys43oxidation had no effect on basal kinase activity orKmandVmaxvalues; however, PKGIα containing the C43S mutation was less responsive to cGMP-induced activation. This reduction in cGMP affinity may in part explain the PKGIα loss-of-function phenotype of the C43S knock-in mouse. In conclusion, disulfide formation at Cys43does not directly activate PKGIα, and the C43S-mutant PKGIα has a higherKafor cGMP. Our results highlight that mutant enzymes should be carefully biochemically characterized before makingin vivoinferences.
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, California 92093.
authors.forename: Hema
authors.initials: H
authors.lastname: Kalyanaraman
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, California 92093.
authors.forename: Shunhui
authors.initials: S
authors.lastname: Zhuang
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, California 92093.
authors.forename: Renate B
authors.initials: RB
authors.lastname: Pilz
authors.affiliation: Department of Medicine, University of California, San Diego, La Jolla, California 92093. Electronic address: dcasteel@ucsd.edu.
authors.forename: Darren E
authors.initials: DE
authors.lastname: Casteel
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D006152:Cyclic GMP / Q000031:analogs & derivatives / Q000494:pharmacology; D062965:Cyclic GMP-Dependent Protein Kinase Type I / Q000235:genetics / Q000378:metabolism*; D004220:Disulfides / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects / Q000502:physiology; D055879:Gene Knock-In Techniques; D006801:Humans; D051379:Mice; D008822:Mice, Transgenic; D020125:Mutation, Missense; D010084:Oxidation-Reduction; D055503:Protein Multimerization / Q000187:drug effects / Q000502:physiology*; D013873:Thionucleotides / Q000494:pharmacology
chemical_list: D004220:Disulfides; D013873:Thionucleotides; C055656:8-((4-chlorophenyl)thio)cyclic-3',5'-GMP; D062965:Cyclic GMP-Dependent Protein Kinase Type I; D006152:Cyclic GMP
keywords: Rho (Rho GTPase); VASP; allosteric regulation; cGMP-dependent protein kinase; cyclic GMP (cGMP); enzyme kinetics; oxidation-reduction (redox)