The activity of cGMP-dependent protein kinase I? 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 PKGI? and PKGI? 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 PKGI? activation has been proposed, in which PKGI? is activated in a cGMP-independent fashion via oxidation of Cys⁴³, resulting in disulfide formation within the PKGI? N-terminal dimerization domain. A "redox-dead" knock-in mouse containing a C43S mutation exhibits phenotypes consistent with decreased PKGI? signaling, but the detailed mechanism of oxidation-induced PKGI? activation is unknown. Therefore, we examined oxidation-induced activation of PKGI?, and in contrast to previous findings, we observed that disulfide formation at Cys⁴³ does not directly activate PKGI? in vitro or 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 PKGI? was induced by H₂O₂ Using purified enzymes, we found that the Cys⁴³ oxidation had no effect on basal kinase activity or K_m and V_max values; however, PKGI? containing the C43S mutation was less responsive to cGMP-induced activation. This reduction in cGMP affinity may in part explain the PKGI? loss-of-function phenotype of the C43S knock-in mouse. In conclusion, disulfide formation at Cys⁴³ does not directly activate PKGI?, and the C43S-mutant PKGI? has a higher K_a for cGMP. Our results highlight that mutant enzymes should be carefully biochemically characterized before making in vivo inferences.

SUBMITTER: Kalyanaraman H 

PROVIDER: S-EPMC5437233 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature