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 Cys⁴³ does not directly activate PKGIα 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 PKGIα 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, 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 Cys⁴³ does not directly activate PKGIα, and the C43S-mutant PKGIα has a higher K_a for cGMP. Our results highlight that mutant enzymes should be carefully biochemically characterized before making in vivo inferences.

Keywords: Rho (Rho GTPase); VASP; allosteric regulation; cGMP-dependent protein kinase; cyclic GMP (cGMP); enzyme kinetics; oxidation-reduction (redox).

Figure 1.

PKGIα is not activated in intact cells by disulfide formation at Cys⁴³. A, 293T cells were transfected with expression vectors for Flag-tagged RhoA (500 ng) and untagged wild-type or C43S-mutant PKGIα (500 ng). The cells were treated for 60 min with 250 μm 8-CPT-cGMP, 100 μm H₂O₂, or vehicle alone, as indicated. RhoA Ser¹⁸⁸ phosphorylation was determined by SDS-PAGE under non-reducing conditions followed by immunoblotting with a RhoA Ser(P)¹⁸⁸ specific antibody. The amount of monomeric reduced (R) and disulfide-linked oxidized (O) PKGIα was determined by blotting with a PKGI specific antibody. B, performed as in A, except 293T cells were transfected with expression vectors for VSV-tagged VASP (200 ng) and wild-type or C43S-mutant PKGIα (200 ng), and blots were probed for VASP Ser(P)²³⁹. These experiments were repeated at least three times with similar results.

Figure 2.

Cys⁴³ oxidation does not activate PKGIα in vitro. A, Coomassie-stained gel demonstrating the purity and integrity of Flag-tagged wild-type and C43S-mutant PKGIα isolated from transiently transfected 293T cells. B, purified wild-type and C43S mutant PKGIα were incubated in the presence or absence of 15 mm DTT and exposed to air for 1 h; the amount of Cys⁴³-cross-linked PKGIα dimer was determined by non-reducing SDS-PAGE/immunoblotting. C and D, in vitro kinase assays were performed in the presence or absence of 10 μm cGMP using either Glasstide (C) or histone H1 (D) as substrates. Reactions were performed in triplicate from single protein preparations.

Figure 3.

Wild-type and C43S-mutant PKGIα enzyme kinetics. A, PKGIα was incubated in the presence or absence of 15 mm DTT for 1 h, and kinase assays were performed in the presence of 10 μm cGMP and increasing amounts of Glasstide. B, C43S PKGIα was incubated in the presence or absence of 15 mm DTT for 1 h, and kinase assays were performed as described in A. C, PKGIα activity was measured as in A, but in the absence of cGMP. D, C43S-mutant PKGIα activity was measured as in B, without cGMP. Reactions were performed in triplicate from single protein preparations, and repeated with two independent protein preps. E, Western blot showing relative amount of reduced (R) and Cys⁴³ oxidized (O) PKGIα in the kinase samples used for the reactions. V_max and K_m values were calculated using GraphPad Prism 7.

Figure 4.

C43S-mutant PKGIα has a decreased affinity for cGMP. A, purified Flag-tagged wild-type and C43S-mutant PKGIα were incubated in the presence or absence of 15 mm DTT, exposed to air for 1 h, and used in in vitro kinase reactions with 500 μm Glasstide as a substrate and increasing amounts of cGMP. Numbers are the averages of four values from two independent protein preparations, with reactions performed in duplicate. K_a values were calculated using GraphPad Prism 7. B, Western blot showing relative amount of reduced (R) and oxidized (O) PKG in the kinase samples used for the reactions.