PKGIα is activated by metal-dependent oxidation in vitro but not in intact cells

Type I cGMP-dependent protein kinases (PKGIs) are important components of various signaling pathways and are canonically activated by nitric oxide- and natriuretic peptide-induced cGMP generation. However, some reports have shown that PKGI alpha can also be activated in vitro by oxidizing agents. Using in vitro kinase assays, here, we found that purified PKGI alpha stored in PBS with Flag peptide became oxidized and activated even in the absence of oxidizing agent; furthermore, once established, this activation could not be reversed by reduction with DTT. We demonstrate that activation was enhanced by addition of Cu2+ before storage, indicating it was driven by oxidation and mediated by trace metals present during storage. Previous reports suggested that PKGI alpha Cys(43), Cys(118), and Cys(196) play key roles in oxidation-induced kinase activation; we show that activation was reduced by C118A or C196V mutations, although C43S PKGI alpha activation was not reduced. In contrast, under the same conditions, purified PKGI beta activity only slightly increased with storage. Using PKGI alpha/PKGI beta chimeras, we found that residues throughout the PKGI alpha-specific autoinhibitory loop were responsible for this activation. To explore whether oxidants activate PKGI alpha in H9c2 and C2C12 cells, we monitored vasodilator-stimulated phosphoprotein phosphorylation downstream of PKGI alpha. While we observed PKGI alpha Cys(43) crosslinking in response to H2O2 (indicating an oxidizing environment in the cells), we were unable to detect increased vasodilator-stimulated phosphoprotein phosphorylation under these conditions. Taken together, we conclude that while PKGI alpha can be readily activated by oxidation in vitro, there is currently no direct evidence of oxidation-induced PKGI alpha activation in vivo.

Automated summary

This study found that purified PKGI alpha can be oxidized and activated during storage, indicating that oxidation and trace metals present during storage play a role in PKGI alpha activation. Previous research suggested that certain amino acids in PKGI alpha are key for oxidation-induced activation. However, there is currently no direct evidence of oxidation-induced PKGI alpha activation in vivo.