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 PKGIα can also be activated in vitro by oxidizing agents. Using in vitro kinase assays, here, we found that purified PKGIα 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 PKGIα Cys⁴³, Cys¹¹⁸, and Cys¹⁹⁶ play key roles in oxidation-induced kinase activation; we show that activation was reduced by C118A or C196V mutations, although C43S PKGIα activation was not reduced. In contrast, under the same conditions, purified PKGIβ activity only slightly increased with storage. Using PKGIα/PKGIβ chimeras, we found that residues throughout the PKGIα-specific autoinhibitory loop were responsible for this activation. To explore whether oxidants activate PKGIα in H9c2 and C2C12 cells, we monitored vasodilator-stimulated phosphoprotein phosphorylation downstream of PKGIα. While we observed PKGIα Cys⁴³ 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 PKGIα can be readily activated by oxidation in vitro, there is currently no direct evidence of oxidation-induced PKGIα activation in vivo.

I 型 cGMP 依赖性蛋白激酶 (PKGI) 是各种信号通路的重要组成部分，并且被一氧化氮和利钠肽诱导的 cGMP 生成典型地激活。然而，一些报道表明，PKGIα 也可以在体外被氧化剂激活。在这里，我们使用体外激酶测定法发现，即使在没有氧化剂的情况下，储存在 PBS 中的纯化 PKGIα 和 Flag 肽也会被氧化和活化。此外，一旦建立，这种激活就不能通过 DTT 的还原来逆转。我们证明了在储存前添加 Cu ²⁺可以增强活化，表明它是由氧化驱动并由储存期间存在的痕量金属介导的。以前的报告表明 PKGIα Cys⁴³、Cys ¹¹⁸和 Cys ¹⁹⁶在氧化诱导的激酶激活中起关键作用；我们显示 C118A 或 C196V 突变降低了激活，尽管 C43S PKGIα 激活没有降低。相反，在相同条件下，纯化的 PKGIβ 活性仅随着储存而略有增加。使用 PKGIα/PKGIβ 嵌合体，我们发现整个 PKGIα 特异性自抑制环的残基负责这种激活。为了探索氧化剂是否激活 H9c2 和 C2C12 细胞中的 PKGIα，我们监测了 PKGIα 下游的血管扩张剂刺激的磷蛋白磷酸化。虽然我们观察到 PKGIα Cys ⁴³响应 H ₂ O _2交联（表明细胞中的氧化环境），我们无法在这些条件下检测到血管扩张剂刺激的磷蛋白磷酸化增加。总之，我们得出结论，虽然 PKGIα 可以很容易地通过体外氧化激活，但目前没有直接证据表明体内氧化诱导的 PKGIα 激活。