Department of Medicine, University of California, San Diego, La Jolla, California, USA.
Verna and Marrs McLean Department of Biochemistry and Molecular Biology and the Department of Pharmacology and Chemical Biology, Baylor College of Medicine, Houston, Texas, USA.
J Biol Chem. 2020 Jul 24;295(30):10394-10405. doi: 10.1074/jbc.RA119.010984. Epub 2020 Jun 5.
Type 1 cGMP-dependent protein kinases (PKGs) play important roles in human cardiovascular physiology, regulating vascular tone and smooth-muscle cell phenotype. A mutation in the human PRKG1 gene encoding cGMP-dependent protein kinase 1 (PKG1) leads to thoracic aortic aneurysms and dissections. The mutation causes an arginine-to-glutamine (RQ) substitution within the first cGMP-binding pocket in PKG1. This substitution disrupts cGMP binding to the pocket, but it also unexpectedly causes PKG1 to have high activity in the absence of cGMP via an unknown mechanism. Here, we identified the molecular mechanism whereby the RQ mutation increases basal kinase activity in the human PKG1α and PKG1β isoforms. Although we found that the RQ substitution (R177Q in PKG1α and R192Q in PKG1β) increases PKG1α and PKG1β autophosphorylation , we did not detect increased autophosphorylation of the PKG1α or PKG1β RQ variant isolated from transiently transfected 293T cells, indicating that increased basal activity of the RQ variants in cells was not driven by PKG1 autophosphorylation. Replacement of Arg-177 in PKG1α with alanine or methionine also increased basal activity. PKG1 exists as a parallel homodimer linked by an N-terminal leucine zipper, and we show that the WT chain in WT-RQ heterodimers partly reduces basal activity of the RQ chain. Using hydrogen/deuterium-exchange MS, we found that the RQ substitution causes PKG1β to adopt an active conformation in the absence of cGMP, similar to that of cGMP-bound WT enzyme. We conclude that the RQ substitution in PKG1 increases its basal activity by disrupting the formation of an inactive conformation.
1 型 cGMP 依赖性蛋白激酶（PKGs）在人类心血管生理学中发挥重要作用，调节血管张力和平滑肌细胞表型。编码 cGMP 依赖性蛋白激酶 1（PKG1）的人 PRKG1 基因突变导致胸主动脉瘤和夹层。该突变导致 PKG1 中第一个 cGMP 结合口袋内的精氨酸到谷氨酰胺（RQ）取代。这种取代破坏了口袋与 cGMP 的结合，但它也通过未知机制导致 PKG1 在没有 cGMP 的情况下具有高活性。在这里，我们确定了 RQ 突变如何增加人 PKG1α 和 PKG1β 同工型的基础激酶活性的分子机制。虽然我们发现 RQ 取代（PKG1α 中的 R177Q 和 PKG1β 中的 R192Q）增加了 PKG1α 和 PKG1β 的自身磷酸化，但我们没有检测到从瞬时转染的 293T 细胞中分离出的 PKG1α 和 PKG1β RQ 变体的自身磷酸化增加，表明细胞中 RQ 变体的基础活性增加不是由 PKG1 自身磷酸化驱动的。PKG1α 中的精氨酸 177 被丙氨酸或甲硫氨酸取代也增加了基础活性。PKG1 作为通过 N 端亮氨酸拉链连接的平行同源二聚体存在，我们表明 WT-RQ 杂二聚体中的 WT 链部分降低了 RQ 链的基础活性。使用氢/氘交换 MS，我们发现 RQ 取代导致 PKG1β 在没有 cGMP 的情况下采用活性构象，类似于 cGMP 结合的 WT 酶。我们得出结论，PKG1 中的 RQ 取代通过破坏无活性构象的形成来增加其基础活性。
