Department of Cardiovascular Diseases, Mayo Medical School, Rochester, Minnesota 55905, USA.
J Biol Chem. 2011 Oct 28;286(43):37274-9. doi: 10.1074/jbc.M111.282905. Epub 2011 Sep 2.
Smooth muscle relaxation in response to NO signaling is due, in part, to a Ca(2+)-independent activation of myosin light chain (MLC) phosphatase by protein kinase G Iα (PKGIα). MLC phosphatase is a trimeric complex of a 20-kDa subunit, a 38-kDa catalytic subunit, and a 110-133-kDa myosin-targeting subunit (MYPT1). Alternative mRNA splicing produces four MYPT1 isoforms, differing by the presence or absence of a central insert and leucine zipper (LZ). The LZ domain of MYPT1 has been shown to be important for PKGIα-mediated activation of MLC phosphatase activity, and changes in LZ+ MYPT1 isoform expression result in changes in the sensitivity of smooth muscle to NO-mediated relaxation. Furthermore, PKGIα has been demonstrated to phosphorylate Ser-694 of MYPT1, but phosphorylation at this site does not always accompany cGMP-mediated smooth muscle relaxation. This study was designed to determine whether MYPT1 isoforms are differentially phosphorylated by PKGIα. The results demonstrate that purified LZ+ MYPT1 fragments are rapidly phosphorylated by PKGIα at Ser-667 and Ser-694, whereas fragments lacking the LZ domain are poor PKGIα substrates. Mutation of Ser-667 and Ser-694 to Ala and/or Asp showed that Ser-667 phosphorylation is more rapid than Ser-694 phosphorylation, suggesting that Ser-667 may play an important role in the activation of MLC phosphatase. These results demonstrate that MYPT1 isoform expression is important for determining the heterogeneous response of vascular beds to NO and NO-based vasodilators, thereby playing a central role in the regulation of vascular tone in health and disease.
平滑肌对 NO 信号的反应性松弛部分归因于蛋白激酶 G Iα（PKGIα）对肌球蛋白轻链（MLC）磷酸酶的钙非依赖性激活。MLC 磷酸酶是一个 20kDa 亚基、一个 38kDa 催化亚基和一个 110-133kDa 肌球蛋白靶向亚基（MYPT1）组成的三聚体复合物。替代 mRNA 剪接产生四种 MYPT1 同工型，区别在于中央插入和亮氨酸拉链（LZ）的存在或缺失。MYPT1 的 LZ 结构域对于 PKGIα 介导的 MLC 磷酸酶活性的激活非常重要，并且 LZ+MYPT1 同工型表达的变化导致平滑肌对 NO 介导的松弛的敏感性发生变化。此外，已经证明 PKGIα 可磷酸化 MYPT1 的 Ser-694 位点，但该位点的磷酸化并不总是伴随着 cGMP 介导的平滑肌松弛。本研究旨在确定 PKGIα 是否会使 MYPT1 同工型发生差异磷酸化。结果表明，纯化的 LZ+MYPT1 片段可被 PKGIα 在 Ser-667 和 Ser-694 位点快速磷酸化，而缺乏 LZ 结构域的片段则是 PKGIα 的不良底物。Ser-667 和 Ser-694 突变为 Ala 和/或 Asp 的突变表明 Ser-667 磷酸化比 Ser-694 磷酸化更快，提示 Ser-667 可能在 MLC 磷酸酶的激活中发挥重要作用。这些结果表明，MYPT1 同工型的表达对于确定血管床对 NO 和基于 NO 的血管扩张剂的异质性反应很重要，从而在健康和疾病中对血管张力的调节中发挥核心作用。
