Interactions between the leucine-zipper motif of cGMP-Dependent protein kinase and the C-terminal region of the targeting subunit of myosin light chain phosphatase
Interactions between the leucine-zipper motif of cGMP-Dependent protein kinase and the C-terminal region of the targeting subunit of myosin light chain phosphatase
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DOI：
10.1016/j.jmb.2007.08.049
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发表时间：
2007-11-09
影响因子：
5.6
通讯作者：
Tao, Terence C.
中科院分区：
文献类型：
--
作者：
Lee, Eunhee;Hayes, David B.;Tao, Terence C.
一氧化氮通过增加cGMP的产生来诱导血管舒张，cGMP是cGMP依赖性蛋白激酶（PKG）的激活剂。PKG随后部分通过激活肌球蛋白轻链磷酸酶（MLCP）引起平滑肌松弛。迄今为止，PKG与MLCP靶向亚基（MYPT 1）之间的相互作用尚未完全了解。一组工作人员的早期研究表明，PKG与MYPT 1的结合分别由两种蛋白质的N端和C端的亮氨酸拉链基序介导。然而，另一个研究小组报道PKG与MYPT 1的结合不需要MYPT 1的亮氨酸拉链基序。在这项工作中，我们充分利用生物物理技术PKG和MYPT 1之间的相互作用的特点。为了这个目的，我们构建了一个重组PKG肽对应于一个预测的卷曲螺旋区，含有亮氨酸拉链基序。我们进一步构建了各种C-末端MYPT 1肽，其具有预测的卷曲螺旋区域、该卷曲螺旋区域之前的延伸和亮氨酸拉链基序的各种组合。我们的研究结果表明，首先，虽然亮氨酸拉链基序在N末端的PKG形成一个同源二聚体卷曲螺旋，在C末端的MYPT 1是单体和非螺旋。其次，PKG的亮氨酸拉链基序与MYPT 1的亮氨酸拉链基序结合形成异二聚体。第三，当MYPT 1的拉链基序不存在时，PKG亮氨酸拉链基序通过形成异源四聚体与MYPT 1的卷曲螺旋区和上游片段结合。这些结果提供了合理化的一些其他人使用替代结合分析的结果。(C)2007爱思唯尔有限公司保留所有权利。
Nitric oxide induces vasodilation by elevating the production of cGMP, an activator of cGMP-dependent protein kinase (PKG). PKG subsequently causes smooth muscle relaxation in part via activation of myosin light chain phosphatase (MLCP). To date, the interaction between PKG and the targeting subunit of MLCP (MYPT1) is not fully understood. Earlier studies by one group of workers showed that the binding of PKG to MYPT1 is mediated by the leucine-zipper motifs at the N and C termini, respectively, of the two proteins. Another group, however, reported that binding of PKG to MYPT1 did not require the leucine-zipper motif of MYPT1. In this work we fully characterized the interaction between PKG and MYPT1 using biophysical techniques. For this purpose we constructed a recombinant PKG peptide corresponding to a predicted coiled coil region that contains the leucine-zipper motif. We further constructed various C-terminal MYPT1 peptides bearing various combinations of a predicted coiled coil region, extensions preceding this coiled coil region, and the leucine-zipper motif. Our results show, firstly, that while the leucine-zipper motif at the N terminus of PKG forms a homodimeric coiled coil, the one at the C terminus of MYPT1 is monomeric and non-helical. Secondly, the leucine-zipper motif of PKG binds to that of MYPT1 to form a heterodimer. Thirdly, when the zipper motif of MYPT1 is absent, the PKG leucine-zipper motif binds to the coiled coil region and upstream segments of MYPT1 via formation of a heterotetramer. These results provide rationalization of some of the findings by others using alternative binding analyses. (C) 2007 Elsevier Ltd. All rights reserved.