Identification of a novel actin binding motif in smooth muscle myosin light chain kinase
Identification of a novel actin binding motif in smooth muscle myosin light chain kinase
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DOI：
10.1074/jbc.274.41.29433
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发表时间：
1999-10-08
影响因子：
4.8
通讯作者：
Stull, JT
中科院分区：
文献类型：
--
作者：
Smith, L;Su, XJ;Stull, JT
由Ca 2 +/钙调蛋白依赖性肌球蛋白轻链激酶催化的肌球蛋白的20-kDa调节轻链的磷酸化在平滑肌收缩和非肌肉细胞中的其他收缩过程的起始中是重要的。先前已经表明，平滑肌肌球蛋白轻链激酶的残基1-142对于高亲和力结合细胞中含肌动蛋白的细丝是必需的（1）。为了进一步定位结合所需的激酶区域，构建了一系列N-末端缺失突变体以及几种N-末端谷胱甘肽S-转移酶融合蛋白。共沉淀实验表明，含有1-75位残基的肽与纯化的平滑肌肌丝结合。此外，N端肽足以在体内与平滑肌细胞中的肌动蛋白应力纤维高亲和力结合。融合蛋白中的丙氨酸扫描诱变鉴定残基Asp-30、Phe-31、Arg-32和Leu-35对于体外结合是重要的。在残基2-7和58-63处存在两个另外的DFRXXL基序。这三个基序中的DFR残基分别被全长激酶中的丙氨酸残基取代。这些突变中的每一个都显著降低了肌球蛋白轻链激酶与肌丝的体外结合，并且每一个都废除了与平滑肌细胞中含肌动蛋白的细丝的高亲和力结合。这些结果确定了一个独特的结构基序组成的三个重复的共识序列在N端的肌球蛋白轻链激酶所需的高亲和力结合肌动蛋白的细丝。
Phosphorylation of the 20-kDa regulatory light chain of myosin catalyzed by a Ca2+/calmodulin-dependent myosin light chain kinase is important in the initiation of smooth muscle contraction and other contractile processes in non-muscle cells. It has been previously shown that residues 1-142 of smooth muscle myosin light chain kinase are necessary for high-affinity binding to actin-containing filaments in cells (1), To further localize the region of the kinase required for binding, a series of N-terminal deletion mutants as well as several N-terminal glutathione S-transferase fusion proteins were constructed. Cosedimentation assays showed that a peptide containing residues 1-75 binds to purified smooth muscle myofilaments, Furthermore, the N-terminal peptide was sufficient for high-affinity binding to actin stress fibers in smooth muscle cells in vivo. Alanine scanning mutagenesis in the fusion protein identified residues Asp-30, Phe-31, Arg-32, and Leu-35 as important for binding in vitro. There are two additional DFRXXL motifs located at residues 2-7 and 58-63. The DFR residues in these three motifs were individually replaced by alanine residues in the full-length kinase, Each of these mutations significantly decreased myosin light chain kinase binding to myofilaments in vitro, and each abolished high-affinity binding to actin-containing filaments in smooth muscle cells in vivo. These results identify a unique structural motif comprised of three repeat consensus sequences in the N terminus of myosin light chain kinase necessary for high-affinity binding to actin-containing filaments.