Autophosphorylation of smooth muscle myosin light chain kinase at its regulatory domain.
Autophosphorylation of smooth muscle myosin light chain kinase at its regulatory domain.
复制标题
平滑肌肌球蛋白轻链激酶在其调节域的自磷酸化。
DOI：
10.1021/bi00015a031
复制
发表时间：
1995
期刊：
影响因子：
2.9
通讯作者：
Ikebe,M
中科院分区：
文献类型：
--
作者：
Tokui,T;Ando,S;Ikebe,M
1995年1月17日接收的修订版Mandarin pt ®摘要：平滑肌肌球蛋白轻链激酶的自磷酸化最初由Foyt et al. [Foyt，H. L.，& Means，A. R.（1985）J. Cyclic NucleotideProtein Phosphorylation Res.260，8978-8983]，然而，自磷酸化对激酶活性以及位点位置的影响尚未阐明。在这里，我们证明，MLCK是自磷酸化的三个网站，Thr 803，Ser 815，和Ser 823，这种磷酸化改变MLCK的活性。两个磷酸化位点位于激酶的调节结构域中，苏氨酸位点朝向自抑制区，丝氨酸位点（Ser 815）靠近钙调蛋白锚定位点。钙调素的结合可显著抑制自磷酸化。Thr 803的自磷酸化是一个分子内的过程，其附近的碱性氨基酸残基与肌球蛋白轻链磷酸化位点高度同源，表明调节位点与三维结构中的催化位点非常接近。苏氨酸位点的磷酸化激活了钙调素非依赖性活性，而丝氨酸位点的磷酸化由于对钙调素的亲和力降低而抑制了钙调素依赖性活性。肌球蛋白轻链激酶（Myosin Light Chain Kinase，MLCK）是钙调蛋白依赖性蛋白激酶家族的一员，广泛存在于脊椎动物细胞中，催化肌球蛋白轻链的磷酸化，分子量为20 000道尔顿。在平滑肌和非肌肉细胞中，这种酶在激活基于肌动球蛋白的细胞收缩性中起重要作用（Hart-shome，1987; Sellers & Adelstein，1987），从而调节平滑肌细胞收缩性和胞质分裂（Warrick & Spudich，1987; Tan & Spudich，1992）。
Revised Manuscript Received January 17, 1995® abstract: Autophosphorylation of smooth muscle myosin light chain kinase was initially reported by Foyt et al.[Foyt, H. L., & Means, A. R.(1985) J. Cyclic NucleotideProtein Phosphorylation Res. 260, 8978—8983], however, the effects of autophosphorylation on the kinase activity as well as the location of the sites have not been elucidated. Here we demonstrate that MLCK is autophosphorylated at three sites, Thr 803, Ser 815, and Ser 823, and this phosphorylation alters MLCK activity. Twophosphorylation sites are located in the regulatory domain of the kinase, thethreonine site toward the autoinhibitory region and the serine site (Ser 815) in close proximity to the calmodulin anchoring site. The autophosphorylation was significantly inhibited by the binding of calmodulin. The autophosphorylation at Thr 803 is an intramolecular process, and the alignment of the basic amino acid residues nearby Thr 803 was highly homologous to the phosphorylation site of myosin light chain, suggesting thatthe regulatory site is in close proximity to the catalytic site in the three-dimensional structure. The phosphorylation at thethreonine site activated the calmodulin-independent activity while the phosphorylation at the serine site inhibited the calmodulin-dependent activity due to a decrease in the affinity for calmodulin. This finding shows another example of the activation of calmodulin-dependent kinases by autophosphorylation at its autoinhibitory region and provides a new clue for understanding the calmodulin/MLCK signalling pathway.Myosin light chain kinase (MLCK), 1 a family of calmodulin-dependent protein kinases, widely distributed in many vertebrate cells, catalyzes the phosphorylation of the 20 000 dalton light chain of myosin. In smooth muscle and nonmuscle cells, this enzyme plays an important role in activating actomyosin-based contractility of the cells (Hart-shome, 1987; Sellers & Adelstein, 1987) and thus regulates smooth muscle cellcontractility and cytokinesis (Warrick & Spudich, 1987; Tan & Spudich, 1992).