Biochemical regulation of the nonmuscle myosin light chain kinase isoform in bovine endothelium.
Biochemical regulation of the nonmuscle myosin light chain kinase isoform in bovine endothelium.
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DOI：
10.1165/ajrcmb.19.5.3126
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发表时间：
1998
影响因子：
6.4
通讯作者：
A. Verin;L. Gilbert-McClain;C. Patterson;J. Garcia
中科院分区：
文献类型：
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作者：
A. Verin;L. Gilbert-McClain;C. Patterson;J. Garcia
血管通透性的特定模型严重依赖于肌球蛋白轻链磷酸化，这一反应是由一种新的高分子量（214 kD） Ca2+/钙调蛋白（CaM）依赖性肌球蛋白轻链激酶（MLCK）异构体催化的，该异构体最近在人内皮细胞中克隆出来。j .和。细胞分子生物学, 1997; 16:489 - 494)。为了探讨丝氨酸蛋白酶凝血酶诱发内皮细胞屏障功能障碍的机制，我们研究了214-kD EC MLCK亚型在牛内皮细胞中的表达调控。EC MLCK异构体以Ca2+依赖的方式结合生物素化CaM，并与肌凝蛋白、肌动蛋白和CaM在功能复合物中共同免疫沉淀。凝血酶迅速增加MLCK的活性，与时间依赖性易位的酶肌动蛋白细胞骨架一致。为了评估EC MLCK活性是否受到直接磷酸化的调节，氨基酸序列分析发现了EC MLCK中多个潜在的丝氨酸/苏氨酸磷酸化位点，包括与cam结合区相邻的环腺苷单磷酸依赖蛋白激酶A （PKA）高度保守的磷酸化位点。pka介导的MLCK磷酸化或丝氨酸/苏氨酸磷酸酶活性抑制（氟或花萼蛋白）均可降低EC MLCK活性，显著增加MLCK磷酸化，同时降低MLCK活性（降低3- 4倍）。总之，尽管EC MLCK亚型表现出该激酶家族固有的多种特征，凝血酶介导的EC收缩和屏障功能障碍需要EC MLCK-肌动蛋白相互作用和MLCK向细胞骨架的易位增加。EC MLCK活性似乎高度依赖于这一关键收缩效应物的磷酸化状态。
Specific models of vascular permeability are critically dependent on myosin light chain phosphorylation, a reaction catalyzed by a novel high molecular-weight (214 kD) Ca2+/calmodulin (CaM)-dependent myosin light chain kinase (MLCK) isoform recently cloned in human endothelium (Am. J. Respir. Cell Mol. Biol., 1997;16:489-494). To evaluate mechanisms of endothelial cell (EC) barrier dysfunction evoked by the serine protease thrombin, we studied the regulation of the 214-kD EC MLCK isoform expressed in bovine endothelium. The EC MLCK isoform bound biotinylated CaM in a Ca2+-dependent manner and co-immunoprecipitated in a functional complex with myosin, actin, and CaM. Thrombin rapidly increased MLCK activity in concert with time-dependent translocation of the enzyme to the actin cytoskeleton. To evaluate whether EC MLCK activity was regulated by direct phosphorylation, amino acid sequence analysis identified multiple potential EC MLCK sites for Ser/Thr phosphorylation, including highly conserved phosphorylation sites for cyclic adenosine monophosphate-dependent protein kinase A (PKA) adjacent to the CaM-binding region. EC MLCK activity was attenuated by either PKA-mediated MLCK phosphorylation or inhibition of Ser/Thr phosphatase activity (fluoride or calyculin), which significantly increased MLCK phosphorylation while decreasing MLCK activity (3- to 4-fold decrease). In summary, although the EC MLCK isoform exhibits multiple features intrinsic to this family of kinases, thrombin-mediated EC contraction and barrier dysfunction requires increased EC MLCK-actin interaction and MLCK translocation to the cytoskeleton. EC MLCK activity appears to be highly dependent upon the phosphorylation status of this key contractile effector.