Verin A D, Gilbert-McClain L I, Patterson C E, Garcia J G
Department of Medicine, Physiology and Biophysics, Indiana University School of Medicine, Richard Roudebush Veterans Administration Center, Indianapolis, Indiana, USA.
Am J Respir Cell Mol Biol. 1998 Nov;19(5):767-76. doi: 10.1165/ajrcmb.19.5.3126.
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.
特定的血管通透性模型严重依赖于肌球蛋白轻链磷酸化，这一反应由一种新的高分子量（214 kD）钙/钙调蛋白（CaM）依赖性肌球蛋白轻链激酶（MLCK）亚型催化，该亚型最近在人内皮细胞中克隆得到（《美国呼吸细胞与分子生物学杂志》，1997年；16:489 - 494）。为了评估丝氨酸蛋白酶凝血酶引发的内皮细胞（EC）屏障功能障碍的机制，我们研究了牛内皮细胞中表达的214-kD EC MLCK亚型的调节。EC MLCK亚型以钙依赖的方式结合生物素化的CaM，并与肌球蛋白、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活性，同时该酶随时间依赖性地转位至肌动蛋白细胞骨架。为了评估EC MLCK活性是否受直接磷酸化调节，氨基酸序列分析确定了多个丝氨酸/苏氨酸磷酸化的潜在EC MLCK位点，包括靠近CaM结合区域的环磷酸腺苷依赖性蛋白激酶A（PKA）的高度保守磷酸化位点。PKA介导的MLCK磷酸化或丝氨酸/苏氨酸磷酸酶活性的抑制（氟化物或花萼海绵诱癌素）均可减弱EC MLCK活性，这显著增加了MLCK磷酸化，同时降低了MLCK活性（降低3至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，但凝血酶介导的EC收缩和屏障功能障碍需要增加EC MLCK - 肌动蛋白相互作用以及MLCK转位至细胞骨架。EC MLCK活性似乎高度依赖于这一关键收缩效应器的磷酸化状态。
