Rho-associated kinase of chicken gizzard smooth muscle
Rho-associated kinase of chicken gizzard smooth muscle
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DOI：
10.1074/jbc.274.6.3744
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发表时间：
1999-02-05
影响因子：
4.8
通讯作者：
Nakano, T
中科院分区：
文献类型：
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作者：
Feng, JH;Ito, M;Nakano, T
从鸡胗平滑肌中提取rho相关激酶（Rho-kinase, Rho-kinase），经sds -聚丙烯酰胺凝胶电泳纯化达到明显的均匀性（160 kDa），鉴定为ROK α亚型。肌球蛋白磷酸酶靶亚基1 （MYPT1）、肌球蛋白和20 kda肌球蛋白轻链的表达率高于其他底物，MYPT1的硫代磷酸化抑制了肌球蛋白磷酸酶的活性。肌凝蛋白丝氨酸19处的磷酸化增加了肌动蛋白激活的Mg+- atp酶活性，即类似于肌凝蛋白轻链激酶。离子强度增大时，肌球蛋白磷酸化增加，可能是由于6s肌球蛋白的形成；离子强度增大时，分离的轻链和肌球蛋白磷酸酶的磷酸化降低。鸟苷5′- o -3-（硫）三磷酸刺激Rhokinase 1.5-2倍。而有限的胰蛋白酶水解引起5-6倍的激活，独立于RhoA。筛选了几种激酶抑制剂，其中最有效的是Y-27632， staurosporine和H-89。几种脂质引起RhoA激酶轻微活化，但花生四烯酸（30-50 μ M）诱导5-6倍的活化，不依赖于RhoA。这些结果表明，平滑肌rho激酶可能通过MYPT1和myosin的磷酸化参与收缩过程，花生四烯酸的激活可能是rho激酶的调控机制。
Rho-associated kinase (Rho-kinase) from chicken gizzard smooth muscle was purified to apparent homogeneity (160 kDa on SDS-polyacrylamide gel electrophoresis) and identified as the ROK alpha isoform, Several substrates were phosphorylated. Rates with myosin phosphatase target subunit 1 (MYPT1), myosin, and the 20-kDa myosin light chain were higher than other substrates, Thiophosphorylation of MYPT1 inhibited myosin phosphatase activity. Phosphorylation of myosin at serine 19 increased actin-activated Mg+-ATPase activity, i.e, similar to myosin light chain kinase. Myosin phosphorylation was increased at higher ionic strengths, possibly by formation of 6 S myosin, Phosphorylation of the isolated light chain and myosin phosphatase was decreased by increasing ionic strength. Rhokinase was stimulated 1.5-2-fold by guanosine 5'-O-3-(thio)triphosphate.RhoA whereas limited tryptic hydrolysis caused a 5-6-fold activation, independent of RhoA. Several kinase inhibitors were screened and most effective were Y-27632, staurosporine, and H-89, Several lipids caused slight activation of Rho-kinase, but arachidonic acid (30-50 mu M) induced a 5-6-fold activation, independent of RhoA. These results suggest that Rho-kinase of smooth muscle may be involved in the contractile process via phosphorylation of MYPT1 and myosin, Activation by arachidonic acid presents a possible regulatory mechanism for Rho-kinase.