The COOH terminus of Rho-kinase negatively regulates Rho-kinase activity
The COOH terminus of Rho-kinase negatively regulates Rho-kinase activity
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DOI：
10.1074/jbc.274.45.32418
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发表时间：
1999-11-05
影响因子：
4.8
通讯作者：
Kaibuchi, K
中科院分区：
文献类型：
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作者：
Amano, M;Chihara, K;Kaibuchi, K
Rho激酶参与Rho下游肌球蛋白轻链的磷酸化，其被认为诱导非肌肉细胞中的平滑肌收缩和应力纤维形成。在这里，我们研究了Rho激酶抑制剂的作用方式。化合物如HA 1077和Y-32885不仅抑制Rho-激酶活性，而且抑制Rho的靶标之一蛋白激酶N的活性，但对强直性肌营养不良激酶相关Cdc 42-结合激酶β（MRCK β）的活性影响较小。含有Rho结合（RB）和普列克底物蛋白同源（PH）结构域的Rho激酶的COOH末端部分（RB/PH（TT）），其中引入点突变以消除Rho结合活性，与Rho-激酶相互作用，从而抑制Rho-激酶活性，而RB/PH（TT）对蛋白激酶N或MRCK β的活性没有影响，表明Rho激酶的COOH末端区域可能是Rho激酶的负调控区域。RB/PH（TT）的表达可特异性阻断Rho或Rho激酶活性形式诱导的NIH 3 T3细胞应力纤维和粘着斑的形成，但不能阻断MRCK β或肌球蛋白轻链活性形式诱导的应力纤维和粘着斑的形成。因此，RB/PH（TT）似乎在体内特异性抑制Rho激酶。
Rho-kinase is implicated in the phosphorylation of myosin Light chain downstream of Rho, which is thought to induce smooth muscle contraction and stress fiber formation in non-muscle cells. Here, we examined the mode of action of inhibitors of Rho-kinase. The chemical compounds such as HA1077 and Y-32885 inhibited not only the Rho-kinase activity but also the activity of protein kinase N, one of the targets of Rho, but had less of an effect on the activity of myotonic dystrophy kinase-related Cdc42-binding kinase beta (MRCK beta). The COOH-terminal portion of Rho-kinase containing Rho-binding (RB) and pleckstrin homology (PH) domains (RB/PH (TT)), in which point mutations were introduced to abolish the Rho binding activity, interacted with Rho-kinase and thereby inhibited the Rho-kinase activity, whereas RB/PH (TT) had no effect on the activity of protein kinase N or MRCK beta, suggesting that the COOH-terminal region of Rho-kinase is a possible negative regulatory region of Rho-kinase. The expression of RB/PH (TT) specifically blocked the stress fiber and focal adhesion formation induced by the active form of Rho or Rho-kinase in NIH 3T3 cells, but not that induced by the active form of MRCK beta or myosin light chain. Thus, RB/PH (TT) appears to specifically inhibit Rho kinase in vivo.