The molecular mechanism of p160ROCK-mediated signaling pathway

我们发现Rho相关的卷曲形成蛋白激酶(P160ROCK)是丝氨酸/苏氨酸激酶。P160ROCK是小分子GTP酶Rho效应蛋白的一员，与激活形式的Rho特异结合，从而激活Rho。在成纤维细胞和花药细胞的转基因实验中，该激酶参与了Rho介导的肌动蛋白应激纤维和局灶性粘连的形成。此外，在神经母细胞瘤N1E-115细胞中，这种激酶通过调节细胞皮质的收缩力量来诱导细胞变圆。因此，人们认为p160ROCK定位于细胞中的几个特定位置，并磷酸化几种底物。最近，我们开发了一种特异性的ROCK抑制剂，我们将其命名为Y-27632。这种化合物与三磷酸腺苷在激活域上竞争。利用该化合物，我们报道了Rho/ROCK通路对高血压模型大鼠血压的调节作用，表明该通路参与了高血压的病理生理过程。本研究通过分离p160ROCK的磷酸化底物，试图了解p160ROCK介导的信号通路(S)的分子机制(S)。结果，我们的主要发现如下：1.p160ROCK通过磷酸化和激活LIM-Kinase来调节Confilin/ADF的磷酸化。P160ROCK与Rho的另一个效应分子MDIA在Rho介导的肌动蛋白应激纤维形成和局部粘连形成上具有协同作用。

We found Rho-associated coiled coil forming protein kinase (p160ROCK) is serine/threonine kinase. p160ROCK is a member of effector protein of small GTPase Rho and specisically binds to activated form of Rho thereby becomes activated upon this binding. During transfction experiments in fibroblast and eptherial cells, this kinase is involved in formation of actin stress fibers and focal adhesions mediated by Rho. In addition to this, in nuroblastama N1E-115 cell this kinase induces cell rounding by regulating contractile force at cell cortex.Thus, it is thought that p160ROCK is localized to several specific sites in the cell and phosphorylates several substrates.Recently, we developed a specific inhibitor of ROCK, we named it Y-27632. This compound competes with ATP at the kinase domain. Using this compound, we reported that Rho/ROCK pathway regulates blood pressure in hypertension model rats, indicating that this pathway is involved in the pathophysiology of hypertension. However, the molecular mechanism of Rho/ROCK pathway is not clearly known.In this study, we try to understand the molecular mechanism (s) of p160ROCK-mediated signaling pathway (s) through the isolation of phosphorylated substrates for p160ROCK.As a result, our major findings are as follows,1. p160ROCK regulates Confilin/ADF phosphorylaytion through phosphorylation and activation of LIM-kinase.2. p160ROCK cooperates with mDia, another effector molecule of Rho, on Rho-mediated actin stress fiber formation and focal adhesion formation.