Different regions of Rho determine Rho-selective binding of different classes of Rho target molecules
Different regions of Rho determine Rho-selective binding of different classes of Rho target molecules
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DOI：
10.1074/jbc.273.30.18943
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发表时间：
1998-07-24
影响因子：
4.8
通讯作者：
Narumiya, S
中科院分区：
文献类型：
--
作者：
Fujisawa, K;Madaule, P;Narumiya, S
根据其Rho结合基序，几种Rho靶分子可分为三类：I类包括蛋白激酶PKN、rhophilin和rhotekin，II类包括蛋白激酶、含有Rho相关卷曲螺旋的蛋白激酶、ROCK-I和ROCK-II，III类包括Citron。利用Rho和Rac之间这些靶标识别的选择性，我们检测了Rho中选择性结合各类Rho靶分子所需的区域。使用Rho/Rac嵌合体和rhophilin、ROCK-1或Citron进行酵母双杂交测定，这项研究表明，在Rho中存在至少两个不同的区域（氨基酸23-40和75-92），这对这些靶标的选择性结合至关重要。前者是与香橼结合所必需的，而后者是与rhophilin结合所必需的。另一方面，两个区域都显示出与ROCK-Ⅰ的亲和力。这通过使用重组ROCK-I和ROCK-II蛋白的配体覆盖测定进一步证实。一致的是，Rho/Rac嵌合体包含任何区域可以诱导应力纤维在转染的HeLa细胞，这种诱导被抑制治疗与Y-27632，ROCK激酶的特异性抑制剂。这些结果表明，不同类别的Rho靶标与Rho的选择性结合是由靶标的不同Rho结合基序与Rho的不同区域之间的相互作用决定的。
Based on their Rho binding motifs several Rho target molecules can be classified into three groups; class I includes the protein kinase PKN, rhophilin, and rhotekin, class II includes the protein kinases, Rho-associated coiled-coil containing protein kinases, ROCK-I and ROCK-II, and class III includes citron, Taking advantage of the selectivity in recognition by these targets between Rho and Rac, we examined the regions in Rho required for selective binding of each class of Rho target molecules, Yeast two-hybrid assays were performed using Rho/Rac chimeras and either rhophilin, ROCK-I, or citron, This study showed the existence of act least two distinct regions in Rho (amino acids 23-40 and 75-92) that are critical for the selective binding of these targets. The former was required for binding: to citron, whereas the latter was necessary for binding to rhophilin, On the other hand, either region showed affinity to ROCK-I. This was further confirmed by ligand overlay assay using both recombinant ROCK-I and ROCK-II proteins. Consistently, Rho/Rac chimeras containing either region can induce stress fibers in transfected HeLa cells, and this induction is suppressed by treatment with Y-27632, a specific inhibitor of ROCK kinases. These results suggest that the selective binding of different classes of Rho targets to Rho is determined by interaction between distinct Rho-binding motifs of the targets and different regions of Rho.