Identification of the Rho-binding domain of p160(ROCK), a Rho-associated coiled-coil containing protein kinase
Identification of the Rho-binding domain of p160(ROCK), a Rho-associated coiled-coil containing protein kinase
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DOI：
10.1074/jbc.271.38.23022
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发表时间：
1996-09-20
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;Fujita, A;Narumiya, S
蛋白丝氨酸/苏氨酸激酶p160（ROCK）已被鉴定为推定的Rho靶蛋白，其在与小GTdR Rho的GTP结合形式结合时被激活（Ishizaki，T.，Maekawa，M.，Fujisawa，K.，Okawa，K.，Iwamatu，A.，Fujita，A.，渡边，N.齐藤，Y.，Kakizuka，A.，Morii，N.，和Narumiya，S. 15，1885-1893）。p160（ROCK）在其NH 2-末端区域具有丝氨酸/苏氨酸激酶结构域，随后是约600个氨基酸长的α-螺旋、富含半胱氨酸的锌指样基序和在COOH末端的普列克底物蛋白同源区。为了鉴定该蛋白的Rho结合结构域，我们将p160分成5个片段，每个片段表达为His标记的重组蛋白，并使用[S-35]鸟苷-5 '-3-O-（硫代）三磷酸（GTP γ S）结合的谷胱甘肽S-转移酶-RhoA进行配体覆盖测定。仅在片段M2中观察到特异性GTP γ S-Rho结合，其覆盖氨基酸727和1021之间的α螺旋的羧基一半的大部分。该片段进一步细分为几个片段，并进行配体覆盖试验以及酵母双杂交系统来鉴定Rho结合区。这些研究将最小Rho结合区定位于氨基酸934-1015。为了鉴定Rho结合的关键氨基酸，我们分析了具有各种点突变的亚片段的Rho结合活性。该分析显示，K934 M、L941 A和E1008 A突变显著削弱了Rho结合，I1009 A突变消除了Rho结合。该区域中的氨基酸序列与Rho效应子基序类别1没有显著的同源性，所述Rho效应子基序类别1由推定的Rho靶标PKN、rhophilin和rhotekin共享。Furuyashiki，T.，石崎，T.，渡边，G.，Watanabe，N.，Fujisawa，K.，Morii，N.，Madaule，P.，和Narumiya，S.（1996）J.Biol.Chem.271，13556-13560），并且可以定义不同类别的Rho效应基序。
A protein serine/threonine kinase, p160(ROCK), has been identified as a putative Rho target protein that is activated when bound to the GTP-bound form of the small GTPase Rho (Ishizaki, T., Maekawa, M., Fujisawa, K., Okawa, K., Iwamatu, A., Fujita, A., Watanabe, N. Saito, Y., Kakizuka, A., Morii, N., and Narumiya, S. (1996) EMBO J. 15, 1885-1893). p160(ROCK) has a serine/threonine kinase domain in its NH2-terminal region, followed by an approximately 600-amino acid-long alpha-helix, a cysteine-rich zinc finger-like motif, and a pleckstrin homology region in the COOH terminus. To identify the Rho binding domain of this protein, we divided p160 into five fragments, expressed each as a His-tagged recombinant protein, and performed a ligand overlay assay using [S-35]guanosine-5'-3-O-(thio)triphosphate (GTP gamma S)-bound glutathione S-transferase-RhoA. Specific GTP gamma S-Rho binding was observed only in the fragment M2, which covered most of the carboxyl half of the a helix between amino acids 727 and 1021. This fragment was further subdivided into several fragments, and the ligand overlay assay as well as the yeast two hybrid system was carried out to identify the Rho binding region. These studies localized the minimum Rho binding region to amino acids 934-1015. To identify critical amino acids for Rho binding, we analyzed the Rho binding activity of the subfragment with various point mutations. This analysis revealed that K934M, L941A, and E1008A mutations significantly weakened Rho binding and an I1009A mutation abolished Rho binding. The amino acid sequence in this region had no significant homology with Rho effector motif class 1, which is shared by putative Rho targets, PKN, rhophilin, and rhotekin, (Held, T., Furuyashiki, T., Ishizaki, T., Watanabe, G., Watanabe, N., Fujisawa, K., Morii, N., Madaule, P., and Narumiya, S. (1996) J. Biol. Chem. 271, 13556-13560) and may define a distinct class of Rho effector motif.