The yeast exchange assay, a new complementary method to screen for Db1-like protein specificity: identification of a novel RhoA exchange factor
The yeast exchange assay, a new complementary method to screen for Db1-like protein specificity: identification of a novel RhoA exchange factor
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DOI：
10.1016/s0014-5793(00)01953-0
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发表时间：
2000-09-01
期刊：
影响因子：
3.5
通讯作者：
Blangy, A
中科院分区：
文献类型：
--
作者：
De Toledo, M;Colombo, K;Blangy, A
Dbl 家族的许多鸟嘌呤核苷酸交换因子 (GEF) 的靶标 Rho GTP 酶仍有待鉴定。在这里，我们报告了一种新方法：酵母交换测定 (YEA)，这是一种快速定性测试，可对 GEF 特异性进行广泛的筛选。在这个基于双杂交系统的测定中，野生型 GTP 酶仅在被特定 GEF 激活后才与其效应子结合。我们通过先前报道的 GEF 激活 GTP 酶来验证 YEA。我们进一步确定一种新型 GEF（GEF337）可激活 YEA 中的 RhoA。 GEF337 在体外促进 RhoA 上的核苷酸交换，并促进成纤维细胞中的 F-肌动蛋白应力纤维组装，这是 RhoA 激活的特征，(C) 2000 年欧洲生化协会联合会。由 Elsevier Science B.V. 出版。保留所有权利。
The target Rho GTPases of many guanine nucleotide exchange factors (GEFs) of the Dbl family remain to be identified. Here we report a new method: the yeast exchange assay (YEA), a rapid qualitative test to perform a wide range screen for GEF specificity. In this assay based on the two-hybrid system, a wild type GTPase binds to its effector only after activation by a specific GEF, We validated the YEA by activating GTPases by previously reported GEFs, We further established that a novel GEF, GEF337, activates RhoA in the YEA. GEF337 promoted nucleotide exchange on RhoA in vitro and promoted F-actin stress fiber assembly in fibroblasts, characteristic of RhoA activation, (C) 2000 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.