ralGDS family members interact with the effector loop of ras p21
ralGDS family members interact with the effector loop of ras p21
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DOI：
10.1128/mcb.14.11.7483-7491.1994
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发表时间：
1994-11
影响因子：
5.3
通讯作者：
Akira Kikuchi;Susan D. Demo;ZHI-HAI Ye;YEN-WEN Chen;Lewis T. Willlams
中科院分区：
文献类型：
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作者：
Akira Kikuchi;Susan D. Demo;ZHI-HAI Ye;YEN-WEN Chen;Lewis T. Willlams
利用酵母双杂交系统，我们鉴定了一种与ras p21相互作用的新蛋白。该蛋白与ralGDS具有69%的氨基酸同源性，ralGDS是ralp24的GDP/GTP交换蛋白。我们将这种蛋白命名为RGL，即ralgds样蛋白。利用酵母双杂交系统，我们发现ras p21的一个效应环突变体在与RGL的ras p21相互作用结构域相互作用时存在缺陷，这表明该结构域通过ras p21的效应环与ras p21结合。由于ralGDS含有一个与RGL的ras p21相互作用域高度同源的区域，我们研究了ralGDS是否可以与ras p21相互作用。在酵母双杂交系统中，ralGDS未能与ras p21的效应环突变体相互作用。在昆虫细胞中，ralGDS与v-ras p21形成复合体，但不与ras p21的显性负突变体形成复合体。ralGDS与ras p21的gtp结合形式相互作用，但不与gdp结合形式相互作用。ralGDS在体外抑制神经纤维瘤病基因产物（NF1） ras p21的gtpase激活活性以及Raf与ras p21的相互作用。这些结果表明，ralGDS特异性地与ras p21的活性形式相互作用，并且ralGDS可以与NF1和Raf竞争与ras p21的效应环结合。因此，ralGDS家族成员可能是ras p21的效应蛋白，也可能抑制ras p21与其效应蛋白之间的相互作用。
Using a yeast two-hybrid system, we identified a novel protein which interacts with ras p21. This protein shares 69% amino acid homology with ral guanine nucleotide dissociation stimulator (ralGDS), a GDP/GTP exchange protein for ral p24. We designated this protein RGL, for ralGDS-like. Using the yeast two-hybrid system, we found that an effector loop mutant of ras p21 was defective in interacting with the ras p21-interacting domain of RGL, suggesting that this domain binds to ras p21 through the effector loop of ras p21. Since ralGDS contained a region highly homologous with the ras p21-interacting domain of RGL, we examined whether ralGDS could interact with ras p21. In the yeast two-hybrid system, ralGDS failed to interact with an effector loop mutant of ras p21. In insect cells, ralGDS made a complex with v-ras p21 but not with a dominant negative mutant of ras p21. ralGDS interacted with the GTP-bound form of ras p21 but not with the GDP-bound form in vitro. ralGDS inhibited both the GTPase-activating activity of the neurofibromatosis gene product (NF1) for ras p21 and the interaction of Raf with ras p21 in vitro. These results demonstrate that ralGDS specifically interacts with the active form of ras p21 and that ralGDS can compete with NF1 and Raf for binding to the effector loop of ras p21. Therefore, ralGDS family members may be effector proteins of ras p21 or may inhibit interactions between ras p21 and its effectors.