IQGAP1 Interaction with RHO Family Proteins Revisited: KINETIC AND EQUILIBRIUM EVIDENCE FOR MULTIPLE DISTINCT BINDING SITES.
IQGAP1 Interaction with RHO Family Proteins Revisited: KINETIC AND EQUILIBRIUM EVIDENCE FOR MULTIPLE DISTINCT BINDING SITES.
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DOI：
10.1074/jbc.m116.752121
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发表时间：
2016-12-16
期刊：
影响因子：
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通讯作者：
Ahmadian MR
中科院分区：
文献类型：
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作者：
Nouri K;Fansa EK;Amin E;Dvorsky R;Gremer L;Willbold D;Schmitt L;Timson DJ;Ahmadian MR
含有IQ基序的GTP酶激活蛋白1（IQGAP 1）在相关信号网络的物理组装中起着核心作用，这些信号网络负责各种细胞过程，包括细胞粘附，极性和迁移。RHO家族蛋白CDC 42和RAC 1已被证明主要与IQGAP 1的GAP相关结构域（GRD）相互作用。然而，其RASGAP C-末端（RGCT）和C-末端结构域在与RHO蛋白相互作用中的作用仍然不清楚。在这里，我们证明了IQGAP 1与RHO蛋白的相互作用是多步结合机制的基础：（i）RGCT与CDC 42或RAC 1的开关区域的高亲和力、GTP依赖性结合，以及（ii）GRD和邻近开关区域的C末端的极低亲和力结合。这些数据通过RGCT内丝氨酸1443至谷氨酸的磷酸模拟突变得到证实，这导致IQGAP 1对CDC 42和RAC 1的亲和力显著降低，清楚地揭示了RGCT对这些相互作用的关键作用。与CDC 42不同的是，RAC 1-GRD相互作用的亲和力极低，表明IQGAP 1与CDC 42相互作用的分子性质部分不同于RAC 1。我们的研究为IQGAP 1与RHO家族蛋白的相互作用特征提供了新的见解，并强调了动力学和平衡分析的互补重要性。我们提出IQGAP 1与RHO蛋白相互作用的能力是基于多步结合过程，这是IQGAP 1作为支架蛋白的动态功能的先决条件，也是IQGAP 1介导的细胞反应的时间调节和整合的关键机制。
IQ motif-containing GTPase activating protein 1 (IQGAP1) plays a central role in the physical assembly of relevant signaling networks that are responsible for various cellular processes, including cell adhesion, polarity, and transmigration. The RHO family proteins CDC42 and RAC1 have been shown to mainly interact with the GAP-related domain (GRD) of IQGAP1. However, the role of its RASGAP C-terminal (RGCT) and C-terminal domains in the interactions with RHO proteins has remained obscure. Here, we demonstrate that IQGAP1 interactions with RHO proteins underlie a multiple-step binding mechanism: (i) a high affinity, GTP-dependent binding of RGCT to the switch regions of CDC42 or RAC1 and (ii) a very low affinity binding of GRD and a C terminus adjacent to the switch regions. These data were confirmed by phosphomimetic mutation of serine 1443 to glutamate within RGCT, which led to a significant reduction of IQGAP1 affinity for CDC42 and RAC1, clearly disclosing the critical role of RGCT for these interactions. Unlike CDC42, an extremely low affinity was determined for the RAC1-GRD interaction, suggesting that the molecular nature of IQGAP1 interaction with CDC42 partially differs from that of RAC1. Our study provides new insights into the interaction characteristics of IQGAP1 with RHO family proteins and highlights the complementary importance of kinetic and equilibrium analyses. We propose that the ability of IQGAP1 to interact with RHO proteins is based on a multiple-step binding process, which is a prerequisite for the dynamic functions of IQGAP1 as a scaffolding protein and a critical mechanism in temporal regulation and integration of IQGAP1-mediated cellular responses.