Abstract
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.
Highlights
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
We observed a rapid change in fluorescence after mixing the GRD1-C-terminal domain (CT) with mGppNHp-bound RAC1 (Fig. 2C) or CDC42 (Fig. 2D), which is directly related to the association reaction
GRD1-CT binding to RAC1-mGppNHp resulted in an increase in fluorescence (Fig. 2C), whereas association with CDC42-mGppNHp led to a fluorescence decay (Fig. 2D)
Summary
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 formation of the active, GTP-bound state of RHO proteins is accompanied by a conformational change in two regions known as switch I and II [2] These regions provide a platform for the selective interaction with structurally and functionally diverse effectors [2, 7, 8], e.g. p21activated kinase 1 (PAK1) [9], Wiskott-Aldrich syndrome proteins (WASP) [10], p67phox, a member of the NADPH oxidase family [11], and semaphorin receptor Plexin B1 [12, 13] as well as the IQ motif-containing GTPase activating proteins (IQGAPs) [14, 15]. Distinct domains of IQGAP1, include an N-terminal calponin homology domain (CHD), a coiled-coil repeat region (CC), a tryptophan-containing proline-rich motif-binding region (WW), four isoleucine/glutamine-containing motifs (IQ), a RAS GAP-related domain (GRD), a RASGAP C-terminal domain (RGCT), and an extreme C-terminal domain (CT) (Fig. 1)
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