IQGAP1 Interaction with RHO Family Proteins Revisited: KINETIC AND EQUILIBRIUM EVIDENCE FOR MULTIPLE DISTINCT BINDING SITES.
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.
From the Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, 40225 Düsseldorf, Germany.
Kazem
K
Nouri
From the Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, 40225 Düsseldorf, Germany.
Eyad K
EK
Fansa
From the Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, 40225 Düsseldorf, Germany.
Ehsan
E
Amin
From the Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, 40225 Düsseldorf, Germany.
Radovan
R
Dvorsky
the Institute of Physical Biology, Heinrich-Heine University, 40225 Düsseldorf, Germany.|Forschungszentrum Jülich, ICS-6, 52428 Jülich, Germany.
Lothar
L
Gremer
the Institute of Physical Biology, Heinrich-Heine University, 40225 Düsseldorf, Germany.|Forschungszentrum Jülich, ICS-6, 52428 Jülich, Germany.
Dieter
D
Willbold
the Institute of Biochemistry, Heinrich-Heine University, 40225 Düsseldorf, Germany, and.
Lutz
L
Schmitt
the School of Pharmacy and Biomolecular Sciences, University of Brighton, Huxley Building, Lewes Road, Brighton BN2 4GJ, United Kingdom.
David J
DJ
Timson
From the Institute of Biochemistry and Molecular Biology II, Medical Faculty of the Heinrich-Heine University, 40225 Düsseldorf, Germany, reza.ahmadian@uni-duesseldorf.de.
Mohammad R
MR
Ahmadian
D001665:Binding Sites; D006801:Humans; D007700:Kinetics; D020764:cdc42 GTP-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020830:rac1 GTP-Binding Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020703:ras GTPase-Activating Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
C101203:IQ motif containing GTPase activating protein 1; C107953:RAC1 protein, human; D020703:ras GTPase-Activating Proteins; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein
CDC42; GTPase activating protein (GAP); IQGAP1; Ras homolog gene family, member A (RHOA); Ras-related C3 botulinum toxin substrate 1 (Rac1); Rho (Rho GTPase); binding affinity; fluorescence anisotropy; protein-protein interaction; signal transduction