IQGAP1 Interaction with RHO Family Proteins Revisited

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.

LGR5 receptor promotes cell–cell adhesion in stem cells and colon cancer cells via the IQGAP1–Rac1 pathway

“…Binding of Rac1/CDC42 to IQGAP1 is regulated by phosphorylation of IQGAP1 at two sites (Ser-1441 and -1443) ( 25 , 26 , 41 ). Detailed kinetic analysis showed that phosphorylation at Ser-1443 led to a decrease in the affinity of human IQGAP1 for Rac1 and CDC42 ( 27 ). To test whether LGR5 regulates IQGAP1–Rac1 interaction through modulation of IQGAP1 phosphorylation, we probed the level of phospho-IQGAP1 with an anti-phosphoserine antibody (no antibody specifically against Ser-1441 or Ser-1443 of IQGAP1 is available ( 42 )).…”

“…Initially, we found that overexpression of LGR5 consistently led to increased binding of Rac1 and actin to IQGAP1 in co-IP analysis. Because it was reported that binding of Rac1/CDC42 to IQGAP1 is inhibited by phosphorylation at Ser-1441/1443 of IQGAP1 ( 26 , 27 , 41 ), we asked whether LGR5 modulates phosphorylation of IQGAP1 at these sites to regulate IQGAP1–Rac1 interaction. Indeed, LGR5 overexpression and KD of LGR5 led to a significant decrease and increase in Ser-1441/1443 phosphorylation, respectively, and LGR5 had no effect on PMA-induced phosphorylation of IQGAP1.…”

“…Specifically, binding of Rac1/CDC42 to IQGAP1 inhibits the IQGAP1–β-catenin interaction, leading to an increase in membrane-bound β-catenin, cell–cell adhesion, and F-actin cross-linking ( 20 , 24 ). Rac1/CDC42 binding to IQGAP1 is inhibited by phosphorylation at two serine sites (Ser-1441 and Ser-1443) of IQGAP1 ( 25 – 27 ). Here, we show that LGR5 interacts with IQGAP1 and decreases phosphorylation at these two serine sites, leading to increased binding of Rac1 and consequently enhancement of cell–cell adhesion.…”

Ras GAP-related and C-terminal domain-dependent localization and tumorigenic activities of IQGAP1 in melanoma cells

“…Reports point to interaction between Rho family small GTPases with IQGAP1 C-terminal domains which could regulate Rho kinase-myosin II dependent retraction. Rho inhibitors disrupt the WRAMP complex in human melanoma cells [ 17 ] and IQGAP1 is known to interact with RhoA [ 43 , 44 ]. While promoting activity of RhoA, IQGAP1 may also decrease activity of Rac1 [ 45 ] in the cell tail during melanoma migration, which may require interactions with C-terminal domains of IQGAP1.…”

The role of Rac in tumor susceptibility and disease progression: from biochemistry to the clinic

“…As an example, IQGAP proteins have been described as a scaffolding platform that assemble small GTPases, GEFs, and effectors. This association is a multi-step process that involves a high affinity binding of IQGAP1 to the switch regions of the small G-protein (a GTP-dependent event), and low affinity binding to an adjacent region (16). The described direct association of IQGAP1 with the Rac-GEF Tiam1 and Rac1 exemplifies the relevance of multiprotein complexes in Rac1 activation (17).…”