IQGAP1: insights into the function of a molecular puppeteer

Abstract

The intracellular spatiotemporal organization of signaling events is critical for normal cellular function. In response to environmental stimuli, cells utilize highly organized signaling pathways that are subject to multiple layers of regulation. However, the molecular mechanisms that coordinate these complex processes remain an enigma. Scaffolding proteins (scaffolins) have emerged as critical regulators of signaling pathways, many of which have well-described functions in immune cells. IQGAP1, a highly conserved cytoplasmic scaffold protein, is able to curb, compartmentalize, and coordinate multiple signaling pathways in a variety of cell types. IQGAP1 plays a central role in cell-cell interaction, cell adherence, and movement via actin/tubulin-based cytoskeletal reorganization. Evidence also implicates IQGAP1 as an essential regulator of the MAPK and Wnt/β-catenin signaling pathways. Here, we summarize the recent advances on the cellular and molecular biology of IQGAP1. We also describe how this pleiotropic scaffolin acts as a true molecular puppeteer, and highlight the significance of future research regarding the role of IQGAP1 in immune cells.

Keywords: IQGAP1; Lymphocytes; Scaffold proteins; Signaling.

Figure 1. IQGAP proteins are evolutionarily conserved from yeast to mammals

A) Phylogenic analysis from aligned amino acid sequences of the mammalian IQGAP proteins (IQGAP1, 2, and 3 in mice and humans) and their homologs, Iqg1p and PES-7, in Saccharomyces cerevisiae and Caenorhabditis elegans, respectively. Red numbers indicate evolutionary distance in millions of years and the mammalian IQGAP1 branch is shaded in grey. B) Graph of the percent identity matrix of IQGAP amino acid sequences. The phylogenic tree and percent identity matrix were generated using Clustal Omega alignment software using amino acid sequences retrieved from NCBI (accession numbers: AAB70827.1, CAB07179.2, NP_057930.2, NP_081987.1, NP_001028656.1, NP_003861.1, NP_006624.2, and AAP06954.1 for Iqg1p, PES-7, Mouse IQGAP1, 2, 3 and Human IQGAP1, 2, 3, respectively).

Figure 2. Protein structure of and specific domain-interacting partners of IQGAP1

IQGAP1 is a 190 kDa (1657 amino acid) protein that contains six distinct protein-interacting domains. The domains and known interacting proteins are shown as well as amino acid sequences (mouse). A) The calponin homology (CH) domain binds the chemokine receptor, CXCR2, and regulates the actin cytoskeleton by binding N-WASp and polymerized filamentous actin (F-actin). B) Six coiled-coil (CC) regions, comprised of highly conserved amino acid repeats, comprise the coiled-coil (CC) domain which binds Ezrin. C) Erk1/2 are recruited to the WW domain. D) The Isoleucine/glutamine-containing (IQ) domain is a binding domain for multiple proteins that include components of MAPK signaling (Rap1a, Rap1b, B-Raf, C-Raf, Mek1 and Mek2), phosphoinositide signaling (PIPKIγ), calcium signaling (S100B and Ca2+-independent interaction of calmodulin and its related proteins), as well as cytoskeletal components (myosin ELC) and cell surface receptors (EGFR and HER2). E) The Ras-GAP domain (GRD) does not function as a GTPase Activating Protein (GAP) but does interact with small GTPases Cdc42, Rac1, and TC10. F) The Ras-GAP C-terminus domain (RGCT) interacts with microtubule-binding proteins CLIP-170 and Clasp2, as well as membrane-resident proteins such as β-catenin, E-cadherin, and APC. The RGCT domain also contains two phosphorylation sites (Ser1441 and Ser1443) of IQGAP1 as well as a polybasic region which binds PIP2. Other proteins previously shown to interact with IQGAP1 (Akt, mTORC1, exorcist, Sec3/8, Lis1, Menin, SMG-2, and Disheveled) are not depicted.

Figure 3. IQGAP1 plays a critical role in actin polymerization

IQGAP1 can regulate actin polymerization by at least three different mechanisms. A) RGCT domain of IQGAP1 can Cdc42 or Rac1 is recruited by autoinhibited WASp through ‘Cdc42-interactive-binding’ (CRIB) motif. This causes a conformational change in the C-terminal verprolin homology domain and central acidic (VCA) region of WASp allowing globular actin to bind through an electrostatic steering mechanism. In addition, IQGAP1 transiently protects Cdc42-GTP in its activated state, reducing its GTP hydrolysis. By delaying the hydrolysis of WASp-bound Cdc42-GTP, IQGAP1 stabilizes the VCA domain of WASp. This sustains the interaction with Arp2/3 and globular actin, promoting the polymerization and branching of actin. B) The CH domain of IQGAP1 interacts with polymerized F-actin. a single N-terminal CH domain facilitates actin polymerization by cross linking filaments into interconnected bundles. C) RGCT domain of IQGAP1 can regulate polymerized actin filament through myosin light chain (MLC) in a co-ordinated effort of MLC kinase and MLC phosphatase by PAK and Rho, respectively. In addition, MLC kinase function can be regulated by IQGAP1-bound calmodulin.

Figure 4. Cytoskeletal remodeling is a conserved function of IQGAP1

The CH and GR domains of IQGAP1 are critical for orchestrating cytoskeletal remodeling and are conserved from yeast Iqg1p. A) Amino acid sequence alignment of the CH domains of yeast Iqg1p (yIqg1p), mouse IQGAP1 (mIQGAP1), and human IQGAP1 (hIQGAP1). Conserved proline residues are outlined in black. Shaded regions indicate alpha-helices previously determined for the CH domain of IQGAP1 (Umemoto et al., 2010) and predicted for Iqg1p using PSIPRED analysis software (UCL Bioinformatics). B) Diagram of Iqg1p-mediated functions involved in cytoskeletal remodeling in yeast mirrors what has been shown for C) IQGAP1 in mammalian cells such as lymphocytes. D) Amino acid sequence alignment of the GR domain. Shaded regions and conserved proline residues are indicated as in (A). Alpha-helical structures were previously determined for the GRD of IQGAP1 (Kurella et al., 2009) and predicted for Iqg1p using PSEPRED analysis software (UCL Bioinformatics). The threonine¹⁰⁴⁶ residue thought to be related to the absence of GAP function is outlined in red. Amino acids involved in the π-helix structure (Kurella et al., 2009), outlined in blue, are located within the variant Ras-GAP motif and contain one of the two conserved tyrosine residues outlined in green.

Figure 5. An IQGAP1 complex tethers F-actin to microtubules

IQGAP1 plays an important role in linking polymerized actin filaments (F-actin) with microtubules. A) The RGCT domain of IQGAP1 links F-actin to plus-end microtubules at the leading edge membrane via a complex including Axin, APC, CLIP170 and Clasp2. GSK-3β acts as a negative regulator of this complex by phosphorylating Clasp2 which causes it to disassociate from IQGAP1 leading to a reduction in cell movement. B) Although the role of IQGAP1 in the formation and function of the MTOC is not well understood, its interaction with Axin and APC may facilitate the organization and multimerization of γ-tubulin molecules that form the core of the centrosome and MTOC. IQGAP1 has also been to localize to the cytoplasmic face of the nuclear envelope, potentially promoting cell cycle-associated assembly and disruption of nuclear envelope

Figure 6. IQGAP1 is a MAPK scaffold that regulates Erk1/2 phosphorylation and facilitates signalosome formation

A) IQGAP1 acts as a scaffold for sequential activation of the Cdc42-Pak1-B/C-Raf-Mek1/2-Erk1/2 MAPK pathway. Receptor-mediated activation of Cdc42, recruited to the GRD domain of IQGAP1, results in the recruitment and activation of Pak1 which binds and activates B-Raf and C-Raf on the IQ domains of IQGAP1. Activated Rafs phosphorylate Mek1/2, also associated with the IQ domains, which in turn phosphorylate Erk1/2 that are recruited to the WW domains of IQGAP1. B) Amino acid sequence alignment of the IQ domain in yeast (yIqg1p), mouse IQGAP1 (mIQGAP1), and human IQGAP1 (hIQGAP1). Shaded regions indicate four individual IQ domains containing the [I/V/L]Qxxx[RG]xxx[RK] motif. C) IQGAP1 facilitates the formation of a MAPK signalosome. Upon receptor-mediated activation and IQGAP1 oligomerization, the IQGAP1-mediated signalosome localizes to the periphery of the nucleus and may promote nuclear translocation of phosphorylated Erk1/2.

Figure 7. Potential mechanism by which IQGAP1 regulates β-catenin-mediated gene transcription

IQGAP1 organizes the N-terminal phosphorylation of β-catenin. A) The RGCT domain of IQGAP1 directly interacts with β-catenin. Under steady state, β-catenin constitutively associates with IQGAP1-RGCT domain-associated APC and Axin as part of the degradation complex. APC associates with Axin via its SAMP domain and facilitates the recruitment of CK1 and GSK3β. APC using its third 15 amino acid repeat grasps the 5th armadillo repeat of β-catenin to facilitate the phosphorylation of β-catenin by CK1 (Ser45) and GSK3β (Thr41, Ser37, Ser33). B) Phosphorylation of these residues leads to the ubiquitination of β-catenin by β-TrCP. At this time, one of the 20 amino acid repeat of APC help to stabilize the association of β-TrCP to β-catenin by associating with the armadillo repeats of β-catenin. C) After activation (such as Wnt3a), β-catenin is rescued from degradation through IQGAP1-RGCT domain-associated PP2A-mediated dephosphorylation and allowed to interact with the TCF/LEF family of transcription factors. β-catenin/TCF/LEF complex is translocated into the nucleus that promotes specific gene transcriptions.