Self-association of IQGAP1: characterization and functional sequelae

The scaffolding protein IQGAP1 participates in numerous cellular functions by binding to target proteins such as actin, calmodulin, E-cadherin, beta-catenin, Cdc42, Rac1, and CLIP-170. IQGAP1 regulates the cytoskeleton, promotes cell motility, and modulates E-cadherin-mediated cell-cell adhesion. However, how IQGAP1 exerts its functions in vivo is still unclear. In this study we investigate the self-association of IQGAP1 and its role in IQGAP1 function. Endogenous IQGAP1 co-immunoprecipitated from MCF-7 cells with IQGAP1 tagged with enhanced green fluorescent protein, indicating that IQGAP1 self-associates in cells. In vitro assays confirmed that IQGAP1 can self-associate and that this effect is mediated by the N-terminal half of the protein. Gel filtration analysis suggested that full-length IQGAP1 exists as a combination of monomers, dimers, and larger oligomers. Analysis performed with multiple fragments of IQGAP1 narrowed the self-association region to amino acids 763-863. In support of this observation, a peptide comprising residues 763-863 disrupted self-association of full-length IQGAP1 in a dose-dependent manner. Similarly, deleting this sequence from IQGAP1 abolished binding to full-length IQGAP1. In addition, the ability of IQGAP1 to increase the amount of active Cdc42 in cells is abrogated upon removal of this region. Consistent with these findings, transfection into cells of a peptide containing the self-association domain significantly reduced the amount of active Cdc42 in cell lysates. These observations define a sequence of IQGAP1 that is necessary for its oligomerization and demonstrate that self-association is required for the normal cellular function of IQGAP1.     

The effect of IQGAP1 on Xenopus embryonic ectoderm requires Cdc42

IQGAP1 contains a number of protein recognition motifs through which it binds to targets. Several in vitro studies have documented that IQGAP1 interacts directly with calmodulin, actin, E-cadherin, beta-catenin, and the small GTPases Cdc42 and Rac. Nevertheless, direct demonstration of in vivo function of mammalian IQGAP1 is limited. Using a novel assay to evaluate in vivo function of IQGAP1, we document here that microinjection of IQGAP1 into early Xenopus embryos generates superficial ectoderm lesions at late blastula stages. This activity was retained by the mutated variants of IQGAP1 in which the calponin homology domain or the WW domain was deleted. By contrast, deletion of the IQ (IQGAP1-DeltaIQ), Ras-GAP-related (IQGAP1-DeltaGRD), or C-terminal (IQGAP1-DeltaC) domains abrogated the effect of IQGAP1 on the embryos. None of the latter mutants bound Cdc42, suggesting that the binding of Cdc42 by IQGAP1 is critical for its function. Moreover, overexpression of IQGAP1, but not IQGAP1-DeltaGRD, significantly increased the amount of active Cdc42 in embryonic cells. Co-injection of wild type IQGAP1 with dominant negative Cdc42, but not the dominant negative forms of Rac or Rho, blocked the effect of IQGAP1 on embryonic ectoderm. Together these data indicate that the activity of IQGAP1 in embryonic ectoderm requires Cdc42 function.     

IQGAP1 promotes cell motility and invasion

The dynamic processes of cell migration and invasion are largely coordinated by Rho family GTPases. The scaffolding protein IQGAP1 binds to Cdc42, increasing the amount of active Cdc42 both in vitro and in cells. Here we show that overexpression of IQGAP1 in mammalian cells enhances cell migration in a Cdc42- and Rac1-dependent manner. Importantly, cell motility was significantly decreased both by knock down of endogenous IQGAP1 using small interfering RNA and by transfection of a dominant negative IQGAP1 construct, IQGAP1DeltaGRD. Cell invasion was similarly altered by manipulating intracellular IQGAP1 concentrations. Moreover, invasion mediated by constitutively active Cdc42 was attenuated by IQGAP1DeltaGRD. Thus, IQGAP1 has a fundamental role in cell motility and invasion.     

Identification and characterization of the Cdc42-binding site of IQGAP1

IQGAP1 is a multi-domained protein that integrates signaling of the Rho family GTPase Cdc42 with regulation of the cytoskeleton. Using SPOT analysis and in vitro peptide competition assays we have identified a 24 amino acid region of IQGAP1 that is necessary for Cdc42 binding. Both in vitro and in vivo analyses reveal that deletion of this sequence abolishes binding of IQGAP1 to Cdc42. In addition, the ability of IQGAP1 to increase the amount of active Cdc42 in cells is abrogated upon removal of this region. An IQGAP1 mutant lacking the Cdc42 binding site mislocalizes to the cell periphery. These observations specifically define a short sequence of IQGAP1 that is required for its interaction with Cdc42 and demonstrate that Cdc42 binding is necessary for the normal subcellular distribution of IQGAP1.     

Polarized distribution of IQGAP proteins in gastric parietal cells and their roles in regulated epithelial cell secretion

Actin cytoskeleton plays an important role in the establishment of epithelial cell polarity. Cdc42, a member of Rho GTPase family, modulates actin dynamics via its regulators, such as IQGAP proteins. Gastric parietal cells are polarized epithelial cells in which regulated acid secretion occurs in the apical membrane upon stimulation. We have previously shown that actin isoforms are polarized to different membrane domains and that the integrity of the actin cytoskeleton is essential for acid secretion. Herein, we show that Cdc42 is preferentially distributed to the apical membrane of gastric parietal cells. In addition, we revealed that two Cdc42 regulators, IQGAP1 and IQGAP2, are present in gastric parietal cells. Interestingly, IQGAP2 is polarized to the apical membrane of the parietal cells, whereas IQGAP1 is mainly distributed to the basolateral membrane. An IQGAP peptide that competes with full-length IQGAP proteins for Cdc42-binding in vitro also inhibits acid secretion in streptolysin-O-permeabilized gastric glands. Furthermore, this peptide disrupts the association of IQGAP and Cdc42 with the apical actin cytoskeleton and prevents the apical membrane remodeling upon stimulation. We propose that IQGAP2 forms a link that associates Cdc42 with the apical cytoskeleton and thus allows for activation of polarized secretion in gastric parietal cells.     

IQGAP1 regulates Salmonella invasion through interactions with actin, Rac1, and Cdc42

To infect host cells, Salmonella utilizes an intricate system to manipulate the actin cytoskeleton and promote bacterial uptake. Proteins injected into the host cell by Salmonella activate the Rho GTPases, Rac1 and Cdc42, to induce actin polymerization. Following uptake, a different set of proteins inactivates Rac1 and Cdc42, returning the cytoskeleton to normal. Although the signaling pathways allowing Salmonella to invade host cells are beginning to be understood, many of the contributing factors remain to be elucidated. IQGAP1 is a multidomain protein that influences numerous cellular functions, including modulation of Rac1/Cdc42 signaling and actin polymerization. Here, we report that IQGAP1 regulates Salmonella invasion. Through its interaction with actin, IQGAP1 co-localizes with Rac1, Cdc42, and actin at sites of bacterial uptake, whereas infection promotes the interaction of IQGAP1 with both Rac1 and Cdc42. Knockdown of IQGAP1 significantly reduces Salmonella invasion and abrogates activation of Cdc42 and Rac1 by Salmonella. Overexpression of IQGAP1 significantly increases the ability of Salmonella to enter host cells and required interaction with both actin and Cdc42/Rac1. Together, these data identify IQGAP1 as a novel regulator of Salmonella invasion.     

Localization of the PAK1-, WASP-, and IQGAP1-specifying regions of Cdc42.

The Rho family small GTPase Cdc42 transmits divergent intracellular signals through multiple effector proteins to elicit cellular responses such as cytoskeletal reorganization. Potential effectors of Cdc42 implicated in mediating its cytoskeletal effect in mammalian cells include PAK1, WASP, and IQGAP1. To investigate the determinants of Cdc42-effector specificity, we utilized recombinant Cdc42 mutants and chimeras made between Cdc42 and RhoA to map the regions of Cdc42 contributing to specific effector p21-binding domain (PBD) interaction. Site-directed mutants of the switch I domain and neighboring regions of Cdc42 demonstrated differential binding patterns toward the PBDs of PAK1, WASP, and IQGAP1, suggesting that switch I provides essential determinants for the effector binding, but recognition of each effector by Cdc42 involves a distinct mechanism. Differing from Rac1, the switch I domain and the surrounding region (amino acids 29 to 55) of Cdc42 appeared to be sufficient for specific binding to PAK1, whereas determinants outside the switch I domain, residues 157-191 and 84-120 in particular, were necessary and sufficient to confer specificity to WASP and IQGAP1, respectively. In addition, IQGAP1, but not PAK1 nor WASP, required the unique "insert region," residues 122-134, of Cdc42 to achieve high affinity binding. Microinjection of the constitutively active Cdc42/RhoA chimeras into serum-starved Swiss 3T3 cells showed that although preserving PAK1- and WASP-binding activity could retain the peripheral actin microspike (PAM)-inducing activity of Cdc42, interaction with PAK1 or WASP was not required for this activity. Moreover, IQGAP1-binding alone by Cdc42 was insufficient for PAM-induction. Thus, Cdc42 utilizes multiple distinct structural determinants to specify different effector recognition and to elicit PAM-inducing effect.     

Lipid raft-associated GTPase signaling controls morphology and CD8+ T cell stimulatory capacity of human dendritic cells

Their eponymous morphology and unique ability to activate naive T cells are hallmark features of dendritic cells (DCs). Specific properties of the actin cytoskeleton may define both characteristics. In search for regulators that coordinate DC phenotype and function, we observed strongly increased expression of the actin-remodeling GTPases Cdc42 and Rac1 during DC development from human stem cells. Cdc42 and Rac1 are constitutively active in immature DCs, and their activity is further up-regulated by maturational stimuli such as LPS or CD40L. Activation of Rac1 is associated with its rapid recruitment into lipid rafts. Cdc42 is not recruited into rafts, but readily activated by raft-associated moieties. The functional interplay of rafts, GTPases, and cortical actin is further shown by GTPase activation and actin remodeling after pharmacological disruption of lipid rafts and by the loss of the actin-based DC morphology by transfection of dominant-negative Cdc42 and Rac1. Both Cdc42 and Rac1 also control the transport of essential immunostimulatory molecules to the DC surface. Transfection with dominant-negative GTPases led to reduced surface expression of MHC class I and CD86. Consecutively, DCs display a reduced stimulatory capacity for CD8(+) T cells, whereas MHC class II-dependent stimulation of CD4(+) T cells remains unperturbed. We conclude that Cdc42 and Rac1 signaling controls DC morphology and conditions DCs for efficient CD8(+) T cell stimulation.     

The Ras GTPase-activating-protein-related human protein IQGAP2 harbors a potential actin binding domain and interacts with calmodulin and Rho family GTPases.

We previously described IQGAP1 as a human protein related to a putative Ras GTPase-activating protein (RasGAP) from the fission yeast Schizosaccharomyces pombe. Here we report the identification of a liver-specific human protein that is 62% identical to IQGAP1. Like IQGAP1, the novel IQGAP2 protein harbors an N-terminal calponin homology motif which functions as an F-actin binding domain in members of the spectrin, filamin, and fimbrin families. Both IQGAPs also harbor several copies of a novel 50- to 55-amino-acid repeat, a single WW domain, and four IQ motifs and have 25% sequence identity with almost the entire S. pombe sar1 RasGAP homolog. As predicted by the presence of IQ motifs, IQGAP2 binds calmodulin. However, neither full-length nor truncated IQGAP2 stimulated the GTPase activity of Ras or its close relatives. Instead, IQGAP2 binds Cdc42 and Racl but not RhoA. This interaction involves the C-terminal half of IQGAP2 and appears to be independent of the nucleotide binding status of the GTPases. Although IQGAP2 shows no GAP activity towards Cdc42 and Rac1, the protein did inhibit both the intrinsic and RhoGAP-stimulated GTP hydrolysis rates of Cdc42 and Rac1, suggesting an alternative mechanism via which IQGAPs might modulate signaling by these GTPases. Since IQGAPs harbor a potential actin binding domain, they could play roles in the Cdc42 and Rac1 controlled generation of specific actin structures.     

Salmonella enterica serotype Typhimurium usurps the scaffold protein IQGAP1 to manipulate Rac1 and MAPK signalling

Salmonella enterica serotype Typhimurium invades eukaryotic cells by re-arranging the host-cell cytoskeleton. However, the precise mechanisms by which Salmonella induces cytoskeletal changes remain undefined. IQGAP1 (IQ motif-containing GTPase-activating protein 1) is a scaffold protein that binds multiple proteins including actin, the Rho GTPases Rac1 and Cdc42 (cell division cycle 42), and components of the MAPK (mitogen-activated protein kinase) pathway. We have shown previously that optimal invasion of Salmonella into HeLa cells requires IQGAP1. In the present paper, we use IQGAP1-null MEFs (mouse embryonic fibroblasts) and selected well-characterized IQGAP1 mutant constructs to dissect the molecular determinants of Salmonella invasion. Knockout of IQGAP1 expression reduced Salmonella invasion into MEFs by 75%. Reconstituting IQGAP1-null MEFs with wild-type IQGAP1 completely rescued invasion. By contrast, reconstituting IQGAP1-null cells with mutant IQGAP1 constructs that specifically lack binding to either Cdc42 and Rac1 (termed IQGAP1ΔMK24), actin, MEK [MAPK/ERK (extracellular-signal-regulated kinase) kinase] or ERK only partially restored Salmonella entry. Cell-permeant inhibitors of Rac1 activation or MAPK signalling reduced Salmonella invasion into control cells by 50%, but had no effect on bacterial entry into IQGAP1-null MEFs. Importantly, the ability of IQGAP1ΔMK24 to promote Salmonella invasion into IQGAP1-null cells was abrogated by chemical inhibition of MAPK signalling. Collectively, these results imply that the scaffolding function of IQGAP1, which integrates Rac1 and MAPK signalling, is usurped by Salmonella to invade fibroblasts and suggest that IQGAP1 may be a potential therapeutic target for Salmonella pathogenesis.     

WIP regulates N-WASP-mediated actin polymerization and filopodium formation

Induction of filopodia is dependent on activation of the small GTPase Cdc42 and on neural Wiskott-Aldrich-syndrome protein (N-WASP). Here we show that WASP-interacting protein (WIP) interacts directly with N-WASP and actin. WIP retards N-WASP/Cdc42-activated actin polymerization mediated by the Arp2/3 complex, and stabilizes actin filaments. Microinjection of WIP into NIH 3T3 fibroblasts induces filopodia; this is inhibited by microinjection of anti-N-WASP antibody. Microinjection of anti-WIP antibody inhibits induction of filopodia by bradykinin, by an active Cdc42 mutant (Cdc42(V12)) and by N-WASP. Our results indicate that WIP and N-WASP may act as a functional unit in filopodium formation, which is consistent with their role in actin-tail formation in cells infected with vaccinia virus or Shigella.     

Interactions among IQGAP1, Cdc42, and the cadherin/catenin protein complex regulate Sertoli-germ cell adherens junction dynamics in the testis

The movement of developing germ cells across the seminiferous epithelium during spermatogenesis involves extensive adherens junction (AJ) restructuring between Sertoli cells, as well as between Sertoli and germ cells. In this report, we show that the intricate interactions between Cdc42 (a Rho family protein of Mr approximately 23 kDa originally identified in membranes of human platelets and placenta, and is the homolog of CDC42Sc, which is known to regulate of bud-site assembly in Saccharomyces cerevisiae) and its effector, IQ motif containing GTPase activating protein (IQGAP1, Mr approximately 189 kDa, it is also an actin-binding protein known to interact with Cdc42 and Rac1 GTPases), regulate Sertoli-germ cell, but not Sertoli-Sertoli cell, AJ dynamics. Using testis lysates for immunoprecipitation (IP), IQGAP1 was shown to associate with E-cadherin, N-cadherin, and beta-catenin (but not beta1-integrin and nectin-2), as well as with actin and vimentin (but not alpha-tubulin). Moreover, IQGAP1 was found to localize to the periphery of both Sertoli and germ cells in the seminiferous epithelium, at sites of cell-cell contacts. Using fluorescent microscopy with dual fluorescent probes, IQGAP1 was found to co-localize, at least in part, with N-cadherin in the seminiferous epithelium consistent with their localization at the basal and apical ES. Using Sertoli-germ cell cocultures, it was demonstrated that AJ assembly associated with a transient induction of Cdc42 and IQGAP1, which was not found when Sertoli cells were cultured alone. Lastly, a shift in the interactions of Cdc42, IQGAP1, beta-catenin, and N-cadherin was detected in Sertoli-germ cell cocultures using an Ca2+-induced AJ disruption model, which was used to examine AJ disassembly and its reassembly. In the presence of Ca2+, IQGAP1 bound preferentially to Cdc42 rather than to beta-catenin. However, when Ca2+ was depleted from cocultures using EGTA, a Ca2+ chelating agent, IQGAP1 lost its affinity for Cdc42 and became tightly associated with beta-catenin, destabilizing cadherin-mediated AJs between Sertoli and germ cells. Yet this shift of protein-protein interaction was not detected in Sertoli cells cultured alone. These results illustrate that the interactions among IQGAP1, Cdc42, and beta-catenin are crucial to the regulation of Sertoli-germ cell, but not Sertoli-Sertoli cell, AJ dynamics in the seminiferous epithelium.     

Regulation of cadherin-mediated cell-cell adhesion by the Rho family GTPases.

Reports in the past two years have shown that Cdc42, Rac1, and Rho - belonging to the Rho small GTPase family - participate in the regulation of cadherin-mediated cell-cell adhesion. IQGAP1, an effector of Cdc42 and Rac1, interacts with cadherin and beta-catenin and induces the dissociation of alpha-catenin from the cadherin-catenins complex leading to disruption of cell-cell adhesion: activated Cdc42 and Rac1 counteract the effect of IQGAP1. Thus, Cdc42 and Rac1 appear to regulate cadherin-mediated cell-cell adhesion acting through IQGAP1.     

Actin pedestal formation by enteropathogenic Escherichia coli is regulated by IQGAP1, calcium, and calmodulin

During infection, enteropathogenic Escherichia coli (EPEC) injects effector proteins into the host cell to manipulate the actin cytoskeleton and promote formation of actin pedestals. IQGAP1 is a multidomain protein that participates in numerous cellular functions, including Rac1/Cdc42 and Ca(2+)/calmodulin signaling and actin polymerization. Here we report that IQGAP1, Ca(2+), and calmodulin modulate actin pedestal formation by EPEC. Infection with EPEC promotes both the interaction of IQGAP1 with calmodulin and the localization of IQGAP1 and calmodulin to actin pedestals while reducing the interaction of IQGAP1 with Rac1 and Cdc42. IQGAP1-null fibroblasts display a reduced polymerization of actin in response to EPEC. In addition, antagonism of calmodulin or chelation of intracellular Ca(2+) reduces EPEC-dependent actin polymerization. Furthermore, IQGAP1 specifically interacts with Tir in vitro and in cells. Together these data identify IQGAP1, Ca(2+), and calmodulin as a novel signaling complex regulating actin pedestal formation by EPEC.     

Interaction with IQGAP1 links APC to Rac1, Cdc42, and actin filaments during cell polarization and migration

Rho family GTPases, particularly Rac1 and Cdc42, are key regulators of cell polarization and directional migration. Adenomatous polyposis coli (APC) is also thought to play a pivotal role in polarized cell migration. We have found that IQGAP1, an effector of Rac1 and Cdc42, interacts directly with APC. IQGAP1 and APC localize interdependently to the leading edge in migrating Vero cells, and activated Rac1/Cdc42 form a ternary complex with IQGAP1 and APC. Depletion of either IQGAP1 or APC inhibits actin meshwork formation and polarized migration. Depletion of IQGAP1 or APC also disrupts localization of CLIP-170, a microtubule-stabilizing protein that interacts with IQGAP1. Taken together, these results suggest a model in which activation of Rac1 and Cdc42 in response to migration signals leads to recruitment of IQGAP1 and APC which, together with CLIP-170, form a complex that links the actin cytoskeleton and microtubule dynamics during cell polarization and directional migration.     

IQGAP1 and calmodulin modulate E-cadherin function

Ca(2+)-dependent cell-cell adhesion is mediated by the cadherin family of transmembrane proteins. Adhesion is achieved by homophilic interaction of the extracellular domains of cadherins on adjacent cells, with the cytoplasmic regions serving to couple the complex to the cytoskeleton. IQGAP1, a novel RasGAP-related protein that interacts with the cytoskeleton, binds to actin, members of the Rho family, and E-cadherin. Calmodulin binds to IQGAP1 and regulates its association with Cdc42 and actin. Here we demonstrate competition between calmodulin and E-cadherin for binding to IQGAP1 both in vitro and in a normal cellular milieu. Immunocytochemical analysis in MCF-7 (E-cadherin positive) and MDA-MB-231 (E-cadherin negative) epithelial cells revealed that E-cadherin is required for accumulation of IQGAP1 at cell-cell junctions. The cell-permeable calmodulin antagonist CGS9343B significantly increased IQGAP1 at areas of MCF-7 cell-cell contact, with a concomitant decrease in the amount of E-cadherin at cell-cell junctions. Analysis of E-cadherin function revealed that CGS9343B significantly decreased homophilic E-cadherin adhesion. On the basis of these data, we propose that disruption of the binding of calmodulin to IQGAP1 enhances the association of IQGAP1 with components of the cadherin-catenin complex at cell-cell junctions, resulting in impaired E-cadherin function.     

IQGAP1, a calmodulin-binding protein with a rasGAP-related domain, is a potential effector for cdc42Hs.

Proteins that associate with the GTP-bound forms of the Ras superfamily of proteins are potential effector targets for these molecular switches. A 195 kDa protein was purified from cell lysates by affinity chromatography on immobilized cdc42Hs-GTP and a corresponding cDNA was isolated. Sequence analysis revealed localized identities to calponin, the WW domain, unconventional myosins and to the rasGAP-related domain (GRD) contained in IRA, NF-1, SAR1 and rasGAP. p195 was found to be identical to IQGAP1, a protein previously reported to bind ras. Purified recombinant p195/IQGAP1 bound to and inhibited the GTPase activity of cdc42Hs and rac whereas no interaction with ras was detected. The C-terminal half of IQGAP1 containing the GRD bound to cdc42 and rac in a GRD-dependent fashion, but a smaller fragment containing only the GRD did not. Cdc42 was also co-immunoprecipitated from cell lysates with antibody specific to p195/IQGAP1. Calmodulin also co-immunoprecipitated with p195/IQGAP1 and was found to associate with fragments containing the IQ domain. Expression of a cDNA fragment encoding the GRD inhibited the CDC24/CDC42 pathway in yeast, but no effect on ras was observed. In mammalian cells, both endogenous and ectopically expressed p195/IQGAP1 were localized to lamellipodia and ruffling cell membranes, where co-localization with actin was apparent. These results suggest that IQGAP1 is an effector target for cdc42Hs and may mediate the effects of this GTPase on cell morphology.     

Cdc42 protein acts upstream of IQGAP1 and regulates cytokinesis in mouse oocytes and embryos

Cdc42 and Rac1 Rho family GTPases, and their interacting protein IQGAP1 are the key regulators of cell polarity. We examined the role of Cdc42 and IQGAP1 in establishing the polarity of mouse oocyte and regulation of meiotic and mitotic divisions. We showed that Cdc42 was localized on the microtubules of meiotic and mitotic spindle and in the cortex of mouse oocytes and cleaving embryos. IQGAP1 was present in the cytoplasm and cortex of growing and fully-grown oocytes. During maturation it disappeared from the cortex and during meiotic and mitotic cytokinesis it concentrated in the contractile ring. Toxin B inhibition of the binding activity of Cdc42 changed the localization of IQGAP1, inhibited emission of the first polar body, and caused disappearance of the cortical actin without affecting the migration of meiotic spindle. This indicates, that in maturing oocytes accumulation of cortical actin is not indispensable for spindle migration. In zygotes treated with toxin B actin cytoskeleton was rearranged and the first and/or subsequent cytokinesis were inhibited. Our results indicate that Cdc42 acts upstream of IQGAP1 and is involved in regulation of cytokinesis in mouse oocytes and cleaving embryos, rather than in establishing the polarity of the oocyte.