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.     

IQGAP1 promotes neurite outgrowth in a phosphorylation-dependent manner

In eukaryotic cells IQGAP1 binds to and alters the function of several proteins, including actin, E-cadherin, beta-catenin, Cdc42, and Rac1. Yeast IQGAP1 homologues have an important role in cytoskeletal organization, suggesting that modulation of the cytoskeleton is a fundamental role of IQGAP1. Phosphorylation is a common mechanism by which cells regulate protein function. Here we demonstrate that endogenous IQGAP1 is highly phosphorylated in MCF-7 human breast epithelial cells. Moreover, incubation of cells with phorbol 12-myristate 13-acetate (PMA) stimulated phosphate incorporation into IQGAP1. By using mass spectrometry, Ser-1443 was identified as the major site phosphorylated on IQGAP1 in intact cells treated with PMA. Ser-1441 was also phosphorylated but to a lesser extent. In vitro analysis with purified proteins documented that IQGAP1 is a substrate for protein kinase Cepsilon, which catalyzes phosphorylation on Ser-1443. Consistent with these findings, inhibition of cellular protein kinase C via bisindolymaleimide abrogated Ser-1443 phosphorylation in response to PMA. To elucidate the biological sequelae of phosphorylation, Ser-1441 and Ser-1443 were converted either to alanine, to create a nonphosphorylatable construct, or to glutamic acid and aspartic acid, respectively, to generate a phosphomimetic IQGAP1. Although overexpression of wild type IQGAP1 promoted neurite outgrowth in N1E-115 neuroblastoma cells, the nonphosphorylatable IQGAP1 S1441A/S1443A had no effect. In contrast, the S1441E/S1443D mutation markedly enhanced the ability of IQGAP1 to induce neurite outgrowth. Our data disclose that IQGAP1 is phosphorylated at multiple sites in intact cells and that phosphorylation of IQGAP1 will alter its ability to regulate the cytoskeleton of neuronal cells.     

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.     

Involvement of IQGAP1, an effector of Rac1 and Cdc42 GTPases, in cell-cell dissociation during cell scattering

We have previously proposed that IQGAP1, an effector of Rac1 and Cdc42, negatively regulates cadherin-mediated cell-cell adhesion by interacting with beta-catenin and by causing the dissociation of alpha-catenin from cadherin-beta-catenin-alpha-catenin complexes and that activated Rac1 and Cdc42 positively regulate cadherin-mediated cell-cell adhesion by inhibiting the interaction of IQGAP1 with beta-catenin. However, it remains to be clarified in which physiological processes the Rac1-Cdc42-IQGAP1 system is involved. We here examined whether the Rac1-IQGAP1 system is involved in the cell-cell dissociation of Madin-Darby canine kidney II cells during 12-O-tetradecanoylphorbol-13-acetate (TPA)- or hepatocyte growth factor (HGF)-induced cell scattering. By using enhanced green fluorescent protein (EGFP)-tagged alpha-catenin, we found that EGFP-alpha-catenin decreased prior to cell-cell dissociation during cell scattering. We also found that the Rac1-GTP level decreased after stimulation with TPA and that the Rac1-IQGAP1 complexes decreased, while the IQGAP1-beta-catenin complexes increased during action of TPA. Constitutively active Rac1 and IQGAP1 carboxyl terminus, a putative dominant-negative mutant of IQGAP1, inhibited the disappearance of alpha-catenin from sites of cell-cell contact induced by TPA. Taken together, these results indicate that alpha-catenin is delocalized from cell-cell contact sites prior to cell-cell dissociation induced by TPA or HGF and suggest that the Rac1-IQGAP1 system is involved in cell-cell dissociation through alpha-catenin relocalization.     

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.     

Characterization of the interactions of the nephrin intracellular domain

Nephrin is a signalling cell-cell adhesion protein of the Ig superfamily and the first identified component of the slit diaphragm that forms the critical and ultimate part of the glomerular ultrafiltration barrier. The extracellular domains of the nephrin molecules form a network of homophilic and heterophilic interactions building the structural scaffold of the slit diaphragm between the podocyte foot processes. The intracellular domain of nephrin is connected indirectly to the actin cytoskeleton, is tyrosine phosphorylated, and mediates signalling from the slit diaphragm into the podocytes. CD2AP, podocin, Fyn kinase, and phosphoinositide 3-kinase are reported intracellular interacting partners of nephrin, although the biological roles of these interactions are unclarified. To characterize the structural properties and protein-protein interactions of the nephrin intracellular domain, we produced a series of recombinant nephrin proteins. These were able to bind all previously identified ligands, although the interaction with CD2AP appeared to be of extremely low stoichiometry. Fyn phosphorylated nephrin proteins efficiently in vitro. This phosphorylation was required for the binding of phosphoinositide 3-kinase, and significantly enhanced binding of Fyn itself. A protein of 190 kDa was found to associate with the immobilized glutathione S-transferase-nephrin. Peptide mass fingerprinting and amino acid sequencing identified this protein as IQGAP1, an effector protein of small GTPases Rac1 and Cdc42 and a putative regulator of cell-cell adherens junctions. IQGAP1 is expressed in podocytes at significant levels, and could be found at the immediate vicinity of the slit diaphragm. However, further studies are needed to confirm the biological significance of this interaction and its occurrence in vivo.     

The IQGAP1-Rac1 and IQGAP1-Cdc42 interactions: interfaces differ between the complexes

IQGAP1 contains a domain related to the catalytic portion of the GTPase-activating proteins (GAPs) for the Ras small G proteins, yet it has no RasGAP activity and binds to the Rho family small G proteins Cdc42 and Rac1. It is thought that IQGAP1 is an effector of Rac1 and Cdc42, regulating cell-cell adhesion through the E-cadherin-catenin complex, which controls formation and maintenance of adherens junctions. This study investigates the binding interfaces of the Rac1-IQGAP1 and Cdc42-IQGAP1 complexes. We mutated Rac1 and Cdc42 and measured the effects of mutations on their affinity for IQGAP1. We have identified similarities and differences in the relative importance of residues used by Rac1 and Cdc42 to bind IQGAP1. Furthermore, the residues involved in the complexes formed with IQGAP1 differ from those formed with other effector proteins and GAPs. Relatively few mutations in switch I of Cdc42 or Rac1 affect IQGAP1 binding; only mutations in residues 32 and 36 significantly decrease affinity for IQGAP1. Switch II mutations also affect binding to IQGAP1 although the effects differ between Rac1 and Cdc42; mutation of either Asp-63, Arg-68, or Leu-70 abrogate Rac1 binding, whereas no switch II mutations affect Cdc42 binding to IQGAP1. The Rho family "insert loop" does not contribute to the binding affinity of Rac1/Cdc42 for IQGAP1. We also present thermodynamic data pertaining to the Rac1/Cdc42-RhoGAP complexes. Switch II contributes a large portion of the total binding energy to these complexes, whereas switch I mutations also affect binding. In addition we identify "cold spots" in the Rac1/Cdc42-RhoGAP/IQGAP1 interfaces. Competition data reveal that the binding sites for IQGAP1 and RhoGAP on the small G proteins overlap only partially. Overall, the data presented here suggest that, despite their 71% identity, Cdc42 and Rac1 appear to have only partially overlapping binding sites on IQGAP1, and each uses different determinants to achieve high affinity binding.     

Regulation of Spine Density and Morphology by IQGAP1 Protein Domains

IQGAP1 is a scaffolding protein that regulates spine number. We now show a differential role for IQGAP1 domains in spine morphogenesis, in which a region of the N-terminus that promotes Arp2/3-mediated actin polymerization and branching stimulates spine head formation while a region that binds to Cdc42 and Rac is required for stalk extension. Conversely, IQGAP1 rescues spine deficiency induced by expression of dominant negative Cdc42 by stimulating formation of stubby spines. Together, our observations place IQGAP1 as a crucial regulator of spine number and shape acting through the N-Wasp Arp2/3 complex, as well as upstream and downstream of Cdc42.     

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.     

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.     

MAPK scaffold IQGAP1 binds the EGF receptor and modulates its activation

Cellular responses produced by EGF are mediated through the receptor (EGFR) and by various enzymes and scaffolds. Recent studies document IQGAP1 as a scaffold for the MAPK cascade, binding directly to B-Raf, MEK, and ERK and regulating their activation in response to EGF. We previously showed that EGF is unable to activate B-Raf in cells lacking IQGAP1. However, the mechanism by which IQGAP1 links B-Raf to EGFR was unknown. Here we report that endogenous EGFR and IQGAP1 co-localize and co-immunoprecipitate in cells. EGF has no effect on the association, but Ca(2+) attenuates binding. In vitro analysis demonstrated a direct association mediated through the IQ and kinase domains of IQGAP1 and EGFR, respectively. Calmodulin disrupts this interaction. Using a mass spectrometry-based assay, we show that EGF induces phosphorylation of IQGAP1 Ser(1443), a residue known to be phosphorylated by PKC. This phosphorylation is eliminated by pharmacological inhibition of either EGFR or PKC and transfection with small interfering RNA directed against the PKCα isoform. In IQGAP1-null cells, EGF-stimulated tyrosine phosphorylation of EGFR is severely attenuated. Normal levels of autophosphorylation are restored by reconstituting wild type IQGAP1 and enhanced by an IQGAP1 S1443D mutant. Collectively, these data demonstrate a functional interaction between IQGAP1 and EGFR and suggest that IQGAP1 modulates EGFR activation.     

The receptor protein-tyrosine phosphatase PTPmu interacts with IQGAP1

The receptor protein-tyrosine phosphatase PTPmu is a member of the Ig superfamily of cell adhesion molecules. The extracellular domain of PTPmu contains motifs commonly found in cell adhesion molecules. The intracellular domain of PTPmu contains two conserved catalytic domains, only the membrane-proximal domain has catalytic activity. The unique features of PTPmu make it an attractive molecule to transduce signals upon cell-cell contact. PTPmu has been shown to regulate cadherin-mediated cell adhesion, neurite outgrowth, and axon guidance. Protein kinase C is a component of the PTPmu signaling pathway utilized to regulate these events. To aid in the further characterization of PTPmu signaling pathways, we used a series of GST-PTPmu fusion proteins, including catalytically inactive and substrate trapping mutants, to identify PTPmu-interacting proteins. We identified IQGAP1, a known regulator of the Rho GTPases, Cdc42 and Rac1, as a novel PTPmu-interacting protein. We show that this interaction is due to direct binding. In addition, we demonstrate that amino acid residues 765-958 of PTPmu, which include the juxtamembrane domain and 35 residues of the first phosphatase domain, mediate the binding to IQGAP1. Furthermore, we demonstrate that constitutively active Cdc42, and to a lesser extent Rac1, enhances the interaction of PTPmu and IQGAP1. These data indicate PTPmu may regulate Rho-GTPase-dependent functions of IQGAP1 and suggest that IQGAP1 is a component of the PTPmu signaling pathway. In support of this, we show that a peptide that competes IQGAP1 binding to Rho GTPases blocks PTPmu-mediated neurite outgrowth.     

Crystal Structure of the GTPase-activating Protein-related Domain from IQGAP1

IQGAP1 is a 190-kDa molecular scaffold containing several domains required for interaction with numerous proteins. One domain is homologous to Ras GTPase-activating protein (GAP) domains. However, instead of accelerating hydrolysis of bound GTP on Ras IQGAP1, using its GAP-related domain (GRD) binds to Cdc42 and Rac1 and stabilizes their GTP-bound states. We report here the crystal structure of the isolated IQGAP1 GRD. Despite low sequence conservation, the overall structure of the GRD is very similar to the GAP domains from p120 RasGAP, neurofibromin, and SynGAP. However, instead of the catalytic “arginine finger” seen in functional Ras GAPs, the GRD has a conserved threonine residue. GRD residues 1099–1129 have no structural equivalent in RasGAP and are seen to form an extension at one end of the molecule. Because the sequence of these residues is highly conserved, this region likely confers a functionality particular to IQGAP family GRDs. We have used isothermal titration calorimetry to demonstrate that the isolated GRD binds to active Cdc42. Assuming a mode of interaction similar to that displayed in the Ras-RasGAP complex, we created an energy-minimized model of Cdc42·GTP bound to the GRD. Residues of the GRD that contact Cdc42 map to the surface of the GRD that displays the highest level of sequence conservation. The model indicates that steric clash between threonine 1046 with the phosphate-binding loop and other subtle changes would likely disrupt the proper geometry required for GTP hydrolysis.The small GTPase Ras functions as a binary switch in cell signaling processes. When bound to GTP, Ras is able to interact with effector proteins, including Raf kinase, and alter their activities. Ras signaling is terminated when bound GTP is hydrolyzed to GDP and inorganic phosphate. The basal rate of GTP hydrolysis on Ras is quite slow (∼1.2 × 10^–4 s^–1), but this rate of hydrolysis can be enhanced ∼10⁵-fold by interaction with a GTPase-activating protein (GAP)² (1). Several RasGAPs have been identified to date including p120 RasGAP and neurofibromin (NF1). The Rho family of Ras-related small GTPases also function as binary switches in cell signaling processes. Whereas the intrinsic rate of GTP hydrolysis on Rho proteins is faster than Ras, this rate can also be stimulated by interaction with a RhoGAP. Examination of the structures of the GAP domains of p120RasGAP (2), neurofibromin (3), SynGAP (4), and the GAP domains from the RhoGAPs p50 RhoGAP and the Bcr homology domain of phosphatidylinositol 3-kinase (5, 6) indicates that although ostensibly different, these all-helical domains are structurally related (7).IQGAP1 was discovered by chance during an attempt to isolate novel matrix metalloproteinases (8). Analysis reveals that the protein contains several discrete domains and motifs including a region containing four isoleucine- and glutamine-rich motifs (IQ repeats) and a region with sequence homology to the Ras-specific GAP domains of p120RasGAP, NF1, and SynGAP (2–4, 8). Subsequently, two homologs, IQGAP2 and IQGAP3, have been discovered. The IQ repeats have been shown to mediate binding to calmodulin and calmodulin-like proteins (e.g. S100, myosin essential light chain), whereas the GAP-related domain (GRD) does not appear to bind to Ras but instead is necessary for binding to the Rho family GTPases Cdc42 and Rac1, primarily in their active forms (9–11). However, instead of accelerating hydrolysis of GTP, IQGAP1 preserves the activated states of Cdc42 and Rac1 to the extent that overexpression of IQGAP1 in cells increases the levels of active GTPase (12). Because IQGAP1 expression increases the level of activated Cdc42, initially there was some confusion as to whether the protein might not represent a novel guanine nucleotide exchange factor. However it now appears that IQGAP1 is an effector of Cdc42 and Rac1 and preserves their activated states by tightly binding to the GTPases and stabilizing them in a conformation not conducive to GTP hydrolysis. IQGAP1 appears to be such an important effector for Cdc42 that abrogation of binding to IQGAP1 not only reduces the levels of active Cdc42, it also reduces membrane-localized Cdc42 and the cellular response to bradykinin (12).A growing body of evidence implicates IQGAP1 in carcinogenesis. Expression of IQGAP1 increases during the transition from a minimally to a highly metastastic form of melanoma, and IQGAP1 has been found to be overexpressed in ovarian, breast, lung, and colorectal cancers (13–17). In vitro, overexpressed IQGAP1 enhances cell motility and invasiveness in a process that requires Cdc42 and Rac (18). β-Catenin is one of the many binding partners of IQGAP1 identified to date. IQGAP1 has been shown to bind to β-catenin and interfere with β-catenin binding to α-catenin, an interaction necessary for stable cell-cell adhesion (19). Another study found that IQGAP2 knock-out mice overexpress IQGAP1 and developage-dependent liver cancer and apoptosis (20).To better understand how a protein domain homologous to others that accelerate GTP hydrolysis can function as an effector and preserve the GTP-bound state, we have determined the x-ray structure of the IQGAP1 GRD. Despite low sequence identity, the GRD structure is quite similar to the GAP domains of p120, neurofibromin, and SynGAP; however, unlike those domains, the GRD possesses a conserved threonine in place of the catalytic arginine finger and has a 31-residue insertion that projects from one end of the molecule. Using the coordinates of Ras·GDP·AlF₃ in complex with the GAP domain of p120, we built a model of Cdc42·GTP bound to the GRD. The model indicates that a steric clash between the conserved Thr¹⁰⁴⁶ and the phosphate-binding loop of Cdc42 and other subtle changes within the active site would likely preclude nucleotide hydrolysis. Sequence conservation mapped to the surface of the GRD indicates that the surface with the highest degree of conservation overlaps with the surface that makes contacts to Cdc42 in the model.     

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.     

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.     

Determination of Dissociation Constants in Living Zebrafish Embryos with Single Wavelength Fluorescence Cross-Correlation Spectroscopy

The quantification of biological interactions is very important in life sciences. Here we report for the first time, to our knowledge, the determination of a biomolecular dissociation constant (K_D) in living zebrafish embryos at physiological protein expression levels. For that purpose, we extend the application of single wavelength fluorescence cross-correlation spectroscopy into small organisms and measure the interaction of Cdc42, a small Rho-GTPase, and IQGAP1, an actin-binding scaffolding protein. Cdc42 and IQGAP1 were labeled with monomeric red fluorescent protein and enhanced green fluorescent protein, respectively. Both fluorophores were excited at a single wavelength of 514 nm, simplifying the fluorescence spectroscopy measurements and allowing quantification. For the determination of the interaction, we used two Cdc42 mutants, the constitutively active Cdc42^G12V which is in a predominantly GTP-bound form and the dominant-negative GDP-bound Cdc42^T17N. While Cdc42^G12V binds to IQGAP1 with an apparent K_D of ∼100 nM, Cdc42^T17N has at least a one-order-of-magnitude lower affinity for the same protein. As a comparison, we measure the same protein-protein interactions in Chinese hamster ovary cell cultures but observe significant differences in protein mobility and K_D from the zebrafish measurements, supporting the notion that bimolecular interactions depend on the biological system under investigation and are best performed under physiologically relevant conditions.     

IQGAPs are differentially expressed and regulated in polarized gastric epithelial cells

IQGAPs, GTPase-activating proteins with an IQ motif, are thought to regulate many actin cytoskeleton-based activities through interactions with Cdc42 and Rac. Recently, Cdc42 was implicated in regulation of gastric parietal cell HCl secretion, and IQGAP2 was immunolocalized with Cdc42 to F-actin-rich intracellular canalicular membranes of isolated gastric parietal cells in primary culture. Here we sought to define distribution and localization of IQGAP1 and IQGAP2 in major oxyntic (acid-secreting) gastric mucosal cell types and to determine whether secretory agonists modulate these proteins. Differential staining protocols were used to identify different cell populations (parietal, chief, surface/pit, and mucous neck cells) in semi-intact glands isolated from rabbit gastric mucosae and to characterize these same cells after dispersion and fractionation on isopycnic density gradients with simultaneous staining for F-actin, H+-K+-ATPase, and GSII lectin-binding sites. There was a pronounced increase in intracellular F-actin staining in dispersed chief cells, apparently from internalization of F-actin-rich apical membranes that normally abut the gland lumen. Therefore, other membrane-associated proteins might also be redistributed by disruption of cell-cell contacts. Western blot analyses were used to quantitate relative concentrations of IQGAPs in defined mucosal cell fractions, and gastric glands were used for in situ localizations. We detected uniform levels of IQGAP2 expression in oxyntic mucosal cells with predominant targeting to regions of cell-cell contact and nuclei of all cell types. IQGAP2 was not detected in parietal cell intracellular canaliculi. IQGAP1 expression was variable and targeted predominantly to the cortex of chief and mucous neck cells. Parietal cells expressed little or no IQGAP1 vs. other mucosal cell types. Phosphoprotein affinity chromatography, isoelectric focusing, and phosphorylation site analyses indicated that both IQGAP1 and IQGAP2 are phosphoproteins potentially regulated by [Ca2+]i/PKC and cAMP signaling pathways, respectively. Stimulation of glands with carbachol, which elevates [Ca2+]i and activates PKC, induced apparent translocation of IQGAP1, but not IQGAP2, to apical poles of chief (zymogen) and mucous neck cells. This response was mimicked by PMA but not by ionomycin or by elevation of [cAMP]i with forskolin. Our observations support a novel, PKC-dependent role for IQGAP1 in regulated exocytosis and suggest that IQGAP2 may play a more general role in regulating cell-cell interactions and possibly migration within the gastric mucosa.     

Distinct Rho GTPase activities regulate epithelial cell localization of the adhesion molecule CEACAM1: involvement of the CEACAM1 transmembrane domain

CEACAM1 is an intercellular adhesion glycoprotein. As CEACAM1 plays an important role in epithelial cell signaling and functions, we have examined its localization in epithelial cells. We have observed that distribution at cell contacts is not always seen in these cells, suggesting that CEACAM1 localization might be regulated. In Swiss 3T3 cells, the targeting of CEACAM1 at cell-cell boundaries is regulated by the Rho GTPases. In the present study, we have used the MDCK epithelial cells to characterize the effects of the Rho GTPases and their effectors on CEACAM1 intercellular targeting. Activated Cdc42 and Rac1 or their downstream effector PAK1 targeted CEACAM1 to sites of cell-cell contacts. On the other hand, neither activated RhoA nor activated Rho kinase directed CEACAM1 to cell boundaries, resulting in a condensed distribution of CEACAM1 at the cell surface. Interestingly, inhibition of this pathway resulted in CEACAM1 intercellular localization suggesting that a tightly regulated balance of Rho GTPase activities is necessary to target CEACAM1 at cell-cell boundaries. In addition, using CEACAM1 mutants and chimeric fusion constructs containing domains of the colony-stimulating factor receptor, we have shown that the transmembrane domain of CEACAM1 is responsible for the Cdc42-induced targeting at cell-cell contacts.     

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.     

Cdc42, Rac1, and their effector IQGAP1 as molecular switches for cadherin-mediated cell-cell adhesion.

Cell-cell adhesion is a dynamic process in various cellular and developmental situations. Cadherins, well-known Ca(2+)-dependent adhesion molecules, are thought to play a major role in the regulation of cell-cell adhesion. However, the molecular mechanism underlying the rearrangement of cadherin-mediated cell-cell adhesion is largely unknown. Cdc42 and Rac1, belonging to the Rho small GTPase family, have recently been shown to be involved in the regulation of cell-cell adhesion. In addition, IQGAP1, an effector for Cdc42 and Rac1, has been shown to regulate the cadherin function through interaction with beta-catenin, a molecule associated with cadherin. In this review, we will summarize the mode of action of Cdc42 and Rac1 as well as IQGAP1 as molecular switches for the cadherin function, and then discuss physiological processes in which the Cdc42/Rac1/IQGAP1 system may be involved.