IQGAP1 is a conserved modular protein overexpressed in cancer and involved in organizing actin and microtubules in motile processes such as adhesion, migration, and cytokinesis. A variety of proteins have been shown to interact with IQGAP1, including the small G proteins Rac1 and Cdc42, actin, calmodulin, ␤-catenin, the microtubule plus end-binding proteins CLIP170 (cytoplasmic linker protein) and adenomatous polyposis coli. However, the molecular mechanism by which IQGAP1 controls actin dynamics in cell motility is not understood. Quantitative co-localization analysis and down-regulation of IQGAP1 revealed that IQGAP1 controls the co-localization of N-WASP with the Arp2/3 complex in lamellipodia. Co-immunoprecipitation supports an in vivo link between IQGAP1 and N-WASP. Pull-down experiments and kinetic assays of branched actin polymerization with N-WASP and Arp2/3 complex demonstrated that the C-terminal half of IQGAP1 activates N-WASP by interacting with its BR-CRIB domain in a Cdc42-like manner, whereas the N-terminal half of IQGAP1 antagonizes this activation by association with a C-terminal region of IQGAP1. We propose that signal-induced relief of the autoinhibited fold of IQGAP1 allows activation of N-WASP to stimulate Arp2/3-dependent actin assembly.Directional cell migration results from the coordination of protrusion formation and cell adhesion. Although the concerted re-organization of actin and microtubules establishes and maintains cell polarization during directional movement, little is known about the molecular mechanisms underlying signal-mediated crosstalk between the two different cytoskeletal arrays (1). In this context, the modular IQGAP1 protein has received intense interest in the past years (2). The multiple partners of IQGAP1, including signaling molecules like Cdc42 or Rac1, calmodulin (3-6), and adhesion/cytoskeletal proteins like ␤-catenin, E-cadherin, actin filaments, and microtubule plus end-tracking proteins (CLIP170 and adenomatous polyposis coli (APC)) strongly suggest that IQGAP1 is an important player in coordinating cell polarity, adhesion, and migration (7-13). Concrete support to this view was brought by evidence showing that IQGAP1 is overexpressed in cancer (14, 15), controls cytokinesis (16 -21), and cell-cell adhesion (22-24). In addition, recent reports showed that IQGAP1 localizes in lamellipodia of motile cells (4,25,26) where it may link microtubule ends to the actin cytoskeleton (12,27) and that overexpression of IQGAP1 increases cell motility, whereas knockdown of the protein reduces cell migration and inhibits the formation of a protrusive actin meshwork at the leading edge (25). Finally, IQGAP1 regulates E-cadherinmediated cell-cell adhesion both positively and negatively (11). However, the functional and molecular link between IQGAP1 and the actin cytoskeleton in cell-cell adhesions and in lamellipodia has remained elusive.Extension of lamellipodia is driven by stimulus-responsive WASP family proteins (N-WASP, WASP, and Scar/WAVE) Cortactin and CARMIL, which act...

The Rho-GTPase Cdc42 is important for the establishment and maintenance of epithelial polarity. Signaling from Cdc42 is propagated via its effector molecules that specifically bind to Cdc42 in the GTP-bound form. The cell-cell contact regulator and actin-binding protein IQGAP1 is described as effector of Cdc42 and Rac. Unexpectedly, we show in this study that IQGAP1 bound also directly nucleotide-depleted Cdc42 (Cdc42-ND). This interaction was enhanced in the presence of phosphatase inhibitors and in epithelial cells without cellcell contacts. Tandem mass spectrometry analysis and immunoprecipitation experiments revealed that IQGAP1 was Ser 1443 -phosphorylated in vivo, potentially by protein kinase C⑀ and upon loss of cell-cell contacts. In addition, we identified two independent domains of the IQGAP1 C terminus that bound exclusively Cdc42-ND. These domains interacted with each other, favoring the binding to Cdc42-GTP. Moreover, phosphorylation on Ser 1443 strongly inhibited this intramolecular interaction. Thus, we unraveled a molecular mechanism that reveals a novel type of Rho-GTPase regulator. We propose that, depending on its phosphorylation state, IQGAP1 might serve as an effector or sequester nucleotide-free Cdc42 to prevent signaling.Epithelial polarity is regulated by the Rho-GTPase Cdc42, which is implicated in signal transduction, gene regulation, cell cycle progression, and cytoskeletal regulation (1). In epithelial cells, Cdc42 regulates establishment and maintenance of basolateral polarity by controlling endocytic and secretory trafficking (2-4). The basolateral membrane of epithelial cells is formed upon establishment of cell-cell contacts. Trans-activation of nectins and formation of E-cadherin mediated cell-cell contacts lead to the activation of Cdc42 (5, 6). However, how cell-cell contact formation favors the activation of Cdc42 is not known.The most direct activators of GTPases are guanine nucleotide exchange factors (GEFs), 1 which are characterized by four properties. GEFs bind to a GDP-bound GTPase, they catalyze the displacement of GDP and stabilize the resulting nucleotidefree GTPase. Due to the high ratio of GTP to GDP in cells, the nucleotide-depleted GTPase-GEF complex dissociates into GEF and GTP-bound (active) GTPase. The active GTPase can then bind selectively to effectors and elicit downstream effects.Activation of GEFs for Cdc42 at sites of cell-cell contacts might involve several modes. Some GEFs are activated by phosphorylation, which abolishes intramolecular inhibition (7-9). Other GEFs need additional factors to exhibit GEF activity. For example, interaction of intersectin with the Cdc42 effector protein N-WASP causes the activation of intersectin GDP displacement activity (10). Also, interaction of DOCK180 with the protein ELMO might be necessary for its activation as a GEF for Rac (11,12).Mammalian IQGAPs are scaffolding proteins with conserved homologues in budding yeast, Dictyostelium, and Caenorhabditis elegans and have been shown to regulate actomyosin ring formation ...