Phosphorylation of IQGAP1 Modulates Its Binding to Cdc42, Revealing a New Type of Rho-GTPase Regulator

Abstract

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 ...

A dual role for IQGAP1 in regulating exocytosis

“…All cells had equal levels of endogenous total CDC42, as demonstrated in the lower panel in Fig. 9, and these results are not unique to ␤-cells, because they confirm similar findings from other cell types (Fukata et al, 2002;Mataraza et al, 2002;Grohmanova et al, 2004), affirming an apparently conserved function for IQGAP1 upstream of CDC42 (Osman et al, 2002). These data help explain why IQGAP1-C disrupts binding, secretion and protein synthesis, confirming that active CDC42 negatively regulates IQGAP1 function in secretion.…”

“…9A). IQGAP1 also binds nucleotidedepleted CDC42 (ND-CDC42) and GDP-CDC42 (Grohmanova et al, 2004), confirming its GEF-like activity on CDC42 and providing an explanation as to why wild-type CDC42 blocked both interaction and secretion (Figs 1 and 8) similar to its dominant active mutants, indicating that increasing the level of CDC42 enhances the pool of its active form, leading to the observed inhibition.…”

“…9C). In support of this model is the finding by independent groups that two different regions on the C-terminus form complexes in vitro (Fukata et al, 2001;Grohmanova et al, 2004) and that phosphorylation of serine 1443 in the second region prevents this interaction, increasing the binding of nucleotide-depleted CDC42 (Grohmanova et al, 2004). This model is generally consistent with that for DBL proteins (CDC42-GEFs) shown to exist in conformational states regulated by tyrosine phosphorylation, which causes an opening of their structure leading to the activation of Rho GTPases (Aghazadeh et al, 2000).…”

MAPK Scaffold IQGAP1 Binds the EGF Receptor and Modulates Its Activation

“…We and others have previously shown that IQGAP1 is phosphorylated in cells in a PKC⑀-dependent manner at Ser 1443 in response to phorbol ester treatment (28,37). It seems reasonable, therefore, to hypothesize that IQGAP1 can be phosphorylated on Ser 1443 in response to EGF-stimulated activation of EGFR.…”

“…EGFR triggers phospholipase C␥ to generate both diacylglycerol and inositol 1,4,5-trisphosphate-mediated calcium fluxes, resulting in the potential activation of multiple PKC isoforms (40,41). Previous studies have shown that Ser 1443 is contained within a PKC⑀ consensus site and that PKC⑀ is able to catalyze IQGAP1 phosphorylation at this site in vitro (28,37). To further characterize PKC involvement in EGF-mediated phosphorylation of IQGAP1, we performed targeted knockdown of the TPA-responsive PKC isoenzymes expressed in HeLa cells (42).…”