Gαs directly drives PDZ-RhoGEF signaling to Cdc42

Gα proteins promote dynamic adjustments of cell shape directed by actin-cytoskeleton reorganization via their respective RhoGEF effectors. For example, Gα₁₃ binding to the RGS-homology (RH) domains of several RH-RhoGEFs allosterically activates these proteins, causing them to expose their catalytic Dbl-homology (DH)/pleckstrin-homology (PH) regions, which triggers downstream signals. However, whether additional Gα proteins might directly regulate the RH-RhoGEFs was not known. To explore this question, we first examined the morphological effects of expressing shortened RH-RhoGEF DH/PH constructs of p115RhoGEF/ARHGEF1, PDZ-RhoGEF (PRG)/ARHGEF11, and LARG/ARHGEF12. As expected, the three constructs promoted cell contraction and activated RhoA, known to be downstream of Gα₁₃. Intriguingly, PRG DH/PH also induced filopodia-like cell protrusions and activated Cdc42. This pathway was stimulated by constitutively active Gα_s (Gα_sQ227L), which enabled endogenous PRG to gain affinity for Cdc42. A chemogenetic approach revealed that signaling by G_s-coupled receptors, but not by those coupled to G_i or G_q, enabled PRG to bind Cdc42. This receptor-dependent effect, as well as CREB phosphorylation, was blocked by a construct derived from the PRG:Gα_s-binding region, PRG-linker. Active Gα_s interacted with isolated PRG DH and PH domains and their linker. In addition, this construct interfered with Gα_sQ227L''s ability to guide PRG''s interaction with Cdc42. Endogenous G_s-coupled prostaglandin receptors stimulated PRG binding to membrane fractions and activated signaling to PKA, and this canonical endogenous pathway was attenuated by PRG-linker. Altogether, our results demonstrate that active Gα_s can recognize PRG as a novel effector directing its DH/PH catalytic module to gain affinity for Cdc42.     

Role of the pleckstrin homology domain in intersectin-L Dbl homology domain activation of Cdc42 and signaling

Intersectin-long (ITSN-L) contains the invariant Dbl homology (DH) and pleckstrin homology (PH) domain structure characteristic of the majority of Dbl family proteins. This strict domain topography suggests that the PH domain serves an essential, conserved function in the regulation of the intrinsic guanine nucleotide exchange activity of the DH domain. We evaluated the role of the PH domain in regulating the DH domain function of ITSN-L. Surprisingly, we found that the PH domain was dispensable for guanine nucleotide exchange activity on Cdc42 in vitro, yet the PH domain enhanced the ability of the DH domain to activate Cdc42 signaling in vivo. PH domains can interact with phosphoinositide substrates and products of phosphatidylinositol 3-kinase (PI3K). However, PI3K activation did not modulate ITSN-L DH domain function in vivo.     

Specific contributions of the small GTPases Rho, Rac, and Cdc42 to Dbl transformation.

Dbl is a representative prototype of a growing family of oncogene products that contain the Dbl homology/pleckstrin homology elements in their primary structures and are associated with a variety of neoplastic pathologies. Members of the Dbl family have been shown to function as physiological activators (guanine nucleotide exchange factors) of the Rho-like small GTPases. Although the expression of GTPase-defective versions of Rho proteins has been shown to induce a transformed phenotype under different conditions, their transformation capacity has been typically weak and incomplete relative to that exhibited by dbl-like oncogenes. Moreover, in some cases (e.g. NIH3T3 fibroblasts), expression of GTPase-defective Cdc42 results in growth inhibition. Thus, in attempting to reconstitute dbl-induced transformation of NIH3T3 fibroblasts, we have generated spontaneously activated ("fast-cycling") mutants of Cdc42, Rac1, and RhoA that mimic the functional effects of activation by the Dbl oncoprotein. When stably expressed in NIH3T3 cells, all three mutants caused the loss of serum dependence and showed increased saturation density. Furthermore, all three stable cell lines were tumorigenic when injected into nude mice. Our data demonstrate that all three Dbl targets need to be activated to promote the full complement of Dbl effects. More importantly, activation of each of these GTP-binding proteins contributes to a different and distinct facet of cellular transformation.     

Cdc42 antagonizes Rho1 activity at adherens junctions to limit epithelial cell apical tension

In epithelia, cells are arranged in an orderly pattern with a defined orientation and shape. Cadherin containing apical adherens junctions (AJs) and the associated actomyosin cytoskeleton likely contribute to epithelial cell shape by providing apical tension. The Rho guanosine triphosphatases are well known regulators of cell junction formation, maintenance, and function. Specifically, Rho promotes actomyosin activity and cell contractility; however, what controls and localizes this Rho activity as epithelia remodel is unresolved. Using mosaic clonal analysis in the Drosophila melanogaster pupal eye, we find that Cdc42 is critical for limiting apical cell tension by antagonizing Rho activity at AJs. Cdc42 localizes Par6–atypical protein kinase C (aPKC) to AJs, where this complex limits Rho1 activity and thus actomyosin contractility, independent of its effects on Wiskott-Aldrich syndrome protein and p21-activated kinase. Thus, in addition to its role in the establishment and maintenance of apical–basal polarity in forming epithelia, the Cdc42–Par6–aPKC polarity complex is required to limit Rho activity at AJs and thus modulate apical tension so as to shape the final epithelium.     

Oncogenic Dbl, Cdc42, and p21-activated kinase form a ternary signaling intermediate through the minimum interactive domains

Activation of many Rho family GTPase pathways involves the signaling module consisting of the Dbl-like guanine nucleotide exchange factors (GEFs), the Rho GTPases, and the Rho GTPase specific effectors. The current biochemical model postulates that the GEF-stimulated GDP/GTP exchange of Rho GTPases leads to the active Rho-GTP species, and subsequently the active Rho GTPases interact with and activate the effectors. Here we report an unexpected finding that the Dbl oncoprotein, Cdc42 GTPase, and PAK1 can form a complex through their minimum functional motifs, i.e., the Dbl-homolgy (DH) and Pleckstrin-homology domains of Dbl, Cdc42, and the PBD domain of PAK1. The Dbl-Cdc42-PAK1 complex is sensitive to the nucleotide-binding state of Cdc42 since either dominant negative or constitutively active Cdc42 readily disrupts the ternary binding interaction. The complex formation depends on the interactions between the DH domain of Dbl and Cdc42 and between Cdc42 and the PBD domain of PAK1 and can be reconstituted in vitro by using the purified components. Furthermore, the Dbl-Cdc42-PAK1 ternary complex is active in generating signaling output through the activated PAK1 kinase in the complex. The GEF-Rho-effector ternary intermediate is also found in other Dbl-like GEF, Rho GTPase, and effector interactions. Finally, PAK1, through the PDB domain, is able to accelerate the GEF-induced GTP loading onto Cdc42. These results suggest that signal transduction through Cdc42 and possibly other Rho family GTPases could involve tightly coupled guanine nucleotide exchange and effector activation mechanisms and that Rho GTPase effector may have a feedback regulatory role in the Rho GTPase activation.     

Recruitment of Dbl by Ezrin and Dystroglycan Drives Membrane Proximal Cdc42 Activation and Filopodia Formation

Dystroglycan is an essential laminin binding cell adhesion molecule which is also an adaptor for several SH2 domain-containing signalling molecules and as a scaffold for the ERK-MAP kinase cascade. Loss of dystroglycan function is implicated in muscular dystrophies and the aetiology of epithelial cancers. We have previously demonstrated a role for dystroglycan and ezrin in the formation of filopodia structures. Here we demonstrate the existence of a dystroglycan:ezrin:Dbl complex that is targeted to the membrane by dystroglycan where it drives local Cdc42 activation and the formation of filopodial. Deletion of an ezrin binding site in dystroglycan prevented the association with ezrin and Dbl and the formation of filopodia. Furthermore, expression of the dystroglycan cytoplasmic domain alone had a dominant-negative effect on filopodia formation and Cdc42 activation by sequestering ezrin and Dbl away from the membrane. Depletion of dystroglycan inhibited Cdc42-induced filopodia formation. For the first time we also demonstrate co-localisation of Cdc42 and dystroglycan at the tips of dynamic filopodia.     

FMNL2 drives actin-based protrusion and migration downstream of Cdc42

Cell migration entails protrusion of lamellipodia, densely packed networks of actin filaments at the cell front. Filaments are generated by nucleation, likely mediated by Arp2/3 complex and its activator Scar/WAVE. It is unclear whether formins contribute to lamellipodial actin filament nucleation or serve as elongators of filaments nucleated by Arp2/3 complex. Here we show that the Diaphanous-related formin FMNL2, also known as FRL3 or FHOD2, accumulates at lamellipodia and filopodia tips. FMNL2 is cotranslationally modified by myristoylation and regulated by interaction with the Rho-guanosine triphosphatase Cdc42. Abolition of myristoylation or Cdc42 binding interferes with proper FMNL2 activation, constituting an essential prerequisite for subcellular targeting. In vitro, C-terminal FMNL2 drives elongation rather than nucleation of actin filaments in the presence of profilin. In addition, filament ends generated by Arp2/3-mediated branching are captured and efficiently elongated by the formin. Consistent with these biochemical properties, RNAi-mediated silencing of FMNL2 expression decreases the rate of lamellipodia protrusion and, accordingly, the efficiency of cell migration. Our data establish that the FMNL subfamily member FMNL2 is a novel elongation factor of actin filaments that constitutes the first Cdc42 effector promoting cell migration and actin polymerization at the tips of lamellipodia.     

Nudel binds Cdc42GAP to modulate Cdc42 activity at the leading edge of migrating cells

Cdc42GAP promotes inactivation of Cdc42, a small GTPase whose activation at the leading edge by guanine nucleotide exchange factors is critical for cell migration. How Cdc42GAP is regulated to ensure proper levels of active Cdc42 is poorly understood. Here we show that Nudel, a cytoplasmic dynein regulator, competes with Cdc42 for binding Cdc42GAP. Consequently, Nudel can inhibit Cdc42GAP-mediated inactivation of Cdc42 in a dose-dependent manner. Both Nudel and Cdc42GAP exhibit leading-edge localization in migrating cells. The localization of Nudel requires its phosphorylation by Erk1/2. Depleting Nudel by RNAi or overexpression of a nonphosphorylatable mutant abolishes Cdc42 activation and cell migration. Our data thus uncover Nudel as a regulator of Cdc42 during cell migration. Nudel facilitates cell migration by sequestering Cdc42GAP at the leading edge to stabilize active Cdc42 in response to extracellular stimuli. Excess active Cdc42 may in turn control its own activity by recruiting Cdc42GAP from Nudel.     

IQGAP1 is a component of Cdc42 signaling to the cytoskeleton

The Ras-GAP related protein IQGAP1 binds several proteins, including actin, calmodulin, E-cadherin and the Rho family GTPase Cdc42. To gain insight into its in vivo function, IQGAP1 was overexpressed in mammalian cells. Transfection of IQGAP1 significantly increased the levels of active, GTP-bound Cdc42, resulting in the formation of peripheral actin microspikes. By contrast, transfection of an IQGAP1 mutant lacking part of the GAP-related domain (IQGAP1deltaGRD) substantially decreased the amount of GTP-bound Cdc42 in cell lysates. Consistent with these findings, IQGAP1DeltaGRD blocked Cdc42 function in cells that stably overexpress constitutively active Cdc42 and abrogated the effect of bradykinin on Cdc42. In cells transfected with IQGAP1deltaGRD, bradykinin was unable to activate Cdc42, translocate Cdc42 to the membrane fraction, or induce filopodia production. IQGAP1deltaGRD transfection altered cellular morphology, producing small, round cells that closely resemble Cdc42-/- cells. Some insight into the mechanism was provided by in vitro analysis, which revealed that IQGAP1deltaGRD increased the intrinsic GTPase activity of Cdc42, thereby increasing the amount of inactive, GDP-bound Cdc42. These data imply that IQGAP1 has a crucial role in transducing Cdc42 signaling to the cytoskeleton.     

Activated ezrin promotes cell migration through recruitment of the GEF Dbl to lipid rafts and preferential downstream activation of Cdc42

Establishment of polarized cell morphology is a critical factor for migration and requires precise spatial and temporal activation of the Rho GTPases. Here, we describe a novel role of the actin-binding ezrin/radixin/moesin (ERM)-protein ezrin to be involved in recruiting Cdc42, but not Rac1, to lipid raft microdomains, as well as the subsequent activation of this Rho GTPase and the downstream effector p21-activated kinase (PAK)1, as shown by fluorescence lifetime imaging microscopy. The establishment of a leading plasma membrane and the polarized morphology necessary for random migration are also dependent on ERM function and Cdc42 in motile breast carcinoma cells. Mechanistically, we show that the recruitment of the ERM-interacting Rho/Cdc42-specific guanine nucleotide exchange factor Dbl to the plasma membrane and to lipid raft microdomains requires the phosphorylated, active conformer of ezrin, which serves to tether the plasma membrane or its subdomains to the cytoskeleton. Together these data suggest a mechanism whereby precise spatial guanine nucleotide exchange of Cdc42 by Dbl is dependent on functional ERM proteins and is important for directional cell migration.     

LTP-triggered cholesterol redistribution activates Cdc42 and drives AMPA receptor synaptic delivery

Neurotransmitter receptor trafficking during synaptic plasticity requires the concerted action of multiple signaling pathways and the protein transport machinery. However, little is known about the contribution of lipid metabolism during these processes. In this paper, we addressed the question of the role of cholesterol in synaptic changes during long-term potentiation (LTP). We found that N-methyl-d-aspartate–type glutamate receptor (NMDAR) activation during LTP induction leads to a rapid and sustained loss or redistribution of intracellular cholesterol in the neuron. A reduction in cholesterol, in turn, leads to the activation of Cdc42 and the mobilization of GluA1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid–type glutamate receptors (AMPARs) from Rab11-recycling endosomes into the synaptic membrane, leading to synaptic potentiation. This process is accompanied by an increase of NMDAR function and an enhancement of LTP. These results imply that cholesterol acts as a sensor of NMDAR activation and as a trigger of downstream signaling to engage small GTPase (guanosine triphosphatase) activation and AMPAR synaptic delivery during LTP.     

Regulation of neurogenesis by Fgf8a requires Cdc42 signaling and a novel Cdc42 effector protein

Fibroblast growth factor (FGF) signaling is required for numerous aspects of neural development, including neural induction, CNS patterning and neurogenesis. The ability of FGFs to activate Ras/MAPK signaling is thought to be critical for these functions. However, it is unlikely that MAPK signaling can fully explain the diversity of responses to FGFs. We have characterized a Cdc42-dependent signaling pathway operating downstream of the Fgf8a splice isoform. We show that a Cdc42 effector 4-like protein (Cdc42ep4-l or Cep4l) has robust neuronal-inducing activity in Xenopus embryos. Furthermore, we find that Cep4l and Cdc42 itself are necessary and sufficient for sensory neurogenesis in vivo. Furthermore, both proteins are involved in Fgf8a-induced neuronal induction, and Cdc42/Cep4l association is promoted specifically by the Fgf8a isoform of Fgf8, but not by Fgf8b, which lacks neuronal inducing activity. Overall, these data suggest a novel role for Cdc42 in an Fgf8a-specific signaling pathway essential for vertebrate neuronal development.     

Rac/Cdc42 and p65PAK regulate the microtubule-destabilizing protein stathmin through phosphorylation at serine 16

We have identified a rapid protein phosphorylation event at residue serine 16 of stathmin using two-dimensional gel electrophoresis coupled to matrix-assisted laser desorption/ionization mass spectrometry in combination with post-source decay analysis, which is induced by the epidermal growth factor receptor. Phosphorylation is specifically mediated by the small GTPases Rac and Cdc42 and their common downstream target, the serine/threonine kinase p65PAK. Both GTPases have previously been shown to regulate the dynamics of actin polymerization. Because stathmin destabilizes microtubules, and this process is inhibited by phosphorylation at residue 16, Rac and Cdc42 can potentially regulate both F-actin and microtubule dynamics.     

Guanylate cyclase and cyclic GMP-dependent protein kinase regulate agrin signaling at the developing neuromuscular junction

During formation of the neuromuscular junction (NMJ), agrin secreted by motor axons signals the embryonic muscle cells to organize a postsynaptic apparatus including a dense aggregate of acetylcholine receptors (AChRs). Agrin signaling at the embryonic NMJ requires the activity of nitric oxide synthase (NOS). Common downstream effectors of NOS are guanylate cyclase (GC), which synthesizes cyclic GMP, and cyclic GMP-dependent protein kinase (PKG). Here we show that GC and PKG are important for agrin signaling at the embryonic NMJ of the frog, Xenopus laevis. Inhibitors of both GC and PKG reduced endogenous AChR aggregation in embryonic muscles by 50-85%, and blocked agrin-induced AChR aggregation in cultured embryonic muscle cells. A cyclic GMP analog, 8-bromo-cyclic GMP, increased endogenous AChR aggregation in embryonic muscles to 3- to 4-fold control levels. Overexpression of either GC or PKG in embryos increased AChR aggregate area by 60-170%, whereas expression of a dominant negative form of GC inhibited endogenous aggregation by 50%. These results indicate that agrin signaling in embryonic muscle cells requires the activity of GC and PKG as well as NOS.     

Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis

Although Rho regulates cytokinesis, little was known about the functions in mitosis of Cdc42 and Rac. We recently suggested that Cdc42 works in metaphase by regulating bi-orient attachment of spindle microtubules to kinetochores. We now confirm the role of Cdc42 by RNA interference and identify the mechanisms for activation and down-regulation of Cdc42. Using a pull-down assay, we found that the level of GTP-Cdc42 elevates in metaphase, whereas the level of GTP-Rac does not change significantly in mitosis. Overexpression of dominant-negative mutants of Ect2 and MgcRacGAP, a Rho GTPase guanine nucleotide exchange factor and GTPase activating protein, respectively, or depletion of Ect2 by RNA interference suppresses this change of GTP-Cdc42 in mitosis. Depletion of Ect2 also impairs microtubule attachment to kinetochores and causes prometaphase delay and abnormal chromosomal segregation, as does depletion of Cdc42 or expression of the Ect2 and MgcRacGAP mutants. These results suggest that Ect2 and MgcRacGAP regulate the activation and function of Cdc42 in mitosis.     

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.     

Apolipoprotein A-I activates Cdc42 signaling through the ABCA1 transporter

It has been suggested that the signal transduction initiated by apolipoprotein A-I (apoA-I) activates key proteins involved in cholesterol efflux. ABCA1 serves as a binding partner for apoA-I, but its participation in apoA-I-induced signaling remains uncertain. We show that the exposure of human fibroblasts to ABCA1 ligands (apolipoproteins and amphipathic helical peptides) results in the generation of intracellular signals, including activation of the small G-protein Cdc42, protein kinases (PAK-1 and p54JNK), and actin polymerization. ApoA-I-induced signaling was abrogated by glyburide, an inhibitor of the ABC transporter family, and in fibroblasts from patients with Tangier disease, which do not express ABCA1. Conversely, induction of ABCA1 expression with the liver X receptor agonist, T0901317, and the retinoid X receptor agonist, R0264456, potentiated apoA-I-induced signaling. Similar effects were observed in HEK293 cells overexpressing ABCA1-green fluorescent protein (GFP) fusion protein, but not ABCA1-GFP (K939M), which fails to hydrolyze ATP, or a nonfunctional ABCA1-GFP with a truncated C terminus. We further found that Cdc42 coimmunoprecipitates with ABCA1 in ABCA1-GFP-expressing HEK293 cells exposed to apoA-I but not in cells expressing ABCA1 mutants. We conclude that ABCA1 transduces signals from apoA-I by complexing and activating Cdc42 and downstream kinases and, therefore, acts as a full apoA-I receptor.