Rnd proteins function as RhoA antagonists by activating p190 RhoGAP

BACKGROUND: The Rnd proteins Rnd1, Rnd2, and Rnd3 (RhoE) comprise a unique branch of Rho-family G-proteins that lack intrinsic GTPase activity and consequently remain constitutively "active." Prior studies have suggested that Rnd proteins play pivotal roles in cell regulation by counteracting the biological functions of the RhoA GTPase, but the molecular basis for this antagonism is unknown. Possible mechanisms by which Rnd proteins could function as RhoA antagonists include sequestration of RhoA effector molecules, inhibition of guanine nucleotide exchange factors, and activation of GTPase-activating proteins (GAPs) for RhoA. However, effector molecules of Rnd proteins with such properties have not been identified. RESULTS: Here we identify p190 RhoGAP (p190), the most abundant GAP for RhoA in cells, as an interactor with Rnd proteins and show that this interaction is mediated by a p190 region that is distinct from the GAP domain. Using Rnd3-RhoA chimeras and Rnd3 mutants defective in p190 binding, as well as p190-deficient cells, we demonstrate that the cellular effects of Rnd expression are mediated by p190. We moreover show that Rnd proteins increase the GAP activity of p190 toward GTP bound RhoA and, finally, demonstrate that expression of Rnd3 leads to reduced cellular levels of RhoA-GTP by a p190-dependent mechanism. CONCLUSIONS: Our results identify p190 RhoGAPs as effectors of Rnd proteins and demonstrate a novel mechanism by which Rnd proteins function as antagonists of RhoA.     

The RhoA GEF Syx Is a Target of Rnd3 and Regulated via a Raf1-Like Ubiquitin-Related Domain

Background

Rnd3 (RhoE) protein belongs to the unique branch of Rho family GTPases that has low intrinsic GTPase activity and consequently remains constitutively active [1], [2]. The current consensus is that Rnd1 and Rnd3 function as important antagonists of RhoA signaling primarily by activating the ubiquitous p190 RhoGAP [3], but not by inhibiting the ROCK family kinases.

Methodology/Principal Findings

Rnd3 is abundant in mouse embryonic stem (mES) cells and in an unbiased two-step affinity purification screen we identified a new Rnd3 target, termed synectin-binding RhoA exchange factor (Syx), by mass spectrometry. The Syx interaction with Rnd3 does not occur through the Syx DH domain but utilizes a region similar to the classic Raf1 Ras-binding domain (RBD), and most closely related to those in RGS12 and RGS14. We show that Syx behaves as a genuine effector of Rnd3 (and perhaps Rnd1), with binding characteristics similar to p190-RhoGAP. Morpholino-oligonucleotide knockdown of Syx in zebrafish at the one cell stage resulted in embryos with shortened anterior-posterior body axis: this phenotype was effectively rescued by introducing mouse Syx1b mRNA. A Rnd3-binding defective mutant of Syx1b mutated in the RBD (E164A/R165D) was more potent in rescuing the embryonic defects than wild-type Syx1b, showing that Rnd3 negatively regulates Syx activity in vivo.

Conclusions/Significance

This study uncovers a well defined Rnd3 effector Syx which is widely expressed and directly impacts RhoA activation. Experiments conducted in vivo indicate that Rnd3 negatively regulates Syx, and that as a RhoA-GEF it plays a key role in early embryonic cell shape changes. Thus a connection to signaling via the planar cell polarity pathway is suggested.     

Rnd1 and Rnd3 targeting to lipid raft is required for p190 RhoGAP activation

The Rnd proteins Rnd1, Rnd2, and Rnd3/RhoE are well known as key regulators of the actin cytoskeleton in various cell types, but they comprise a distinct subgroup of the Rho family in that they are GTP bound and constitutively active. Functional differences of the Rnd proteins in RhoA inhibition signaling have been reported in various cell types. Rnd1 and Rnd3 antagonize RhoA signaling by activating p190 RhoGAP, whereas Rnd2 does not. However, all the members of the Rnd family have been reported to bind directly to p190 RhoGAP and equally induce activation of p190 RhoGAP in vitro, and there is no evidence that accounts for the functional difference of the Rnd proteins in RhoA inhibition signaling. Here we report the role of the N-terminal region in signaling. Rnd1 and Rnd3, but not Rnd2, have a KERRA (Lys-Glu-Arg-Arg-Ala) sequence of amino acids in their N-terminus, which functions as the lipid raft-targeting determinant. The sequence mediates the lipid raft targeting of p190 RhoGAP correlated with its activation. Overall, our results demonstrate a novel regulatory mechanism by which differential membrane targeting governs activities of Rnd proteins to function as RhoA antagonists.     

p190RhoGAP has cellular RacGAP activity regulated by a polybasic region

p190RhoGAP is a GTPase-activating protein (GAP) known to regulate actin cytoskeleton dynamics by decreasing RhoGTP levels through activation of the intrinsic GTPase activity of Rho. Although the GAP domain of p190RhoGAP stimulates the intrinsic' GTPase activity of several Rho family members (Rho, Rac, Cdc42) under in vitro conditions, p190RhoGAP is generally regarded as a GAP for RhoA in the cell. The cellular RacGAP activity of the protein has not been proven directly. We have previously shown that the in vitro RacGAP and RhoGAP activity of p190RhoGAP was inversely regulated through a polybasic region of the protein. Here we provide evidence that p190RhoGAP shows remarkable GAP activity toward Rac also in the cell. The cellular RacGAP activity of p190RhoGAP requires an intact polybasic region adjacent to the GAP domain whereas the RhoGAP activity is inhibited by the same domain. Our data indicate that through its alternating RacGAP and RhoGAP activity, p190RhoGAP plays a more complex role in the Rac–Rho antagonism than it was realized earlier.     

The BNIP-2 and Cdc42GAP Homology (BCH) Domain of p50RhoGAP/Cdc42GAP Sequesters RhoA from Inactivation by the Adjacent GTPase-activating Protein Domain

The BNIP-2 and Cdc42GAP homology (BCH) domain is a novel regulator for Rho GTPases, but its impact on p50-Rho GTPase-activating protein (p50RhoGAP or Cdc42GAP) in cells remains elusive. Here we show that deletion of the BCH domain from p50RhoGAP enhanced its GAP activity and caused drastic cell rounding. Introducing constitutively active RhoA or inactivating GAP domain blocked such effect, whereas replacing the BCH domain with endosome-targeting SNX3 excluded requirement of endosomal localization in regulating the GAP activity. Substitution with homologous BCH domain from Schizosaccharomyces pombe, which does not bind mammalian RhoA, also led to complete loss of suppression. Interestingly, the p50RhoGAP BCH domain only targeted RhoA, but not Cdc42 or Rac1, and it was unable to distinguish between GDP and the GTP-bound form of RhoA. Further mutagenesis revealed a RhoA-binding motif (residues 85-120), which when deleted, significantly reduced BCH inhibition on GAP-mediated cell rounding, whereas its full suppression also required an intramolecular interaction motif (residues 169-197). Therefore, BCH domain serves as a local modulator in cis to sequester RhoA from inactivation by the adjacent GAP domain, adding to a new paradigm for regulating p50RhoGAP signaling.     

Detection of Rho GEF and GAP activity through a sensitive split luciferase assay system

Rho GTPases regulate the assembly of cellular actin structures and are activated by GEFs (guanine-nucleotide-exchange factors) and rendered inactive by GAPs (GTPase-activating proteins). Using the Rho GTPases Cdc42, Rac1 and RhoA, and the GTPase-binding portions of the effector proteins p21-activated kinase and Rhophilin1, we have developed split luciferase assays for detecting both GEF and GAP regulation of these GTPases. The system relies on purifying split luciferase fusion proteins of the GTPases and effectors from bacteria, and our results show that the assays replicate GEF and GAP specificities at nanomolar concentrations for several previously characterized Rho family GEFs (Dbl, Vav2, Trio and Asef) and GAPs [p190, Cdc42 GAP and PTPL1-associated RhoGAP]. The assay detected activities associated with purified recombinant GEFs and GAPs, cell lysates expressing exogenous proteins, and immunoprecipitates of endogenous Vav1 and p190. The results demonstrate that the split luciferase system provides an effective sensitive alternative to radioactivity-based assays for detecting GTPase regulatory protein activities and is adaptable to a variety of assay conditions.     

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.     

Rich, a rho GTPase-activating protein domain-containing protein involved in signaling by Cdc42 and Rac1.

A previously unidentified Rho GTPase-activating protein (GAP) domain-containing protein was found in a yeast two-hybrid screen for cDNAs encoding proteins binding to the Src homology 3 domain of Cdc42-interacting protein 4 (CIP4). The protein was named RICH-1 (RhoGAP interacting with CIP4 homologues), and, in addition to the RhoGAP domain, it contained an N-terminal domain with endophilin homology and a C-terminal proline-rich domain. Transient transfections of RICH-1 indicated that it bound to CIP4 in vivo, as shown by co-immunoprecipitation experiments, as well as co-localization assays. In vitro assays demonstrated that the RhoGAP domain of RICH-1 catalyzed GTP hydrolysis on Cdc42 and Rac1, but not on RhoA. Ectopic expression of the RhoGAP domain as well as the full-length protein interfered with platelet-derived growth factor BB-induced membrane ruffling, but not with serum-induced stress fiber formation, further emphasizing the notion that, in vivo, RICH-1 is a GAP for Cdc42 and Rac1.     

Localized suppression of RhoA activity by Tyr31/118-phosphorylated paxillin in cell adhesion and migration

RhoA activity is transiently inhibited at the initial phase of integrin engagement, when Cdc42- and/or Rac1-mediated membrane spreading and ruffling predominantly occur. Paxillin, an integrin-assembly protein, has four major tyrosine phosphorylation sites, and the phosphorylation of Tyr31 and Tyr118 correlates with cell adhesion and migration. We found that mutation of Tyr31/118 caused enhanced activation of RhoA and premature formation of stress fibers with substantial loss of efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells. These phenotypes were similar to those induced by RhoA(G14V) in parental cells, and could be abolished by expression of RhoA(T19N), Rac1(G12V), or p190RhoGAP in the mutant-expressing cells. Phosphorylated Tyr31/118 was found to bind to two src homology (SH)2 domains of p120RasGAP, with coprecipitation of endogenous paxillin with p120RasGAP. p190RhoGAP is known to be a major intracellular binding partner for the p120RasGAP SH2 domains. We found that Tyr31/118-phosphorylated paxillin competes with p190RhoGAP for binding to p120RasGAP, and provides evidence that p190RhoGAP freed from p120RasGAP efficiently suppresses RhoA activity during cell adhesion. We conclude that Tyr31/118-phosphorylated paxillin serves as a template for the localized suppression of RhoA activity and is necessary for efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells.     

Drosophila RhoGAP68F is a putative GTPase activating protein for RhoA participating in gastrulation

The Rho family small GTPases Rho, Rac, and Cdc42 regulate cell shape and motility through the actin cytoskeleton. These proteins cycle between a GTP-bound “on” state and a GDP-bound “off” state and are negatively regulated by GTPase-activating proteins (GAPs), which accelerate the small GTPase’s intrinsic hydrolysis of bound GTP to GDP. Drosophila RhoGAP68F is similar to the mammalian protein p50RhoGAP/Cdc42GAP, which exhibits strong GAP activity toward Cdc42. We find that, despite the strong similarities between RhoGAP68F and p50RhoGAP/Cdc42GAP, RhoGAP68F is most effective as a GAP for RhoA. These in vitro data are supported by the in vivo analysis of mutants in RhoGAP68F. We demonstrate through the characterization of two alleles of the RhoGAP68F gene that RhoGAP68F participates in gastrulation of the embryo, a morphogenetic event driven by cell constriction that involves RhoA signaling. We propose that RhoGAP68F functions as a regulator of RhoA signaling during gastrulation.     

Fluorescence approaches for monitoring interactions of Rho GTPases with nucleotides, regulators, and effectors

To understand the manner in which biological macromolecules interact with each other, we need not only structural information, but also details of kinetics and thermodynamics of the processes involved. This is particularly important for key proteins acting in signal transduction such as the small GTPases of the Ras superfamily. The complexity of their roles is constantly increasing since a large number of GTPases have been identified and each of these in turn interacts with a variety of regulatory and signaling proteins such as GAPs, GEFs, and downstream effectors. There are a number of methods that can be used to characterize the specificity, strength, and stoichiometry of such intermolecular interactions, to understand the effect of binding on the protein structure, and, ultimately, to obtain insights into their biological functions. This article discusses the use of fluorescence spectroscopic methods, which allows real-time monitoring of ligand- and protein-protein interactions at submicromolar concentrations, and quantification of the kinetic and equilibrium constants. Fluorescently labeled guanine nucleotides serve as fluorescence reporter groups to investigate the interactions of GTPases of the Rho family (e.g., RhoA, Rac1, and Cdc42). We present examples for quantitative characterization of (i) Rac1 x GDP interaction, (ii) Cdc42-interaction with the GTPase binding domain of the Wiskott Aldrich syndrome protein (three alternative approaches), (iii) accelerated nucleotide exchange reaction of RhoA by the catalytic domains of p190RhoGEF, and (iv) intrinsic and stimulated GTP-hydrolysis reaction by the catalytic domain of p50RhoGAP.     

Concerted regulation of cell dynamics by BNIP-2 and Cdc42GAP homology/Sec14p-like,proline-rich,and GTPase-activating protein domains of a novel Rho GTPase-activating protein,BPGAP1

RhoA, Cdc42, and Rac1 are small GTPases that regulate cytoskeletal reorganization leading to changes in cell morphology and cell motility. Their signaling pathways are activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins (GAPs). We have identified a novel RhoGAP, BPGAP1 (for BNIP-2 and Cdc42GAP Homology (BCH) domain-containing, Proline-rich and Cdc42GAP-like protein subtype-1), that is ubiquitously expressed and shares 54% sequence identity to Cdc42GAP/p50RhoGAP. BP-GAP1 selectively enhanced RhoA GTPase activity in vivo although it also interacted strongly with Cdc42 and Rac1. "Pull-down" and co-immunoprecipitation studies indicated that it formed homophilic or heterophilic complexes with other BCH domain-containing proteins. Fluorescence studies of epitope-tagged BPGAP1 revealed that it induced pseudopodia and increased migration of MCF7 cells. Formation of pseudopodia required its BCH and GAP domains but not the proline-rich region, and was differentially inhibited by coexpression of the constitutively active mutant of RhoA, or dominant negative mutants of Cdc42 and Rac1. However, the mutant without the proline-rich region failed to confer any increase in cell migration despite the induction of pseudopodia. Our findings provide evidence that cell morphology changes and migration are coordinated via multiple domains in BPGAP1 and present a novel mode of regulation for cell dynamics by a RhoGAP protein.     

Role of prenylation in the interaction of Rho-family small GTPases with GTPase activating proteins

The role of prenylation in the interaction of Rho-family small GTPases with their GTPase activating proteins (GAPs) was investigated. Prenylated and nonprenylated small GTPases were expressed in Sf9 insect cells and Escherichia coli, respectively. Nucleotide binding to and hydrolysis by prenylated and nonprenylated proteins were identical, but three major differences were observed in their reactions with GAPs. (1) Membrane-associated GAPs accelerate GTP hydrolysis only on prenylated Rac1 and RhoA, but they are inactive on the nonprenylated form of these proteins. The difference is independent of the presence of detergents. In contrast to Rac1 and RhoA, nonprenylated Cdc42 is able to interact with membrane-localized GAPs. (2) Full-length p50RhoGAP and p190RhoGAP react less intensely with nonprenylated Rac1 than with the prenylated protein, whereas no difference was observed in the reaction of isolated GAP domains of either p50RhoGAP or Bcr with the different types of Rac1. (3) Fluoride exerts a significant inhibitory effect only on the interaction of prenylated Rac1 with the isolated GAP domains of p50RhoGAP or Bcr. The effect of fluoride is not influenced by addition or chelation of Al(3+). This is the first detailed study demonstrating that prenylation of the small GTPase is an important factor in determining its reaction with GAPs. It is suggested that both intramolecular interactions and membrane targeting of GAP proteins represent potential mechanisms regulating Rac signaling.     

Models of the cooperative mechanism for Rho effector recognition: implications for RhoA-mediated effector activation

Activated GTPases of the Rho family regulate a spectrum of functionally diverse downstream effectors, initiating a network of signal transduction pathways by interaction and activation of effector proteins. Although effectors are defined as proteins that selectively bind the GTP-bound state of the small GTPases, there have been also several indications for a nucleotide-independent binding mode. By characterizing the molecular mechanism of RhoA interaction with its effectors, we have determined the equilibrium dissociation constants of several Rho-binding domains of three different effector proteins (Rhotekin, ROCKI/ROK beta/p160ROCK, PRK1/PKNalpha where ROK is RhoA-binding kinase) for both RhoA.GDP and RhoA.GTP using fluorescence spectroscopy. In addition, we have identified two novel Rho-interacting domains in ROCKI, which bind RhoA with high affinity but not Cdc42 or Rac1. Our results, together with recent structural data, support the notion of multiple effector-binding sites in RhoA and strongly indicate a cooperative binding mechanism for PRK1 and ROCKI that may be the molecular basis of Rho-mediated effector activation.     

Integrin engagement suppresses RhoA activity via a c-Src-dependent mechanism

The Rho family GTPases Cdc42, Rac1 and RhoA control many of the changes in the actin cytoskeleton that are triggered when growth factor receptors and integrins bind their ligands [1] [2]. Rac1 and Cdc42 stimulate the formation of protrusive structures such as membrane ruffles, lamellipodia and filopodia. RhoA regulates contractility and assembly of actin stress fibers and focal adhesions. Although prolonged integrin engagement can stimulate RhoA [3] [4] [5], regulation of this GTPase by early integrin-mediated signals is poorly understood. Here we show that integrin engagement initially inactivates RhoA, in a c-Src-dependent manner, but has no effect on Cdc42 or Rac1 activity. Additionally, early integrin signaling induces activation and tyrosine phosphorylation of p190RhoGAP via a mechanism that requires c-Src. Dynamic modulation of RhoA activity appears to have a role in motility, as both inhibition and activation of RhoA hinder migration [6] [7] [8]. Transient suppression of RhoA by integrins may alleviate contractile forces that would otherwise impede protrusion at the leading edge of migrating cells.     

Regulating axon branch stability: the role of p190 RhoGAP in repressing a retraction signaling pathway.

Mechanisms that regulate axon branch stability are largely unknown. Genome-wide analyses of Rho GTPase activating protein (RhoGAP) function in Drosophila using RNA interference identified p190 RhoGAP as essential for axon stability in mushroom body neurons, the olfactory learning and memory center. p190 inactivation leads to axon branch retraction, a phenotype mimicked by activation of GTPase RhoA and its effector kinase Drok and modulated by the level and phosphorylation of myosin regulatory light chain. Thus, there exists a retraction pathway from RhoA to myosin in maturing neurons, which is normally repressed by p190. Local regulation of p190 could control the structural plasticity of neurons. Indeed, genetic evidence supports negative regulation of p190 by integrin and Src, both implicated in neural plasticity.     

Integrin signaling through Arg activates p190RhoGAP by promoting its binding to p120RasGAP and recruitment to the membrane

The Rho family GTPases RhoA (Rho), Rac1, and Cdc42 are essential effectors of integrin-mediated cell attachment and spreading. Rho activity, which promotes formation of focal adhesions and actin stress fibers, is inhibited upon initial cell attachment to allow sampling of the new adhesive environment. The Abl-related gene (Arg) tyrosine kinase mediates adhesion-dependent inhibition of Rho through phosphorylation and activation of the Rho inhibitor p190RhoGAP-A (p190). p190 phosphorylation promotes its binding to p120RasGAP (p120). Here, we elucidate the mechanism by which p120 binding regulates p190 activation after adhesion. We show that p190 requires its p120-binding domain to undergo Arg-dependent activation in vivo. However, p120 binding does not activate p190RhoGAP activity in vitro. Instead, activation of p190 requires recruitment to the cell periphery. Integrin-mediated adhesion promotes relocalization of p190 and p120 to the cell periphery in wild-type fibroblasts, but not in arg(-/-) fibroblasts. A dominant-negative p120 fragment blocks p190:p120 complex formation, prevents activation of p190 by adhesion, and disrupts the adhesion-dependent recruitment of p190 to the cell periphery. Our results demonstrate that integrin signaling through Arg activates p190 by promoting its association with p120, resulting in recruitment of p190 to the cell periphery where it inhibits Rho.     

RhoA inactivation by p190RhoGAP regulates cell spreading and migration by promoting membrane protrusion and polarity

The binding of extracellular matrix proteins to integrins triggers rearrangements in the actin cytoskeleton by regulating the Rho family of small GTPases. The signaling events that mediate changes in the activity of Rho proteins in response to the extracellular matrix remain largely unknown. We have demonstrated in previous studies that integrin signaling transiently suppresses RhoA activity through stimulation of p190RhoGAP. Here, we investigated the biological significance of adhesion-dependent RhoA inactivation by manipulating p190RhoGAP signaling in Rat1 fibroblasts. The inhibition of RhoA activity that is induced transiently by adhesion was antagonized by expression of dominant negative p190RhoGAP. This resulted in impaired cell spreading on a fibronectin substrate, reduced cell protrusion, and premature assembly of stress fibers. Conversely, overexpression of p190RhoGAP augmented cell spreading. Dominant negative p190RhoGAP elevated RhoA activity in cells on fibronectin and inhibited migration, whereas overexpression of the wild-type GAP decreased RhoA activity, promoted the formation of membrane protrusions, and enhanced motility. Cells expressing dominant negative p190RhoGAP, but not control cells or cells overexpressing the wild-type GAP, were unable to establish polarity in the direction of migration. Taken together, these data demonstrate that integrin-triggered RhoA inhibition by p190RhoGAP enhances spreading and migration by regulating cell protrusion and polarity.     

Signaling Pathways That Control Rho Kinase Activity Maintain the Embryonic Epicardial Progenitor State

This study identifies signaling pathways that play key roles in the formation and maintenance of epicardial cells, a source of progenitors for coronary smooth muscle cells (SMCs). After epithelial to mesenchymal transition (EMT), mesenchymal cells invade the myocardium to form coronary SMCs. RhoA/Rho kinase activity is required for EMT and for differentiation into coronary SMCs, whereas cAMP activity is known to inhibit EMT in epithelial cells by an unknown mechanism. We use outgrowth of epicardial cells from E9.5 isolated mouse proepicardium (PE) explants, wild type and Epac1 null E12.5 mouse heart explants, adult rat epicardial cells, and immortalized mouse embryonic epicardial cells as model systems to identify signaling pathways that regulate RhoA activity to maintain the epicardial progenitor state. We demonstrate that RhoA activity is suppressed in the epicardial progenitor state, that the cAMP-dependent Rap1 GTP exchange factor (GEF), Epac, known to down-regulate RhoA activity through activation of Rap1 GTPase activity increased, that Rap1 activity increased, and that expression of the RhoA antagonistic Rnd proteins known to activate p190RhoGAP increased and associated with p190RhoGAP. Finally, EMT is associated with increased p63RhoGEF and RhoGEF-H1 protein expression, increased GEF-H1 activity, with a trend in increased p63RhoGEF activity. EMT is suppressed by partial silencing of p63RhoGEF and GEF-H1. In conclusion, we have identified new signaling molecules that act together to control RhoA activity and play critical roles in the maintenance of coronary smooth muscle progenitor cells in the embryonic epicardium. We suggest that their eventual manipulation could promote revascularization after myocardial injury.     

Probing the cellular effects of bacterial effector proteins with the Yersinia toolbox

The type 3 secretion system (T3SS) is a powerful bacterial nanomachine that is able to modify the host cellular immune defense in favor of the pathogen by injection of effector proteins. In this regard, cellular Rho GTPases such as Rac1, RhoA or Cdc42 are targeted by a large group of T3SS effectors by mimicking cellular guanine exchange factors or GTPase-activating proteins. However, functional analysis of one type of T3SS effector that is translocated by bacterial pathogens is challenging because the T3SS effector repertoire can comprise a large number of proteins with redundant or interfering functions. Therefore, we developed the Yersinia toolbox to either analyze singular effector proteins of Yersinia spp. or different bacterial species in the context of bacterial T3SS injection into cells. Here, we focus on the WxxxE guanine exchange factor mimetic proteins IpgB1, IpgB2 and Map, which activate Rac1, RhoA or Cdc42, respectively, as well as the Rho GTPase inactivators YopE (a GTPase-activating mimetic protein) and YopT (cysteine protease), to generate a toolbox module for Rho GTPase manipulation.