Cadherin engagement inhibits RhoA via p190RhoGAP

Cadherins are transmembrane receptors that mediate cell-cell adhesion in epithelial cells. A number of changes occur during cadherin-mediated junction formation, one of which is a rearrangement of the actin cytoskeleton. Key regulators of actin cytoskeletal dynamics in cells are the Rho family of GTPases. We have demonstrated in previous studies that cadherin signaling suppresses RhoA activity and activates Rac1. The signaling events downstream of cadherins that modulate the activity of Rho family proteins remain unknown. Here we have identified a pathway by which RhoA becomes inactivated by cadherins. To determine whether cadherins regulate RhoA through activation of a GTPase-activating protein (GAP) for RhoA, we used constitutively active RhoA to isolate activated GAPs. Using this assay, we have identified the RhoA-specific GAP, p190RhoGAP, downstream from engaged cadherins. We found that cadherin engagement induced tyrosine phosphorylation of p190RhoGAP and increased its binding to p120RasGAP. The increased precipitation of p190RhoGAP with 63LRhoA was blocked by addition of PP2 suggesting that Src family kinases are required downstream from cadherin signaling. The inhibition of RhoA activity by cadherins was antagonized by expression of a dominant negative p190RhoGAP. Taken together, these data demonstrate that p190RhoGAP activity is critical for RhoA inactivation by cadherins.     

The cAMP-responsive Rap1 guanine nucleotide exchange factor, Epac, induces smooth muscle relaxation by down-regulation of RhoA activity

Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC(20)) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2'-O-Me-cAMP ("007"), significantly reduced agonist-induced contractile force, RLC(20), and myosin light chain phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI(2) analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca(2+) desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.     

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.     

Direct interaction of focal adhesion kinase with p190RhoGEF

Focal adhesion kinase (FAK) is a protein-tyrosine kinase that associates with multiple cell surface receptors and signaling proteins through which it can modulate the activity of several intracellular signaling pathways. FAK activity can influence the formation of distinct actin cytoskeletal structures such as lamellipodia and stress fibers in part through effects on small Rho GTPases, although the molecular interconnections of these events are not well defined. Here, we report that FAK interacts with p190RhoGEF, a RhoA-specific GDP/GTP exchange factor, in neuronal cells and in brain tissue extracts by co-immunoprecipitation and co-localization analyses. Using a two-hybrid assay and deletion mutagenesis, the binding site of the FAK C-terminal focal adhesion targeting (FAT) domain was identified within the C-terminal coiled-coil domain of p190RhoGEF. Binding was independent of a LD-like binding motif within p190RhoGEF, yet FAK association was disrupted by a mutation (Leu-1034 to Ser) that weakens the helical bundle structure of the FAK FAT domain. Neuro-2a cell binding to laminin increased endogenous FAK and p190RhoGEF tyrosine phosphorylation, and co-transfection of a dominant-negative inhibitor of FAK activity, termed FRNK, inhibited lamininstimulated p190RhoGEF tyrosine phosphorylation and p21 RhoA GTP binding. Overexpression of FAK in Neuro-2a cells increased both endogenous p190RhoGEF tyrosine phosphorylation and RhoA activity, whereas these events were inhibited by FRNK co-expression. Because insulin-like growth factor 1 treatment of Neuro-2a cells increased FAK tyrosine phosphorylation and enhanced p190RhoGEF-mediated activation of RhoA, our results support the conclusion that FAK association with p190RhoGEF functions as a signaling pathway downstream of integrins and growth factor receptors to stimulate Rho activity.     

Neurite outgrowth from PC12 cells by basic fibroblast growth factor (bFGF) is mediated by RhoA inactivation through p190RhoGAP and ARAP3

The rat pheochromocytoma cell line PC12 has been widely used as a model to study neuronal differentiation. PC12 cells give rise to neurites in response to basic fibroblast growth factor (bFGF). However, it is unclear whether bFGF promotes neurite outgrowth by inducing RhoA inactivation, and a mechanism for RhoA inactivation in PC12 cells in response to bFGF has not been reported. Lysophosphatidic acid (LPA) treatment and the expression of constitutively active (CA)‐RhoA (RhoA V14) impaired neurite formation in response to bFGF, while Tat‐C3 exoenzyme and the expression of dominant negative (DN)‐RhoA (RhoA N19) stimulated neurite outgrowth. GTP‐bound RhoA levels were reduced in response to bFGF, which suggests that the inactivation of RhoA is essential to neurite outgrowth in response to bFGF. To investigate the mechanism of RhoA inactivation, this study examined the roles of p190RhoGAP and Rap‐dependent RhoGAP (ARAP3). DN‐p190RhoGAP prevented neurite outgrowth, while WT‐p190RhoGAP and Src synergistically stimulated neurite outgrowth; these findings suggest that bFGF promotes the inactivation of RhoA and subsequent neurite outgrowth through p190RhoGAP and Src. Furthermore, DN‐Rap1 and DN‐ARAP3 reduced neurite formation in PC12 cells. These results suggest that RhoA is likely to be inactivated by p190RhoGAP and ARAP3 during neurite outgrowth in response to bFGF. J. Cell. Physiol. 224: 786–794, 2010. © 2010 Wiley‐Liss, Inc.     

Suppression of RhoA activity by focal adhesion kinase-induced activation of p190RhoGAP: role in regulation of endothelial permeability

The interaction of endothelial cells with extracellular matrix proteins at focal adhesions sites contributes to the integrity of vascular endothelial barrier. Although focal adhesion kinase (FAK) activation is required for the recovery of the barrier function after increased endothelial junctional permeability, the basis for the recovery remains unclear. We tested the hypothesis that FAK activates p190RhoGAP and, thus, negatively regulates RhoA activity and promotes endothelial barrier restoration in response to the permeability-increasing mediator thrombin. We observed that thrombin caused a transient activation of RhoA but a more prolonged FAK activation temporally coupled to the recovery of barrier function. Thrombin also induced tyrosine phosphorylation of p190RhoGAP, which coincided with decrease in RhoA activity. We further showed that FAK was associated with p190RhoGAP, and importantly, recombinant FAK phosphorylated p190RhoGAP in vitro. Inhibition of FAK by adenoviral expression of FRNK (a dominant negative FAK construct) in monolayers prevented p190RhoGAP phosphorylation, increased RhoA activity, induced actin stress fiber formation, and produced an irreversible increase in endothelial permeability in response to thrombin. We also observed that p190RhoGAP was unable to attenuate RhoA activation in the absence of FAK activation induced by FRNK. The inhibition of RhoA by the C3 toxin (Clostridium botulinum toxin) restored endothelial barrier function in the FRNK-expressing cells. These findings in endothelial cells were recapitulated in the lung microcirculation in which FRNK expression in microvessel endothelia increased vascular permeability. Our studies demonstrate that FAK-induced down-modulation of RhoA activity via p190RhoGAP is a crucial step in signaling endothelial barrier restoration after increased endothelial permeability.     

Transforming growth factor beta regulates the expression of the M2 muscarinic receptor in atrial myocytes via an effect on RhoA and p190RhoGAP

Transforming growth factor beta (TGFbeta) signaling is involved in the development and regulation of multiple organ systems and cellular signaling pathways. We recently demonstrated that TGFbeta regulates the response of atrial myocytes to parasympathetic stimulation. Here, TGFbeta(1) is shown to inhibit expression of the M(2) muscarinic receptor (M(2)), which plays a critical role in the parasympathetic response of the heart. This effect is mimicked by overexpression of a dominant negative mutant of RhoA and by the RhoA kinase inhibitor Y27632, whereas adenoviral expression of a dominant activating-RhoA reverses TGFbeta inhibition of M(2) expression. TGFbeta(1) also mediates a decrease in GTP-bound RhoA and a reciprocal increase in the expression of the RhoA GTPase-activating protein, p190RhoGAP, whereas total RhoA is unchanged. Inhibition of M(2) promoter activity by TGFbeta(1) is mimicked by overexpression of p190RhoGAP, whereas a dominant negative mutant of p190RhoGAP reverses this effect of TGFbeta(1). In contrast to atrial myocytes, in mink lung epithelial cells, in which TGFbeta signaling through activation of RhoA has been previously identified, TGFbeta(1) stimulated an increase in GTP-bound RhoA in association with a reciprocal decrease in the expression of p190RhoGAP. Both effects demonstrated a similar dose dependence on TGFbeta(1). Thus TGFbeta regulation of M(2) muscarinic receptor expression is dependent on RhoA, and TGFbeta regulation of p190RhoGAP expression may be a cell type-specific mechanism for TGFbeta signaling through RhoA.     

VCAM-1 inhibits TGFbeta stimulated epithelial-mesenchymal transformation by modulating Rho activity and stabilizing intercellular adhesion in epicardial mesothelial cells

Regulation of epithelial-mesenchymal transformation (EMT) is of central importance both in normal development and in disease. During heart development, cells of the superficial epicardial mesothelium undergo EMT to give rise to precursor cells of the coronary vasculature and cardiac fibroblasts. Here we report that the alpha(4)beta(1) integrin ligand, VCAM-1, inhibits EMT of chick epicardial mesothelial cells stimulated by TGFbeta isoforms. We further investigated the molecular basis of this inhibition using cultured chick embryonic and rat adult epicardial mesothelial cells. We observed that VCAM-1 increased cortical actin filaments at intercellular junctions and reduced stress fibers across epicardial cells. VCAM-1 inhibited stress fiber formation by TGFbeta1, TGFbeta2, TGFbeta3 and lysophosphatidic acid and altered Rho activity stimulated by TGFbeta3. This was accompanied by an increase in tyrosine phosphorylation of p190RhoGAP. All three TGFbeta isoforms weakened intercellular adhesion, reduced membrane localization of beta-catenin and E-cadherin and stimulated epicardial EMT in chick hearts. Each of these effects was restricted by simultaneous VCAM-1 treatment. Our data support the hypothesis that VCAM-1 can alter epicardial EMT at two key points: it limits Rho-dependent events such as stress fiber formation and it maintains the association of beta-catenin and E-cadherin with the adherens junction.     

Role of p190RhoGAP in beta 2 integrin regulation of RhoA in human neutrophils

We found that engagement of beta(2) integrins on human neutrophils induced activation of RhoA, as indicated by the increased ratio of GTP:GTP + GDP recovered on RhoA and translocation of RhoA to a membrane fraction. The clustering of beta(2) integrins also induced a time-dependent increase in GDP bound to RhoA, which correlated with beta(2) integrin-induced activation of p190RHOGAP: The activation of p190RhoGAP was completely blocked by [4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] (PP1), a selective inhibitor of Src family tyrosine kinases. However, clustering of beta(2) integrins did not increase the basal tyrosine phosphorylation of p190RhoGAP, nor did it affect the amount of p120RasGAP bound to p190RHOGAP: Instead, the beta(2) integrin-induced activation of p190RhoGAP was accompanied by increased tyrosine phosphorylation of a p190RhoGAP-associated protein, p120RasGAP, and accumulation of both p120RasGAP and p190RhoGAP in a membrane fraction. PP1 blocked the beta(2) integrin-induced phosphorylation of p120RasGAP, as well as the translocation of p190RhoGAP and p120RasGAP, but it did not affect the accumulation of RhoA in the membrane fraction. In agreement with the mentioned findings, PP1 also increased the GTP:GTP + GDP ratio recovered on RhoA immunoprecipitated from beta(2) integrin-stimulated cells. Thus, in neutrophils, beta(2) integrin-induced activation of p190RhoGAP requires a signal from a Src family tyrosine kinase, but it does not occur via the signaling pathway responsible for activation of RHOA:     

HIV-1 Nef disrupts the podocyte actin cytoskeleton by interacting with diaphanous interacting protein

The ability of the human immunodeficiency virus, type 1 (HIV-1) protein Nef to induce cytoskeleton changes in infected host cells is a key event in viral replication. In renal podocytes, we found that Nef induced loss of stress fibers and increased lamellipodia, pathological changes leading to proteinuria in HIV-associated nephropathy. These morphological changes were mediated by Nef-induced Rac1 activation and RhoA inhibition. We identified a new interaction between Nef and diaphanous interacting protein (DIP), a recently described regulator of Rho and Rac signaling. We found that the Src homology 3 binding domain of DIP and the Nef PXXP motif were required for this interaction. Nef also interacts with Vav2 in podocytes. DIP and Vav2 both interact directly with Nef in a competitive manner. DIP interacts with p190RhoGAP, and intact DIP was required for Nef-induced phosphorylation of p190RhoGAP. DIP also interacts with Vav2, and although DIP enhanced baseline phosphorylation of Vav2, it was not required for Nef-induced Vav2 activation. In Nef-infected podocytes, Src kinase induces phosphorylation of DIP, p190RhoGAP, and Vav2, leading to RhoA inhibition and Rac1 activation. Inhibition of the Nef-induced signaling pathway by using a dominant negative of either Src or DIP or siRNA for DIP or p190RhoAGAP restored RhoA activity and stress fiber formation in Nef-infected podocytes, whereas siRNA for Vav2 reduced Rac1 activity and formation of lamellipodia. We conclude that in HIV-infected podocytes, Nef, through the recruitment of DIP and p190RhoAGAP to Nef-Src complex, activates p190RhoAGAP and down-regulates RhoA activity.     

Control of neurite outgrowth by RhoA inactivation

cAMP induces neurite outgrowth in the rat pheochromocytoma cell line 12 (PC12). In particular, di-butyric cAMP (db-cAMP) induces a greater number of primary processes with shorter length than the number induced by nerve growth factor (NGF). db-cAMP up- and down-regulates GTP-RhoA levels in PC12 cells in a time-dependent manner. Tat-C3 toxin stimulates neurite outgrowth, whereas lysophosphatidic acid (LPA) and constitutively active (CA)-RhoA reduce neurite outgrowth, suggesting that RhoA inactivation is essential for the neurite outgrowth from PC12 cells stimulated by cAMP. In this study, the mechanism by which RhoA is inactivated in response to cAMP was examined. db-cAMP induces phosphorylation of RhoA and augments the binding of RhoA with Rho guanine nucleotide dissociation inhibitor (GDI). Moreover, RhoA (S188D) mimicking phosphorylated RhoA induces greater neurite outgrowth than RhoA (S188A) mimicking dephosphorylated form does. Additionally, db-cAMP increases GTP-Rap1 levels, and dominant negative (DN)-Rap1 and DN-Rap-dependent RhoGAP (ARAP3) block neurite outgrowth induced by db-cAMP. DN-p190RhoGAP and the Src inhibitor PP2 suppress neurite outgrowth, whereas transfection of c-Src and p190RhoGAP cDNAs synergistically stimulate neurite outgrowth. Taken together, RhoA is inactivated by phosphorylation of itself, by p190RhoGAP which is activated by Src, and by ARAP3 which is activated by Rap1 during neurite outgrowth from PC12 cells in response to db-cAMP.     

β3 integrin-EGF receptor cross-talk activates p190RhoGAP in mouse mammary gland epithelial cells

Active RhoA localizes to plasma membrane, where it stimulates formation of focal adhesions and stress fibers. RhoA activity is inhibited by p190RhoGAP following integrin-mediated cell attachment to allow sampling of new adhesive environments. p190RhoGAP is itself activated by Src-dependent tyrosine phosphorylation, which facilitates complex formation with p120RasGAP. This complex then translocates to the cell surface, where p190RhoGAP down-regulates RhoA. Here we demonstrate that the epidermal growth factor receptor (EGFR) cooperates with β3 integrin to regulate p190RhoGAP activity in mouse mammary gland epithelial cells. Adhesion to fibronectin stimulates tyrosine phosphorylation of the EGFR in the absence of receptor ligands. Use of a dominant inhibitory EGFR mutant demonstrates that fibronectin-activated EGFR recruits p120RasGAP to the cell periphery. Expression of an inactive β3 integrin subunit abolishes p190RhoGAP tyrosine phosphorylation, demonstrating a mechanistic link between β3 integrin-activated Src and EGFR regulation of the RhoA inhibitor. The β3 integrin/EGFR pathway also has a positive role in formation of filopodia. Together our data suggest that EGFR constitutes an important intrinsic migratory cue since fibronectin is a key component of the microenvironment in normal mammary gland development and breast cancer. Our data also suggest that EGFR expressed at high levels has a role in eliciting cell shape changes associated with epithelial-to-mesenchymal transition.     

SHP-2 positively regulates myogenesis by coupling to the Rho GTPase signaling pathway

Myogenesis is an intricate process that coordinately engages multiple intracellular signaling cascades. The Rho family GTPase RhoA is known to promote myogenesis, however, the mechanisms controlling its regulation in myoblasts have yet to be fully elucidated. We show here that the SH2-containing protein tyrosine phosphatase, SHP-2, functions as an early modulator of myogenesis by regulating RhoA. When MyoD was expressed in fibroblasts lacking functional SHP-2, muscle-specific gene activity was impaired and abolition of SHP-2 expression by RNA interference inhibited muscle differentiation. By using SHP-2 substrate-trapping mutants, we identified p190-B RhoGAP as a SHP-2 substrate. When dephosphorylated, p190-B RhoGAP has been shown to stimulate the activation of RhoA. During myogenesis, p190-B RhoGAP was tyrosyl dephosphorylated concomitant with the stimulation of SHP-2's phosphatase activity. Moreover, overexpression of a catalytically inactive mutant of SHP-2 inhibited p190-B RhoGAP tyrosyl dephosphorylation, RhoA activity, and myogenesis. These observations strongly suggest that SHP-2 dephosphorylates p190-B RhoGAP, leading to the activation of RhoA. Collectively, these data provide a mechanistic basis for RhoA activation in myoblasts and demonstrate that myogenesis is critically regulated by the actions of SHP-2 on the p190-B Rho GAP/RhoA pathway.     

A Blk–p190RhoGAP signaling module downstream of activated Gα13 functionally opposes CXCL12-stimulated RhoA activation and cell invasion

Activation of the GTPase RhoA linked to cell invasion can be tightly regulated following Gα₁₃ stimulation. We have used a cellular model displaying Gα₁₃-dependent inhibition of RhoA activation associated with defective cell invasion to the chemokine CXCL12 to characterize the molecular players regulating these processes. Using both RNAi transfection approaches and protein overexpression experiments here we show that the Src kinase Blk is involved in Gα₁₃-activated tyrosine phosphorylation of p190RhoGAP, which causes RhoA inactivation and ultimately leads to deficient cell invasion. Characterization of molecular interplays between Gα₁₃, Blk and p190RhoGAP revealed that Blk binds Gα₁₃, and that Blk-mediated p190RhoGAP phosphorylation upon Gα₁₃ activation correlates with weakening of Gα₁₃–Blk association connected to increased Blk–p190RhoGAP assembly. These results place Blk upstream of the p190RhoGAP–RhoA pathway in Gα₁₃-activated cells, overall representing an opposing signaling module during CXCL12-triggered invasion. In addition, analyses with Blk- or Gα₁₃-knockdown cells indicated that Blk can also mediate CXCL12-triggered phosphorylation of p190RhoGAP independently of Gα₁₃. However, even if CXCL12 induces the Blk-mediated GAP phosphorylation, the simultaneous stimulation of the guanine-nucleotide exchange factor Vav1 by the chemokine, as earlier reported, leads to a net increase in RhoA activation. Therefore, when Gα₁₃ is concurrently stimulated with CXCL12 there appears to be sufficient Blk activity to promote adequate levels of p190RhoGAP tyrosine phosphorylation to inactivate RhoA and to impair cell invasiveness.     

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.     

Tension on JAM-A activates RhoA via GEF-H1 and p115 RhoGEF

Junctional adhesion molecule A (JAM-A) is a broadly expressed adhesion molecule that regulates cell–cell contacts and facilitates leukocyte transendothelial migration. The latter occurs through interactions with the integrin LFA-1. Although we understand much about JAM-A, little is known regarding the protein’s role in mechanotransduction or as a modulator of RhoA signaling. We found that tension imposed on JAM-A activates RhoA, which leads to increased cell stiffness. Activation of RhoA in this system depends on PI3K-mediated activation of GEF-H1 and p115 RhoGEF. These two GEFs are further regulated by FAK/ERK and Src family kinases, respectively. Finally, we show that phosphorylation of JAM-A at Ser-284 is required for RhoA activation in response to tension. These data demonstrate a direct role of JAM-A in mechanosignaling and control of RhoA and implicate Src family kinases in the regulation of p115 RhoGEF.     

The GTPase-deficient Rnd Proteins Are Stabilized by Their Effectors

Rnd proteins are Rho family GTP-binding proteins with cellular functions that antagonize RhoA signaling. We recently described a new Rnd3 effector Syx, also named PLEKHG5, that interacts with Rnds via a Raf1-like "Ras-binding domain." Syx is a multidomain RhoGEF that participates in early zebrafish development. Here we demonstrated that Rnd1, Rnd2, and Rnd3 stability is acutely dependent on interaction with their effectors such as Syx or p190 RhoGAP. Although Rnd3 turnover is blocked by treatment of cells with MG132, we provide evidence that such turnover is mediated indirectly by effects on the Rnd3 effectors, rather than on Rnd3 itself, which is not significantly ubiquitinated. The minimal regions of Syx and p190 RhoGAP that bind Rnd3 are not sequence-related but have similar effects. We have identified features that allow for Rnd3 turnover including a conserved Lys-45 close to the switch I region and the C-terminal membrane-binding domain of Rnd3, which cannot be substituted by the equivalent Cdc42 CAAX sequence. By contrast, an effector binding-defective mutant of Rnd3 when overexpressed undergoes turnover at normal rates. Interestingly the activity of the RhoA-regulated kinase ROCK stimulates Rnd3 turnover. This study suggests that Rnd proteins are regulated through feedback mechanisms in cells where the level of effectors and RhoA activity influence the stability of Rnd proteins. This effector feedback behavior is analogous to the ability of ACK1 and PAK1 to prolong the lifetime of the active GTP-bound state of Cdc42 and Rac1.     

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.