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Characterization of the interaction between Rnd3 and Syx. (A) Schematic of domains identified in Syx: including zinc-finger, Rnd3binding (in cyan), Dbl homology (DH), pleckstrin homology (PH) and PDZ-binding motif. Sequence alignment of Raf1-like RBDs in mouse Syx (AAU04953), zebrafish Syx (XM_686228.1), human RGS12 (NP_002917), RGS14 (NP_006471) and Raf1 (AAA60247). Conserved residues are shaded in grey; a key helix is underlined; amino acid side chains contacting Ras are in cyan and residues mutated in this study are in yellow. The catalytic residues T559/K560 in Syx DH domain are denoted by yellow arrowheads. (B) Rnd3 interacts with the N-terminal of Syx. 293T cells were cotransfected with SBP-Flag Rnd3 and indicated Flag-Syx constructs. The purified protein complex was immunoblotted to visualize bound Syx. (C) R178 and K179 of Syx are critical Rnd3 binding residues. 293T cells were co-transfected as for (B) with the indicated constructs and assessed for interaction. M1 and M2 refer to the mutant constructs, E156AT157A and R178EK179D. (D) Rnd3 and RhoA (WT) interact with Syx via the RBD and DH domain, respectively. The mutant constructs, R178EK179D and T559EK560E are represented by Syx RBDmut and Syx DHmut, respectively. (E) Rnd3 interacts with Syx(145-216). doi:10.1371/journal.pone.0012409.g003
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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...
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Context 1
... Syx protein has a number of domains that show homology to other proteins including the DH domain (407-596) that accelerates the exchange of nucleotides on Rho family GTPases, and the PH domain (658-749), a PDZ-binding domain at the C- terminus, and a putative zinc finger region with little sequence similarity outside the cysteine-histidine residues ( Figure 3A Figure S1). Thus the N-terminal of Syx is necessary and sufficient for its interaction with Rnd3. ...
Context 2
... was surprising given that only the DH domain of this protein would be expected to interact with Rho proteins. We found that N-terminal truncations Syx(77-800) and Syx(112-800) could bind to Rnd3 while Syx(162-800) could not ( Figure 3B), implicating residues downstream of amino acid 112 in binding, a region that lies outside the zinc finger domain. ...
Context 3
... determined visually that a 63 amino acid region (154-216) has significant homology with the putative Ras binding domains (RBD) of RGS12 and RGS14 ( Figure 3A). Based on the crystal structure of Rap1a complexed with the RBD of Raf1 [24], the side chains of T68, K84 and R89 of Raf1 (marked in blue) are known to directly contact K-Ras. ...
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... then created two mutants, Syx(1-800) E156A/T157A (exposed side-chains not directly involved in effector binding) and R178E/K179D (equivalent to the Raf1 K84/R89 side-chains involved with Ras binding). In Figure 3C we see that Syx(136-800) fragment lacking sequences N-terminal to the putative RBD bound well to Rnd3, as did the Syx(1-800) E156A/T157A; it was clear however that the R178E/K179D substitutions prevented Syx binding to Rnd3. These support the notion that Syx RBD extends from 145-216 and is involved in Rnd3 binding. ...
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... Rnd3 interaction site is quite distinct from the RhoA exchange domain. The binding of RhoA to the Syx DH domain ( Figure 3D) was confirmed by the Syx T559E/K560E substitutions in the RhoGEF DH domain which abolishe RhoA but not Rnd3 binding. We also confirmed that this RBD-like motif alone Syx(145-216) binds well to Rnd3 ( Figure 3E), demonstrating that the Syx ubiquitin-fold RBD is involved in binding Rnd3 in a manner similar to the effector Raf1 with Ras. ...
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... binding of RhoA to the Syx DH domain ( Figure 3D) was confirmed by the Syx T559E/K560E substitutions in the RhoGEF DH domain which abolishe RhoA but not Rnd3 binding. We also confirmed that this RBD-like motif alone Syx(145-216) binds well to Rnd3 ( Figure 3E), demonstrating that the Syx ubiquitin-fold RBD is involved in binding Rnd3 in a manner similar to the effector Raf1 with Ras. This contrasts with the Rac1/Rnd3/RhoD binding domain of plexin B1 which is ubiquitin-like in nature but binds to these GTPases with a completely different 'face' [25]. ...
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... the early phenotype associated with the Syx is consistent with it playing an important role in vertebrate gastrulation. It is notable that a Syx-like protein is found also in primitive lancelet chordates ( Figure S3). ...
Context 8
... Ral, Rit) [26]. Based on our Syx RBD mutagenesis analysis (Figure 3), Syx likely binds Rnd3 in a manner resembling the Rap1A-Raf1 complex [24]. Other RhoGEFs have been found with classical RBDs. ...
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Citations
... While not located in a specific known domain or motif, there is a putative zinc-finger motif upstream and the Ras binding domain (RBD) domain downstream of the Arg97 residue. The physiological function of the arginine residue at position 97 is not yet known, but the location near the RBD binding domain suggests that this arginine residue could be important for binding to rnd3, a RhoA effector [20]. Subsequently, due to changes in PLEKHG5 binding with rnd3, a direct change in RhoA activation could occur. ...
Mutations in PLEKHG5, a pleckstrin homology domain containing member of the GEF family, are associated with distal spinal muscular atrophy and intermediate Charcot-Marie-Tooth disease. Here, we describe an isolated case with distal intermediate neuropathy with scapular winging. By whole exome sequencing, we identified the homozygous PLEKHG5 Arg97Gln missense mutation, located in the N-terminal region of the protein. This mutation resides between a zinc-finger motif and a RBD domain, involved in binding rnd3, a RhoA effector protein. We conclude that based on the characteristic phenotype presented by the patient and the supportive genetic findings, the PLEKHG5 mutation is the causative variant.
... RhoE often exerts its functions through interacting with other effectors as well. We tested the interaction of the isoforms with two additional well-known RhoE effectors, p190RhoGAP and synectin-binding RhoA exchange factor (Syx), which were involved in the regulation of cell protrusion and migration 34 , and embryonic cell shape 35 , respectively. Again, the co-IP assays indicated that RhoE and RhoEα bound to the two effectors (Fig. 5c), suggesting a possible functional redundancy of the two isoforms. ...
The Rho family of GTPases consists of 20 members including RhoE. Here, we discover the existence of a short isoform of RhoE designated as RhoEα, the first Rho GTPase isoform generated from alternative translation. Translation of this new isoform is initiated from an alternative start site downstream of and in-frame with the coding region of the canonical RhoE. RhoEα exhibits a similar subcellular distribution while its protein stability is higher than RhoE. RhoEα contains binding capability to RhoE effectors ROCK1, p190RhoGAP and Syx. The distinct transcriptomes of cells with the expression of RhoE and RhoEα, respectively, are demonstrated. The data propose distinctive and overlapping biological functions of RhoEα compared to RhoE. In conclusion, this study reveals a new Rho GTPase isoform generated from alternative translation. The discovery provides a new scope of understanding the versatile functions of small GTPases and underlines the complexity and diverse roles of small GTPases. Dai et al. report the identification and characterization of a new isoform of RhoE (RhoEα), a member of the Rho GTPase family, which is generated from the same gene by alternative translation initiation at the downstream ATG codon 46. Compared to RhoE, RhoEα does not differ in the subcellular localization but has increased protein stability and distinct molecular signalling profile.
... The effect of Syx on Rnd3 is also observed on the other Rnd proteins, but the interaction of Syx with Rnd1 or Rnd2 is weaker than the one with Rnd3 [24]. Syx is also considered as an Rnd effector as Rnd proteins potentially modulate RhoA activation through this RhoGEF [26]. ...
... Over past years, members of the Rho GTPase family have been demonstrated as principal actors in many cellular processes including cytoskeletal [24,26] organization, cell cycle, proliferation, cell shape and movement. Rnd proteins have also been implicated in these processes, as they are known to act mainly as antagonists of the RhoA/ROCK pathway, even if they also function through specific effectors. ...
Rnd proteins constitute a subfamily of Rho GTPases represented in mammals by Rnd1, Rnd2 and Rnd3. Despite their GTPase structure, their specific feature is the inability to hydrolyse GTP-bound nucleotide. This aspect makes them atypical among Rho GTPases. Rnds are regulated for their expression at the transcriptional or post-transcriptional levels and they are activated through post-translational modifications and interactions with other proteins. Rnd proteins are mainly involved in the regulation of the actin cytoskeleton and cell proliferation. Whereas Rnd3 is ubiquitously expressed, Rnd1 and 2 are tissue-specific. Increasing data has described their important role during development and diseases. Herein, we describe their involvement in physiological and pathological conditions with a focus on the neuronal and vascular systems, and summarize their implications in tumorigenesis.
... One significance of this is that it defines novel connections between FOXC1 and the RHO pathway. PLEKHG5 encodes a multidomain RHO-guanine nucleotide exchange factor and participates in negative feedback regulation of the RHO pathway (80)(81)(82)(83)(84)(85)(86)(87). The feedback mechanism includes RHOA activation by PLEKHG5, ROCK1 (a RHO kinase) activation by RHOA, stabilization and activation of Rnd proteins (members of Rho family GTP-binding proteins) by ROCK1 and antagonistic effects of Rnd proteins on PLEKHG5 activation (80)(81)(82)(83)(84)(85)(86)(87). ...
... PLEKHG5 encodes a multidomain RHO-guanine nucleotide exchange factor and participates in negative feedback regulation of the RHO pathway (80)(81)(82)(83)(84)(85)(86)(87). The feedback mechanism includes RHOA activation by PLEKHG5, ROCK1 (a RHO kinase) activation by RHOA, stabilization and activation of Rnd proteins (members of Rho family GTP-binding proteins) by ROCK1 and antagonistic effects of Rnd proteins on PLEKHG5 activation (80)(81)(82)(83)(84)(85)(86)(87). There exists ample data that evidence the importance of RHOA and RHO signaling in TM functions and glaucoma pathogenesis. ...
Glaucoma is a leading cause of blindness. We aimed in this study to identify genes that may make subtle and cumulative contributions to glaucoma pathogenesis. To this end, we identified molecular interactions and pathways that include transcription factors (TFs) FOXC1, PITX2, PAX6, and NFKB1, and various microRNAs including miR-204 known to have relevance to trabecular meshwork (TM) functions and/or glaucoma. TM tissue is involved in glaucoma pathogenesis. In-house microarray transcriptome results and data sources were used to identify target genes of the regulatory molecules. Bioinformatics analyses were done to filter TM and glaucoma relevant genes. These were submitted to network creating softwares to define interactions, pathways, and a network that would include the genes. The network was stringently scrutinized and minimized, then expanded by addition of microarray data and data on TF and microRNA binding sites. Selected features of the network were confirmed by empirical studies such as dual luciferase assays, real time PCR and Western blot experiments, and apoptosis assays. MYOC, WDR36, LTPBP2, RHOA, ،CYP1B1, OPA1, SPARC, MEIS2, PLEKHG5, RGS5, BBS5, ALDH1A1, NOMO2, CXCL6, FMNL2, ADAMTS5, CLOCK, and DKK1 were among genes included in the final network. Pathways identified included those that affect ECM properties, IOP, ciliary body functions, retinal ganglion cell viability, apoptosis, focal adhesion, and oxidative stress response. The identification of many genes potentially involved in glaucoma pathology is consistent with its being a complex disease. The inclusion of several known glaucoma related genes validates the approach used.
... The regulation of actin cytoskeleton by RND1 could also involve RND1 partners. RND1 can bind Syx [30], which activates RHOA. In the same way as for RND3 [30], binding of RND1 to Syx may cause Syx inhibition and in turn RHOA inhibition. ...
... RND1 can bind Syx [30], which activates RHOA. In the same way as for RND3 [30], binding of RND1 to Syx may cause Syx inhibition and in turn RHOA inhibition. A RND1 partner, Socius, was identified by the yeast two-hybrid technique using RND1 as bait [18]. ...
The Rho GTPase family can be classified into classic and atypical members. Classic members cycle between an inactive Guanosine DiPhosphate -bound state and an active Guanosine TriPhosphate-bound state. Atypical Rho GTPases, such as RND1, are predominantly in an active GTP-bound conformation. The role of classic members in oncogenesis has been the subject of numerous studies, while that of atypical members has been less explored. Besides the roles of RND1 in healthy tissues, recent data suggest that RND1 is involved in oncogenesis and response to cancer therapeutics. Here, we present the current knowledge on RND1 expression, subcellular localization, and functions in healthy tissues. Then, we review data showing that RND1 expression is dysregulated in tumors, the molecular mechanisms involved in this deregulation, and the role of RND1 in oncogenesis. For several aggressive tumors, RND1 presents the features of a tumor suppressor gene. In these tumors, low expression of RND1 is associated with a bad prognosis for the patients. Finally, we highlight that RND1 expression is induced by anticancer agents and modulates their response. Of note, RND1 mRNA levels in tumors could be used as a predictive marker of both patient prognosis and response to anticancer agents.
... RhoE is the best studied member of these GTPase-deficient Rho members, and it is likely that what has been shown in studies on RhoE is also applicable to the other Rnd members, and probably also to RhoH. RhoE was shown to bind the RhoGEF Syx, although not through the DH/PH domain, but instead through a Ras-binding-domain-like motif in the N-terminus of Syx, which suggests that Syx is a RhoE effector rather than an activator [29]. RhoGDI does not appear to form an inhibitory complex with RhoE, while RhoGAP domains cannot stimulate the GTPase activity of RhoE, as the catalytically important so-called 'arginine finger' in the RhoGAP domain is sterically hindered, which prevents a functional interaction with RhoE [28]. ...
Involvement of Rho GTPases in cancer has been a matter of debate since the identification of the first members of this branch of the Ras superfamily of small GTPases. The Rho GTPases were ascribed important roles in the cell, although these were restricted to regulation of cytoskeletal dynamics, cell morphogenesis, and cell locomotion, with initially no clear indications of direct involvement in cancer progression. This paradigm has been challenged by numerous observations that Rho-regulated pathways are often dysregulated in cancers. More recently, identification of point mutants in the Rho GTPases Rac1, RhoA, and Cdc42 in human tumors has finally given rise to a new paradigm, and we can now state with confidence that Rho GTPases serve as oncogenes in several human cancers. This article provides an exposé of current knowledge of the roles of activated Rho GTPases in cancers.
... However, it also acts upstream of RhoA by binding and activating p190RhoGAP (Wennerberg et al., 2003) leading to the inactive GDP-bound form of RhoA. Additionally, Rnd3 binds to and inhibits the activation of the RhoA GEF Syx (Goh and Manser, 2010). Investigating p190RhoGAP, we found that it was expressed equally in normal pulmonary and IPF fibroblasts, but its activity was significantly lower in the IPF lines, consistent with these cells having higher RhoA activity. ...
Idiopathic pulmonary fibrosis (IPF) is an incurable disease of the lung that is characterized by excessive deposition of extracellular matrix (ECM), resulting in disruption of normal lung function. The signals regulating fibrosis include both TGFβ and tissue rigidity, and a major signaling pathway implicated in fibrosis involves activation of the GTPase RhoA. During studies exploring how elevated RhoA activity is sustained in IPF, we discovered that not only is RhoA activated by profibrotic stimuli, but also that the expression of Rnd3, a major antagonist of RhoA activity, and the activity of p190RhoGAP (p190), a Rnd3 effector, are both suppressed in IPF fibroblasts. Restoration of Rnd3 levels in IPF fibroblasts results in an increase in p190 activity, a decrease in RhoA activity and a decrease in the overall fibrotic phenotype. We also find that treatment with IPF drugs nintedanib and pirfenidone decreases the fibrotic phenotype and RhoA activity through upregulation of Rnd3 expression and p190 activity. These data provide evidence for a pathway in IPF where fibroblasts downregulate Rnd3 levels and p190 activity to enhance RhoA activity and drive the fibrotic phenotype.
... Tout comme RND1, RND3 est connu pour inhiber l'activation de RhoA via son interaction avec p190RhoGAP (Wennerberg et al., 2003). De façon similaire, Goh and Manser ont déterminé que l'inhibition de RhoA-GTP pouvait également résulter de la régulation négative de Syx par RND3 (Goh and Manser, 2010) d'effet sur l'inhibition des fibres de stress par RND1 mais il réverse l'effet sur la morphologie cellulaire ( Figure 9). RND1 peut également interagir avec le domaine SH2 de Grb7 (Growth factor receptor-bound protein 7), une protéine appartenant à la famille des protéines adaptatrices impliquées dans la transduction de signal (Vayssiere et al., 2000). ...
La famille des GTPases Rho, comprenant 20 membres, contrôle la dynamique du cytosquelette d'actine et différents processus cellulaires comme la migration. En plus de leurs rôles bien établis, certaines GTPases Rho, notamment RhoB et Rac1, ont émergé en tant que gènes de réponse précoce aux dommages à l'ADN. En effet, RhoB est induite en réponse à divers stress génotoxiques, y compris la camptothécine (CPT), les UV et le cisplatine, et protège principalement les cellules de l'apoptose. Le rôle des autres GTPases Rho en réponse précoce aux génotoxiques reste largement méconnu. Dans ce projet, nous avons utilisé la camptothécine, un inhibiteur de la topoisomérase I (TOP1), qui stabilise sélectivement les complexes de clivage TOP1-ADN (TOP1cc) sur la chromatine, afin de cribler les GTPases Rho induites de façon précoce par les dommages à l'ADN. En plus de RhoB, nous avons identifié RND1 comme un gène rapidement induit par la CPT. L'induction de RND1 est réversible et étroitement corrélée à la présence de TOP1cc induit par la CPT. En accord avec ces observations, les rayons UV et le péroxyde d'hydrogène, qui stabilisent indirectement les TOP1cc, induisent également RND1. La CPT augmente la transcription de RND1 indépendamment de l'activité de son promoteur minimal. De plus, la CPT augmente l'activité de la poly ADP-ribose polymérase (PARP1), dont l'inhibition prévient la transcription de RND1. La surexpression de RND1 augmente également l'expression de PARP1, suggérant une régulation positive entre PARP1 et RND1 en réponse aux TOP1cc. Ainsi, nous proposons qu'en réponse à la CPT, les TOP1cc activent PARP1, qui à son tour favorise la transcription de RND1, initiant ainsi une boucle de rétrocontrôle positive. Enfin, nous avons montré que RND1 protège les cellules contre l'apoptose induite par la CPT et entraîne leur résistance à la CPT. L'ensemble de ces résultats ont permis d'identifier RND1 comme nouvelle GTPase Rho impliquée dans la réponse au stress et proposent un nouveau mécanisme de régulation de la transcription des gènes en réponse aux TOP1cc via l'activation de PARP1. Ces résultats suggèrent par ailleurs qu'inhiber la signalisation de RND1 pourrait sensibiliser les cellules tumorales aux dérivés cliniques de la CPT.
... The formation of larger cells in Plekhg5-depeleted osteoclasts is attributed to the loss of polarity of macrophages, rather than multinucleation. Several studies on other cell types have shown that Plekhg5 regulates cell polarity and morphological changes [33,34]. A recent study with breast cancer cells has reported that Plekhg5 is implicated in RhoA-mediated cell polarity, and that Plekhg5-depleted cells exhibit a rounded, flattened shape [35]. ...
... Originally, Plekhg5 was known to be a RhoA-GEF [14,36,37]. Recently, however, Plekhg5 has been shown to interact with the Rho-GTPases Rnd3 not through the Dbl homology, but rather through a region similar to the classic Raf1 Ras-binding domain [33]. Myosin VI and synectin form a complex with Plekhg5 that likely associates with the actin cytoskeleton and could facilitate retrograde translocation of the complex along actin filaments [33,36,39]. ...
... Recently, however, Plekhg5 has been shown to interact with the Rho-GTPases Rnd3 not through the Dbl homology, but rather through a region similar to the classic Raf1 Ras-binding domain [33]. Myosin VI and synectin form a complex with Plekhg5 that likely associates with the actin cytoskeleton and could facilitate retrograde translocation of the complex along actin filaments [33,36,39]. The 14-3-3 family of proteins are associated with Plekhg5 at the N-and C-terminal regions in a phosphorylation-dependent manner [40]. ...
Osteoclasts are multinucleated bone-resorbing cells that are formed by fusion of monocyte/macrophage lineage. Osteoclasts and macrophages generate podosomes that are actin-based dynamic organelles implicated in cell adhesion, spreading, migration, and degradation. However, the detailed mechanisms of podosome organization remain unknown. Here, we identified the Rho-specific guanine-nucleotide exchange factor (Rho-GEF) Plekhg5 as an up-regulated gene during differentiation of osteoclasts from macrophages. Knockdown of Plekhg5 with small interfering RNA in both macrophages and osteoclasts induced larger cell formation with impaired cell polarity and resulted in an elongated and flattened shape. In macrophages, Plekhg5 depletion enhanced random migration, but impaired directional migration, adhesion, and matrix degradation. Plekhg5 in osteoclasts affected random migration, podosome organization, and bone resorption. Plekhg5 depletion affected signaling and localization of several Rho downstream effectors. In fact, end-binding protein 1 (EB1), cofilin and vinculin were abnormally localized in Plekhg5-depleted cells, and mDia1 and LIM kinase (LIMK)1 were upregulated in Plekhg5-depleted cells compared with control cells. However, overexpression of Plekhg5 in macrophages induced an increase in its mRNA level, but failed to increase the protein level, indicating that overexpressed Plekhg5 was degraded in macrophages but not HEK293T cells. Thus, Plekhg5 affects cell polarity, migration, adhesion, degradation, and podosome organization in macrophages and osteoclasts.
... Recently, Yoshida et al. (2015) showed that Syx, a novel RhoA guanine exchange factor, activates RhoA downstream of Nrp1 signaling. However, because Syx-deficient zebrafish embryos die during gastrulation (Goh and Manser, 2010), we could not test this hypothesis. ...
During cardiovascular development, tight spatiotemporal regulation of molecular cues is essential for controlling endothelial cell (EC) migration. Secreted class III Semaphorins play an important role in guidance of neuronal cell migration and were lately linked to regulating cardiovascular development. Recently, SEMA3D gene disruptions were associated with cardiovascular defects in patients; however, the mechanisms of action were not revealed. Here we show for the first time that Sema3d regulates collective EC migration in zebrafish through two separate mechanisms. Mesenchymal Sema3d guides outgrowth of the common cardinal vein via repulsion and signals through PlexinD1. Additionally, within the same ECs, we identified a novel function of autocrine Sema3d signaling in regulating Actin network organization and EC morphology. We show that this new function requires Sema3d signaling through Neuropilin1, which then regulates Actin network organization through RhoA upstream of Rock, stabilizing the EC sheet. Our findings are highly relevant for understanding EC migration and the mechanisms of collective migration in other contexts.
