Visualizing the subcellular localization of RHOB-GTP and GTPase-Effector complexes using a split-GFP/nanobody labelling assay.

Small GTPases are highly regulated proteins that control essential signaling pathways through the activity of their effector proteins. Among the RHOA subfamily, RHOB regulates peculiar functions that could be associated with the control of the endocytic trafficking of signaling proteins. Here, we used an optimized assay based on tripartite split-GFP complementation to localize GTPase-effector complexes with high-resolution. The detection of RHOB interaction with the Rhotekin Rho binding domain (RBD) that specifically recognizes the active GTP-bound GTPase, is performed in vitro by the concomitant addition of recombinant GFP1-9 and a GFP nanobody. Analysis of RHOB-RBD complexes localization profiles combined with immunostaining and live cell imaging indicated a serum-dependent reorganization of the endosomal and membrane pool of active RHOB. We further applied this technology to the detection of RHO-effector complexes that highlighted their subcellular localization with high resolution among the different cellular compartments.

Tensor Decomposition of Largest Convolutional Eigenvalues Reveals Pathologic Predictive Power of RhoB in Rectal Cancer Biopsy.

RhoB protein belongs to the Rho GTPase family, which plays an important role in governing cell signaling and tissue morphology. Its expression is known to have implications in pathologic processes of diseases. In particular, the role of RhoB in rectal cancer is not well understood. Investigation in the regulation and communication of this protein, detected by immunohistochemical staining on the microscope, can help gain insightful information leading to optimal disease treatment options. Herein, deep learning-based image analysis and the decomposition of multiway arrays were used to study the predictive factor of RhoB in two cohorts of patients with rectal cancer having survival rates of <5 and >5 years. The results show distinctions between the tensor decomposition factors of the two cohorts.

The RhoB small GTPase in physiology and disease.

RhoB is a Rho family GTPase that is highly similar to RhoA and RhoC, yet has distinct functions in cells. Its unique C-terminal region is subject to specific post-translational modifications that confer different localization and functions to RhoB. Apart from the common role with RhoA and RhoC in actin organization and cell migration, RhoB is also implicated in a variety of other cellular processes including membrane trafficking, cell proliferation, DNA-repair and apoptosis. RhoB is not an essential gene in mice, but it is implicated in several physiological and pathological processes. Its multiple roles will be discussed in this review.

RhoB is a component of the human cytomegalovirus assembly complex and is required for efficient viral production.

Human Cytomegalovirus (HCMV), an ubiquitous ß-herpesvirus, is a significant pathogen that causes medically severe diseases in immunocompromised individuals and in congenitally infected neonates. RhoB belongs to the family of Rho GTPases, which regulates diverse cellular processes. Rho proteins are implicated in the entry and egress from the host cell of mainly α- and γ-herpesviruses, whereas ß-herpesviruses are the least studied in this regard. Here, we studied the role of RhoB GTPase during HCMV lytic infection. Microscopy analysis, both in fixed and live infected cells showed that RhoB was translocated to the assembly complex/compartment (AC) of HCMV, a cytoplasmic zone in infected cells where many viral structural proteins are known to accumulate and assembly of new virions takes place. Furthermore, RhoB was localized at the AC even when the expression of the late HCMV AC proteins was inhibited. At the very late stages of infection, cellular projections were formed containing RhoB and HCMV virions, potentially contributing to the successful viral spread. Interestingly, the knockdown of RhoB in HCMV-infected cells resulted in a significant reduction of the virus titer and could also affect the accumulation of AC viral proteins at this subcellular compartment. RhoB knockdown also affected actin fibers' structure. Actin reorganization was observed at late stages of infection originating from the viral AC and surrounding the cellular projections, implying a potential interplay between RhoB and actin during HCMV assembly and egress. In conclusion, our results demonstrate for the first time that RhoB is a constituent of the viral AC and is required for HCMV productive infection.

Generation of a single chain antibody variable fragment (scFv) to sense selectively RhoB activation.

Determining the cellular level of activated form of RhoGTPases is of key importance to understand their regulatory functions in cell physiopathology. We previously reported scFvC1, that selectively bind to the GTP-bound form of RhoA, RhoB and RhoC. In this present study we generate, by molecular evolution, a new phage library to isolate scFvs displaying high affinity and selectivity to RhoA and RhoB. Using phage display affinity maturation against the GTP-locked mutant RhoAL63, we isolated scFvs against RhoA active conformation that display Kd values at the nanomolar range, which corresponded to an increase of affinity of three orders of magnitude compared to scFvC1. Although a majority of these evolved scFvs remained selective towards the active conformation of RhoA, RhoB and RhoC, we identified some scFvs that bind to RhoA and RhoC but not to RhoB activated form. Alternatively, we performed a substractive panning towards RhoB, and isolated the scFvE3 exhibiting a 10 times higher affinity for RhoB than RhoA activated forms. We showed the peculiar ability of scFvE3 to detect RhoB but not RhoA GTP-bound form in cell extracts overexpressing Guanine nucleotide Exchange Factor XPLN as well as in EGF stimulated HeLa cells. Our results demonstrated the ability of scFvs to distinguish RhoB from RhoA GTP-bound form and provide new selective tools to analyze the cell biology of RhoB GTPase regulation.

Divergence of Rho residue 43 impacts GEF activity.

RhoA, RhoB and RhoC GTPases are over 85% identical at the amino acid level, with RhoA and RhoC differing at only one residue (43) across the initial two-thirds of their sequences. A putative regulatory distinction between the molecules is their capacity to be uniquely activated by guanine nucleotide exchange factors (GEFs). We hypothesize that variation of amino acid residue 43 between RhoA/B (valine) and RhoC (isoleucine) impacts GEF activity. Direct participation of residue 43 in GEF-catalyzed exchange was confirmed by the observation that mutation of this position to a threonine reduced GEF-catalyzed nucleotide exchange activity in vitro (Vav2, XPLN, GEFT, Dbl and Dbs) and greatly depressed RhoA and RhoC GTP-loading profiles in cell lysates. Using a residue swap approach, substitution of RhoA Val 43 with an Ile was found to significantly promote basal nucleotide exchange activity and enhance GTP-loading in cells. Substitution of Val 43 with an Ile in RhoB negatively affected nucleotide exchange in vitro. Substitution of RhoC Ile 43 with a Val increased GEF-catalyzed exchange in vitro. In addition, RhoC-I43V was more efficacious at driving ovarian cancer cell invasion through matrigrel than wild-type RhoC, RhoC-I43T, wild-type RhoA, RhoA-V43I or RhoA-V43T GTPases. These findings suggest that a divergence between RhoA/B and RhoC at residue 43 impacts basal and GEF-stimulated nucleotide exchange activity.

The C-terminal sequence of RhoB directs protein degradation through an endo-lysosomal pathway.

BACKGROUND: Protein degradation is essential for cell homeostasis. Targeting of proteins for degradation is often achieved by specific protein sequences or posttranslational modifications such as ubiquitination. METHODOLOGY/PRINCIPAL FINDINGS: By using biochemical and genetic tools we have monitored the localization and degradation of endogenous and chimeric proteins in live primary cells by confocal microscopy and ultra-structural analysis. Here we identify an eight amino acid sequence from the C-terminus of the short-lived GTPase RhoB that directs the rapid degradation of both RhoB and chimeric proteins bearing this sequence through a lysosomal pathway. Elucidation of the RhoB degradation pathway unveils a mechanism dependent on protein isoprenylation and palmitoylation that involves sorting of the protein into multivesicular bodies, mediated by the ESCRT machinery. Moreover, RhoB sorting is regulated by late endosome specific lipid dynamics and is altered in human genetic lipid traffic disease. CONCLUSIONS/SIGNIFICANCE: Our findings characterize a short-lived cytosolic protein that is degraded through a lysosomal pathway. In addition, we define a novel motif for protein sorting and rapid degradation, which allows controlling protein levels by means of clinically used drugs.

Phosphorylation of RhoB by CK1 impedes actin stress fiber organization and epidermal growth factor receptor stabilization.

RhoB is a small GTPase implicated in cytoskeletal organization, EGF receptor trafficking and cell transformation. It is an immediate-early gene, regulated at many levels of its biosynthetic pathway. Herein we show that the serine/threonine protein kinase CK1 phosphorylates RhoB in vitro but not RhoA or RhoC. With the use of specific CK1 inhibitors, IC261 and D4476, we show that the kinase phosphorylates also RhoB in HeLa cells. Mass spectrometry analysis demonstrates that RhoB is monophosphorylated by CK1, in its C-terminal end, on serine 185. The substitution of Ser185 by Ala dramatically inhibited the phosphorylation of RhoB in cultured cells. Lastly we show that the inhibition of CK1 activates RhoB and promotes RhoB dependent actin fiber formation and EGF-R level. Our data provide the first demonstration of RhoB phosphorylation and indicate that this post-translational maturation would be a novel critical mechanism to control the RhoB functions.

Expression of RhoB in the developing Xenopus laevis embryo.

Rho GTPases are signaling components that participate to the control of cell morphology, adhesion and motility through the regulation of F-actin cytoskeleton dynamics. In this paper, we report the identification of RhoB in Xenopus laevis (XRhoB) and its expression pattern during early development. Whole-mount in situ hybridization analysis indicated that XrhoB is expressed at high levels in the dorsal marginal zone early in gastrula and in the dorsal midline at later stages. At mid-neurula stages, XrhoB expression extends to the central nervous system, presomitic mesoderm and somites. Later during development, rhoB mRNA is detected in the eyes, the migrating neural crest cells as well as the dorso-lateral part of the somites.

Transformation of hydrogel-based inverse opal photonic sensors from FCC to L1(1) during swelling.

The structural evolution of Bragg diffracting inverse opal hydrogel sensors during swelling is directly observed by two-photon laser scanning fluorescence microscopy and compared to predictions from finite element analysis. A fluorescently labeled pH-sensitive hydrogel is UV-polymerized in a dried polystyrene colloidal crystal template, which is etched to yield an inverse opal. Fluorescence imaging of the hydrogel at different pH values reveals an inhomogeneous deformation of the FCC array of aqueous pores. The pores elongate along the sample normal direction and collapse along the sample parallel directions, consistent with the Bragg response, which indicates a 1-D increase in the interlayer distance. Interconnects between the pores serve as anchor points during hydrogel expansion into the pores. Pinning of the hydrogel to the substrate causes a change of the hydrogel lattice symmetry during deformation, from FCC (ABC stacking) to L1(1) (ABCA'B'C' stacking). Reconstructed cross-sections confirm that a 1-D increase in the interlayer distance along the substrate normal direction is responsible for the diffraction response of an inverse opal hydrogel sensor. Comparison with predictions from finite element analysis shows qualitative agreement, although the experimental mesostructure is significantly more deformed than the calculated data, due to buckling in the experimental system that is not captured by the model.

Palmitoylated cysteine 192 is required for RhoB tumor-suppressive and apoptotic activities.

RhoA and RhoB share 86% amino acid sequence identity, yet RhoA promotes whereas RhoB suppresses malignant transformation. Amino acids 29, 100, 116, 123, 129, 140-143, 141, 146, 152, 154, 155, 173, 181, 183-187, 189, 190, 191, 192, and 193 in RhoB were mutated to the corresponding RhoA residues to determine those critical for RhoB tumor-suppressive activity. Of all the mutants made, only the cysteine 192 (one of two palmitoylation sites) and cysteine 193 (the prenylation site) point mutations abolish RhoB functions. In contrast, mutation of the other palmitoylation site, cysteine 189, did not affect RhoB functions. Moving cysteine 192 to position 190 did not affect RhoB function either. Mutation of cysteine 192 to glycine, alanine, or serine blocks the ability of RhoB to suppress transforming growth factor beta type II receptor, p2lwaf, and AP-1 promoter transcriptional activities. Furthermore, mutations of cysteines 192 and 193, but not 189, mislocalize RhoB and prevent RhoB from inhibiting anchorage-dependent and anchorage-independent tumor growth and colony formation as well as prevent it from inducing apoptosis. The cysteine 192 RhoB mutant is farnesylated and geranylgeranylated as efficiently as wild type RhoB. A RhoA-(1-180)/RhoB-(181-196) chimera inhibited tumor cell proliferation and induced apoptosis as efficiently as RhoB. These results demonstrate that the presence of neither cysteine 193 nor cysteine 192 alone is sufficient and that both palmitoylated cysteine 192 and prenylated cysteine 193, but not palmitoylated cysteine 189, are required for RhoB tumor-suppressive and proapoptotic activities.

RhoB mRNA is stabilized by HuR after UV light.

RhoB is a small GTP-binding protein that is involved in apoptotic signal transduction. We have cloned the mouse RhoB mRNA including a 1377 nucleotide 3'-untranslated region (UTR) that contains six AU-rich elements (AREs) as well as several uridine-rich stretches. There is 94% homology overall between the mouse and rat RhoB genes and 92% homology between the mouse and a putative human clone. Ultraviolet light (UVL) induces RhoB production through regulated changes in gene transcription and mRNA stabilization although the latter mechanism is unknown. We observed that UVL increased the half-life of RhoB mRNA from 63 min to 3.3 h in NIH/3T3 cells and from 87 min to 2.7 h in normal human keratinocyte cells. In vitro mobility shift assays demonstrated that HuR bound the 3'-UTR of RhoB at three distinct locations (nucleotides 1342-1696, 1765-1920 and 1897-1977) suggesting a regulatory role for this RNA-binding protein. HuR immunoprecipitations were positive for RhoB mRNA indicating an in vivo association, and Western blot analysis and immunofluorescence demonstrated that HuR rapidly partitions from the nucleus to the cytoplasm after UVL. Therefore, we propose a model in which UVL induces stress-activated signal transduction leading to nuclear/cytoplasmic shuttling of HuR and subsequent stabilization of RhoB mRNA.

Mitogen-responsive expression of RhoB is regulated by RNA stability.

The small GTPase-encoding gene RhoB is strongly induced as part of the immediate early response of serum-stimulated fibroblasts. In this report, we have characterized the mechanism for growth factor responsiveness of RhoB in Rat-2 fibroblasts. By Northern blotting and ribonuclease protection, we observed low or barely detectable levels of RhoB mRNA in quiescent cells, but expression was transiently induced in response to serum stimulation, such that the mRNA peaked within 30 min and then declined over the next hour. Analysis of the rat promoter revealed cis-elements conserved with the mouse and human genes, including a pair of CEBP sites near the transcriptional start site. However, in contrast to the analysis of RNA, RhoB promoter fusions were constitutively expressed in quiescent cells in transient transfections, and were unaffected by serum. Similarly, stable RhoB promoter integrants were highly expressed in quiescent cells, and growth factor caused a slight decrease in activity. This indicates that growth factor-inducible RhoB expression cannot be mediated by transcriptional activation. We then examined decay of the RhoB mRNA and found that serum caused significant stabilization. Additionally, fusion of the 3' RhoB untranslated region (UTR) to a constitutively expressed reporter gene caused serum and growth factor as well as DNA damage-inducible expression. These observations are consistent with the view that RhoB mRNA is produced constitutively but its abundance is controlled in response to growth factors, and other signals including DNA damage, by stabilization through elements within the 3' UTR.

A novel strategy for specifically down-regulating individual Rho GTPase activity in tumor cells.

The Rho family GTPases RhoA, RhoB, and RhoC regulate the actin cytoskeleton, cell movement, and cell growth. Unlike Ras, up-regulation or overexpression of these GDP/GTP binding molecular switches, but not activating point mutations, has been associated with human cancer. Although they share over 85% sequence identity, RhoA, RhoB, and RhoC appear to play distinct roles in cell transformation and metastasis. In NIH 3T3 cells, RhoA or RhoB overexpression causes transformation whereas RhoC increases the cell migration rate. To specifically target RhoA, RhoB, or RhoC function, we have generated a set of chimeric molecules by fusing the RhoGAP domain of p190, a GTPase-activating protein that accelerates the intrinsic GTPase activity of all three Rho GTPases, with the C-terminal hypervariable sequences of RhoA, RhoB, or RhoC. The p190-Rho chimeras were active as GTPase-activating proteins toward RhoA in vitro, co-localized with the respective active Rho proteins, and specifically down-regulated Rho protein activities in cells depending on which Rho GTPase sequences were included in the chimeras. In particular, the p190-RhoA-C chimera specifically inhibited RhoA-induced transformation whereas p190-RhoC-C specifically reversed the migration phenotype induced by the active RhoC. In human mammary epithelial-RhoC breast cancer cells, p190-RhoC-C, but not p190-RhoA-C or p190-RhoB-C, reversed the anchorage-independent growth and invasion phenotypes caused by RhoC overexpression. In the highly metastatic A375-M human melanoma cells, p190-RhoC-C specifically reversed migration, and invasion phenotypes attributed to RhoC up-regulation. Thus, we have developed a novel strategy utilizing RhoGAP-Rho chimeras to specifically down-regulate individual Rho activity and demonstrate that this approach may be applied to multiple human tumor cells to reverse the growth and/or invasion phenotypes associated with disregulation of a distinct subtype of Rho GTPase.

Isoprenylation of RhoB is necessary for its degradation. A novel determinant in the complex regulation of RhoB expression by the mevalonate pathway.

Statins improve vascular functions by mechanisms independent from their cholesterol-lowering effect. Rho GTPases are emerging as key targets for the vascular effects of statins. RhoB is a short-lived, early-response inducible protein involved in receptor endocytosis, apoptosis, and gene expression. Here we show that statins regulate RhoB expression by acting at multiple levels. Simvastatin increased RhoB protein levels by 8- to 10-fold. This effect was related to a depletion of isoprenoid intermediates, as deduced from the observation that several metabolites of the cholesterol biosynthetic pathway, namely, mevalonate and geranylgeranyl-pyrophosphate, attenuated simvastatin-induced RhoB up-regulation. Moreover, prenyltransferase inhibitors mimicked simvastatin effect. Cholesterol supplementation did not prevent simvastatin-elicited up-regulation but increased RhoB levels per se. Simvastatin moderately augmented RhoB transcript levels, but markedly impaired the degradation of RhoB protein, which accumulated in the cytosol in its non-isoprenylated form. Inhibition of RhoB isoprenylation was apparently required for simvastatin-induced up-regulation, because levels of an isoprenylation-deficient RhoB mutant were not affected by simvastatin. Moreover, this mutant was found to be markedly more stable than the wild-type protein. These results show that RhoB isoprenylation is necessary for rapid turnover of this protein and identify a novel link between the cholesterol biosynthetic pathway and the regulation of G-protein expression.

RhoB, not RhoA, represses the transcription of the transforming growth factor beta type II receptor by a mechanism involving activator protein 1.

The transforming growth factor-beta (TGF-beta) type I (T beta R-I) and type II (T beta R-II) receptors are responsible for transducing TGF-beta signals. We have previously shown that inhibition of farnesyltransferase activity results in an increase in T beta R-II expression, leading to enhanced TGF-beta binding, signaling, and inhibition of tumor cell growth, suggesting that a farnesylated protein(s) exerts a repressive effect on T beta R-II expression. Likely candidates are farnesylated proteins such as Ras and RhoB, which are both farnesylated and involved in cell growth control. Neither a dominant negative Ha-Ras, constitutively activated Ha-Ras, or a pharmacological inhibitor of MEK1 affected T beta R-II transcription. However, ectopic expression of RhoB, but not the closely related family member RhoA, resulted in a 5-fold decrease of T beta R-II promoter activity. Furthermore, ectopic expression of RhoB, but not RhoA, resulted in a significant decrease of T beta R-II protein expression and resistance of tumor cells to TGF-beta-mediated cell growth inhibition. Deletion analysis of the T beta R-II promoter identified a RhoB-responsive region, and mutational analysis of this region revealed that a site for the transcription factor activator protein 1 (AP1) is critical for RhoB-mediated repression of T beta R-II transcription. Electrophoretic mobility shift assays clearly showed that the binding of AP1 to its DNA-binding site is strongly inhibited by RhoB. Consequently, transcription assays using an AP1 reporter showed that AP1-mediated transcription is down-regulated by RhoB. Altogether, these results identify a mechanism by which RhoB antagonizes TGF-beta action through transcriptional down-regulation of AP1 in T beta R-II promoter.

RhoB prenylation is driven by the three carboxyl-terminal amino acids of the protein: evidenced in vivo by an anti-farnesyl cysteine antibody.

Protein isoprenylation is a lipid posttranslational modification required for the function of many proteins that share a carboxyl-terminal CAAX motif. The X residue determines which isoprenoid will be added to the cysteine. When X is a methionine or serine, the farnesyl-transferase transfers a farnesyl, and when X is a leucine or isoleucine, the geranygeranyl-transferase I, a geranylgeranyl group. But despite its CKVL motif, RhoB was reported to be both geranylgeranylated and farnesylated. Thus, the determinants of RhoB prenylation appear more complex than initially thought. To determine the role of RhoB CAAX motif, we designed RhoB mutants with modified CAAX sequence expressed in baculovirus-infected insect cells. We demonstrated that RhoB was prenylated as a function of the three terminal amino acids, i.e., RhoB bearing the CAIM motif of lamin B or CLLL motif of Rap1A was farnesylated or geranylgeranylated, respectively. Next, we produced a specific polyclonal antibody against farnesyl cysteine methyl ester allowing prenylation analysis avoiding the metabolic labeling restrictions. We confirmed that the unique modification of the RhoB CAAX box was sufficient to direct the RhoB distinct prenylation in mammalian cells and, inversely, that a RhoA-CKVL chimera could be alternatively prenylated. Moreover, the immunoprecipitation of endogenous RhoB from cells with the anti-farnesyl cysteine antibody suggested that wild-type RhoB is farnesylated in vivo. Taken together, our results demonstrated that the three last carboxyl amino acids are the main determinants for RhoB prenylation and described an anti-farnesyl cysteine antibody as a useful tool for understanding the cellular control of protein farnesylation.