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Dimitris Kardassis - One of the best experts on this subject based on the ideXlab platform.
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Transcriptional and post-transcriptional regulation of the genes encoding the small GTPases RHOA, RhoB, and RhoC: implications for the pathogenesis of human diseases
Cellular and Molecular Life Sciences, 2018Co-Authors: Eirini Nomikou, Christos Stournaras, Melina Livitsanou, Dimitris KardassisAbstract:Rho GTPases are highly conserved proteins that play critical roles in many cellular processes including actin dynamics, vesicular trafficking, gene transcription, cell-cycle progression, and cell adhesion. The main mode of regulation of Rho GTPases is through guanine nucleotide binding (cycling between an active GTP-bound form and an inactive GDP-bound form), but transcriptional, post-transcriptional, and post-translational modes of Rho regulation have also been described. In the present review, we summarize recent progress on the mechanisms that control the expression of the three members of the Rho-like subfamily (RHOA, RhoB, and RhoC) at the level of gene transcription as well as their post-transcriptional regulation by microRNAs. We also discuss the progress made in deciphering the mechanisms of cross-talk between Rho proteins and the transforming growth factor β signaling pathway and their implications for the pathogenesis of human diseases such as cancer metastasis and fibrosis.
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Modulation of TGFβ/Smad signaling by the small GTPase RhoB
Cellular Signalling, 2018Co-Authors: Melina Livitsanou, Christos Stournaras, Eleftheria Vasilaki, Dimitris KardassisAbstract:We have shown previously that the small GTPases RHOA and RhoB play important roles in early TGF beta-induced actin cytoskeleton reorganization and that RhoB is transcriptionally activated by TGF be ...
Jae-bong Park - One of the best experts on this subject based on the ideXlab platform.
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Regulation of RHOA GTPase and various transcription factors in the RHOA pathway.
Journal of cellular physiology, 2018Co-Authors: Jae-gyu Kim, Rokibul Islam, Jung Y Cho, Hwalrim Jeong, Kim-cuong Cap, Yohan Park, Abu Jubayer Hossain, Jae-bong ParkAbstract:RHOA GTPase plays a variety of functions in regulation of cytoskeletal proteins, cellular morphology, and migration along with various proliferation and transcriptional activity in cells. RHOA activity is regulated by guanine nucleotide exchange factors (GEFs), GTPase activating proteins (GAPs), and the guanine nucleotide dissociation factor (GDI). The RHOA-RhoGDI complex exists in the cytosol and the active GTP-bound form of RHOA is located to the membrane. GDI displacement factors (GDFs) including IκB kinase γ (IKKγ) dissociate the RHOA-GDI complex, allowing activation of RHOA through GEFs. In addition, modifications of Tyr42 phosphorylation and Cys16/20 oxidation in RHOA and Tyr156 phosphorylation and oxidation of RhoGDI promote the dissociation of the RHOA-RhoGDI complex. The expression of RHOA is regulated through transcriptional factors such as c-Myc, HIF-1α/2α, Stat 6, and NF-κB along with several reported microRNAs. As the role of RHOA in regulating actin-filament formation and myosin-actin interaction has been well described, in this review we focus on the transcriptional activity of RHOA and also the regulation of RHOA message itself. Of interest, in the cytosol, activated RHOA induces transcriptional changes through filamentous actin (F-actin)-dependent ("actin switch") or-independent means. RHOA regulates the activity of several transcription regulators such as serum response factor (SRF)/MAL, AP-1, NF-κB, YAP/TAZ, β-catenin, and hypoxia inducible factor (HIF)-1α. Interestingly, RHOA also itself is localized to the nucleus by an as-yet-undiscovered mechanism.
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Regulation of RHOA GTPase and novel target proteins for ROCK.
Small GTPases, 2017Co-Authors: Eun-kyoung Choi, Jae-gyu Kim, Rokibul Islam, Jung Y Cho, Hwalrim Jeong, Yohan Park, Hee-jun Kim, Cuong Kim Cap, Jae-bong ParkAbstract:Rho GTPases play significant roles in cellular function and their activity is regulated by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), providing activation and inactivation of these GTPases, respectively. Active GTP-bound form of RHOA activates its effector proteins while the inactive GDP-bound form of RHOA exists in a RHOA-RhoGDI (guanine nucleotide dissociation inhibitor) complex in the cytosol. In particular, IκB kinase γ IKKγ/NF-κB essential modulator (NEMO) plays a role as a GDI displacement factor (GDF) for RHOA activation through binding to RHOA-RhoGDI complex. Meanwhile, prion protein inactivates RHOA despite RHOA/RhoGDI association. Novel target proteins for Rho-associated kinase (ROCK) such as glycogen synthase kinase (GSK)-3β and IKKβ are recently discovered. Here, we elaborate on a post-translationally modified version of RHOA, phosphorylated at Tyr42 and oxidized at Cys16/20. This form of RHOA dissociates from RHOA-RhoGDI complex and activates IKKβ on IKKγ/NEMO, thus providing possibly a critical role for tumourigenesis.
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Regulation of RHOA activity by the cellular prion protein.
Cell death & disease, 2017Co-Authors: Hee-jun Kim, Jae-bong Park, Hong-seok Choi, Jeong-ho Park, Mo Jong Kim, Hyoung-gon Lee, Robert B. Petersen, Yong-sun Kim, Eun-kyoung ChoiAbstract:The cellular prion protein (PrPC) is a highly conserved glycosylphosphatidylinositol (GPI)-anchored membrane protein that is involved in the signal transduction during the initial phase of neurite outgrowth. The Ras homolog gene family member A (RHOA) is a small GTPase that is known to have an essential role in regulating the development, differentiation, survival, and death of neurons in the central nervous system. Although recent studies have shown the dysregulation of RHOA in a variety of neurodegenerative diseases, the role of RHOA in prion pathogenesis remains unclear. Here, we investigated the regulation of RHOA-mediated signaling by PrPC using both in vitro and in vivo models and found that overexpression of PrPC significantly induced RHOA inactivation and RHOA phosphorylation in hippocampal neuronal cells and in the brains of transgenic mice. Using siRNA-mediated depletion of endogenous PrPC and overexpression of disease-associated mutants of PrPC, we confirmed that PrPC induced RHOA inactivation, which accompanied RHOA phosphorylation but reduced the phosphorylation levels of LIM kinase (LIMK), leading to cofilin activation. In addition, PrPC colocalized with RHOA, and the overexpression of PrPC significantly increased neurite outgrowth in nerve growth factor-treated PC12 cells through RHOA inactivation. However, the disease-associated mutants of PrPC decreased neurite outgrowth compared with wild-type PrPC. Moreover, inhibition of Rho-associated kinase (ROCK) substantially facilitated neurite outgrowth in NGF-treated PC12 cells, similar to the effect induced by PrPC. Interestingly, we found that the induction of RHOA inactivation occurred through the interaction of PrPC with RHOA and that PrPC enhanced the interaction between RHOA and p190RhoGAP (a GTPase-activating protein). These findings suggest that the interactions of PrPC with RHOA and p190RhoGAP contribute to neurite outgrowth by controlling RHOA inactivation and RHOA-mediated signaling and that disease-associated mutations of PrPC impair RHOA inactivation, which in turn leads to prion-related neurodegeneration.
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iκb kinase γ nuclear factor κb essential modulator ikkγ nemo facilitates RHOA gtpase activation which in turn activates rho associated kinase rock to phosphorylate ikkβ in response to transforming growth factor tgf β1
Journal of Biological Chemistry, 2014Co-Authors: Hee-jun Kim, Jae-gyu Kim, Miyoung Moon, Seolhye Park, Jae-bong ParkAbstract:Transforming growth factor (TGF)-β1 plays several roles in a variety of cellular functions. TGF-β1 transmits its signal through Smad transcription factor-dependent and -independent pathways. It was reported that TGF-β1 activates NF-κB and RHOA, and RHOA activates NF-κB in several kinds of cells in a Smad-independent pathway. However, the activation molecular mechanism of NF-κB by RHOA upon TGF-β1 has not been clearly elucidated. We observed that RHOA-GTP level was increased by TGF-β1 in RAW264.7 cells. RHOA-GDP and RhoGDI were bound to N- and C-terminal domains of IKKγ, respectively. Purified IKKγ facilitated GTP binding to RHOA complexed with RhoGDI. Furthermore, Dbs, a guanine nucletotide exchange factor of RHOA much more enhanced GTP binding to RHOA complexed with RhoGDI in the presence of IKKγ. Indeed, si-IKKγ abolished RHOA activation in response to TGF-β1 in cells. However, TGF-β1 stimulated the release of RHOA-GTP from IKKγ and Rho-associated kinase (ROCK), an active RHOA effector protein, directly phosphorylated IKKβ in vitro, whereas TGF-β1-activated kinase 1 activated RHOA upon TGF-β1 stimulation. Taken together, our data indicate that IKKγ facilitates RHOA activation via a guanine nucletotide exchange factor, which in turn activates ROCK to phosphorylate IKKβ, leading to NF-κB activation that induced the chemokine expression and cell migration upon TGF-β1.
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p190RhoGAP and Rap-dependent RhoGAP (ARAP3) inactivate RHOA in response to nerve growth factor leading to neurite outgrowth from PC12 cells
Experimental & Molecular Medicine, 2010Co-Authors: Chan-young Jeon, Jae-bong ParkAbstract:Rat pheochromocytoma (PC12) cells have been used to investigate neurite outgrowth. Nerve growth factor (NGF) has been well known to induce neurite outgrowth from PC12 cells. RHOA belongs to Ras-related small GTP-binding proteins, which regulate a variety of cellular processes, including cell morphology alteration, actin dynamics, and cell migration. NGF suppressed GTP-RHOA levels after 12 h in PC12 cells and was consistently required for a long time to induce neurite outgrowth. Constitutively active (CA)-RHOA suppressed neurite outgrowth from PC12 cells in response to NGF, whereas dominant-negative (DN)-RHOA stimulated it, suggesting that RHOA inactivation is essential for neurite outgrowth. Here, we investigated the mechanism of RHOA inactivation. DN-p190RhoGAP abrogated neurite outgrowth, whereas wild-type (WT)-p190RhoGAP and WT-Src synergistically stimulated it along with accelerating RHOA inactivation, suggesting that p190RhoGAP, which can be activated by Src, is a major component in inhibiting RHOA in response to NGF in PC12 cells. Contrary to RHOA, Rap1 was activated by NGF, and DN-Rap1 suppressed neurite outgrowth, suggesting that Rap1 is also essential for neurite outgrowth. RHOA was co-immunoprecipitated with Rap1, suggesting that Rap1 interacts with RHOA. Furthermore, a DN-Rap-dependent RhoGAP (ARAP3) prevented RHOA inactivation, abolishing neurite formation from PC12 cells in response to NGF. These results suggest that NGF activates Rap1, which, in turn, up-regulates ARAP3 leading to RHOA inactivation and neurite outgrowth from PC12 cells. Taken together, p190RhoGAP and ARAP3 seem to be two main factors inhibiting RHOA activity during neurite outgrowth in PC12 cells in response to NGF.
Pierre Pacaud - One of the best experts on this subject based on the ideXlab platform.
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RHOA Phosphorylation Induces Rac1 Release from Guanine Dissociation Inhibitor α and Stimulation of Vascular Smooth Muscle Cell Migration
Molecular and cellular biology, 2010Co-Authors: Malvyne Rolli-derkinderen, Gilles Toumaniantz, Pierre Pacaud, Gervaise LoirandAbstract:Although overactivation of RHOA is recognized as a common component of vascular disorders, the molecular mechanisms regulating RHOA activity in vascular smooth muscle cells (VSMC) are still unclear. We have previously shown that in VSMC, RHOA is phosphorylated on Ser188 by nitric oxide (NO)-stimulated cGMP-dependent kinase (PKG), which leads to RHOA-Rho kinase pathway inhibition. In this study, we showed that expression of phosphoresistant RHOA mutants prevented the stimulation of VSMC migration and adhesion induced by NO-PKG pathway activation. In contrast, under basal conditions, phosphomimetic RHOA mutants stimulated VSMC adhesion and migration through a signaling pathway requiring Rac1 and the Rho exchange factor Vav3. RHOA phosphorylation or phosphomimetic RHOA mutants induced Rac1 activation but did not activate Vav3. Indeed, phosphorylated RHOA or phosphomimetic mutants trapped guanine dissociation inhibitor α (GDIα), leading to the release of Rac1 and its translocation to the membrane, where it was then activated by the basal Vav3 nucleotide exchange activity. In vivo, RHOA phosphorylation induced by PKG activation in the aortas of rats treated with sildenafil induced dissociation of Rac1 from GDIα and activation of the Rac1 signaling pathway. These results suggest that the phosphorylation of RHOA represents a novel potent and physiological GDIα displacement factor that leads to Rac1 activation and regulation of Rac1-dependent VSMC functions.
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Phosphorylation of serine 188 protects RHOA from ubiquitin/proteasome-mediated degradation in vascular smooth muscle cells.
Circulation Research, 2005Co-Authors: Malvyne Rolli-derkinderen, Gervaise Loirand, Vincent Sauzeau, Laurent Boyer, Emmanuel Lemichez, Céline Baron, Daniel Henrion, Pierre PacaudAbstract:cAMP and cyclic GMP-dependent kinases (PKA and PKG) phosphorylate the small G protein RHOA on Ser188. We have previously demonstrated that phosphorylation of Ser188 inhibits RHOA-dependent functions and positively regulates RHOA expression, and that the nitric oxide (NO)/cGMP-dependent protein kinase pathway plays an essential role, both in vitro and in vivo, in the regulation of RHOA protein expression and functions in vascular smooth muscle cells. Here we analyze the consequences of Ser188 phosphorylation on RHOA protein degradation. By expressing Ser188 phosphomimetic wild-type (WT-RHOA-S188E) and active RHOA proteins (Q63L-RHOA-S188E), we show that phosphorylation of Ser188 of RHOA protects RHOA, particularly its active form, from ubiquitin-mediated proteasomal degradation. Coimmunoprecipitation experiments indicate that the resistance of the phosphorylated active form of RHOA to proteasome-mediated degradation is because of its cytoplasmic sequestration through enhanced RhoGDI interaction. In rat aortic smooth muscle cells, stimulation of PKG and inhibition of proteasome by lactacystin, induce nonadditive increases in RHOA protein expression. In addition, stimulation of PKG leads to the accumulation of GTP-bound RHOA in the cytoplasm. In vivo stimulation of the NO/PKG signaling by treating rats with sildenafil increased RHOA level and RHOA phosphorylation, and enhanced its association to RhoGDI in the pulmonary artery, whereas opposite effects are induced by chronic inhibition of NO synthesis in N-omega-nitro-L-arginine-treated rats. Our results thus suggest that Ser188 phosphorylation-mediated protection against degradation is a physiological process regulating the level of endogenous RHOA and define a novel function for RhoGDI, as an inhibitor of Rho protein degradation.
Gervaise Loirand - One of the best experts on this subject based on the ideXlab platform.
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RHOA Phosphorylation Induces Rac1 Release from Guanine Dissociation Inhibitor α and Stimulation of Vascular Smooth Muscle Cell Migration
Molecular and cellular biology, 2010Co-Authors: Malvyne Rolli-derkinderen, Gilles Toumaniantz, Pierre Pacaud, Gervaise LoirandAbstract:Although overactivation of RHOA is recognized as a common component of vascular disorders, the molecular mechanisms regulating RHOA activity in vascular smooth muscle cells (VSMC) are still unclear. We have previously shown that in VSMC, RHOA is phosphorylated on Ser188 by nitric oxide (NO)-stimulated cGMP-dependent kinase (PKG), which leads to RHOA-Rho kinase pathway inhibition. In this study, we showed that expression of phosphoresistant RHOA mutants prevented the stimulation of VSMC migration and adhesion induced by NO-PKG pathway activation. In contrast, under basal conditions, phosphomimetic RHOA mutants stimulated VSMC adhesion and migration through a signaling pathway requiring Rac1 and the Rho exchange factor Vav3. RHOA phosphorylation or phosphomimetic RHOA mutants induced Rac1 activation but did not activate Vav3. Indeed, phosphorylated RHOA or phosphomimetic mutants trapped guanine dissociation inhibitor α (GDIα), leading to the release of Rac1 and its translocation to the membrane, where it was then activated by the basal Vav3 nucleotide exchange activity. In vivo, RHOA phosphorylation induced by PKG activation in the aortas of rats treated with sildenafil induced dissociation of Rac1 from GDIα and activation of the Rac1 signaling pathway. These results suggest that the phosphorylation of RHOA represents a novel potent and physiological GDIα displacement factor that leads to Rac1 activation and regulation of Rac1-dependent VSMC functions.
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Phosphorylation of serine 188 protects RHOA from ubiquitin/proteasome-mediated degradation in vascular smooth muscle cells.
Circulation Research, 2005Co-Authors: Malvyne Rolli-derkinderen, Gervaise Loirand, Vincent Sauzeau, Laurent Boyer, Emmanuel Lemichez, Céline Baron, Daniel Henrion, Pierre PacaudAbstract:cAMP and cyclic GMP-dependent kinases (PKA and PKG) phosphorylate the small G protein RHOA on Ser188. We have previously demonstrated that phosphorylation of Ser188 inhibits RHOA-dependent functions and positively regulates RHOA expression, and that the nitric oxide (NO)/cGMP-dependent protein kinase pathway plays an essential role, both in vitro and in vivo, in the regulation of RHOA protein expression and functions in vascular smooth muscle cells. Here we analyze the consequences of Ser188 phosphorylation on RHOA protein degradation. By expressing Ser188 phosphomimetic wild-type (WT-RHOA-S188E) and active RHOA proteins (Q63L-RHOA-S188E), we show that phosphorylation of Ser188 of RHOA protects RHOA, particularly its active form, from ubiquitin-mediated proteasomal degradation. Coimmunoprecipitation experiments indicate that the resistance of the phosphorylated active form of RHOA to proteasome-mediated degradation is because of its cytoplasmic sequestration through enhanced RhoGDI interaction. In rat aortic smooth muscle cells, stimulation of PKG and inhibition of proteasome by lactacystin, induce nonadditive increases in RHOA protein expression. In addition, stimulation of PKG leads to the accumulation of GTP-bound RHOA in the cytoplasm. In vivo stimulation of the NO/PKG signaling by treating rats with sildenafil increased RHOA level and RHOA phosphorylation, and enhanced its association to RhoGDI in the pulmonary artery, whereas opposite effects are induced by chronic inhibition of NO synthesis in N-omega-nitro-L-arginine-treated rats. Our results thus suggest that Ser188 phosphorylation-mediated protection against degradation is a physiological process regulating the level of endogenous RHOA and define a novel function for RhoGDI, as an inhibitor of Rho protein degradation.
Masafumi Muratani - One of the best experts on this subject based on the ideXlab platform.
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Activation of RHOA-VAV1 signaling in angioimmunoblastic T-cell lymphoma.
Leukemia, 2017Co-Authors: Motoko Fujisawa, Mamiko Sakata-yanagimoto, Shoko Nishizawa, Daisuke Komori, P. Gershon, M. Kiryu, K. Fukumoto, Terukazu Enami, S Tanzima, Masafumi MurataniAbstract:Somatic G17V RHOA mutations were found in 50–70% of angioimmunoblastic T-cell lymphoma (AITL). The mutant RHOA lacks GTP binding capacity, suggesting defects in the classical RHOA signaling. Here, we discovered the novel function of the G17V RHOA: VAV1 was identified as a G17V RHOA-specific binding partner via high-throughput screening. We found that binding of G17V RHOA to VAV1 augmented its adaptor function through phosphorylation of 174Tyr, resulting in acceleration of T-cell receptor (TCR) signaling. Enrichment of cytokine and chemokine-related pathways was also evident by the expression of G17V RHOA. We further identified VAV1 mutations and a new translocation, VAV1–STAP2, in seven of the 85 RHOA mutation-negative samples (8.2%), whereas none of the 41 RHOA mutation-positive samples exhibited VAV1 mutations. Augmentation of 174Tyr phosphorylation was also demonstrated in VAV1–STAP2. Dasatinib, a multikinase inhibitor, efficiently blocked the accelerated VAV1 phosphorylation and the associating TCR signaling by both G17V RHOA and VAV1–STAP2 expression. Phospho-VAV1 staining was demonstrated in the clinical specimens harboring G17V RHOA and VAV1 mutations at a higher frequency than those without. Our findings indicate that the G17V RHOA–VAV1 axis may provide a new therapeutic target in AITL.
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ACTIVATION OF RHOA‐VAV1 SIGNALING IN ANGIOIMMUNOBLASTIC T‐CELL LYMPHOMA
Hematological Oncology, 2017Co-Authors: Mamiko Sakata-yanagimoto, Motoko Fujisawa, Shoko Nishizawa, Daisuke Komori, P. Gershon, M. Kiryu, T. Swarna, K. Fukumoto, Terukazu Enami, Masafumi MurataniAbstract:Somatic G17V RHOA mutations were found in 50-70% of angioimmunoblastic T-cell lymphoma (AITL). The mutant RHOA lacks GTP binding capacity, suggesting defects in the classical RHOA signaling. Here, we discovered the novel function of the G17V RHOA: VAV1 was identified as a G17V RHOA-specific binding partner via high throughput screening. We found that binding of G17V RHOA to VAV1 augmented its adaptor function through phosphorylation of 174Tyr, resulting in acceleration of T-cell receptor (TCR) signaling. Enrichment of cytokine and chemokine-related pathways was also evident by the expression of G17V RHOA. We further identified VAV1 mutations and a new translocation, VAV1-STAP2, in seven of the 85 RHOA mutation-negative samples (8.2%), while none of the 41 RHOA mutation-positive samples exhibited VAV1 mutations. Augmentation of 174Tyr phosphorylation was demonstrated also in VAV1-STAP2. Dasatinib, a multi-kinase inhibitor, efficiently blocked the accelerated VAV1 phosphorylation and the associating TCR signaling by both G17V RHOA and VAV1-STAP2 expression. Phsopho-VAV1 staining was demonstrated in the clinical specimens harboring G17V RHOA and VAV1 mutations at a higher frequency than those without. Our findings indicate that the G17V RHOA-VAV1 axis may provide a new therapeutic target in AITL.Leukemia accepted article preview online, 23 August 2017. doi:10.1038/leu.2017.273.
