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Activated Rac1, but not the tumorigenic variant Rac1b, is ubiquitinated on Lys 147 through a JNK-regulated process
The FEBS Journal
Abstract
Unlabelled:
Ubiquitination and proteasomal degradation have recently emerged as an additional level of regulation of activated forms of Rho GTPases. To characterize this novel regulatory pathway and to gain insight into its biological significance, we studied the ubiquitination of two constitutively activated forms of Rac1, i.e. the mutationally activated Rac1L61, and the tumorigenic splice variant Rac1b, which is defective for several downstream signaling pathways, including JNK activation. Whereas Rac1L61 undergoes polyubiquitination and subsequent proteasomal degradation in HEK293 cells, Rac1b is poorly ubiquitinated and appears to be much more resistant to proteasomal degradation than Rac1L61. Mutational analysis of all lysine residues in Rac1 revealed that the major target site for Rac1 ubiquitination is Lys147, a solvent-accessible residue that has a similar conformation in Rac1b. Like Rac1L61, Rac1b was found to be largely associated with plasma membrane, a known prerequisite for Rac1 ubiquitination. Interestingly, Rac1b ubiquitination could be stimulated by coexpression of Rac1L61, suggesting positive regulation of Rac1 ubiquitination by Rac1 downstream signaling. Indeed, ubiquitination of Rac1L61 is critically dependent on JNK activation.
In conclusion:
(a) Rac1b appears to be more stable than Rac1L61 with regard to the ubiquitin-proteasome system, and this may be of importance for the expression and tumorigenic capacity of Rac1b; and (b) ubiquitination of activated Rac1 occurs through a JNK-activated process, which may explain the defective ubiquitination of Rac1b. The JNK-dependent activation of Rac1 ubiquitination would create a regulatory loop allowing the cell to counteract excessive activation of Rac1 GTPase.
... Both the use of Rac1-activating stimuli (e.g., growth factors or bacterial toxins) and of 'constitutively active' mutants have facilitated studies on Rac1 signaling [18]. The constitutively active Rac1 Q61L mutant, which is incapable of GTP hydrolysis, has been used widely in fundamental studies on the cellular consequences of Rac1 signaling [19][20][21]. However, experiments with this Rac1 mutant may give an incomplete view, as GTP/GDP cycling is an important aspect of Rho GTPase regulation and signaling [22]. ...
... We next performed a ubiquitination assay for the same Rac1 mutants ( Fig 2D) and found that, as shown before [19], Rac1 Q61L ubiquitination is significantly increased (2.2-fold) compared to Rac1 WT. In addition, we observed a significant 1.4-fold increased ubiquitination of Rac1 G12V and N92I compared to WT. Rac1 R66A was less ubiquitinated than WT, namely 0.8-fold, comparable to T17N. ...
... We observed a significant increase in total ubiquitination of the myc-tagged Rac1 Q61L compared to WT as was shown previously [19] (Fig 5C). This increase was absent when lysines 16 or 147 in Rac1 Q61L were mutated (0.4-and 0.6-fold compared to Rac1 Q61L). ...
Signaling by the Rho GTPase Rac1 is key to the regulation of cytoskeletal dynamics, cell spreading and adhesion. It is widely accepted that the inactive form of Rac1 is bound by Rho GDI, which prevents Rac1 activation and Rac1-effector interactions. In addition, GDI-bound Rac1 is protected from proteasomal degradation, in line with data showing that Rac1 ubiquitination occurs exclusively when Rac1 is activated. We set out to investigate how Rac1 activity, GDI binding and ubiquitination are linked. We introduced single amino acid mutations in Rac1 which differentially altered Rac1 activity, and compared whether the level of Rac1 activity relates to Rac1 ubiquitination and GDI binding. Results show that Rac1 ubiquitination and the active Rac1 morphology is proportionally increased with Rac1 activity. Similarly, we introduced lysine-to-arginine mutations in constitutively active Rac1 to inhibit site-specific ubiquitination and analyze this effect on Rac1 signaling output and ubiquitination. These data show that the K16R mutation inhibits GTP binding, and consequently Rac1 activation, signaling and–ubiquitination, while the K147R mutation does not block Rac1 signaling, but does inhibits its ubiquitination. In both sets of mutants, no direct correlation was observed between GDI binding and Rac1 activity or -ubiquitination. Taken together, our data show that a strong, positive correlation exists between Rac1 activity and its level of ubiquitination, but also that GDI dissociation does not predispose Rac1 to ubiquitination.
... Rac1 is a signaling G protein that regulates a number of cellular events, among which, besides cytoskeletal reorganization, there are cell growth and activation of kinases. Rac1 signaling is also modulated through post-translational modifications [2][3][4][5][6][7][8][9] (Table 1). In fact, Rac1 is prenylated and subsequently palmitoylated at Cys178, and these post-translational modifications affect Rac1 localization and activity [2]. ...
... In fact, Rac1 is prenylated and subsequently palmitoylated at Cys178, and these post-translational modifications affect Rac1 localization and activity [2]. Furthermore, several studies have shown that active Rac1 bound to the plasma membrane can be ubiquitinated and subjected to proteasome-mediated degradation, leading to disassembly of epithelial cell-cell contacts and to a regulatory feedback on its activity [3,4]. The major target site for Rac1 ubiquitination is Lys147 and seems to be dependent on JNK (Jun N-terminal Kinase) activation [4], although also the tumor suppressor HACE1 has a role in the regulation of Rac1 ubiquitylation and activity [5,9]. ...
... Furthermore, several studies have shown that active Rac1 bound to the plasma membrane can be ubiquitinated and subjected to proteasome-mediated degradation, leading to disassembly of epithelial cell-cell contacts and to a regulatory feedback on its activity [3,4]. The major target site for Rac1 ubiquitination is Lys147 and seems to be dependent on JNK (Jun N-terminal Kinase) activation [4], although also the tumor suppressor HACE1 has a role in the regulation of Rac1 ubiquitylation and activity [5,9]. Among the Rac1 post-translational modifications, there is SUMOylation by a small ubiquitin-like protein (SUMO) that is covalently linked to lysine residues. ...
The small GTPases of the Rho family regulate many aspects of actin dynamics, but are functionally connected to many other cellular processes. Rac1, a member of this family, besides its known function in the regulation of actin cytoskeleton, plays a key role in the production of reactive oxygen species, in gene transcription, in DNA repair, and also has been proven to have specific roles in neurons. This review focuses on the cooperation between Rac1 and Rab proteins, analyzing how the coordination between these GTPases impact on cells and how alterations of their functions lead to disease.
... was modulated by ubiquitination and SUMOylation. The ubiquitin pathway is the most common method used by cells to regulate protein activation, localization and function 57,58 . Therefore, to identify whether ubiquitination was involved in the NS1-mediated regulation of Rac1, we co-transfected Myc-Rac1 and HA-Ub with pcD-NA3.0-NS1 or pcDNA3.0 in 293T cells. ...
... The C-terminus of Rac1 is crucial for binding to several regulatory proteins and the proper localization of Rac1 in the plasma membrane, and the C-terminal 21 aa of Rac1 were confirmed as the nuclear localization sequence (NLS) 58 . To confirm the location of the ubiquitin reaction in the cell, we constructed a mutant plasmid with a deletion of the C-terminal 21aa of Rac1 (called Rac1 Δ WT). ...
... Our experimental results showed that the Rac1 protein can be modified by Lys63-linked ubiquitination and SUMO1-conjugated SUMOylation. Lysine 147 of Rac1 was most likely the main acceptor site for ubiquitin, which was previously reported by Orane Visvikis 58 ; however, we demonstrated that this site of the Rac1 protein was primarily responsible for K63-linked ubiquitination during influenza virus infection. In addition to this finding, the lysine 184 and 186 sites also contributed to ubiquitination at a certain extent. ...
Influenza A virus (IAV) is a major human pathogen with the potential to become pandemic. IAV contains only eight RNA segments; thus, the virus must fully exploit the host cellular machinery to facilitate its own replication. In an effort to comprehensively characterize the host machinery taken over by IAV in mammalian cells, we generated stable A549 cell lines with over-expression of the viral non-structural protein (NS1) to investigate the potential host factors that might be modulated by the NS1 protein. We found that the viral NS1 protein directly interacted with cellular Rac1 and facilitated viral replication. Further research revealed that NS1 down-regulated Rac1 activity via post-translational modifications. Therefore, our results demonstrated that IAV blocked Rac1-mediated host cell signal transduction through the NS1 protein to facilitate its own replication. Our findings provide a novel insight into the mechanism of IAV replication and indicate new avenues for the development of potential therapeutic targets.
... When GTP-bound, it interacts with various molecules that elicit downstream responses. 1 Multiple mechanisms control Rac1 activity including nucleotide binding and hydrolysis catalysed by guanine nucleotide exchange factors and GTPase activating proteins (GAPs), regulation of subcellular localization, modulation of protein levels and post-translational modifications such as prenylation 2 as well as ubiquitin-like (Ubl)-type modifications, including ubiquitylation [3][4][5] and SUMOylation. 6 Rac1 degradation by the ubiquitin-proteasome system occurs early during hepatocyte growth factor (HGF)-induced cell scattering 4 and in response to cytotoxic necrotizing factor-1 (CNF-1), 7 both of which activate Rac1. ...
... Rac1 ubiquitylation at Lys147 is also stimulated by downstream signalling through JNK, creating a negative feedback loop to terminate Rac1 signalling. 3 More recently, it was shown that ubiquitylation and subsequent degradation of active Rac1 is regulated by caveolin1. 5 Together, these studies imply a mechanism for terminating Rac1 signalling downstream of multiple stimuli. ...
... HACE1 overexpression promoted degradation of Rac1 selectively in the membrane fraction ( Figure 2f) consistent with the observation that a Rac1 mutant that cannot go to the membrane is not ubiquitylated. 3 Live-cell imaging revealed co-localization of Rac1 and HACE1 at membrane ruffles in MDCKII cells treated with HGF ( Figure 2g) despite the majority of HACE1 being cytoplasmic as previously described. 9 These results demonstrate that HACE1 is required for the poly-ubiquitylation and degradation of active Rac1 associated with membranes in response to HGF. ...
The small GTPase Rac1 is a key regulator of cell motility. Multiple mechanisms regulate Rac1 activity including its ubiquitylation and subsequent degradation. Here, we identify the tumour suppressor HACE1 (HECT domain and Ankyrin repeat Containing E3 ubiquitin-protein ligase 1) as an E3 ubiquitin ligase responsible for Rac1 degradation following activation by a migration stimulus. We show that HACE1 and Rac1 interaction is enhanced by hepatocyte growth factor (HGF) signalling, a Rac activator and potent stimulus of cell migration. Furthermore, HACE1 catalyses the poly-ubiquitylation of Rac1 at lysine 147 following its activation by HGF, resulting in its proteasomal degradation. This negative feedback mechanism likely restricts cell motility. Consistent with this, HACE1 depletion is accompanied by increased total Rac1 levels and accumulation of Rac1 in membrane ruffles. Moreover, HACE1-depletion enhances cell migration independently of growth factor stimulation, which may have significance for malignant conversion. A non-ubiquitylatable Rac1 rescues the migration defect of Rac1-null cells to a greater extent than wild-type Rac1. These findings identify HACE1 as an antagonist of cell migration through its ability to degrade active Rac1.Oncogene advance online publication, 21 May 2012; doi:10.1038/onc.2012.189.
... One possibility is that the RAC1B protein was stabilized so that its levels increased. Recent data have provided evidence that RAC1 can be proteolytically degraded following its ubiquitinylation on lysine 147 [90][91][92]; however, RAC1B was found to be a poor substrate for this modification [90]. The ubiquitinylation of RAC1 was proposed to occur in a regulatory loop to counteract excessive activation of RAC1 following the activation of protein kinase JNK by GTP-loaded RAC1, which RAC1B cannot activate. ...
... One possibility is that the RAC1B protein was stabilized so that its levels increased. Recent data have provided evidence that RAC1 can be proteolytically degraded following its ubiquitinylation on lysine 147 [90][91][92]; however, RAC1B was found to be a poor substrate for this modification [90]. The ubiquitinylation of RAC1 was proposed to occur in a regulatory loop to counteract excessive activation of RAC1 following the activation of protein kinase JNK by GTP-loaded RAC1, which RAC1B cannot activate. ...
Simple Summary
Tumours are now known to develop more quickly when the tumour cell mass is located in a tissue that shows signs of chronic inflammation. Under such conditions, inflammatory cells from the surrounding tumour microenvironment provide survival signals to which cancer cells respond. We have previously found that some colorectal tumours overexpress the protein RAC1B that sustains tumour cell survival. Here we used a colon mucosa-like in vitro cell model and found that the presence of cancer-associated fibroblasts and pro-inflammatory macrophages stimulated colorectal cells to increase their RAC1B levels. Under these conditions, the secreted survival signals were analysed, and interleukin-6 identified as the main trigger for the increase in RAC1B levels. The results contribute to understand the tumour-promoting effect of inflammation at the molecular level.
Abstract
An inflammatory microenvironment is a tumour-promoting condition that provides survival signals to which cancer cells respond with gene expression changes. One example is the alternative splicing variant Rat Sarcoma Viral Oncogene Homolog (Ras)-Related C3 Botulinum Toxin Substrate 1 (RAC1)B, which we previously identified in a subset of V-Raf Murine Sarcoma Viral Oncogene Homolog B (BRAF)-mutated colorectal tumours. RAC1B was also increased in samples from inflammatory bowel disease patients or in an acute colitis mouse model. Here, we used an epithelial-like layer of polarized Caco-2 or T84 colorectal cancer (CRC) cells in co-culture with fibroblasts, monocytes or macrophages and analysed the effect on RAC1B expression in the CRC cells by RT-PCR, Western blot and confocal fluorescence microscopy. We found that the presence of cancer-associated fibroblasts and M1 macrophages induced the most significant increase in RAC1B levels in the polarized CRC cells, accompanied by a progressive loss of epithelial organization. Under these conditions, we identified interleukin (IL)-6 as the main trigger for the increase in RAC1B levels, associated with Signal Transducer and Activator of Transcription (STAT)3 activation. IL-6 neutralization by a specific antibody abrogated both RAC1B overexpression and STAT3 phosphorylation in polarized CRC cells. Our data identify that pro-inflammatory extracellular signals from stromal cells can trigger the overexpression of tumour-related RAC1B in polarized CRC cells. The results will help to understand the tumour-promoting effect of inflammation and identify novel therapeutic strategies.
... The defective ubiquitination of RAC1B may thus be explained by RAC1B's inability to activate JNK (see section 6.1.). Interestingly, RAC1B ubiquitination could be stimulated by coexpression of RAC1-Q61L [16], suggesting positive regulation of RAC1B ubiquitination by RAC1 downstream signaling. ...
... Although the mechanistic basis for this is still elusive, it indicates that RAC1B can act to suppress expression of RAC1. A reverse interaction which impacted protein stability has been observed in that degradation of RAC1B via ubiquitination is enhanced upon coexpression of the constitutively active RAC1-Q61L mutant [16] (see section 4.3.). Mutual negative regulation of RAC1 and RAC1B may be involved in one or more of the cellular processes described in sections 5.4.-5.10. ...
RAC1B is an alternatively spliced isoform of the monomeric GTPase RAC1. It differs from RAC1 by a 19 amino acid in frame insertion, termed exon 3b, resulting in an accelerated GDP/GTP-exchange and an impaired GTP-hydrolysis. Although RAC1B has been ascribed several protumorigenic functions such as cell cycle progression and apoptosis resistance, its role in malignant transformation, and other functions driving tumor progression like epithelial-mesenchymal transition, migration/invasion and metastasis are less clear. Insertion of exon 3b endows RAC1B with specific biochemical properties that, when compared to RAC1, encompass both loss-of-functions and gain-of-functions with respect to the type of upstream activators, downstream targets, and binding partners. In its extreme, this may result in RAC1B and RAC1 acting in an antagonistic fashion in regulating a specific cellular response with RAC1B behaving as an endogenous inhibitor of RAC1. In this review, we strive to provide the reader with a comprehensive overview, rather than critical discussions, on various aspects of RAC1B biology in eukaryotic cells.
... However, ubiquitination of RhoU had not been previously reported. We now find that RhoU can form RhoU-ubiquitin (Ub) conjugates (Fig. 3 E; HA-RhoU(Ub)n, visualized as multiple HA-positive bands that increase in size as RhoU-Ub levels are increased, rendering a gel smear; Rolli-Derkinderen et al., 2005;Visvikis et al., 2008). This shift in molecular weight is entirely consistent with increased ubiquitination (Rolli-Derkinderen et al., 2005;Visvikis et al., 2008). ...
... We now find that RhoU can form RhoU-ubiquitin (Ub) conjugates (Fig. 3 E; HA-RhoU(Ub)n, visualized as multiple HA-positive bands that increase in size as RhoU-Ub levels are increased, rendering a gel smear; Rolli-Derkinderen et al., 2005;Visvikis et al., 2008). This shift in molecular weight is entirely consistent with increased ubiquitination (Rolli-Derkinderen et al., 2005;Visvikis et al., 2008). The linkage specificity of ubiquitination can determine the stability of the target protein, where K48-linked ubiquitination targets substrates for proteasomal degradation, whereas K63-linked ubiquitination serves as a regulatory signal (Weissman, 2001). ...
P21-activated kinase 4 (PAK4) is a Cdc42 effector protein thought to regulate cell adhesion disassembly in a kinase-dependent manner. We found that PAK4 expression is significantly higher in high-grade human breast cancer patient samples, whereas depletion of PAK4 modifies cell adhesion dynamics of breast cancer cells. Surprisingly, systematic analysis of PAK4 functionality revealed that PAK4-driven adhesion turnover is neither dependent on Cdc42 binding nor kinase activity. Rather, reduced expression of PAK4 leads to a concomitant loss of RhoU expression. We report that RhoU is targeted for ubiquitination by the Rab40A-Cullin 5 complex and demonstrate that PAK4 protects RhoU from ubiquitination in a kinase-independent manner. Overexpression of RhoU rescues the PAK4 depletion phenotype, whereas loss of RhoU expression reduces cell adhesion turnover and migration. These data support a new kinase-independent mechanism for PAK4 function, where an important role of PAK4 in cellular adhesions is to stabilize RhoU protein levels. Thus, PAK4 and RhoU cooperate to drive adhesion turnover and promote cell migration.
... Monoubiquitination is a dynamic and reversible modification that can orchestrate cellular events including DNA repair, gene expression, endocytosis, and nuclear export 10 . Emerging evidence suggests that monoubiquitination regulates large and small GTPases, including Ras [11][12][13][14] . Monoubiquitination of K-Ras at position 147 has been shown to promote tumorigenesis 15 ; mutation of oncogenic K-Ras to prevent monoubiquitination (Ras K147L ) impaired its ability to promote tumor growth when ectopically expressed in NIH 3T3 mouse fibroblasts. ...
... Monoubiquitination disrupts interactions of Rap2A with effector proteins and inhibits the ability of Rap2A to promote dendrite development 12 . Monoubiquitination has also been observed in Rac1, although the biological consequence of this modification is not yet known 13,14 . Our chemical ligation strategy and multidimensional approach will be useful for the study of these targets, particularly in cases where the relevant ubiquitin ligase has not been identified. ...
Cell growth and differentiation are controlled by growth factor receptors coupled to the GTPase Ras. Oncogenic mutations disrupt GTPase activity, leading to persistent Ras signaling and cancer progression. Recent evidence indicates that monoubiquitination of Ras leads to Ras activation. Mutation of the primary site of monoubiquitination impairs the ability of activated K-Ras (one of the three mammalian isoforms of Ras) to promote tumor growth. To determine the mechanism of human Ras activation, we chemically ubiquitinated the protein and analyzed its function by NMR, computational modeling and biochemical activity measurements. We established that monoubiquitination has little effect on the binding of Ras to guanine nucleotide, GTP hydrolysis or exchange-factor activation but severely abrogates the response to GTPase-activating proteins in a site-specific manner. These findings reveal a new mechanism by which Ras can trigger persistent signaling in the absence of receptor activation or an oncogenic mutation.
... For instance, Rac1 can also be poly-ubiquitinated leading to its degradation by the proteasome, but a splice variant of Rac1 termed Rac1b, is not ubiquitinated and therefore is more stable. 72 As Rac1b is constitutively active and is often highly expressed in colorectal and breast tumors, this increased stability may facilitate increased tumorigenicity. However, the ubiquitination seems to specifically target active GTP-bound Rac1, 72,73 suggesting that in polar regions of the cell, containing pools of active and inactive Rac1, active Rac1 would be preferentially ubiquitin-conjugated and thus reduced motility would ensue. ...
... 72 As Rac1b is constitutively active and is often highly expressed in colorectal and breast tumors, this increased stability may facilitate increased tumorigenicity. However, the ubiquitination seems to specifically target active GTP-bound Rac1, 72,73 suggesting that in polar regions of the cell, containing pools of active and inactive Rac1, active Rac1 would be preferentially ubiquitin-conjugated and thus reduced motility would ensue. Interestingly, although ubiquitination of Rac1 results in its degradation and would therefore decrease migration, sumoylation of Rac1 may be necessary for maintenance of Rac1 activation following HGF stimulation and the subsequent formation of membrane ruffles. ...
The regulation of the small GTPases leading to their membrane localization has long been attributed to processing of their C-terminal CAAX box. As deregulation of many of these GTPases have been implicated in cancer and other disorders, prenylation and methylation of this CAAX box has been studied in depth as a possibility for drug targeting, but unfortunately, to date no drug has proved clinically beneficial. However, these GTPases also undergo other modifications that may be important for their regulation. Ubiquitination has long been demonstrated to regulate the fate of numerous cellular proteins and recently it has become apparent that many GTPases, along with their GAPs, GeFs and GDis, undergo ubiquitination leading to a variety of fates such as re-localization or degradation. in this review we focus on the recent literature demonstrating that the regulation of small GTPases by ubiquitination, either directly or indirectly, plays a considerable role in controlling their function and that targeting these modifications could be important for disease treatment.
... The ubiquitination of RAC1 at its major ubiquitination site K147, which is the Ub-site regulated in this study, is dependent on the activity of c-Jun N-terminal kinases (MAPK8, MAPK9, MAPK10) (73). The kinase activity of MAPK8 and MAPK9 was markedly increased by BaP-treatment and double-stimulation with LPS and AhR-ligand. ...
Emerging studies revealed that the Aryl hydrocarbon receptor (AhR), a receptor sensing environmental contaminants, is executing an immunomodulatory function. However, it is an open question to which extent this is achieved by its role as a transcription factor or via non-genomic signaling. We utilized a multi-post-translational modification-omics approach to examine non-genomic AhR-signaling after activation with endogenous (FICZ) or exogenous (BaP) ligand in endotoxin-activated (LPS) monocyte-derived macrophages. While AhR activation affected abundances of few proteins, regulation of ubiquitination and phosphorylation were highly pronounced. Although the number and strength of effects depended on the applied AhR-ligand, both ligands increased ubiquitination of Rac1, which participates in PI3K/AKT-pathway-dependent macrophage activation, resulting in a pro-inflammatory phenotype. In contrast, co-treatment with ligand and LPS revealed a decreased AKT activity mediating an anti-inflammatory effect. Thus, our data show an immunomodulatory effect of AhR activation through a Rac1ubiquitination-dependent mechanism that attenuated AKT-signaling, resulting in a mitigated inflammatory response.
... However, earlier studies emphasize that Rac1b did not activate p21 protein kinase nor its downstream protein kinase JNK (Matos et al., 2003;Esufali et al., 2007). An interesting difference between Rac1 and Rac1b, not analyzed in this study, has been described regarding their sensitivity toward ubiquitinylation which is involved in regulation of protein stability (Visvikis et al., 2008). Lys147 of Rac1 has been identified to be modified by the ubiquitin E3 ligases HACE1, whereas Rac1b was poorly ubiquitinated and more resistant against proteasomal degradation. ...
Rac1 is a ubiquitously expressed Rho GTPase and an important regulator of the actin cy-toskeleton. Its splice variant Rac1b exhibits a 19aa in-frame insertion and is predominantly active. Both proteins were described in tumorigenesis or metastasis. We investigated the contribution of Rac1 and Rac1b to tumor progression of human non-small-cell-lungadenocarcinoma (NSCLA). Rac1 protein was present in 8/8 NSCLA cell lines analyzed, whereas Rac1b was expressed in only 6/8. In wound healing assays, EGFP-Rac1 slightly de-creased cell migration, whereas proliferation was increased in both, Rac1- and Rac1bexpressing cells. In the in vivo chorioallantoic invasion model, EGFP-Rac1-expressing cells formed more invasive tumors compared to EGFP-Rac1b. This increased invasiveness correlated with enhanced phosphorylation of p38α, AKT, and GSK3β and activation of serum response- and Smad-dependent gene promoters by Rac1. In contrast, Rac1b solely activated the MAP kinase JNK2, together with TCF/LEF1- and NFκB-responsive gene reporters. Rac1b, as Rac1 phosphorylated p38α, AKT, and GSK3β. Knockdown of the splicing factor ESRP1, which mediates out-splicing of exon 3b from RAC1, resulted in increased Rac1b mRNA and suppression of the EMT-associated transcription factor ZEB1. Our data demonstrate different signaling and functional activities of Rac1 and Rac1b and an important role for Rac1 in lung cancer metastasis.
... Inhibition of Rac1 degradation increases ROS production and disrupts the endothelial barrier; this occurs through various mechanisms, including disruption of the plasma and mitochondrial membrane through membrane lipid peroxidation, which reduces ATP generation and decreases metabolism and cell survival (Daugaard et al., 2013;Farber, 1994;Kovacic et al., 2001;van Wetering et al., 2002). To inhibit ROS production, the HECT E3 ubiquitin ligase HACE1 targets active Rac1 for degradation by ubiquitylation at K147 (Daugaard et al., 2013;Mettouchi and Lemichez, 2012;Torrino et al., 2011;Visvikis et al., 2008). In contrast, Rac1-mediated ROS production is increased by the E3 ligase TRAF6. ...
Endothelial cell-cell contacts are essential for vascular integrity and physiology, protecting tissues and organs from edema and uncontrolled invasion of inflammatory cells. The vascular endothelial barrier is dynamic, but its integrity is preserved through a tight control at different levels. Inflammatory cytokines and G-protein-coupled receptor agonists, such as histamine, reduce endothelial integrity and increase vascular leakage. This is due to elevated myosin-based contractility, in conjunction with phosphorylation of proteins at cell-cell contacts. Conversely, reducing contractility stabilizes or even increases endothelial junctional integrity. Rho GTPases are key regulators of such cytoskeletal dynamics and endothelial cell-cell contacts. In addition to signaling-induced regulation, the expression of junctional proteins, such as occludin, claudins and vascular endothelial cadherin, also controls endothelial barrier function. There is increasing evidence that, in addition to protein phosphorylation, ubiquitylation (also known as ubiquitination) is an important and dynamic post-translational modification that regulates Rho GTPases, junctional proteins and, consequently, endothelial barrier function. In this Review, we discuss the emerging role of ubiquitylation and deubiquitylation events in endothelial integrity and inflammation. The picture that emerges is one of increasing complexity, which is both fascinating and promising given the clinical relevance of vascular integrity in the control of inflammation, and of tissue and organ damage.
... The monoubiquitylation of ICAP-1 prevents binding to β1 integrin and regulates β1 integrin-dependent adhesion According to structure predictions and crystallographic data , the monoubiquitylation site is located in the β1 integrin-binding domain of ICAP-1 facing the isoleucine residue important for the binding to β1 integrin ( Fig. 2A). As this monoubiquitylation could interfere with the interaction between ICAP-1 and β1 integrin, we used two classical methods to produce an ubiquitylated form of a protein (Torrino et al., 2011;Visvikis et al., 2008), first by co-transfecting ICAP-1 with His-tagged ubiquitin and second by creating a chimera made of ubiquitin fused to the C-terminal tail of ICAP-1 (ICAP-1-Ubi) (Fig. 2B). We tested the ability of WT, non-ubiquitylatable (K158R) and monoubiquitylated ICAP-1 (endogenous ubiquitylation or chimera) to interact with the cytoplasmic domain of either β1 integrin or β3 integrin fused with GST or with GST alone by pulldown assay (Fig. S2A) or by ELISA assay (Fig. 2C). ...
ICAP-1 is involved in integrin dynamics and force generation by controlling integrin endocytosis through nm23-dependent scission of endocytic chlatrin coated pits.ICAP-1 has been identified as a specific partner of b1 integrin (Degani et al., 2002; Zhang and Hemler, 1999). We have previously shown that ICAP-1 is involved in cell mechanoresponse and cell differentiation in a b1 integrin dependent manner (Bouvard et al., 2007; Brunner et al., 2011; Faurobert et al., 2013; Millon-Frémillon et al., 2008; Renz et al., 2015). However, as ICAP-1 is also able to adapt cell migration in response to substrate stiffness in a β1-integrin-independent manner (Bouin et al., 2017), we speculated on a more general role of ICAP-1 in cell adhesion and focal adhesion dynamics. For this purpose we have created cellular environment where b1 integrin and/or ICAP-1 were absent by using four cell lines: WT osteoblast, b1 integrin KO osteoblast cells, ICAP-1 KO osteoblast cells and double KO b1/ICAP-1 osteoblast cells in order to monitor b3 integrin behavior. As expected, depletion of b1 integrin is associated with the loss of cell spreading and force generation according traction force microscopy measurement. Surprisingly, the supplementary deletion of ICAP-1 (b1 integrin and ICAP-1 KO) leads to restoration of cell spreading and force generation which are dependent on b3 integrin. These b3 integrin-mediated forces are correlated with slow diffusion of b3 integrin within adhesion sites and slow turnover of b3 integrin containing focal adhesion (FRAP/TIRF/videomicroscopy). We addressed the question whether ICAP-1 might regulate b3 integrin endocytosis since ICAP-1 interacts with nm23-H2 (Fournier et al., 2002), a nucleoside diphosphate kinases (NDPKs) involved in dynamin-mediated endocytosis by producing GTP through adenosine triphosphate (ATP)–driven conversion of guanosine diphosphate (GDP) (Boissan et al., 2014). We show that the deletion of either nm23 or dynamin or chlatrin in cells depleted in b1 integrin is able to mimic the combined loss of b1 integrin and ICAP-1 by restoring cell spreading, force generation and b3 integrin dynamics. To confirm the involvement of ICAP-1 in b3 integrin endocytosis, we show that the b3 integrin antibody uptake is efficiently blocked in cells depleted in ICAP-1. Our results suggest that ICAP-1 might be involved in integrin dynamics and force generation by controlling integrin endocytosis through nm23-dependent scission of endocytic chlatrin coated pits.
... Ces résultats sont en accord avec ceux de l'équipe du Dr. ) entraîne un changement conformationnel du CNF1 et l'ancrage du domaine médian du CNF1 dans la bicouche lipidique de la vésicule endosomale(4). Le domaine catalytique est ensuite relargué au niveau du cytosol (5) exerçant son activité déamidase sur les RhoGTPases (6). 1) Contrôle de Rac1 par ubiquitylation et dégradation par le protéasome L'ubiquitylation de Rac1 a été initialement décrite en réponse au CNF1 et a été par la suite étendue à une réponse de l'activation de Rac1 par des GEFs (11) Rac1 est ubiquitylée au niveau de la lysine 147(23). Bien que Rac1 interagisse avec plusieurs ubiquitine ligases, telles que POSH, Cbl ou le complexe SCF, aucune étude n'a montré que Rac1 est une cible de ces enzymes(12). ...
Nous cherchons à comprendre le mode de fonctionnement d’HACE1, un suppresseur de tumeur majeur, dont l’expression est altérée dans 50% des tumeurs humaines. Notre équipe a démontré que cette ubiquitine-ligase cible et provoque la dégradation de Rac1 au niveau du protéasome. Rac1 est une protéine oncogénique promouvant la croissance tumorale. En ciblant Rac1, HACE1 permet de restreindre le stress oxydatif des cellules, diminuant ainsi leurs dommages à l’ADN. Pour comprendre les voies de signalisation cellulaires ciblées par HACE1, nous avons ensuite recherché des protéines interagissant avec elle. Mes travaux révèlent que l'optineurine (OPTN), une protéine jusqu’alors connue dans des désordres neuro-dégénératifs, forme un complexe avec HACE1. Nous avons montré que ce complexe régule transcriptionnellement et traductionnellement la Cycline-D1. Nous avons également montré que l’OPTN se localise dans les points d'ancrage des cellules à la matrice extracellulaire (MEC) et régule leur formation. En réponse à l’élasticité de la MEC, nous avons montré que le complexe HACE1-OPTN réprime, au travers du métabolisme, la division cellulaire. Une étude que nous avons effectuée sur des données cliniques indiquerait l'importance d’une perte d’expression d’OPTN dans le cancer du sein, associée à des dérégulations des tensions de la MEC tumorale
... [1][2][3] Additionally, Rac1 signaling is also modulated through post-translational modifications that dictate its activation status, abundance and localization. [20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36] It has also become apparent that several factors influence Rac1 downstream signaling, via coupling active Rac1 to specific downstream effectors, thereby selectively activating certain Rac1-driven functions. 3,31,33,[37][38][39][40][41][42][43][44][45][46] Given the diverse modes involved in Rac1 regulation, studies focused on deciphering the underlying mechanisms implicated in governing Rac1 signaling promise to provide insight into novel therapeutic avenues for effective Rac1 targeting. ...
Abnormal Rac1 signaling is linked to a number of debilitating human diseases, including, cancer, cardiovascular diseases and neurodegenerative disorders. As such, Rac1 represents an attractive therapeutic target, yet the search for effective Rac1 inhibitors is still underway. Given the adverse effects associated with Rac1 signaling perturbation, cells have evolved several mechanisms to ensure the tight regulation of Rac1 signaling. Thus, characterizing these mechanisms can provide invaluable information regarding major cellular events that lead to aberrant Rac1 signaling. Importantly, this information can be utilized to further facilitate the development of effective pharmacological modulators that can restore normal Rac1 signaling. In this review, we focus on the pathological role of Rac1 signaling, highlighting the benefits and potential drawbacks of targeting Rac1 in a clinical setting. Additionally, we provide an overview of available compounds that target key Rac1 regulatory mechanisms and discuss future therapeutic avenues arising from our understanding of these mechanisms.
... Based on our immunoprecipitation results in rat cells, Rac1b expressed at even very low levels in normal colon mucosa epithelia is capable of moderating the AKT2/MCL1 pathway. We postulated that a low level of Rac1b exerts important function in normal cells, tissues or organs, due to Rac1b's constitutive activity and resistance to degradation [58]. However, the functional consequences of knocking out Rac1 gene exon-3b, especially changes in the incidences of tumors and inflammatory diseases, such as Crohn's disease, still need to be evaluated carefully in Rac1b -/rats. ...
Rac1b is a constitutively activated, alternatively spliced form of the small GTPase Rac1. Previous studies showed that Rac1b promotes cell proliferation and inhibits apoptosis. In the present study, we used microarray analysis to detect genes differentially expressed in HEK293T cells and SW480 human colon cancer cells stably overexpressing Rac1b. We found that the pro-proliferation genes JNK2, c-JUN and cyclin-D1 as well as anti-apoptotic AKT2 and MCL1 were all upregulated in both lines. Rac1b promoted cell proliferation and inhibited apoptosis by activating the JNK2/c-JUN/cyclin-D1 and AKT2/MCL1 pathways, respectively. Very low Rac1b levels were detected in the colonic epithelium of wild-type Sprague-Dawley rats. Knockout of the rat Rac1 gene exon-3b or knockdown of endogenous Rac1b in HT29 human colon cancer cells downregulated only the AKT2/MCL1 pathway. Our study revealed that very low levels of endogenous Rac1b inhibit apoptosis, while Rac1b upregulation both promotes cell proliferation and inhibits apoptosis. It is likely the AKT2/MCL1 pathway is more sensitive to Rac1b regulation.
... These kinases are known to be essential for stress responses such as ultraviolet, heat, and hypotonicity, cell cycle regulation, differentiation, growth and so on [22][23][24][25][26]. It is reported that JNK directly phosphorylates some transcri tion factors, and regulates p stability of the target transcription factors through ubiquitin-proteasome system [32,33]. Furthermore, DNA binding activities of Jun and c-Myc transcription factors are controlled via phosphorylation process by CKII [34,35]. ...
... In the Absence of Coro1C, Caveolin Targets Rac1 toward Proteasomal Degradation-It has been reported that Rac1 is subject to proteasomal degradation (24,25) and also that caveolar endocytosis of Rac1 leads to degradation (13). It is noticeable that, when Coro1C knockdown cells were stimulated with H/0, Rac1 moved from pellet to soluble fractions at 30 min but disappeared from the soluble fraction at 60 min without returning to the pellet (Fig. 1C). ...
Sustained, directional fibroblast migration requires both polarised activation of the protrusive signal, Rac1, and redistribution of inactive Rac1 from the rear of the cell so that it can be redistributed or degraded. In this manuscript, we determine how alternative endocytic mechanisms dictate the fate of Rac1 in response to the extracellular matrix environment. We discover that both coronin-1C and caveolin retrieve Rac1 from similar locations at the rear and sides of the cell. We find that coronin-1C-mediated extraction, which is responsible for Rac1 recycling, is a constitutive process that maintains Rac1 protein levels within the cell. In the absence of coronin-1C, the effect of caveolin-mediated endocytosis, which targets Rac1 for proteosomal degradation, becomes apparent. Unlike constitutive, coronin-1C-mediated trafficking, caveolin-mediated Rac1 endocytosis is induced by engagement of the fibronectin receptor, syndecan-4. Such an inducible endocytic/degradation mechanism would predict that, in the presence of fibronectin, caveolin defines regions of the cell that are resistant to Rac1 activation, but in the absence of fibronectin leaves more of the membrane susceptible to Rac1 activation and protrusion. Indeed, we demonstrate that fibronectin-stimulated activation of Rac1 is accelerated in the absence of caveolin, and that when caveolin is knocked down, polarisation of active Rac1 is lost in FRET experiments and culminates in shunting migration in a fibrous fibronectin matrix. While the concept of polarised Rac1 activity in response to chemoattractants has always been apparent, our understanding of the balance between recycling and degradation explains how polarity can be maintained once the chemotactic gradient has faded.
Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
... Owing to the slow intrinsic rates of nucleotide exchange and hydrolysis, temporal regulation of Rac1 activity requires modulatory factors, such as guanine nucleotide exchange factors (GEFs) 2 , which facilitate exchange of GDP for GTP; GTPase-activating proteins (GAPs), which catalyze GTP hydrolysis; and guanine nucleotide dissociation inhibitors (GDIs), which prevent GDP dissociation and sequester Rac1 away from cell membranes [2,3]. In addition, Rac1 is spatially and temporally regulated by various posttranslational modifications, including Cterminal lipidation [4][5][6], phosphorylation [7], ubiquitination [8,9], and SUMOylation [10]. Rac1 is an essential protein [11] that plays a critical role in regulating multiple cellular processes. ...
The Rac1 GTPase is an essential and ubiquitous protein that signals through numerous pathways to control critical cellular processes, including cell growth, morphology, and motility. Rac1 deletion is embryonic lethal, and its dysregulation or mutation can promote cancer, arthritis, cardiovascular disease, and neurological disorders. Rac1 activity is highly regulated by modulatory proteins and post-translational modifications. While much attention has been devoted to guanine nucleotide exchange factors that act on Rac1 to promote GTP loading and Rac1 activation, cellular oxidants may also regulate Rac1 activation by promoting guanine nucleotide exchange. Herein, we show that Rac1 contains a redox-sensitive cysteine (Cys(18)) that can be selectively oxidized at physiological pH due to its lowered pKa. Consistent with these observations, we show that Rac1 is glutathiolated in primary chondrocytes. Oxidation of Cys(18) by glutathione greatly perturbs Rac1 guanine nucleotide binding and promotes nucleotide exchange. As aspartate substitutions have been previously used to mimic cysteine oxidation, we characterized the biochemical properties of Rac1(C18D). We also evaluated Rac1(C18S) as a redox-insensitive variant and find that it retains structural and biochemical properties similar to Rac1(WT) but is resistant to thiol oxidation. In addition, Rac1(C18D), but not Rac1(C18S), shows greatly enhanced nucleotide exchange, similar to that observed for Rac1 oxidation by glutathione. We employed Rac1(C18D) in cell-based studies to assess whether this fast-cycling variant, which mimics Rac1 oxidation by glutathione, affects Rac1 activity and function. Expression of Rac1(C18D) in Swiss 3T3 cells showed greatly enhanced GTP-bound Rac1 relative to Rac1(WT) and the redox-insensitive Rac1(C18S) variant. Moreover, expression of Rac1(C18D) in HEK-293T cells greatly promoted lamellipodia formation. Our results suggest that Rac1 oxidation at Cys(18) is a novel post-translational modification that upregulates Rac1 activity.
... [29][30][31] Rac1 is the only Rho family GTPase shown to be monoubiquitinated, and this modification affects Rac1 localization. 32 Ubiquitin is a highly regulated and reversible modification that can be removed by deubiquitinating enzymes (DUBs); however, Ras-and Rho-specific DUBs have yet to be discovered. ...
While numerous studies support regulation of Ras GTPases by reactive oxygen and nitrogen species, the Rho subfamily has received considerably less attention. Over the last few years, increasing evidence is emerging that supports the redox sensitivity of Rho GTPases. Moreover, as Rho GTPases regulate the cellular redox state by controlling enzymes that generate and convert reactive oxygen and nitrogen species, redox feedback loops likely exist. Here, we provide an overview of cellular oxidants, Rho GTPases, and their inter-dependence.
... However, when the insert is absent Rho can bind, but no longer activate, its downstream effector Rho kinase (69). The Rho insert in the small GTPase Rac1 was recently shown to be monoubiquitinated (70). Although no function has yet been assigned to monoubiquitination of Rac1, it is possible that this modification is involved in the mechanism by which Rho interacts with downstream effectors. ...
Heterotrimeric G proteins are well known to transmit signals from cell surface receptors to intracellular effector proteins. There is growing appreciation that G proteins are also present at endomembrane compartments, where they can potentially interact with a distinct set of signaling proteins. Here, we examine the cellular trafficking function of the G protein α subunit in yeast, Gpa1. Gpa1 contains a unique 109 amino acid insert, within the α-helical domain, that undergoes a variety of posttranslational modifications. Among these is monoubiquitination, catalyzed by the NEDD4 family ubiquitin ligase Rsp5. Using a newly optimized method for G protein purification, together with biophysical measures of structure and function, we show that the ubiquitination domain does not influence enzyme activity. By screening a panel of 39 gene deletion mutants, each lacking a different ubiquitin-binding domain protein, we identify seven that are necessary to deliver Gpa1 to the vacuole compartment including four proteins (Ede1, Bul1, Ddi1 and Rup1) previously not known to be involved in this process. Finally, we show that proper endocytosis of the G protein is needed for sustained cellular morphogenesis and mating in response to pheromone stimulation. We conclude that a cascade of ubiquitin-binding proteins serves to deliver the G protein to its final destination within the cell. In this instance, and in contrast to the previously characterized visual system, endocytosis from the plasma membrane is needed for proper signal transduction rather than for signal desensitization.
... Surprisingly, our data in colorectal cells revealed that AKT2 and AKT3 did not affect Rac1b at the transcript but rather at the protein level (Fig. 3A,B). It remains to be investigated whether this effect is due to changes in translation efficiency or proteolytic degradation of Rac1b, in which case conflicting results on Rac1b ubiquitylation were reported (Esufali et al. 2007;Visvikis et al. 2008). Although AKT can phosphorylate and inhibit GSK3β, this mechanism is apparently not used for regulating Rac1b in colorectal cells because only GSK3β but not AKT depletion affected Rac1b transcript levels. . ...
The premessenger RNA of the majority of human genes can generate various transcripts through alternative splicing, and different tissues or disease states show specific patterns of splicing variants. These patterns depend on the relative concentrations of the splicing factors present in the cell nucleus, either as a consequence of their expression levels or of post-translational modifications, such as protein phosphorylation, which are determined by signal transduction pathways. Here, we analyzed the contribution of protein kinases to the regulation of alternative splicing variant Rac1b that is overexpressed in certain tumor types. In colorectal cells, we found that depletion of AKT2, AKT3, GSK3β, and SRPK1 significantly decreased endogenous Rac1b levels. Although knockdown of AKT2 and AKT3 affected only Rac1b protein levels suggesting a post-splicing effect, the depletion of GSK3β or SRPK1 decreased Rac1b alternative splicing, an effect mediated through changes in splicing factor SRSF1. In particular, the knockdown of SRPK1 or inhibition of its catalytic activity reduced phosphorylation and subsequent translocation of SRSF1 to the nucleus, limiting its availability to promote the inclusion of alternative exon 3b into the Rac1 pre-mRNA. Altogether, the data identify SRSF1 as a prime regulator of Rac1b expression in colorectal cells and provide further mechanistic insight into how the regulation of alternative splicing events by protein kinases can contribute to sustain tumor cell survival.
... The interaction of RhoA with Smurf 1 or Cullin-3 promotes its ubiquitylation and degradation by the proteasome (50,51). Rac1 also can be ubiquitylated at Lys 147 through a JNK-dependent process to promote its degradation (52). Tumorigenic variants of Rac1 are poorly ubiquitylated, resulting in a higher resistance to proteasomal degradation. ...
RhoE/Rnd3 is an atypical member of the Rho family of small GTPases. In addition to regulating actin cytoskeleton dynamics,
RhoE is involved in the regulation of cell proliferation, survival, and metastasis. We examined RhoE expression levels during
cell cycle and investigated mechanisms controlling them. We show that RhoE accumulates during G1, in contact-inhibited cells, and when the Akt pathway is inhibited. Conversely, RhoE levels rapidly decrease at the G1/S transition and remain low for most of the cell cycle. We also show that the half-life of RhoE is shorter than that of other
Rho proteins and that its expression levels are regulated by proteasomal degradation. The expression patterns of RhoE overlap
with that of the cell cycle inhibitor p27. Consistently with an involvement of RhoE in cell cycle regulation, RhoE and p27
levels decrease after overexpression of the F-box protein Skp2. We have identified a region between amino acids 231 and 240
of RhoE as the Skp2-interacting domain and Lys235 as the substrate for ubiquitylation. Based on our results, we propose a mechanism according to which proteasomal degradation
of RhoE by Skp2 regulates its protein levels to control cellular proliferation.
... Post-translational modifications, including ubiquitination and phosphorylation, regulate the function of key signaling biomolecules by modulating their activity, localization, and protein stability. Ubiquitination of small GTPases controls their behavior in cells, including migratory ability and cell cycle progression [20,29,30]. Here, RhoA lifespan is regulated by the SCF FBXL19 E3 ligase complex by mediating RhoA ubiquitination. ...
RhoA is a small GTPase multifunctional protein that regulates cell proliferation and cytoskeletal reorganization. Regulation of its protein stability plays an important role in its biological functions. We have shown that a Skp1-Cul1-F-box (SCF) FBXL19 E3 ubiquitin ligase targets Rac1, a related member of the Rho family for ubiquitination and degradation. Here, SCF(FBXL19) mediates RhoA ubiquitination and proteasomal degradation in lung epithelial cells. Ectopically expressed FBXL19 decreased RhoA wild type, active, and inactive forms. Cellular depletion of FBXL19 increased RhoA protein levels and extended its half-life. FBXL19 bound the small GTPase in the cytoplasm leading to RhoA ubiquitination at Lys(135). A RhoA(K135R) mutant protein was resistant to SCF(FBXL19)-mediated ubiquitination and degradation and exhibited a longer lifespan. Protein kinase Erk2-mediated phosphorylation of RhoA was both sufficient and required for SCF(FBXL19)-mediated RhoA ubiquitination and degradation. Thus, SCF(FBXL19) targets RhoA for its disposal, a process regulated by Erk2. Ectopically expressed FBXL19 reduced phosphorylation of p27 and cell proliferation, a process mediated by RhoA. Further, FBXL19 cellular expression diminished lysophosphatidic acid (LPA)-induced phosphorylation of myosin light chain (MLC) and stress fiber formation. Hence, SCF(FBXL19) functions as a RhoA antagonist during cell proliferation and cytoskeleton rearrangement. These results provide the first evidence of an F-box protein targeting RhoA thereby modulating its cellular lifespan that impacts cell proliferation and cytoskeleton rearrangement.
... Regarding the tumorogenic role of Rac1b, Rac1b was discovered as a splice variant of Rac1 over-expressed in malignant colorectal cancer cells (Jordan, et al. 1999), and has also been shown to activate NFĸβ activation leading to EMT induction and ROS formation in mouse mammary carcinoma cells (SCp2 cells) (Radisky, et al. 2005), as well as increase tumor cell survival in colorectal cells (Caco-2) via transcriptional activation of CyclinD1 and the canonical Wnt pathway along with other mechanisms (Esufali, et al. 2007, Matos andJordan 2005). In addition the concentration of Rac1b protein is known to increase in the cell because it does not undergo normal proteosomal cycling as is the case with Rac1a protein, which is ubiquitinated leading to degradation and turn-over (Visvikis, et al. 2008). ...
Emerging trends in cell-therapy based tissue repair have focused on the renewable source of adult stem cells including human bone marrow-derived mesenchymal stromal cells (hMSCs). Due to immunomodulatory properties as well as a potential to differentiate into cells characteristic of all three germ layers, hMSCs provide a source of immature cells for utilization in cell-therapy based treatments. Marrow isolated adult multilineage inducible (MIAMI) cells are a homogeneous sub-population of hMSCs which maintain self-renewal potential during ex vivo expansion, in addition to efficiently undergoing trans-differentiation into neuron-like cells in vitro. Even though hMSCs have the potential to be used for neural tissue repair, the molecular mechanisms by which they are stimulated to become neuron-like cells have not been fully characterized. Therefore the work described herein focuses on the molecular mechanisms by which MIAMI cells undergo NT-3 dependent neuronal commitment. MIAMI cells express both the full length (FL-) and tyrosine kinase deficient (TKd-) isoforms of the NTRK3 receptor, the primary NT-3 receptor, at the protein level. NT-3 stimulation of MIAMI cells during neuronal commitment induced the phosphorylation of FL-NTRK3, degradation of TKd-NTRK3, downstream activation of the Mek1/2-Erk1/2 signaling cascade, and subsequent up-regulation of a limited number of pro-neuronal genes. These findings were verified using chemical inhibitors to block NTRK autophosphorylation (K252a) and Erk1/2 activation (U0126). TKd-NTRK3 is hypothesized to activate Rac1 upon NT-3 stimulation. Rac1 was found to suppress NT-3 stimulated Erk1/2 phosphorylation, as well as downstream gene expression, as determined using a Rac1 chemical inhibitor. Further characterization confirmed that Rac1b is the predominant Rac1 isoform in MIAMI cells. Rac1b siRNA mediated knock-down resulted in increased expression of the pro-neuronal genes NGN2, MAP2, NFH and NFL during NT-3 stimulation via regulation of Mek1/2-Erk1/2. Rac1b is also involved in NT-3 stimulated cell proliferation, as well as repression of CCND1 and CCNB1 mRNA expression. In an attempt to enhance neuronal differentiation of MIAMI cells, EGF and bFGF were used to pretreat MIAMI cells prior to NT-3 stimulated neuronal commitment. EGF/bFGF pretreatment increased NTRK3 and NTRK1 protein levels along with NT-3 stimulated Erk1/2 phosphorylation. In addition, bFGF versus EGF/bFGF pretreatment restricted the expression of the pro-neuronal transcription factors Ngn2 and Prox1 versus the neural stem cells self-renewal transcription factor Musashi-1, respectively. The culmination of this work provides a model for the NT-3 induced neuronal commitment of MIAMI cells in vitro, as well as insight into the neurogenic potential of MSCs for future applications in cell-therapy based tissue repair.
... 137 This post-translational modification is distinct from Rac1 ubiquitylation, which occurs on Lys147 and triggers its degradation. 88 Preventing Rac1 sumoylation did not alter its localization to membranes, nor the binding to GEFs and effector proteins, but did reduce GTP binding. Rac1 binding to GTP, as well as its partitioning in "liquid-ordered plasma membrane domains," is also stimulated by palmitoylation of Cys178, which is immediately N-terminal of the HVR. ...
Cellular signaling by small GTPases is critically dependent on proper spatio-temporal orchestration of activation and output. In addition to their core G (guanine nucleotide binding)-domain, small GTPases comprise a hypervariable region (HVR) and a lipid anchor that are generally accepted to control subcellullar localization. The HVR encodes in many small GTPases a polybasic region (PBR) that permits charge-mediated association to the inner leaflet of the plasma membrane or to intracellular organelles. Over the past 15-20 years, evidence has accumulated for specific protein-protein interactions, mediated by the HVR, that control both targeting and signaling specificity of small GTPases. Using the RhoGTPase Rac1 as a paradigm we here review a series of protein partners that require the Rac1 HVR for association and that control various aspects of localized Rac1 signaling. Some of these proteins represent Rac1 activators, whereas others mediate Rac1 inactivation and degradation and yet others potentiate Rac1 downstream signaling. Finally, evidence is discussed which shows that the HVR of Rac1 also contributes to effector interactions, co-operating with the N-terminal effector domain. The complexity of localized Rac1 signaling, reviewed here, is most likely exemplary for many other small GTPases as well, representing a challenge to identify and define similar mechanisms controlling the specific signaling induced by small GTPases.
... To determine whether Rnd3 is ubiquitinated, we transfected HeLa cells with plasmids encoding streptavidin-binding peptide (SBP)-Rnd3 (and HAubiquitin); lysates and purified proteins were probed with anti-HA to detect the ubiquitinated proteins. We were unable to detect any ubiquitinated species of Rnd3 whereas polyubiquitinated Rac1 (29,38) was easily observed (Fig. 3C). To determine whether prolonged inhibition of proteasome function could stabilize putative ubiquitinated species of Rnd3, overnight treatment was performed, but again no ubiquitinated Rnd3 was detected (data not shown). ...
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.
... 42 The lysine 147 of Rac1 is most likely the acceptor site of ubiquitin. 43 This lysine is located in the vicinity of a polybasic stretch of amino-acids residues at the C-terminal part of Rac1. This region is crucial for binding to several regulatory proteins as well as for the proper localization of Rac1 at the membrane. ...
Rho GTPases undergo ubiquitylation and degradation via the ubiquitin-proteasome pathway. We now report in the November issue of Developmental Cell that the E3 ubiquitin-ligase HACE1 catalyzes the ubiquitylation of GTP-bound Rac1. Depletion of HACE1 leads to an increase of Rac1 activity. We have proposed that HACE1 limits Rac1 activity in cells, a regulation that is usurped by some pathogenic bacteria for efficient invasion of host cell monolayers. We here review these findings in parallel with the regulation of RhoA by the ubiquitin and proteasome system (UPS) and discuss the impact of these regulations on the capacity of Rho GTPases to signal.
... Moreover, Cav1 deletion leads to an increase in non-and mono-ubiquitylated Rac1, suggesting that Cav1 selectively regulates degradation of polyubiquitylated activated Rac1 [87,88]. Mutational analysis of all lysine residues in Rac1 revealed that ubiquitin chains are preferentially cross-linked to lysine-147, a solventaccessible residue with a similar conformation in Rac1b, an alternative splice form of Rac1 [89]. The HECT-domain-containing E3-ubiquitin-ligase tumor suppressor HACE1 preferentially binds to GTP-bound Rac1 for ubiquitylation [90]. ...
To date, most studies of Rho GTPase regulation have focused on the classic GTPase cycle - GTP binding and hydrolysis - controlled by guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and GDP-dissociation inhibitors (GDIs). Recent investigations have unveiled important additional regulatory mechanisms: microRNA (miRNA) regulating post-transcriptional processing of Rho GTPase-encoding mRNAs; palmitoylation and nuclear targeting affecting intracellular distribution; post-translational phosphorylation, transglutamination and AMPylation impacting Rho GTPase signaling; and ubiquitination controlling Rho GTPase protein stability and turnover. These modes of regulation add to the complexity of the Rho GTPase signaling network and allow precise spatiotemporal control of individual Rho GTPases. This review discusses these 'unconventional' modes of regulation and their contribution to cellular function, focusing on post-transcriptional and post-translational events beyond the classic GTPase cycle regulatory model.
... A proteasomal degradation resistant and thus constitutively active mutant of Rac1 (Rac1b), is found in colorectal and breast cancer tumour cells (Jordan, et al., 1999;Schnelzer, et al., 2000). Interestingly, RNAi mediated silencing of this mutant results in a failure of cancer cells to undergo an epithelial to mesenchymal transition (Radisky, et al., 2005) suggesting a role for ubiquitination of Rac1 in controlling cell motility (Visvikis, et al., 2008). Rho GTPases can also be regulated by the ubiquitination of their GEF activators. ...
... One feasible idea is that CYLD may deubiquitinate RAC1 and thereby promote RAC1 activation to enable efficient cell migration. This notion is supported by observations that RAC1 activity is regulated by ubiquitination (28,29). As CYLD is involved in melanoma cell migration, further studies of post-transcriptional regulation of MT and/or RAC1 could provide new targets for future therapeutic intervention to control melanoma cell motility. ...
The cylindromatosis gene (CYLD) encodes a deubiquitinase that was initially identified as a tumor suppressor and has recently been investigated in connection with a variety of normal physiological processes. In contrast to its cell-proliferative activity, the effect of CYLD protein on cell migration has been a matter of debate. We investigated the effect of CYLD-siRNA on the migration activity of malignant melanoma cells. Expression of CYLD mRNA/protein was lower in 6 of 8 malignant melanoma cell lines than in 3 sets of primary-cultured normal human epidermal melanocytes. Knockdown of CYLD significantly increased the proliferation activities of two melanoma cell lines (p<0.05), along with BCL3 nuclear translocation followed by CCND1 overexpression. In contrast to the proliferation-related activity, CYLD knockdown significantly decreased the cell migration of all the melanoma cell lines (n=7, p<0.05), and we demonstrated that the mechanism regulating melanoma cell migration was activation of RAC1 through the action of CYLD. Our findings provide new insight into the role of CYLD-induced RAC1 activation in melanoma cell migration.
... In contrast with RhoA, an E3 ubiquitin ligase for Rac1 has yet to be described. Active, but not inactive, Rac1 is ubiquitylated at Lys 147 [103] and this requires interaction with Rac1 effector proteins [104,105]. This is followed by polyubiquitylation and proteasomal degradation, a process that requires the Rac1 hypervariable C-terminus [104]. ...
Post-translational modifications are used by cells to link additional information to proteins. Most modifications are subtle and concern small moieties such as a phosphate group or a lipid. In contrast, protein ubiquitylation entails the covalent attachment of a full-length protein such as ubiquitin. The protein ubiquitylation machinery is remarkably complex, comprising more than 15 Ubls (ubiquitin-like proteins) and several hundreds of ubiquitin-conjugating enzymes. Ubiquitin is best known for its role as a tag that induces protein destruction either by the proteasome or through targeting to lysosomes. However, addition of one or more Ubls also affects vesicular traffic, protein-protein interactions and signal transduction. It is by now well established that ubiquitylation is a component of most, if not all, cellular signalling pathways. Owing to its abundance in controlling cellular functions, ubiquitylation is also of key relevance to human pathologies, including cancer and inflammation. In the present review, we focus on its role in the control of cell adhesion, polarity and directional migration. It will become clear that protein modification by Ubls occurs at every level from the receptors at the plasma membrane down to cytoskeletal components such as actin, with differential consequences for the pathway's final output. Since ubiquitylation is fast as well as reversible, it represents a bona fide signalling event, which is used to fine-tune a cell's responses to receptor agonists.
... Indeed, inhibition of proteasomes with two different inhibitors prevented IAP-mediated degradation of Rac1 ( Figure 5A; Supplementary Figure S5B). Previous studies have revealed that activated Rac1 is predisposed to ubiquitin-dependent degradation and lysine 147 was identified as a potential ubiquitin acceptor for Rac1 ( Doye et al, 2002;Lerm et al, 2002;Visvikis et al, 2008). As expected, activated Rac1 was more susceptible to XIAPmediated degradation (Supplementary Figure S5C). ...
Inhibitors of apoptosis proteins (IAPs) are a highly conserved class of multifunctional proteins. Rac1 is a well-studied Rho GTPase that controls numerous basic cellular processes. While the regulation of nucleotide binding to Rac1 is well understood, the molecular mechanisms controlling Rac1 degradation are not known. Here, we demonstrate X-linked IAP (XIAP) and cellular IAP1 (c-IAP1) directly bind to Rac1 in a nucleotide-independent manner to promote its polyubiquitination at Lys147 and proteasomal degradation. These IAPs are also required for degradation of Rac1 upon CNF1 toxin treatment or RhoGDI depletion. Consistently, downregulation of XIAP or c-IAP1 by various strategies led to an increase in Rac1 protein levels in primary and tumour cells, leading to an elongated morphology and enhanced cell migration. Further, XIAP counteracts Rac1-dependent cellular polarization in the developing zebrafish hindbrain and promotes the delamination of neurons from the normal tissue architecture. These observations unveil an evolutionarily conserved role of IAPs in controlling Rac1 stability thereby regulating the plasticity of cell migration and morphogenesis.
... Active, GTP-bound Rho GTPases transduce upstream signals by interacting with effector molecules (Van Aelst and D'Souza-Schorey, 1997;Bishop and Hall, 2000;Gundersen, 2002;Bokoch, 2003;Burridge and Wennerberg, 2004;Govek et al., 2005;Bustelo et al., 2007;Kurisu and Takenawa, 2009). Spatial and temporal specificity of Rho GTPase signaling are achieved in part via spatio-temporal regulation of regulatory molecules (Etienne-Manneville and Hall, 2002), GEFs that link Rho GTPases to specific effectors through scaffolding molecules (Buchsbaum et al., 2002;Jaffe et al., 2004), post-translational modifications such as lipid modification and phosphorylation (Ward et al., 2004;Roberts et al., 2008), and by ubiquitin-mediated proteosome degradation (Senadheera et al., 2001;Doye et al., 2002;Wang et al., 2003;Ward et al., 2004;Visvikis et al., 2008). In general during migration, Cdc42 plays an integral role in polarizing the centrosome and defining the direction of movement, Rac and Cdc42 are classic regulators of actin-based lamellipodia and filopodia formation, respectively, and Rho regulates cell contractility. ...
The architectonics of the mammalian brain arise from a remarkable range of directed cell migrations, which orchestrate the emergence of cortical neuronal layers and pattern brain circuitry. At different stages of cortical histogenesis, specific modes of cell motility are essential to the stepwise formation of cortical architecture. These movements range from interkinetic nuclear movements in the ventricular zone, to migrations of early-born, postmitotic polymorphic cells into the preplate, to the radial migration of precursors of cortical output neurons across the thickening cortical wall, and the vast, tangential migrations of interneurons from the basal forebrain into the emerging cortical layers. In all cases, actomyosin motors act in concert with cell adhesion receptor systems to provide the force and traction needed for forward movement. As key regulators of actin and microtubule cytoskeletons, cell polarity, and adhesion, the Rho GTPases play critical roles in CNS neuronal migration. This review will focus on the different types of migration in the developing neocortex and cerebellar cortex, and the role of the Rho GTPases, their regulators and effectors in these CNS migrations, with particular emphasis on their involvement in radial migration.
HACE1 is an ankyrin repeat (AKR) containing HECT‐type E3 ubiquitin ligase that interacts with and ubiquitinates multiple substrates. While HACE1 is a well‐known tumor suppressor, its structure and mode of ubiquitination are not understood. The authors present the cryo‐EM structures of human HACE1 along with in vitro functional studies that provide insights into how the enzymatic activity of HACE1 is regulated. HACE1 comprises of an N‐terminal AKR domain, a middle (MID) domain, and a C‐terminal HECT domain. Its unique G‐shaped architecture interacts as a homodimer, with monomers arranged in an antiparallel manner. In this dimeric arrangement, HACE1 ubiquitination activity is hampered, as the N‐terminal helix of one monomer restricts access to the C‐terminal domain of the other. The in vitro ubiquitination assays, hydrogen‐deuterium exchange mass spectrometry (HDX–MS) analysis, mutagenesis, and in silico modeling suggest that the HACE1 MID domain plays a crucial role along with the AKRs in RAC1 substrate recognition.
Statins are commonly used in the middle-aged and elderly people for treatment of hyperlipidemia. Both alpha lipoic acid (ALA) and ellagic acid (EA) are natural antioxidants found in a normal diet. They can protect against cellular damage and induce cellular apoptosis in many types of cancer cells. Fluvastatin (FLV) was combined with ALA and EA in a nanostructured lipid carrier (NLC) formula. The prepared NLCs were imaged with a transmission electron microscope (TEM). Particle size and zeta potential and FLV entrapment efficiency (%EE) were measured, and the FLV release profile was constructed. Cellular viability, caspase-3 enzyme levels, and cellular cycle were analyzed. The prepared NLCs were spherical, with a size of 85.2 ± 4.1 nm, and had a zeta potential of − 25.1 ± 3.4 mV and a %EE of 98.2 ± 1.1%. FLV IC50 was decreased by half by the formula and by about 30% when compared with the three drugs together. According to cell-cycle analysis, treatment with FLV-ALA-EA NLCs caused a significant increase in pre-G1 phase by about 1.44-fold in comparison with FLV-ALA-EA. These findings demonstrate that ALA and EA induced cell death, which makes their combination with FLV a candidate for prostate cancer therapy.
The E3 ubiquitin ligase HACE1 is a key regulator of cellular homeostasis best-characterized for its ability to control the activity of the Rho GTPase Rac1. This GTPase is encoded by an essential gene whose product controls a wide array of cellular processes such as cell adhesion, migration and proliferation. Accordingly, the repression of HACE1 expression due to genetic and epigenetic alterations has been associated with numerous pathologies, including cancer, neurodegenerative and developmental diseases. However, nothing is known about the posttranslational regulation of HACE1 activity. Here, we unveiled that HACE1 gets phosphorylated at serine Ser-385 by Group-I Pak kinases in response to Rac1/Cdc42 activation. Mechanistically, we define that the phospho-mimetic mutant HACE1(S385E) displays a lower capacity to ubiquitinate Rac1 in cells. In addition, our work attributes to the phosphorylation of Ser-385 a pivotal role in the state of HACE1 oligomerization, which sets the basis for deciphering the relationship between HACE1 structure and activity. In parallel, we have found that the loss of HACE1 expression leads to the disruption of epithelial monolayer cohesion characterized by disrupted of cell-cell junctions. Accordingly, we determined that loss of HACE1 results in the acquisition of epithelial-mesenchymal transition (EMT) features, including a transcriptionally regulated switch of expression between E-cadherin and N-cadherin. Altogether, this work reveals a phospho-mediated regulation of HACE1 activity that is under the control of Group I PAKs and implicates HACE1 in the balance between epithelium integrity versus EMT.
The small RhoGTPase Rac1 is implicated in a variety of events related to actin cytoskeleton rearrangement. Remarkably, another event that is completely different from those related to actin regulation has the same relevance; the Rac1-mediated production of reactive oxygen species (ROS) through NADPH oxidases (NOX). Each outcome involves different Rac1 downstream effectors; on one hand, events related to the actin cytoskeleton require Rac1 to bind to WAVEs proteins and PAKs that ultimately promote actin branching and turnover, on the other, NOX-derived ROS production demands active Rac1 to be bound to a cytosolic activator of NOX. How Rac1-mediated signaling ends up promoting actin-related events, NOX-derived ROS, or both is poorly understood. Rac1 regulators, including scaffold proteins, are known to exert tight control over its functions. Hence, evidence of Rac1 regulatory events leading to both actin remodeling and NOX-mediated ROS generation are discussed. Moreover, cellular functions linked to physiological and pathological conditions that exhibit crosstalk between Rac1 outcomes are analyzed, while plausible roles in neuronal functions (and dysfunctions) are highlighted. Together, discussed evidence shed light on cellular mechanisms which requires Rac1 to direct either actin- and/or ROS-related events, helping to understand crucial roles of Rac1 dual functionality.
The E3 ubiquitin ligase HACE1 is a potent tumor suppressor that controls cell proliferation and ubiquitylates the small GTPase Rac1 to target it to proteasomal degradation. Whether and how the activity of HACE1 is regulated by the N-terminal ankyrin (ANK) and the middle (MID) domains is ill defined. Here, we identified in the version 64 of the Catalogue of Somatic Mutations in Cancer (COSMIC) 13 missense mutations of hace1 located outside the HECT domain, and found that all lead to defective control of cell proliferation. In addition, several mutations located in the ankyrin domain displayed a dramatic reduction in Rac1 ubiquitylation associated with a decrease of colony formation in soft agar. 3D structure modelling of the 7 ankyrin-repeats coupled to functional analysis identified a surface epitope centered on one of the mutated residue, Gly-175, which is critical for controlling Rac1 binding and ubiquitylation. We also identified a role for the MID domain in conferring the specificity of association of HACE1 to the active form of Rac1. Our study of the functional interplay between HACE1 and Rac1 in cancer thus sheds a new light on the molecular mechanism of Rac1 ubiquitylation by HACE1 and the impact of its cancer-associated mutations in cell proliferation.
Cell migration is a complex process requiring density and rigidity sensing of the microenvironment to adapt cell migratory speed through focal adhesion and actin cytoskeleton regulation. ICAP-1, a β1 integrin partner, is essential for ensuring integrin activation cycle and focal adhesion formation. We show that ICAP-1 is monoubiquitinated by Smurf1, preventing ICAP-1 binding to β1 integrin. The non-ubiquitinable form of ICAP-1 modifies β1 integrin focal adhesion organization and interferes with fibronectin density sensing. ICAP-1 is also required for adapting cell migration in response to substrate stiffness in a β1 integrin-independent manner. ICAP-1 monoubiquitination regulates rigidity sensing by increasing MRCKα-dependent cell contractility through myosin phosphorylation independently of substrate rigidity. We provide evidence that ICAP-1 monoubiquitination helps in switching from ROCK2-mediated to MRCKα-mediated cell contractility. ICAP-1 monoubiquitination serves as a molecular switch to coordinate extracellular matrix density and rigidity sensing thus acting as a critical modulator of cell migration and mechanosensing.
Rho GTPases are well known for their roles in regulating cell migration, and also contribute to a variety of other cellular responses. They are subdivided into two groups: typical and atypical. The typical Rho family members, including RhoA, Rac1 and Cdc42, cycle between an active GTP-bound and inactive GDP-bound conformation, and are regulated by GEFs, GAPs and GDIs, whereas atypical Rho family members have amino acid substitutions that alter their ability to interact with GTP/GDP and hence are regulated by different mechanisms. Both typical and atypical Rho GTPases contribute to cancer progression. In a few cancers, RhoA or Rac1 are mutated, but in most cancers expression levels and/or activity of Rho GTPases is altered. Rho GTPase signaling could therefore be therapeutically targeted in cancer treatment.
Studies on deamidase toxins continue to reveal unexpected molecular mechanisms that are relevant to many fields of biology. This holds true for the discovery of the regulation of small GTPases by ubiquitin-mediated proteasomal degradation, as well as the identification of key factors controlling these cellular regulations. A growing body of evidence has highlighted the importance of these regulations in infection, inflammatory disorders, and cancer. As an example of the complexity of host-pathogen interactions, several groups have firmly established that the corruption of the activity of GTPases is perceived by the host as signaling a pathogen attack. Together, these findings open up new avenues for using specific toxin properties to stimulate Rho proteins, and thus protective responses against microbes. The discovery of new deamidase toxins targeting different cellular factors further demonstrates the importance of this type of posttranslational modification and key cellular targets.
The proto-oncogene SET/I2PP2A, an inhibitor of the phosphatase PP2A and a potential therapeutic target for cancer, interacts with the RhoGTPase Rac1 and regulates cell motility. SET is primarily nuclear but can readily translocate to the cytoplasm. Here, we investigated this translocation in more detail. Using an image analysis method to analyse nucleo-cytoplasmic shuttling of YFP-SET, we find that the protein shows repetitive shuttling in a seemingly random fashion. We found that Rac1 activity increases the frequency of these nuclear exit events of SET. In search for cellular activators of this event, we found FTY720 (fingolimod), an immunomodulator and activator of PP2A, to rapidly induce nucleo-cytoplasmic translocation of SET. Subsequently, SET accumulates in cytoplasmic aggregates of unknown nature. Moreover, we observed that the nuclear pool of Rac1 translocates simultaneously with SET, both during spontaneous as well as FTY720-induced translocation. Finally, FTY720-induced nuclear exit is dependent on the nuclear exporter CRM1, on PP2A activity as well as on microtubule dynamics. These results show that the immunomodulator and PP2A activator FTY720, induces rapid nucleo-cytoplasmic shuttling of SET, suggesting that SET translocation is part of a negative feedback loop. This data may be relevant to the potential use of FTY720 in the treatment of leukemias and inflammatory disorders.
Colorectal cancer (CRC) is the second largest cause of cancer mortality in Western countries, mostly due to metastasis. Understanding the natural history and prognostic factors in patients with metastatic CRC (mCRC) is essential for the optimal design of clinical trials. The main prognostic factors currently used in clinical practice are related to tumor behavior (e.g., white blood counts, levels of lactate dehydrogenase, levels of alkaline phosphatase) disease extension (e.g., presence of extrahepatic spread, number of organs affected) and general functional status (e.g., performance status as defined by the Eastern Cooperative Oncology Group). However, these parameters are not always sufficient to establish appropriate therapeutic strategies. First-line therapy in mCRC combines conventional chemotherapy (CHT) (e.g., FOLFOX, FOLFIRI, CAPOX) with a number of agents targeted to specific signaling pathways (TA) (e.g., panitumumab and cetuximab for cases KRAS/NRAS WT, and bevacizumab). Although the response rate to this combination regime exceeds 50%, progression of the disease is almost universal and only less than 10% of patients are free of disease at 2 years. Current clinical trials with second and third line therapy include new TA, such as tyrosin-kinase receptors inhibitors (MET, HER2, IGF-1R), inhibitors of BRAF, MEK, PI3K, AKT, mTORC, NOTCH and JAK1/JAK2, immunotherapy modulators and check point inhibitors (anti-PD-L1 and anti- PD1). Despite the identification of multiple prognostic and predictive biomarkers and signatures, it is still unclear how expression of many of these biomarkers is modulated by CHT and/or TA, thus potentially affecting response to treatment. In this review we analyzed how certain biomarkers in tumor cells and microenvironment influence the response to new TA and immune-therapies strategies in mCRC pre-treated patients.
PTC3 and PTC5 are tripartite Tc (toxin complex) toxins from Photorhabdus luminescens, which consist of the binding component TcdA1, the linker component TcdB2 and the enzyme components TccC3 and TccC5, respectively. While PTC5 ADP-ribosylates Rho proteins at Gln61/63 resulting in constitutive activation of the GTPases, PTC3 ADP-ribosylates actin at Thr148 thereby inducing actin polymerization. Here, we identified amino acids involved in ADP-ribosyltransferase activity of TccC3 and TccC5 and analyzed the substrate specificity of Rho-activating TccC5. We compared the time-dependency of Rho protein activation by PTC5 in HeLa cells with the effects of Escherichia coli cytotoxic necrotizing factor 1 (CNF1), which activates Rho GTPases by deamidation of Gln61/63. Using a luciferase reporter assay, we show that PTC5 and PTC3 stimulated gene transcription via myocardin-related transcription factor A (also called MAL) and AP1. MAL activation by PTC5 involved Rho kinase and formins. Activation of AP1 by PTC5 occurred via two MAP kinase pathways involving ERK and Jun kinase, respectively.
Chemotherapy is the principal treatment in metastatic colorectal cancer (mCRC) patients. RAC1b, a RAC1 spliced variant, is over-expressed in colorectal cancer (CRC), and impairs apoptosis by activation of nuclear-factor-KB. Since RAC1b has been associated with the BRAF(V600E) mutation, associated with poor prognosis in CRC, we evaluated the role of RAC1b expression as a predictor of chemotherapy efficacy in mCRC.
We analysed KRAS and BRAF mutation, microsatellite instability and RAC1b expression in 157 mCRC patients treated with FOLFOX/XELOX in first-line therapy.
KRAS mutations were detected in 46 patients (34%), 10 patients were BRAF mutant (7%) and 79 were WT for both, KRAS and BRAF (59%). RAC1b overexpression was found in 30 patients (19%). In the multivariate analysis, BRAF mutational status was a poor prognostic factor for overall survival (OS); hazard ratio (HR), 2.78 (95% confidence interval (CI), 1.35-5.72; p=0.0057). RAC1b overexpression was a poor survival factor for OS (HR, 2.35; 95% CI, 1.2-4.59; p=0.01) and progression-free survival (PFS) (HR, 2.4; 95% CI, 1.2-4.78; p=0.01) in KRAS/BRAF WT mCRC patients.
RAC1b overexpression constitutes a marker of poor prognosis in KRAS/BRAF WT mCRC patients treated with first-line FOLFOX/XELOX therapy.
Rho GTPases are a class of evolutionarily conserved proteins comprising 20 members, which are predominantly known for their role in regulating the actin cytoskeleton. They are primarily regulated by binding of GTP/GDP, which is again controlled by regulators like GEFs, GAPs, and RhoGDIs. Rho GTPases are thus far well known for their role in the regulation of actin cytoskeleton and migration. Here we present an overview on the role of Rho GTPases in regulating cell shape and plasticity of cell migration. Finally, we discuss the emerging roles of ubiquitination and sumoylation in regulating Rho GTPases and cell migration.
Inhibitors of Apoptosis Proteins (IAPs) are well-studied E3 ubiquitin ligases predominantly known for regulation of apoptosis. We uncovered that IAPs can function as a direct E3 ubiquitin ligase of RhoGTPase Rac1. cIAP1 and XIAP directly conjugate polyubiquitin chains to Lysine 147 of activated Rac1 and target it for proteasomal degradation. Consistently, loss of these IAPs by various strategies led to stabilization of Rac1 and mesenchymal mode of migration in tumor cells. IAPs also regulate Rac1 degradation upon RhoGDI1 depletion and CNF1 toxin treatment. Our observations revealed an evolutionarily conserved role of IAPs in regulating Rac1 stability shedding light on to the mechanisms behind ubiquitination-dependent inactivation of Rac1 signaling.
The Rho family small GTPases of the Ras superfamily play key roles in regulating diverse signaling pathways that control a myriad of fundamental cellular processes such as cytoskeletal dynamics, cell cycle progression, gene expression, cell polarity, migration and cell transformation. The Rho GTPases cycle between an active GTP-bound and an inactive GDP-bound form, which is controlled by many regulators including GEFs, GAPs and GDIs. Recent studies have revealed a new layer of regulation for Rho GTPases, indicating that several members of the Rho family of small GTPases including RhoA, Rac1, and RhoBTB, as well as the Ras family member Rap1B, are also regulated by the ubiquitin-proteasome pathway, which plays important roles in controlling cell polarity, migration, cell transformation and actin dynamics. Importantly, regulators for Rho GTP-GDP cycling such as RhoGDI and Rho-GEF ECT2 were also found to be modulated by the ubiquitin pathway. In this review, we focus on how ubiquitin signaling guides the fate and function of Rho GTPases and their regulators, especially how the E3 ubiquitin ligase Smurf1 regulates cell polarity and motility through targeting RhoA for ubiquitination and degradation.
Rac is a member of the Rho family of small GTPases, which act as molecular switches to control a wide array of cellular functions. In particular, Rac signaling has been implicated in the control of cell-cell adhesions, cell-matrix adhesions, cell migration, cell cycle progression and cellular transformation. As a result of its functional diversity, Rac signaling can influence several aspects of tumorigenesis. Consistent with this, in vivo evidence that Rac signaling contributes to tumorigenesis is continuously emerging. Additionally, our understanding of the mechanisms by which Rac signaling is regulated is rapidly expanding and consequently adds to the complexity of how Rac signaling could be modulated during tumorigenesis. Here we review the numerous biological functions and regulatory mechanisms of Rac signaling and discuss how they could influence the different stages of tumorigenesis.
It is generally assumed that a specific ubiquitin ligase (E3) links protein substrates to polyubiquitin chains containing a single type of isopeptide linkage, and that chains composed of linkages through Lys(48), but not through Lys(63), target proteins for proteasomal degradation. However, when we carried out a systematic analysis of the types of ubiquitin (Ub) chains formed by different purified E3s and Ub-conjugating enzymes (E2s), we found, using Ub mutants and mass spectrometry, that the U-box E3, CHIP, and Ring finger E3s, MuRF1 and Mdm2, with the E2, UbcH5, form a novel type of Ub chain that contains all seven possible linkages, but predominantly Lys(48), Lys(63), and Lys(11) linkages. Also, these heterogeneous chains contain forks (bifurcations), where two Ub molecules are linked to the adjacent lysines at Lys(6) + Lys(11), Lys(27) + Lys(29), or Lys(29) + Lys(33) on the preceding Ub molecule. However, the HECT domain E3s, E6AP and Nedd4, with the same E2, UbcH5, form homogeneous chains exclusively, either Lys(48) chains (E6AP) or Lys(63) chains (Nedd4). Furthermore, with other families of E2s, CHIP and MuRF1 synthesize homogeneous Ub chains on the substrates. Using the dimeric E2, UbcH13/Uev1a, they attach Lys(63) chains, but with UbcH1 (E2-25K), MuRF1 synthesizes Lys(48) chains on the substrate. We then compared the capacity of the forked heterogeneous chains and homogeneous chains to support proteasomal degradation. When troponin I was linked by MuRF1 to a Lys(48)-Ub chain or, surprisingly, to a Lys(63)-Ub chain, troponin I was degraded rapidly by pure 26S proteasomes. However, when linked to the mixed forked chains, troponin I was degraded quite poorly, and its polyUb chain, especially the forked linkages, was disassembled slowly by proteasome-associated isopeptidases. Because these Ring finger and U-box E3s with UbcH5 target proteins for degradation in vivo, but Lys(63) chains do not, cells probably contain additional factors that prevent formation of such nondegradable Ub-conjugates and that protect proteins linked to Lys(63)-Ub chains from proteasomal degradation.
The Rho, Rac and Cdc42 GTPases coordinately regulate the organization of the actin cytoskeleton and the JNK MAP kinase pathway. Mutational analysis of Rac has previously shown that these two activities are mediated by distinct cellular targets, though their identity is not known. Two Rac targets, p65(PAK) and MLK, are ser/thr kinases that have been reported to be capable of activating the JNK pathway. We present evidence that neither is the Rac target mediating JNK activation in Cos-1 cells. We have used yeast two-hybrid selection and identified a new target of Rac, POSH. This protein consists of four SH3 domains and ectopic expression leads to the activation of the JNK pathway and to nuclear translocation of NF-kappaB. When overexpressed in fibroblasts, POSH is a strong inducer of apoptosis. We propose that POSH acts as a scaffold protein and contributes to Rac-induced signal transduction pathways leading to diverse gene transcriptional changes.
One of the most powerful ways of studying the function of a protein is
to specifically block its activity within cells. Over the past decade, dominant-inhibitory
proteins have emerged as popular tools with which to accomplish this task;
these mutated proteins interfere with the function of their normal cellular
counterparts or with proteins that interact with them. This approach has been
used extensively in the elucidation of signal-transduction cascades, such
as those involving Ras-family proteins. Here I discuss the power and potential
pitfalls of using dominant-inhibitory Ras proteins.
Rac1 is a member of the Ras superfamily of small GTPases involved in signal transduction pathways that induce the formation of lamellipodia, stimulate cell proliferation and activate the JNK/SAPK protein kinase cascade. Here we describe that amplification by RT-PCR of the entire Rac1 coding sequence from a series of human adult and fetal tissues revealed beside the expected Rac1 cDNA, a variant product which contained additional 57 nucleotides between codons 75 and 76. This variant resulted in an in-frame insertion of 19 new amino acids immediately behind the switch II region, including two potential threonine phosphorylation sites for casein kinase II and protein kinase C. Primers designed within and downstream of the inserted nucleotide sequence allowed isolation of a genomic clone with intronic consensus sequences demonstrating that the insertion corresponds to a novel, yet undescribed exon 3b. This Rac1 splice variant, designated Rac1b, was predominantly identified in skin and epithelial tissues from the intestinal tract. Most notably, the expression of rac1b versus rac1 was found to be elevated in colorectal tumors at various stages of neoplastic progression, as compared to their respective adjacent tissues. We suggest that the 19 amino acid-insertion following the switch II region may create a novel effector binding site in rac1b, and thus participate in signaling pathways related to the normal or neoplastic growth of the intestinal mucosa.
Rac1 is a member of the Ras superfamily of small guanosine triphosphatases (GTPases) that act as molecular switches to control cytoskeletal rearrangements and cell growth. Analogous to Ras, constitutively activating point mutations of Rac1 cause tumorigenic transformation of cell lines. However, there is no information about whether Rac1 is also mutated in vivo. After RT - PCR of Rac1, several clones of seven benign and 10 malignant breast cancer tissues as well as eight breast cancer cell lines were sequenced. Only single-nucleotide polymorphisms of Rac1 could be detected, and none of these corresponded to constitutively activating point mutations that have been used in cell lines for transformation. While sequencing Rac1 in breast tissues, a new Rac1 isoform with an insertion of 19 codons within the reading frame of Rac1 close to switch region II was identified and named Rac1b. The Rac1b protein acts like a fast cycling GTPase in GTP binding and hydrolysis assays. In Northern and Western blot experiments both Rac1 RNA and Rac1 protein had a significantly higher expression in breast cancer tissues compared to normal breast tissue samples. Immunohistochemical staining of Rac1 showed weak Rac1 expression in benign breast disease but high expression level in ductal carcinoma-in-situ, primary breast cancer, and lymph node metastases. In addition, breast tumor cells from patients with recurrent disease had Rac1 expression at the plasma membrane, suggesting activation of Rac1, in patients with aggressive breast cancer. Oncogene (2000).
The principal guanine nucleotide exchange factors for Rho family G proteins contain tandem Dbl-homology (DH) and pleckstrin-homology (PH) domains that catalyse nucleotide exchange and the activation of G proteins. Here we have determined the crystal structure of the DH and PH domains of the T-lymphoma invasion and metastasis factor 1 (Tiam1) protein in complex with its cognate Rho family G protein, Rac1. The two switch regions of Rac1 are stabilized in conformations that disrupt both magnesium binding and guanine nucleotide interaction. The resulting cleft in Rac1 is devoid of nucleotide and highly exposed to solvent. The PH domain of Tiam1 does not contact Rac1, and the position and orientation of the PH domain is markedly altered relative to the structure of the uncomplexed, GTPase-free DH/PH element from Sos1. The Tiam1/Rac1 structure highlights the interactions that catalyse nucleotide exchange on Rho family G proteins, and illustrates structural determinants dictating specificity between individual Rho family members and their associated Dbl-related guanine nucleotide exchange factors.
The cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli has been shown to activate members of the Rho family by deamidation of glutamine 63. This amino acid is essential for hydrolysis
of GTP, and any substitution results in a constitutively active Rho. Activation of Rho induces the formation of stress fibers,
filopodia, and membrane ruffles due to activation of RhoA, Cdc42, and Rac, respectively. Here we show that the level of endogenous
Rac decreased in CNF1-treated HEK293 and HeLa cells. The amount of mRNA remained unaffected, leaving the possibility that
Rac is subject to proteolytic degradation. Treatment of cells with lactacystin, an inhibitor of the 26S proteasome, protected
Rac from degradation. We have previously shown that CNF1 activates the c-Jun N-terminal kinase (JNK) only transiently in HeLa
cells (M. Lerm, J. Selzer, A. Hoffmeyer, U. R. Rapp, K. Aktories, and G. Schmidt, Infect. Immun. 67:496-503, 1998). Here we show that CNF1-induced JNK activation is stabilized in the presence of lactacystin. The data indicate
that Rac is degraded by a proteasome-dependent pathway in CNF1-treated cells.
Rho GTPases are molecular switches that control a wide variety of signal transduction pathways in all eukaryotic cells. They are known principally for their pivotal role in regulating the actin cytoskeleton, but their ability to influence cell polarity, microtubule dynamics, membrane transport pathways and transcription factor activity is probably just as significant. Underlying this biological complexity is a simple biochemical idea, namely that by switching on a single GTPase, several distinct signalling pathways can be coordinately activated. With spatial and temporal activation of multiple switches factored in, it is not surprising to find Rho GTPases having such a prominent role in eukaryotic cell biology.
We report that the multidomain protein POSH (plenty of SH3s) acts as a scaffold for the JNK pathway of neuronal death. This pathway consists of a sequential cascade involving activated Rac1/Cdc42, mixed-lineage kinases (MLKs), MAP kinase kinases (MKKs) 4 and 7, c-Jun N-terminal kinases (JNKs) and c-Jun, and is required for neuronal death induced by various means including nerve growth factor (NGF) deprivation. In addition to binding GTP-Rac1 as described previously, we find that POSH binds MLKs both in vivo and in vitro, and complexes with MKKs 4 and 7 and with JNKs. POSH overexpression promotes apoptotic neuronal death and this is suppressed by dominant-negative forms of MLKs, MKK4/7 and c-Jun, and by an MLK inhibitor. Moreover, a POSH antisense oligonucleotide and a POSH small interfering RNA (siRNA) suppress c-Jun phosphorylation and neuronal apoptosis induced by NGF withdrawal. Thus, POSH appears to function as a scaffold in a multiprotein complex that links activated Rac1 and downstream elements of the JNK apoptotic cascade.
The Rho family of small guanosine triphosphatases regulates actin cytoskeleton dynamics that underlie cellular functions such
as cell shape changes, migration, and polarity. We found that Smurf1, a HECT domain E3 ubiquitin ligase, regulated cell polarity
and protrusive activity and was required to maintain the transformed morphology and motility of a tumor cell. Atypical protein
kinase C zeta (PKCζ), an effector of the Cdc42/Rac1-PAR6 polarity complex, recruited Smurf1 to cellular protrusions, where
it controlled the local level of RhoA. Smurf1 thus links the polarity complex to degradation of RhoA in lamellipodia and filopodia
to prevent RhoA signaling during dynamic membrane movements.
The cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli activates members of the Rho family by deamidation of glutamine 61/63. Because this amino acid is crucial for GTP hydrolysis,
deamidation of glutamine 61/63 results in constitutively active Rho proteins. Recently, it was shown that the level of CNF1-activated
Rac is rapidly diminished in CNF1-treated cells by proteolytic degradation. Here, we studied the requirements for CNF1-induced
Rac degradation. By overexpressing His-tagged activated Rac mutants we show that constitutive activation is necessary for
degradation of Rac. However, permanent activation is not sufficient for degradation, because Rac that is constitutively activated
by transamidation at glutamine 61 by the Bordetella dermonecrotic toxin is not degraded. Overexpression of His-tagged Rac mutants deficient in interaction with GTPase-activating
protein (Rac(N92D) and Rac(Y64H)) and guanosine nucleotide dissociation inhibitor (Rac(H103E)) were degraded after activation
by CNF1, whereas Rac(Y40C), which is not able to interact with CRIB domain effectors or plenty of SH3, was not degraded. Isoprenylation
and the presence of a putative mitotic destruction box are essential for CNF-induced degradation. In contrast to Rac1, Rac2,
and Rac3 were not degraded following constitutive activation by CNF1. Using site-directed mutagenesis, we defined the polybasic
region and amino acids 90, 107, 147, and 151 as responsible for isotype-specific degradation.
The turnover of Jun proteins, like that of other transcription factors, is regulated through ubiquitin-dependent proteolysis.
Usually, such processes are regulated by extracellular stimuli through phosphorylation of the target protein, which allows
recognition by F box–containing E3 ubiquitin ligases. In the case of c-Jun and JunB, we found that extracellular stimuli also
modulate protein turnover by regulating the activity of an E3 ligase by means of its phosphorylation. Activation of the Jun
amino-terminal kinase (JNK) mitogen-activated protein kinase cascade after T cell stimulation accelerated degradation of c-Jun
and JunB through phosphorylation-dependent activation of the E3 ligase Itch. This pathway modulates cytokine production by
effector T cells.
A novel splice variant of Rac1, designated Rac1b, is expressed in human breast and colon carcinoma cells. Rac1b contains an additional 19 amino-acid insert immediately behind the switch II domain, a region important for Rac1 interaction with regulators and effectors. Recent studies showed that Rac1b exhibited the biochemical properties of a constitutively activated GTPase, yet it showed impaired interaction with downstream effectors, suggesting that Rac1b may be defective in biological activity. Whether Rac1b is a biologically active protein was not addressed. Therefore, we evaluated the biochemical, signaling and growth-promoting properties of authentic Rac1b. Similar to previous observations, we found that Rac1b showed enhanced intrinsic guanine nucleotide exchange activity, impaired intrinsic GTPase activity, and failed to interact with RhoGDI. Surprisingly, we found that Rac1b, like the constitutively-activated and transforming Rac1(Q61L) mutant, promoted growth transformation of NIH3T3 cells. Rac1b-expressing cells also showed a loss of density-dependent and anchorage-dependent growth. Surprisingly, unlike activated Rac1(61L), Rac1b did not show enhanced activation of the nuclear factor kappaB (NF-kappaB) transcription factor or stimulate cyclin D1 expression, the signaling activities that best correlate with Rac1 transforming activity. However, Rac1b did promote activation of the AKT serine/threonine kinase. Therefore, we suggest that Rac1b selectively activates a subset of Rac1 downstream signaling pathways to facilitate cellular transformation.
The transition of cells from an epithelial to a mesenchymal phenotype is a critical event during morphogenesis in multicellular
organisms and underlies the pathology of many diseases, including the invasive phenotype associated with metastatic carcinomas.
Transforming growth factor β (TGFβ) is a key regulator of epithelial-to-mesenchymal transition (EMT). However, the molecular
mechanisms that control the dissolution of tight junctions, an early event in EMT, remain elusive. We demonstrate that Par6,
a regulator of epithelial cell polarity and tight-junction assembly, interacts with TGFβ receptors and is a substrate of the
type II receptor, TβRII. Phosphorylation of Par6 is required for TGFβ-dependent EMT in mammary gland epithelial cells and
controls the interaction of Par6 with the E3 ubiquitin ligase Smurf1. Smurf1, in turn, targets the guanosine triphosphatase
RhoA for degradation, thereby leading to a loss of tight junctions. These studies define how an extracellular cue signals
to the polarity machinery to control epithelial cell morphology.
The tumour microenvironment can be a potent carcinogen, not only by facilitating cancer progression and activating dormant cancer cells, but also by stimulating tumour formation. We have previously investigated stromelysin-1/matrix metalloproteinase-3 (MMP-3), a stromal enzyme upregulated in many breast tumours, and found that MMP-3 can cause epithelial-mesenchymal transition (EMT) and malignant transformation in cultured cells, and genomically unstable mammary carcinomas in transgenic mice. Here we explain the molecular pathways by which MMP-3 exerts these effects: exposure of mouse mammary epithelial cells to MMP-3 induces the expression of an alternatively spliced form of Rac1, which causes an increase in cellular reactive oxygen species (ROS). The ROS stimulate the expression of the transcription factor Snail and EMT, and cause oxidative damage to DNA and genomic instability. These findings identify a previously undescribed pathway in which a component of the breast tumour microenvironment alters cellular structure in culture and tissue structure in vivo, leading to malignant transformation.
Ubiquitylation is an emerging mechanism implicated in a variety of nonproteolytic cellular functions. The attachment of a single ubiquitin (Ub) or poly-Ub (lysine 63) chains to proteins control gene transcription, DNA repair and replication, intracellular trafficking and virus budding. In these processes, protein ubiquitylation exhibits inducibility, reversibility and recognition by specialized domains, features similar to protein phosphorylation, which enable Ub to act as a signaling device. Here, we highlight several recent examples on how Ub regulates signaling and how signaling regulates ubiquitylation during physiological and pathological cellular processes.
Epithelial cells disassemble their adherens junctions and "scatter" during processes such as tumor cell invasion as well as some stages of embryonic development. Control of actin polymerization is a powerful mechanism for regulating the strength of cell-cell adhesion. In this regard, studies have shown that sustained activation of Rac1, a well-known regulator of actin dynamics, results in the accumulation of polymerized actin at cell-cell contacts in epithelia and an increase in E-cadherin-mediated adhesion. Here we show that active Rac1 is ubiquitinated and subject to proteasome-mediated degradation during the early stages of epithelial cell scattering. These findings delineate a mechanism for the down-regulation of Rac1 in the disassembly of epithelial cell-cell contacts and support the emerging theme that UPS-mediated degradation of the Rho family GTPases may serve as an efficient mechanism for GTPase deactivation in the sustained presence of Dbl-exchange factors.
Ubiquitylation of RhoA has emerged as an important aspect of both the virulence of Escherichia coli producing cytotoxic necrotizing factor (CNF) 1 toxin and the establishment of the polarity of eukaryotic cells. Owing to the molecular activity of CNF1, we have investigated the relationship between permanent activation of RhoA catalyzed by CNF1 and subsequent ubiquitylation of RhoA by Smurf1. Using Smurf1-deficient cells and by RNA interference (RNAi)-mediated Smurf1 knockdown, we demonstrate that Smurf1 is a rate-limiting and specific factor of the ubiquitin-mediated proteasomal degradation of activated RhoA. We further show that the cancer cell lines HEp-2, human embryonic kidney 293 and Vero are specifically deficient in ubiquitylation of either activated Rac, Cdc42, or Rho, respectively. In contrast, CNF1 produced the cellular depletion of all three isoforms of Rho proteins in the primary human cell types we have tested. We demonstrate that ectopic expression of Smurf1 in Vero cells, deficient for RhoA ubiquitylation, restores ubiquitylation of the activated forms of RhoA. We conclude here that Smurf1 ubiquitylates activated RhoA and that, in contrast to human primary cell types, some cancer cell lines have a lower ubiquitylation capacity of specific Rho proteins. Thus, both CNF1 and transforming growth factor-beta trigger activated RhoA ubiquitylation through Smurf1 ubiquitin-ligase.
Rac1b was recently identified in malignant colorectal tumors as an alternative splice variant of Rac1 containing a 19-amino
acid insertion next to the switch II region. The structures of Rac1b in the GDP- and the GppNHp-bound forms, determined at
a resolution of 1.75 Å, reveal that the insertion induces an open switch I conformation and a highly mobile switch II. As
a consequence, Rac1b has an accelerated GEF-independent GDP/GTP exchange and an impaired GTP hydrolysis, which is restored
partially by GTPase-activating proteins. Interestingly, Rac1b is able to bind the GTPase-binding domain of PAK but not full-length
PAK in a GTP-dependent manner, suggesting that the insertion does not completely abolish effector interaction. The presented
study provides insights into the structural and biochemical mechanism of a self-activating GTPase.
Ubiquitination is known to regulate early stages of intracellular vesicular transport, without proteasomal involvement. We now show that, in yeast, ubiquitination regulates a late-stage, membrane fusion, with proteasomal involvement. A known proteasome mutant had a vacuolar fragmentation phenotype in vivo often associated with vacuolar membrane fusion defects, suggesting a proteasomal role in fusion. Inhibiting vacuolar proteasomes interfered with membrane fusion in vitro, showing that fusion cannot occur without proteasomal degradation. If so, one would expect to find ubiquitinated proteins on vacuolar membranes. We found a small number of these, identified the most prevalent one as Ypt7 and mapped its two major ubiquitination sites. Ubiquitinated Ypt7 was linked to the degradation event that is necessary for fusion: vacuolar Ypt7 and vacuolar proteasomes were interdependent, ubiquitinated Ypt7 became a proteasomal substrate during fusion, and proteasome inhibitors reduced fusion to greater degree when we decreased Ypt7 ubiquitination. The strongest model holds that fusion cannot proceed without proteasomal degradation of ubiquitinated Ypt7. As Ypt7 is one of many Rab GTPases, ubiquitin-proteasome regulation may be involved in membrane fusion elsewhere.
Rho GTPases participate in various cellular processes, including normal and tumor cell migration. It has been reported that RhoA is targeted for degradation at the leading edge of migrating cells by the E3 ubiquitin ligase Smurf1, and that this is required for the formation of protrusions. We report that Smurf1-dependent RhoA degradation in tumor cells results in the down-regulation of Rho kinase (ROCK) activity and myosin light chain 2 (MLC2) phosphorylation at the cell periphery. The localized inhibition of contractile forces is necessary for the formation of lamellipodia and for tumor cell motility in 2D tissue culture assays. In 3D invasion assays, and in in vivo tumor cell migration, the inhibition of Smurf1 induces a mesenchymal-amoeboid-like transition that is associated with a more invasive phenotype. Our results suggest that Smurf1 is a pivotal regulator of tumor cell movement through its regulation of RhoA signaling.
Rac1b is a tumor-specific splice variant of the Rac1 GTPase that displays limited functional similarities to Rac1. We have shown previously a novel cross-talk between Rac1 and beta-catenin, which induces canonical Wnt pathway activation in colorectal cancer cells. This prompted us to investigate if Rac1b, frequently overexpressed in colon tumors, contributes to Wnt pathway dysregulation. We show that Rac1b overexpression stimulates Tcf-mediated gene transcription, whereas depletion of Rac1b results in decreased expression of the Wnt target gene cyclin D1. Reconstitution experiments revealed an important difference between Rac1 and Rac1b such that Rac1b was capable of functionally interacting with Dishevelled-3 (Dvl-3) but not beta-catenin to mediate synergistic induction of Wnt target genes. In agreement, Dvl-3 but not beta-catenin caused increased activation of Rac1b levels, which may explain the functional cooperativity displayed in transcription assays. Furthermore, we show that Rac1b negatively regulates E-cadherin expression and results in decreased adhesion of colorectal cancer cells. RNA interference-mediated suppression of Rac1b resulted in reduced expression of Slug, a specific transcriptional repressor of E-cadherin, and a concomitant increase in E-cadherin transcript levels was observed. Intriguingly, mutation of the polybasic region of Rac1b resulted in complete loss of Rac1b stimulatory effects on transcription and suppressive effects on adhesion, indicating the importance of nuclear and membrane localization of Rac1b. Our results suggest that Rac1b overexpression may facilitate tumor progression by enhancing Dvl-3-mediated Wnt pathway signaling and induction of Wnt target genes specifically involved in decreasing the adhesive properties of colorectal cancer cells.
Rac1 is a member of the Rho family of small GTPases, which control signaling pathways that regulate actin cytoskeletal dynamics and gene transcription. Rac1 is activated by guanine nucleotide exchange factors and inactivated by GTPase-activating proteins. In addition, Rho-GDP dissociation inhibitors (Rho-GDIs) can inhibit Rac1 by sequestering it in the cytoplasm. We have found previously that colorectal tumors express an alternatively spliced variant, Rac1b, containing 19 additional amino acids following the switch II region. Here we characterized the regulation and downstream signaling of Rac1b. Although little Rac1b protein is expressed in cells, the amount of activated Rac1b protein often exceeds that of activated Rac1, suggesting that Rac1b contributes significantly to the downstream signaling of Rac in cells. The regulation of both Rac1 and Rac1b activities is dependent on guanine nucleotide exchange factors and GTPase-activating proteins, but the difference in their activation is mainly determined by the inability of Rac1b to interact with Rho-GDI. As a consequence, most Rac1b remains bound to the plasma membrane and is not sequestered by Rho-GDI in the cytoplasm. Unlike Rac1, activated Rac1b is unable to induce lamellipodia formation and is unable to bind and activate p21-activated protein kinase nor activate the downstream protein kinase JNK. However, both Rac1 and Rac1b are able to activate NFkappaB to the same extent. These data suggest that alternative splicing of Rac1 leads to a highly active Rac variant that differs in regulation and downstream signaling.
Rac1 is a member of the Ras superfamily of small guanosine triphosphatases (GTPases) that act as molecular switches to control cytoskeletal rearrangements and cell growth. Analogous to Ras, constitutively activating point mutations of Rac1 cause tumorigenic transformation of cell lines. However, there is no information about whether Rac1 is also mutated in vivo. After RT–PCR of Rac1, several clones of seven benign and 10 malignant breast cancer tissues as well as eight breast cancer cell lines were sequenced. Only single-nucleotide polymorphisms of Rac1 could be detected, and none of these corresponded to constitutively activating point mutations that have been used in cell lines for transformation. While sequencing Rac1 in breast tissues, a new Rac1 isoform with an insertion of 19 codons within the reading frame of Rac1 close to switch region II was identified and named Rac1b. The Rac1b protein acts like a fast cycling GTPase in GTP binding and hydrolysis assays. In Northern and Western blot experiments both Rac1 RNA and Rac1 protein had a significantly higher expression in breast cancer tissues compared to normal breast tissue samples. Immunohistochemical staining of Rac1 showed weak Rac1 expression in benign breast disease but high expression level in ductal carcinoma-in-situ, primary breast cancer, and lymph node metastases. In addition, breast tumor cells from patients with recurrent disease had Rac1 expression at the plasma membrane, suggesting activation of Rac1, in patients with aggressive breast cancer.
Remodeling of the endothelial cell (EC) cytoskeleton is central to many functions of the endothelium. The Rho family of small GTP-binding proteins have been identified as key regulators of F-actin cytoskeletal dynamics in a variety of cell types. They integrate signals from soluble mediators interacting with cytokine, growth factor tyrosine kinase, and G-protein–coupled receptors (GPCRs); as well as signals from cell–cell, and cell–matrix protein adhesion molecules. Recently, it has become appreciated that effector molecules downstream of Rho GTP-binding proteins also modulate several other well described cell signaling pathways. We review the role these molecules play in the cell, with a particular focus on the EC. HISTORY The Rho family of small GTP-binding proteins, which consists of 22 members, is part of the larger Ras GTP-binding protein superfamily. These approximately 21-kDa proteins cycle between inactive GDP-and active GTP-bound forms to act as a molecular switch in signal transduction pathways. The members of this family are grouped by virtue of a shared structural motif, the Rho insert loop, that is present in the GTPase domain and contributes to the binding specificity for downstream effector molecules (1,2). In addition to this shared structural feature, most Rho family members undergo post-translational modification to link farnesyl or geranylgeranyl groups to the cysteine in a CAAX motif at the C-terminus of the molecule. Subcellular localization of the molecule is directed by the lipid moiety and, in some family members, is also influenced by additional domains in the C-terminus.
p67phox is an essential part of the NADPH oxidase, a multiprotein enzyme complex that produces superoxide ions in response to microbial infection. Binding of the small GTPase Rac to p67phox is a key step in the assembly of the active enzyme complex. The structure of Rac·GTP bound to the N-terminal TPR (tetratrico-peptide repeat) domain of p67phox reveals a novel mode of Rho family/effector interaction and explains the basis of GTPase specificity. Complex formation is largely mediated by an insertion between two TPR motifs, suggesting an unsuspected versatility of TPR domains in target recognition and in their more general role as scaffolds for the assembly of multiprotein complexes.
Salmonella spp. utilize a specialized protein secretion system to deliver a battery of effector proteins into host cells. Several of these effectors stimulate Cdc42- and Rac1-dependent cytoskeletal changes that promote bacterial internalization. These potentially cytotoxic alterations are rapidly reversed by the effector SptP, a tyrosine phosphatase and GTPase activating protein (GAP) that targets Cdc42 and Rac1. The 2.3 Å resolution crystal structure of an SptP–Rac1 transition state complex reveals an unusual GAP architecture that mimics host functional homologs. The phosphatase domain possesses a conserved active site but distinct surface properties. Binding to Rac1 induces a dramatic stabilization in SptP of a four-helix bundle that makes extensive contacts with the Switch I and Switch II regions of the GTPase.
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The selective degradation of many short-lived proteins in eukaryotic cells is carried out by the ubiquitin system. In this pathway, proteins are targeted for degradation by covalent ligation to ubiquitin, a highly conserved small protein. Ubiquitin-mediated degradation of regulatory proteins plays important roles in the control of numerous processes, including cell-cycle progression, signal transduction, transcriptional regulation, receptor down-regulation, and endocytosis. The ubiquitin system has been implicated in the immune response, development, and programmed cell death. Abnormalities in ubiquitin-mediated processes have been shown to cause pathological conditions, including malignant transformation. In this review we discuss recent information on functions and mechanisms of the ubiquitin system. Since the selectivity of protein degradation is determined mainly at the stage of ligation to ubiquitin, special attention is focused on what we know, and would like to know, about the mode of action of ubiquitin-protein ligation systems and about signals in proteins recognized by these systems.
Rho family-specific guanine nucleotide dissociation inhibitors (RhoGDIs) decrease the rate of nucleotide dissociation and release Rho proteins such as RhoA, Rac and Cdc42 from membranes, forming tight complexes that shuttle between cytosol and membrane compartments. We have solved the crystal structure of a complex between the RhoGDI homolog LyGDI and GDP-bound Rac2, which are abundant in leukocytes, representing the cytosolic, resting pool of Rho species to be activated by extracellular signals. The N-terminal domain of LyGDI (LyN), which has been reported to be flexible in isolated RhoGDIs, becomes ordered upon complex formation and contributes more than 60% to the interface area. The structure is consistent with the C-terminus of Rac2 binding to a hydrophobic cavity previously proposed as isoprenyl binding site. An inner segment of LyN forms a helical hairpin that contacts mainly the switch regions of Rac2. The architecture of the complex interface suggests a mechanism for the inhibition of guanine nucleotide dissociation that is based on the stabilization of the magnesium (Mg2+) ion in the nucleotide binding pocket.
Several bacterial toxins target Rho GTPases, which constitute molecular switches in several signaling processes and master regulators of the actin cytoskeleton. The biological activities of Rho GTPases are blocked by C3-like transferases, which ADP-ribosylate Rho at Asn41, but not Rac or Cdc42. Large clostridial cytotoxins (e. g., Clostridium difficile toxin A and B) glucosylate Rho GTPases at Thr37 (Rho) or Thr35 (Rac/Cdc42), thereby inhibiting Rho functions by preventing effector coupling. The 'injected' toxins ExoS, YopE and SptP from Pseudomonas aeruginosa, Yersinia and Salmonella ssp., respectively, which are transferred into the eukaryotic target cells by the type-III secretion system, inhibit Rho functions by acting as Rho GAP proteins. Rho GTPases are activated by the cytotoxic necrotizing factors CNF1 and CNF2 from Escherichia coli and by the dermonecrotizing toxin DNT from B. bronchiseptica. These toxins deamidate/transglutaminate Gln63 of Rho to block the intrinsic and GAP-stimulated GTP hydrolysis, thereby constitutively activating the GTPases. Rho GTPases are also activated by SopE, a type-III system injected protein from Salmonella ssp., that acts as a GEF protein.
The most damaging change during cancer progression is the switch from a locally growing tumour to a metastatic killer. This switch is believed to involve numerous alterations that allow tumour cells to complete the complex series of events needed for metastasis. Relatively few genes have been implicated in these events. Here we use an in vivo selection scheme to select highly metastatic melanoma cells. By analysing these cells on DNA arrays, we define a pattern of gene expression that correlates with progression to a metastatic phenotype. In particular, we show enhanced expression of several genes involved in extracellular matrix assembly and of a second set of genes that regulate, either directly or indirectly, the actin-based cytoskeleton. One of these, the small GTPase RhoC, enhances metastasis when overexpressed, whereas a dominant-negative Rho inhibits metastasis. Analysis of the phenotype of cells expressing dominant-negative Rho or RhoC indicates that RhoC is important in tumour cell invasion. The genomic approach allows us to identify families of genes involved in a process, not just single genes, and can indicate which molecular and cellular events might be important in complex biological processes such as metastasis.
p67phox is an essential part of the NADPH oxidase, a multiprotein enzyme complex that produces superoxide ions in response to microbial infection. Binding of the small GTPase Rac to p67phox is a key step in the assembly of the active enzyme complex. The structure of Rac.GTP bound to the N-terminal TPR (tetratricopeptide repeat) domain of p67phox reveals a novel mode of Rho family/effector interaction and explains the basis of GTPase specificity. Complex formation is largely mediated by an insertion between two TPR motifs, suggesting an unsuspected versatility of TPR domains in target recognition and in their more general role as scaffolds for the assembly of multiprotein complexes.
Small GTP-binding proteins (G proteins) exist in eukaryotes from yeast to human and constitute a superfamily consisting of more than 100 members. This superfamily is structurally classified into at least five families: the Ras, Rho, Rab, Sar1/Arf, and Ran families. They regulate a wide variety of cell functions as biological timers (biotimers) that initiate and terminate specific cell functions and determine the periods of time for the continuation of the specific cell functions. They furthermore play key roles in not only temporal but also spatial determination of specific cell functions. The Ras family regulates gene expression, the Rho family regulates cytoskeletal reorganization and gene expression, the Rab and Sar1/Arf families regulate vesicle trafficking, and the Ran family regulates nucleocytoplasmic transport and microtubule organization. Many upstream regulators and downstream effectors of small G proteins have been isolated, and their modes of activation and action have gradually been elucidated. Cascades and cross-talks of small G proteins have also been clarified. In this review, functions of small G proteins and their modes of activation and action are described.
Salmonella spp. utilize a specialized protein secretion system to deliver a battery of effector proteins into host cells. Several of these effectors stimulate Cdc42- and Rac1-dependent cytoskeletal changes that promote bacterial internalization. These potentially cytotoxic alterations are rapidly reversed by the effector SptP, a tyrosine phosphatase and GTPase activating protein (GAP) that targets Cdc42 and Rac1. The 2.3 A resolution crystal structure of an SptP-Rac1 transition state complex reveals an unusual GAP architecture that mimics host functional homologs. The phosphatase domain possesses a conserved active site but distinct surface properties. Binding to Rac1 induces a dramatic stabilization in SptP of a four-helix bundle that makes extensive contacts with the Switch I and Switch II regions of the GTPase.
Pseudomonas aeruginosa is an opportunistic bacterial pathogen of great medical relevance. One of its major toxins, exoenzyme S (ExoS), is a dual function protein with a C-terminal Ras-ADP-ribosylation domain and an N-terminal GTPase activating protein (GAP) domain specific for Rho-family proteins. We report here the three-dimensional structure of the N-terminal domain of ExoS determined by X-ray crystallography to 2.4 A resolution. Its fold is all helical with a four helix bundle core capped by additional irregular helices. Loops that are known to interact with Rho-family proteins show very large mobility. Considering the importance of ExoS in Pseudomonas pathogenicity, this structure could be of interest for drug targeting.
Mutations in X-linked genes are likely to account for the observation that more males than females are affected by mental retardation. Causative mutations have recently been identified in both syndromic X-linked mental retardation (XLMR) and in the genetically heterogeneous 'nonspecific' forms of XLMR, for which cognitive impairment is the only defining clinical feature. Proteins that function in chromatin remodelling are affected in three important syndromic forms of XLMR. In nonspecific forms of the disorder, defects have been found in signal-transduction pathways that are believed to function during neuronal maturation. These findings provide important insights into the molecular and cellular defects that underlie mental retardation.
The functionality and efficacy of Rho GTPase signaling is pivotal for a plethora of biological processes. Due to the integral nature of these molecules, the dysregulation of their activities can result in diverse aberrant phenotypes. Dysregulation can, as will be described below, be based on an altered signaling strength on the level of a specific regulator or that of the respective GTPase itself. Alternatively, effector pathways emanating from a specific Rho GTPase may be under- or overactivated. In this review, we address the role of the Rho-type GTPases as a subfamily of the Ras-superfamily of small GTP-binding proteins in the development of various disease phenotypes. The steadily growing list of genetic alterations that specifically impinge on proper Rho GTPase function corresponds to pathological categories such as cancer progression, mental disabilities and a group of quite diverse and unrelated disorders. We will provide an overview of disease-rendering mutations in genes that have been positively correlated with Rho GTPase signaling and will discuss the cellular and molecular mechanisms that may be affected by them.
The 26S proteasome is recognized as the principal mediator of intracellular proteolysis in eukaryotes. As a consequence, its influence on cellular metabolism is as complex and manifold as are the proteins degraded by this protease, and new natural substrates are being discovered in ever increasing numbers. It has long been realized that the modulation of the steady-state levels of proteins can occur at the level of their synthesis as well as their degradation (Schimke 1973). Thus, one major area of proteasome function is the control of basic cellular processes such as cell cycle progression, signal transduction, and transcription via the degradation of short-lived regulatory factors. In addition, the proteasome plays a central role in the removal of misfolded, aberrant, or damaged proteins, which is a critical aspect of the cellular stress response. Last but not least, the mammalian proteasome is responsible for the generation of antigenic peptides presented on the cell surface by major histocompatibility complex (MHC) class I molecules as an integral part of the immune system (see the chapter by Niedermann, this volume). Tight control of proteasome activity is essential to guarantee the correctly timed removal of short-lived regulatory proteins but at the same time prevent the untimely destruction of other important cellular components not targeted for degradation. The system that distinguishes between stable proteins and those destined for breakdown and thus ensures the fidelity of selective proteolysis is the ubiquitin system. Accordingly, malfunctions or absence of components of this intricate enzymatic machinery lead to a variety of inherited or acquired diseases (Schwartz and Ciechanover 1999).
CNF1 toxin is a virulence factor produced by uropathogenic Escherichia coli. Upon cell binding and introduction into the cytosol, CNF1 deamidates glutamine 63 of RhoA (or 61 of Rac and Cdc42), rendering constitutively active these GTPases. Unexpectedly, we measured in bladder cells a transient CNF1-induced activation of Rho GTPases, maximal for Rac. Deactivation of Rac correlated with the increased susceptibility of its deamidated form to ubiquitin/proteasome-mediated degradation. Sensitivity to ubiquitylation could be generalized to other permanent-activated forms of Rac and to its sustained activation by Dbl. Degradation of the toxin-activated Rac allowed both host cell motility and efficient cell invasion by uropathogenic bacteria. CNF1 toxicity thus results from a restricted activation of Rho GTPases through hijacking the host cell proteasomal machinery.
The RAS oncogenes were identified almost 20 years ago. Since then, we have learnt that they are members of a large family of small GTPases that bind GTP and hydrolyse it to GDP. This is then exchanged for GTP and the cycle is repeated. The switching between these two states regulates a wide range of cellular processes. A branch of the RAS family--the RHO proteins--is also involved in cancer, but what is the role of these proteins and would they make good therapeutic targets?
In the past few years, an important question in microbiology has arisen from reports indicating that several pathogenic bacteria have evolved virulence factors directed towards a Ras subfamily of GTPases, namely the Rho GTPases. Progress made in studying both the virulence factors and the signaling pathways involving Rho GTPases has shed light on this crosstalk. One central question is raised by the findings that both activating and inactivating virulence factors that target Rho GTPases coexist in some pathogenic bacteria. Further studies on this peculiar aspect of the bacteria-host cell interactions, which leads to the outbreak of infectious diseases, might clarify whether this aspect of Rho GTPase activation or inactivation represents a finely adapted response of the pathogen for its own benefit or might lead to a reaction of the host against the bacteria.
The 76-residue protein ubiquitin exists within eukaryotic cells both as a monomer and in the form of isopeptide-linked polymers called polyubiquitin chains. In two well-described cases, structurally distinct polyubiquitin chains represent functionally distinct intracellular signals. Recently, additional polymeric structures have been detected in vivo and in vitro, and several large families of proteins with polyubiquitin chain-binding activity have been discovered. Although the molecular mechanisms governing specificity in chain synthesis and recognition are still incompletely understood, the scope of signaling by polyubiquitin chains is likely to be broader than originally envisioned.
The E3 ubiquitin (Ub) ligase Itch is a critical regulator of T helper 2 (Th2) cytokine production through its ability to induce Ub-dependent JunB degradation. After T cell receptor engagement, Itch undergoes JNK1-mediated phosphorylation that greatly enhances its enzymatic activity. To investigate how phosphorylation activates an E3 Ub ligase we have identified the JNK1 phosphorylation sites within Itch as S199, S232, and T222, which are located within a Pro-rich region. Phosphorylation of these sites is necessary and sufficient for disrupting an inhibitory interaction between the WW domain of Itch and its catalytic HECT (Homologous to E6-AP C Terminus) domain and induces a conformational change that greatly enhances the catalytic activity of Itch, a HECT E3 ligase found to be directly activated upon its phosphorylation.
• JNK
The CNF1 toxin is produced by uropathogenic and meningitis-causing Escherichia coli. CNF1 catalyzes the constitutive activation of Rho proteins by deamidation. The threshold of activation of Rho proteins by CNF1 is, however, attenuated because of a concomitant decrease of their cellular levels. Depletion of activated-Rac1 is catalyzed by ubiquitin-mediated proteasomal degradation. Consequently, we show by effector-binding pull-down that co-treatment of intoxicated cells with the MG132 proteasome-inhibitor results in a higher level of activation of Rac, as well as RhoA and Cdc42. We show that CNF1 induces the transient recruitment of Rho proteins to cellular membranes. Interestingly, at the difference of Rac and Cdc42, the inhibition of the proteasome during CNF1 treatment does not result in a significant accumulation of RhoA to cellular membranes. Using an in vivo ubiquitylation assay, we evidence that mutation of the geranylgeranyl acceptor cysteine of Rac1 (Rac1C189G) abolished the sensitivity of permanently activated-Rac1 to ubiquitylation, whereas Rac1C189G remained able to bind to the effector-binding domain of p21-PAK. Collectively, these results indicate that association with the cellular membranes is a necessary step for activated-Rac1 ubiquitylation.
Rho GTPases are well known to regulate actin dynamics. They activate two types of actin nucleators, WASP/WAVE proteins and Diaphanous-related formins (DRFs), which induce different types of actin organization. Their ability to interact with membranes allows them to target actin polymerization to discrete sites on the plasma membrane and to intracellular membrane compartments and thereby induce membrane protrusions or regulate vesicle movement. Most studies have concentrated on just three of the 22 mammalian Rho proteins, RhoA, Rac1 and Cdc42. However, recent research indicates that several other members of the Rho family, including Rif, RhoD, TC10 and Wrch1, and also related Rho-of-plants proteins (ROPs) in plants, stimulate actin polymerization and affect plasma membrane protrusion and/or vesicular traffic.
Cloning of a novel human Rac1b splice variant with increased expression in colorectal tumors
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Regulation of the polarity protein Par6 by TGFbeta receptors controls epithelial cell plasticity
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