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The FWD1/beta-TrCP-mediated degradation pathway establishes a 'turning off switch' of a Cdc42 guanine nucleotide exchange factor, FGD1
Abstract
FWD1/beta-TrCP is the F-box protein that functions as the receptor subunit of the SCF(FWD1/beta-TrCP) ubiquitin ligase and has been shown to be responsible for the degradation of important signaling molecules such as IkappaBs and beta-catenin. Protein substrates of FWD1/beta-TrCP contain a consensus DSGPsiXS motif (where Psi represents a hydrophobic residue and X represents any amino acid). Recognition by FWD1/beta-TrCP requires phosphorylation of the conserved serines in that motif. Here we show that FGD1, a Cdc42 guanine nucleotide exchange factor (GEF), is a novel target of the SCF(FWD1/beta-TrCP) ubiquitin ligase. A mutant FGD1 protein, FGD1(SA), in which both of the critical serine residues in the DSGPsiXS motif have been replaced by alanines, does not interact with FWD1/beta-TrCP and exhibits increased stability. Morphological changes induced by wild-type FGD1 (FGD1(WT)) are reduced by the co-expression of SCF(FWD1/beta-TrCP) whereas those induced by FGD1(SA) are not affected. FGD1(SA)-expressing cells show a higher level of cell motility than FGD1(WT)-expressing cells. We present a novel 'turning off' mechanism for the inactivation of FGD1, an upstream regulator for Cdc42.
... How GEFs are inactivated is largely unknown, however, in our previous report, we presented a unique mechanism for the inactivation of FGD1, a GEF for Cdc42 and known as the faciogenital dysplasia gene product, by the ubiquitin/proteasome system (Hayakawa et al . 2005). Ubiquitin-dependent proteolysis by the proteasome plays an essential role in a number of key biological processes, including cell cycle progression, transcription and signal transduction (Karin & Ben-Neriah 2000;Glickman & Ciechanover 2002). Protein ubiquitination usually requires three processes, those involving an ubiquitin-activatin ...
... e consensus sequence of DSG Ψ XS ( Ψ represents a hydrophobic residue and X represents any amino acid) (Karin & Ben-Neriah 2000;Mantovani & Banks 2003). Earlier we found that FGD1 is recognized by SCF FWD1/ β -TrCP through the phosphorylation of two serine residues in its DS 283 GIDS 287sequence, leading to degradation of the GEF by the proteasome (Hayakawa et al . 2005). ...
... In our previous study, we showed that FGD1, known as a GEF for Cdc42, is recognized by ubiquitin ligase SCF FWD1/ β -TrCP through the phosphorylation of serine residues in the conserved motif, that is, DS 283 GIDS 287 (Hayakawa et al . 2005). We found that FGD3, earlier identified as a homologue of FGD1 (Pasteris et al . 2000), also conserves this same motif, that is, DS 72 GIDS 76 (Fig. 1a). Sequence similarity between FGD1 and FGD3 is also observed in their DH and adjacent PH domain (i.e. amino acid residue identity is 70.0% and 60.6%, respectively) (Pasteris et al . 2000 ...
We previously demonstrated that FGD1, the Cdc42 guanine nucleotide exchange factor (GEF) responsible for faciogenital dysplasia, is targeted by the ubiquitin ligase SCF(FWD1/beta-TrCP) upon phosphorylation of two serine residues in its DSGIDS motif and subsequently degraded by the proteasome. Here we show that FGD3, which was identified as a homologue of FGD1 but has been poorly characterized, has conserved the same motif and is down-regulated similarly by SCF(FWD1/beta-TrCP). Although FGD3 and FGD1 share strikingly similar Dbl homology (DH) domains and adjacent pleckstrin homology (PH) domains, both of which are responsible for guanine nucleotide exchange, there also exist remarkable differences in their structures. Indeed, FGD1 and FGD3 induced significantly different morphological changes in HeLa Tet-Off cells: whereas FGD1 induced long finger-like protrusions, FGD3 induced broad sheet-like protrusions when the level of GTP-bound Cdc42 was significantly increased by the inducible expression of FGD3. Furthermore, FGD1 and FGD3 reciprocally regulated cell motility: when inducibly expressed in HeLa Tet-Off cells, FGD1 stimulated cell migration whereas FGD3 inhibited it. Thus we demonstrate that the highly homologous GEFs, FGD1 and FGD3 play different roles to regulate cellular functions but that their intracellular levels are tightly controlled by the same destruction pathway through SCF(FWD1/beta-TrCP).
... However, in the case of FGD4/Frabin, in addition to the direct activation of Cdc42 through the GEF domain, Rac-1 is also activated via indirect pathway(s) (Nakanishi et al. 2008). Another challenge is the tightly and temporally regulated expression of GEFs and their fast degradation as seen for FGD1 and FGD3 (Hayakawa et al. 2005;Hayakawa et al. 2008). Therefore, the detection of endogenous levels ...
... An addition, FGD1 (S205A) was not phosphorylated after EGF induction which confirmed the previous finding that the Ser 205 is the trigger for membrane localization upon growth factor stimulation. The same group has also investigated the degradation pathway of FGD1 in HeLa cells (Hayakawa et al. 2005). FGD1 is phosphorylated at two serine residues in the DSGIDS linker region (see domain structure on Figure Growth factors stimulate/induce FGD1/Fgd1by, for example, including phosphorylation at serine 205, which leads to the activation of both in CDC42/Cdc42 dependent and independent signaling cascades. ...
... For instance, whereas FGD1 stimulates directed cell migration, the FGD1 (S205I) mutant fails to do so (Oshima et al., 2011). Furthermore, the same mutation appears to confer increased stability to the protein (Hayakawa et al., 2005). Thus, the naturally occurring mutations identified in FGDY patients aid the study of FGD1 by pointing to specific residues that can be tested for their involvement in the regulation of FGD1 function. ...
... The FGD1 SH3-binding domain binds directly to the SH3 domain of mAbp1 or cortactin (Hou et al., 2003), and FGD1–cortactin binding promotes CDC42-independent actin assembly by the Arp2/3 complex (Kim et al., 2004). Phosphorylation at serine 205 by glycogen synthase kinase 3-b (GSK3-b) mediates the degradation of FGD1 at the proteasome (Hayakawa et al., 2005). binding of FGD1 to the multi-domain protein cortactin stimulates actin nucleation independently of the FGD1 guanine nucleotide exchange activity (Kim et al., 2004). ...
Disabling mutations in the FGD1 gene cause faciogenital dysplasia (also known as Aarskog-Scott syndrome), a human X-linked developmental disorder that results in disproportionately short stature, facial, skeletal and urogenital anomalies, and in a number of cases, mild mental retardation. FGD1 encodes the guanine nucleotide exchange factor FGD1, which is specific for the Rho GTPase cell division cycle 42 (CDC42). CDC42 controls cytoskeleton-dependent membrane rearrangements, transcriptional activation, secretory membrane trafficking, G1 transition during the cell cycle and tumorigenic transformation. The cellular mechanisms by which FGD1 mutations lead to the hallmark skeletal deformations of faciogenital dysplasia remain unclear, but the pathology of the disease, as well as some recent discoveries, clearly show that the protein is involved in the regulation of bone development. Two recent studies unveiled new potential functions of FGD1, in particular, its involvement in the regulation of the formation and function of invadopodia and podosomes, which are cellular structures devoted to degradation of the extracellular matrix in tumour and endothelial cells. Here, we discuss the hypothesis that FGD1 might be an important regulator of events controlling extracellular matrix remodelling and possibly cell invasion in physiological and pathological settings. Additionally, we focus on how studying the cell biology of FGD1 might help us to connect the dots that link CDC42 signalling with remodelling of the extracellular matrix (ECM) in physiology and complex diseases, while, at the same time, furthering our understanding of the pathogenesis of faciogenital dysplasia.
... Immunoprecipitation of detectable amounts of GEF16 initially proved difficult, which suggested that the protein could have a high rate of turnover. Previous studies have indicated that GEFs can be unstable (Hayakawa et al, 2005) and proteasomal degradation has been identified as a regulatory mechanism for some Rho GEFs such as the proto-oncogene Dbl (Kamynina et al, 2007). Also like Dbl, GEF16 has a stretch of proline, glutamic acid, serine and threonine residues (PEST sequence) at residues 222 -246. ...
... Indeed, MG132 is known to stabilise the interaction of the Rho-GEF Pbl/ECT2 with the E3 ligase E6AP (Reiter et al, 2006), and the suggestion that GEF levels may be regulated by the proteasome has been previously reported by others. Examples include the Cdc42-specific GEF h-PEM2, which is subject to proteasomal degradation via the HECT-type E3 ligase Smurf1 (Yamaguchi et al, 2008) and the Cdc42-specific GEF FWD1, which is stabilized in the presence of MG132 (Hayakawa et al, 2005). Indeed, we have shown that the full-length GEF16 protein is unstable in yeast cells and it contains PEST sequences, which have previously been shown to signal rapid degradation in yeast (Marchal et al, 1998). ...
Guanidine exchange factor (GEF)-catalysed activation of Rho proteins such as Cdc42 has been shown to have a crucial role in cellular transformation, malignant progression and invasion. We have previously shown that the HPV16 E6 oncoprotein binds to the PDZ domain protein Tax-interacting-protein 1 (Tip-1) and we now report identification and functional analysis of a novel Tip-1 binding GEF.
Yeast two-hybrid, in vitro pull-down, site-directed mutagenesis, semiquantitative PCR, co-immunoprecipitation and western blotting were used to identify/confirm novel Tip-1 binding partners and analyse cellular expression levels. In vitro kinetic analyses of recombinant proteins, siRNA gene silencing and in cell assays were used to measure Rho protein activation.
Tax-interacting-protein 1 was shown to interact with ARHGEF16 by its carboxyl PDZ binding motif. Levels of ARHGEF16 were increased in transformed and immortalised cells expressing ectopic HPV16 E6 and Cdc42 was co-immunoprecipitated by ARHGEF16 in the presence of high-risk HPV E6. In vitro kinetic analysis confirmed that recombinant ARHGEF16 activates Cdc42 and this was increased by the addition of recombinant Tip-1 and E6. Cells expressing HPV16 E6 had higher levels of Cdc42 activation, which was decreased by siRNA silencing of either Tip-1 or ARHGEF16.
These data suggest that HPV16 E6, Tip-1 and ARHGEF16 may cooperate to activate Cdc42 and support a potential link between the expression of HPV16 E6 and Cdc42 activation.
... FGD1 contains a DSGXXS motif and is recognized by b-TrCP when the two serine residues in this sequence are phosphorylated. This promotes ubiquitination and subsequent degradation of GEF by the proteasome [69]. The ubiquitination and degradation of GEF is regulated by GSK3 inhibitors. ...
Glycogen Synthase Kinase-3 (GSK3) was originally reported as a key enzyme of glucose homeostasis through regulation of the rate of glycogen synthesis. It has subsequently been found to influence most cellular processes, including growth, differentiation and death, as part of its role in modulating response to hormonal, nutritional and cellular stress stimuli. More than 100 protein targets for GSK3 have been proposed although only a small fraction of these have been convincingly validated in physiological cell systems. The effects of GSK3 phosphorylation on substrates includes alteration of enzyme activity, protein localisation, protein:protein interaction and protein stability. This latter form of regulation of GSK3 substrates is the focus of this review. There is an ever-growing list of GSK3 substrates that upon phosphorylation are targeted to the beta-transducin repeat containing protein (β-TrCP), thereby allowing ubiquitination of bound protein by cullin-1 and so initiating destruction at the proteasome. We propose the existence of a GSK3- β -TrCP 'destruction hit-list' that allows the co-ordinated removal (or stabilisation) of a set of proteins with a common physiological purpose, through control of GSK3. We identify 29 proteins where there is relatively strong evidence for regulation by a GSK3- β -TrCP axis and note common features of regulation and pathophysiology. Furthermore, we assess the potential of pre-phosphorylation (priming) of these targets (normally a prerequisite for GSK3 recognition) to provide a second layer of regulation delineated by the priming kinase that allows GSK3 to mark them for destruction. Finally, we discuss whether this knowledge improves options for therapeutic intervention.
... Fgd1 is a central regulator of extracellular matrix (ECM) remodeling. The proteasome degradation of fgd1 is mediated by phosphorylation at serine 205 via glycogen synthase kinase 3- (21). ...
The FGD1 gene encodes for a guanine exchange factor (GEF) protein which specifically activates the Rho GTPase Cdc42. For cellular migration, Cdc42 is a key molecular switch that regulates cytoskeleton restructuring, gene transcription, cellular morphology, extension, and cell adhesion. In the past decade, germline mutations in the FGD1 gene have been associated with a rare X-linked disorder known as faciogenital dysplasia (FGDY). Malformations are consistent with a loss of cellular migration during embryonic development. Insertion and deletion mutations in FGD1 result in a frameshift causing inactivation of fgd1 protein. Since Cdc42 is a key molecular switch in cytoskeletal restructuring and cell adhesion, the loss of fgd1 is postulated to attenuate Cdc42 mediated cellular migration in embryonic development. In metastatic tumors, Cdc42 modulates migration and invasiveness. Fgd1 overexpression has been found in infiltrating and poorly differentiated breast and invasive prostate tumors. Amplification at Xp11.21, the FGD1 gene locus, has been reported in several cancers. Sequencing analyses in numerous types of cancer have found missense mutations in the FGD1 gene in metastatic tumors. FGDY and cancer studies suggest that the germline and somatic changes downregulate or upregulate the FGD1 gene playing a key in the development of diseases.
... In further support of an oncogenic role, SKP2 has been reported to be overexpressed in various types of human tumour samples (TABLE 3), including lymphoma 84 , prostate cancer 85 , colorectal cancer 86 , melanoma 87 , nasopharyngeal carcinoma 88 , pancreatic cancer 89 and breast carcinoma 90,91 . Moreover, SKP2 might function as a prognostic marker for various human cancers. ...
F-box proteins, which are the substrate-recognition subunits of SKP1-cullin 1-F-box protein (SCF) E3 ligase complexes, have pivotal roles in multiple cellular processes through ubiquitylation and subsequent degradation of target proteins. Dysregulation of F-box protein-mediated proteolysis leads to human malignancies. Notably, inhibitors that target F-box proteins have shown promising therapeutic potential, urging us to review the current understanding of how F-box proteins contribute to tumorigenesis. As the physiological functions for many of the 69 putative F-box proteins remain elusive, additional genetic and mechanistic studies will help to define the role of each F-box protein in tumorigenesis, thereby paving the road for the rational design of F-box protein-targeted anticancer therapies.
... It is yet to be established whether Smurf–mediated regulation these two Rho GTPases occurs in a coordinated manner. In addition to Smurf, Cdc42 activity could be regulated by ubiquitination and or proteasomal degradation of its GEFs, FGD1 and FGD3 by E3 ligase SCF FWD1/β-TrCP (Hayakawa, et al., 2005;Hayakawa, et al., 2008). There is an interesting interplay between the regulatory effects of ubiquitination and phosphorylation. ...
... Cbl escapes this inhibition by promoting the down-regulation of -Pix, liberating Cbl for EGFR attenuation and reducing the exchange activity available for Cdc42 [55,57]. Other members of the pool of Cdc42 GEFs are also targets for degradation; Faciogenital Dysplacias (FGDs) 1 and 3 and h-PEM2 are subject to TrCP and Smurf1 mediated ubiquitylation and degradation respectively [58][59][60]. ...
A variety of post-translational modifications such as phosphorylation, acetylation and ubiquitylation transduce cellular signals, which culminate in changes in gene transcription. In this article we examine the ways in which selective protein degradation provides an extra dimension to the regulation of such signalling cascades. We discuss (i) how both lysosomal and proteasomal systems are used to attenuate kinase and rho family GTPase signalling, thereby coupling activation with degradation, (ii) signal propagation contingent upon the selective degradation of inhibitory components, exemplified by the degradation of IκB to activate NF-κB signalling, and (iii) tonic suppression of signalling pathways by turnover of the transcription factors β-catenin and p53.
... complex (FWD1/β-TrCP), but with different functions since FGD1 stimulates, while FGD3 inhibits cell migration. 98,99 Moreover, EGF stimulation can translocate FGD1 but not FGD3 to the plasma membrane, further suggesting that the ubiquitin modification plays a substrate specific role in the EGF signaling pathway. 100 Hence, the ubiquitination of Ras and Rho family GEFs and GAPs play a critical role in regulating the activation of GTPases, and since many of these GEFs and GAPs can regulate multiple GTPases, targeting one of these proteins may have additional effects. ...
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.
... It has been shown that multisite phosphorylation of Sic1 and Tec1 triggers their recognition by the F-box adaptor protein Cdc4 of the SCF E3 ubiquitin ligase complex (Deshaies and Joazeiro, 2009). SCF-dependent degradation has also been shown for the mammalian Cdc42 GEFs FGD1 and FGD3 (Hayakawa et al., 2005(Hayakawa et al., , 2008. Thus we speculate that in U. maydis phosphorylated Cdc24 is recognized by a yet-uncharacterized ubiquitin ligase that targets Cdc24 for ubiquitin dependent degradation. ...
Dimorphic switching from budding to filamentous growth is a characteristic feature of many pathogenic fungi. In the fungal model organism Ustilago maydis polarized growth is induced by the multiallelic b mating type locus and requires the Rho family GTPase Rac1. Here we show that mating type-induced polarized growth involves negative feedback regulation of the Rac1-specific guanine nucleotide exchange factor (GEF) Cdc24. Although Cdc24 is essential for polarized growth, its concentration is drastically diminished during filament formation. Cdc24 is part of a protein complex that also contains the scaffold protein Bem1 and the PAK kinase Cla4. Activation of Rac1 results in Cla4-dependent degradation of the Rac1-GEF Cdc24, thus creating a regulatory negative feedback loop. We generated mutants of Cdc24 that are resistant to Cla4-dependent destruction. Expression of stable Cdc24 variants interfered with filament formation, indicating that negative feedback regulation of Cdc24 is critical for the establishment of polarized growth.
... Sin embargo, la conexión mecanística entre GSK-3 y Nrf2 no se conoce. Un gran número de estudios han demostrado que GSK-3 dirige la ubiquitinación y degradación proteasómica de varios factores de transcripción y de otras proteínas, que incluyen Snail (Zhou et al., 2004), β-catenina (Papkoff et al., 1998), Gli2 y Gli3 (Pan et al., 2006; Wang et al., 2006), Xom (Zhu et al., 2002), Cdc25a (Kang et al., 2008), FGD1 y 3 (Hayakawa et al., 2005; Hayakawa et al., 2008) y Mcl-1 (Ding et al., 2007Huang et al., 2000; Cullinan et al., 2003; Numazawa et al., 2003; Papaiahgari et al., 2004; Jain et al., 2006; Xu et al., 2006; Yuan et al., 2006; Jain et al., 2007; Pi et al., 2007; Apopa et al., 2008; Sun et al., 2009). Como respuesta al estrés oxidativo, la cascada de señalizaciones mediada por la fosfatidilinositol 3-quinasa (PI3K) produce la despolimerización de los microfilamentos de actina, facilitando la translocación nuclear de Nrf2 (Lee et al., 2004). ...
Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 22 de Octubre de 2010
... This is part of a negative feedback mechanism for EGF-induced signalling, as inactive Cdc42 fails to sequester Cbl, which allows Cbl-mediated degradation of the EGF receptor. For Cdc42, inhibition of its signalling by targeting a GEF appears an important pathway, as Smurf-1 can ubiquitylate the Cdc42 GEF hPEM-2 but not Cdc42 itself (Yamaguchi et al., 2008) ( and Rbx-1 inhibits Cdc42 activation by targeting its GEFs FGD1 and FGD3 for ubiquitylation (Hayakawa et al., 2005; Hayakawa et al., 2008). Thus, while the exact underlying mechanisms of UPS targeting of Cdc42 remain to be elucidated, a growing number of studies indicate that the UPS controls Cdc42 through regulating the available pool of Cdc42 GEFs (Fig. 4A). ...
Rho-like guanosine triphosphatases (RhoGTPases) control many aspects of cellular physiology through their effects on the actin cytoskeleton and on gene transcription. Signalling by RhoGTPases is tightly coordinated and requires a series of regulatory proteins, including guanine-nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs) and guanine-nucleotide dissociation inhibitors (GDIs). GEFs and GAPs regulate GTPase cycling between the active (GTP-bound) and inactive (GDP-bound) states, whereas GDI is a cytosolic chaperone that binds inactive RhoGTPases. Like many other proteins, RhoGTPases are subject to degradation following the covalent conjugation of ubiquitin. There have been increasing indications that ubiquitylation of small GTPases occurs in a regulated fashion, primarily upon activation, and is an important means to control signalling output. Recent work has identified cellular proteins that control RasGTPase and RhoGTPase ubiquitylation and degradation, allowing us to amend the canonical model for GTPase (in)activation. Moreover, accumulating evidence for indirect regulation of GTPase function through the ubiquitylation of GTPase regulators makes this post-translational modification a key feature of GTPase-dependent signalling pathways. Here, we will discuss these recent insights into the regulation of RhoGTPase ubiquitylation and their relevance for cell signalling.
Colorectal tumorigenesis is driven by alterations in genes and proteins responsible for cancer initiation, progression, and invasion. This multistage process is based on a dense network of protein–protein interactions (PPIs) that become dysregulated as a result of changes in various cell signaling effectors. PPIs in signaling and regulatory networks are known to be mediated by short linear motifs (SLiMs), which are conserved contiguous regions of 3–10 amino acids within interacting protein domains. SLiMs are the minimum sequences required for modulating cellular PPI networks. Thus, several in silico approaches have been developed to predict and analyze SLiM-mediated PPIs. In this review, we focus on emerging evidence supporting a crucial role for SLiMs in driver pathways that are disrupted in colorectal cancer (CRC) tumorigenesis and related PPI network alterations. As a result, SLiMs, along with short peptides, are attracting the interest of researchers to devise small molecules amenable to be used as novel anti-CRC targeted therapies. Overall, the characterization of SLiMs mediating crucial PPIs in CRC may foster the development of more specific combined pharmacological approaches.
Within the 69 identified putative F-box proteins in the human genome, besides the FBXW subfamily (Chap. 2) and the FBXL subfamily (Chap. 3), the remaining 36 F-box proteins were designated as F-box only (FBXO) proteins. It is noteworthy that the FBXO subfamily proteins have been identified to contain the F-box motif in its N-terminus and various types of functional domains in its C-terminus that mediate substrate binding. Unlike FBXW proteins with the WD40 repeats motif and FBXL proteins with the LRR motif, most of the functional domains in FBXO proteins have yet to be fully characterized. Compared to the FBXW and FBXL subfamilies, the FBXO subfamily is more diversified with 21 functional homology domains identified within the FBXO subfamily (Fig. 4.1).
In this chapter, we focus our discussion on the recent genetic, pathological as well as the biochemical evidence suggesting possible tumor suppressor or oncogenic roles for FBXO subfamily members (Tables 4.1 and 4.3). As stated in previous chapters, given the fact that physiological evidence (mouse modeling results) is considered as the strongest supportive data to implicate any given F-box protein in tumorigenesis (Table 4.2), we limit our discussion to those FBXO members with available mouse genetic models.
Nuclear factor (erythroid-derived 2)-like 2 (NRF2) is a master regulator of cellular homeostasis that controls the expression of more than 1% human genes related to biotransformation reactions, redox homeostasis, energetic metabolism, DNA repair and proteostasis. Its activity has a tremendous impact on physiology and pathology and therefore it is very tightly regulated mainly at the level of protein stability. In addition to the very well-established regulation by the ubiquitin E3 ligase adapter KEAP1, recent advances have identified a novel mechanism based on signaling pathways that regulate glycogen synthase kinse-3 (GSK-3). This kinase phosphorylates specific serine residues in the Neh6 domain of NRF2 to create a degradation domain that is then recognized by the ubiquitin ligase adapter β-TrCP and tagged for proteasome degradation by a Cullin1/Rbx1 complex. Here we review the mechanistic elements and the signaling pathways that participate in this regulation by GSK-3/β-TrCP. These pathways include those activated by ligands of tyrosine kinase, G protein coupled, metabotropic and ionotropic receptors that activate phosphatidyl inositol 3-kinase (PI3K)/ATK and by the canonical WNT signaling pathway where a fraction of NRF2 interacts with Axin1/GSK-3. Considering that free NRF2 protein is localized in the nucleus, we propose a model termed "double flux controller" to explain how KEAP1 and β-TrCP coordinate the stability of NRF2 under several scenarios. The GSK-3/β-TrCP axis provides a novel therapeutic strategy to modulate NRF2 activity.
Copyright © 2015. Published by Elsevier Inc.
Approximately 30% of autogenous vein grafts develop luminal narrowing and fail because of intimal hyperplasia or negative remodeling. We previously found that vein graft cells from patients who later develop stenosis proliferate more in vitro in response to growth factors than cells from patients who maintain patent grafts. To discover novel determinants of vein graft outcome, we have analyzed gene expression profiles of these cells using a systems biology approach to cluster the genes into modules by their coexpression patterns and to correlate the results with growth data from our prior study and with new studies of migration and matrix remodeling.
RNA from 4-hour serum- or platelet-derived growth factor (PDGF)-BB-stimulated human saphenous vein cells obtained from the outer vein wall (20 cell lines) was used for microarray analysis of gene expression, followed by weighted gene coexpression network analysis. Cell migration in microchemotaxis chambers in response to PDGF-BB and cell-mediated collagen gel contraction in response to serum were also determined. Gene function was determined using short-interfering RNA to inhibit gene expression before subjecting cells to growth or collagen gel contraction assays. These cells were derived from samples of the vein grafts obtained at surgery, and the long-term fate of these bypass grafts was known.
Neither migration nor cell-mediated collagen gel contraction showed a correlation with graft outcome. Although 1188 and 1340 genes were differentially expressed in response to treatment with serum and PDGF, respectively, no single gene was differentially expressed in cells isolated from patients whose grafts stenosed compared with those that remained patent. Network analysis revealed four unique groups of genes, which we term modules, associated with PDGF responses, and 20 unique modules associated with serum responses. The "yellow" and "skyblue" modules, from PDGF and serum analyses, respectively, correlated with later graft stenosis (P = .005 and P = .02, respectively). In response to PDGF, yellow was also associated with increased cell growth. For serum, skyblue was also associated with inhibition of collagen gel contraction. The hub genes for yellow and skyblue (ie, the gene most connected to other genes in the module), scavenger receptor class A member 5 (SCARA5) and suprabasin (SBSN), respectively, were tested for effects on proliferation and collagen contraction. Knockdown of SCARA5 increased proliferation by 29.9% ± 7.8% (P < .01), whereas knockdown of SBSN had no effect. Knockdown of SBSN increased collagen gel contraction by 24.2% ± 8.6% (P < .05), whereas knockdown of SCARA5 had no effect.
Using weighted gene coexpression network analysis of cultured vein graft cell gene expression, we have discovered two small gene modules, which comprise 42 genes, that are associated with vein graft failure. Further experiments are needed to delineate the venous cells that express these genes in vivo and the roles these genes play in vein graft healing, starting with the module hub genes SCARA5 and SBSN, which have been shown to have modest effects on cell proliferation or collagen gel contraction.
Copyright © 2015 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.
Cellular proteins are degraded by the ubiquitin-proteasome system (UPS) in a precise and timely fashion. Such precision is conferred by the high substrate specificity of ubiquitin ligases. Identification of substrates of ubiquitin ligases is crucial not only to unravel the molecular mechanisms by which the UPS controls protein degradation but also for drug discovery purposes because many established UPS substrates are implicated in disease. We developed a combined bioinformatics and affinity purification– mass spectrometry (AP-MS) workflow for the system-wide identification of substrates of SCF bTrCP , a member of the SCF family of ubiquitin ligases. These ubiquitin ligases are characterized by a multi-subunit architecture typically consisting of the invariable subunits Rbx1, Cul1, and Skp1, and one of 69 F-box proteins. The F-box protein of this member of the family is bTrCP. SCF bTrCP binds, through the WD40 repeats of bTrCP, to the DpSGXX(X)pS diphosphorylated motif in its substrates. We recovered 27 pre-viously reported SCF bTrCP substrates, of which 22 were verified by two independent statistical proto-cols, thereby confirming the reliability of this approach. In addition to known substrates, we identified 221 proteins that contained the DpSGXX(X)pS motif and also interacted specifically with the WD40 repeats of bTrCP. Thus, with SCF bTrCP , as the example, we showed that integration of structural infor-mation, AP-MS, and degron motif mining constitutes an effective method to screen for substrates of ubiquitin ligases. INTRODUCTION
Regulation of transcription factor Nrf2 (NF-E2-related factor 2) involves redox-sensitive proteasomal degradation via the
E3 ubiquitin ligase Keap1/Cul3. However, Nrf2 is controlled by other mechanisms that have not yet been elucidated. We now
show that glycogen synthase kinase 3 (GSK-3) phosphorylates a group of Ser residues in the Neh6 domain of mouse Nrf2 that
overlap with an SCF/β-TrCP destruction motif (DSGIS, residues 334 to 338) and promotes its degradation in a Keap1-independent
manner. Nrf2 was stabilized by GSK-3 inhibitors in Keap1-null mouse embryo fibroblasts. Similarly, an Nrf2ΔETGE mutant, which cannot be degraded via Keap1, accumulated when GSK-3 activity was blocked. Phosphorylation of a Ser cluster
in the Neh6 domain of Nrf2 stimulated its degradation because a mutant Nrf2ΔETGE 6S/6A protein, lacking these Ser residues, exhibited a longer half-life than Nrf2ΔETGE. Moreover, Nrf2ΔETGE 6S/6A was insensitive to β-TrCP regulation and exhibited lower levels of ubiquitination than Nrf2ΔETGE. GSK-3β enhanced ubiquitination of Nrf2ΔETGE but not that of Nrf2ΔETGE 6S/6A. The Nrf2ΔETGE protein but not Nrf2ΔETGE 6S/6A coimmunoprecipitated with β-TrCP, and this association was enhanced by GSK-3β. Our results show for the first time that Nrf2
is targeted by GSK-3 for SCF/β-TrCP-dependent degradation. We propose a “dual degradation” model to describe the regulation
of Nrf2 under different pathophysiological conditions.
In the absence of crystallographic data, NMR has emerged as the best way to define protein-ligand interactions. Using NMR experiments based on magnetization transfer, one can sort bound from unbound molecules, estimate the dissociation constant, identify contacts implied in the binding, characterize the structure of the bound ligand and conduct ligand competition assays.
FGD1 encodes a guanine nucleotide exchange factor for Cdc42. Mutations in the FGD1 gene are responsible for an X-linked disorder known as Aarskog-Scott syndrome (AAS). While most mutations were found in the catalytic region, which consists of Dbl homology (DH) domain and adjacent pleckstrin homology (PH) domain, a missense mutation in the proline-rich domain is also found in a patient with typical clinical features as AAS. In this mutant FGD1, the serine residue at 205 is replaced with isoleucine. We recently demonstrated that FGD1 translocated to the membrane in response to extracellular stimuli such as epidermal growth factor (EGF) whereas FGD1 with S(205)/I substitution did not. Here we show that the proline-rich domain is critical for FGD1-induced directionally persistent cell migration. When inducibly expressed in HeLa Tet-Off cells, FGD1 stimulates directional migration whereas FGD1 with S(205)/I substitution does not affect it. We further demonstrate that FGD1 augments EGF-stimulated c-Jun NH(2)-terminal kinase (JNK) activation. In the presence of JNK inhibitor SP600125, motility of FGD1-expressing cells is significantly impaired, indicating a critical role of JNK in cell migration. However, FGD3, an FGD1 homologue lacking the proline-rich domain, and FGD1 with S(205)/I substitution augment EGF-stimulated JNK activation similarly to FGD1, suggesting that the proline-rich domain is not involved in the regulation of JNK. Finally, we show that FGD1, but not FGD1 with S(205)/I substitution, is phosphorylated in response to EGF, suggesting that the phosphorylation of S(205) may trigger the FGD1 translocation to the leading edge membrane and enable cells to undergo directional migration.
Sumoylation is a posttranslational modification process in which SUMO proteins are covalently and reversibly conjugated to their targets via enzymatic cascade reactions. Since the discovery of SUMO-1 in 1996, the SUMO pathway has garnered increased attention due to its role in a number of important biological activities such as cell cycle progression, epigenetic modulation, signal transduction, and DNA replication/repair, as well as its potential implication in human pathogenesis such as in cancer development and metastasis, neurodegenerative disorders and craniofacial defects. The role of the SUMO pathway in regulating cardiogenic gene activity, development and/or disorders is just emerging. Our review is based on recent advances that highlight the regulation of cardiac gene activity in cardiac development and disease by the SUMO conjugation pathway.
We previously demonstrated that FGD1, the Cdc42 guanine nucleotide exchange factor (GEF) responsible for faciogenital dysplasia, and its homologue FGD3 are targeted by the ubiquitin ligase SCF(FWD1) upon phosphorylation of two serine residues in their DSGIDS motif and subsequently degraded by the proteasome. FGD1 and FGD3 share highly homologous Dbl homology (DH) and adjacent pleckstrin homology (PH) domains, both of which are responsible for GEF activity. However, their function and regulation are remarkably different. Here we demonstrate extracellular signal-responsive translocation of FGD1, but not FGD3. During the wound-healing process, translocation of FGD1 to the leading edge membrane occurs in cells facing to the wound. Furthermore, epidermal growth factor (EGF) stimulates the membrane translocation of FGD1, but not FGD3. As the most striking difference, FGD3 lacks the N-terminal proline-rich domain that is conserved in FGD1, indicating that proline-rich domain may play a crucial role in signal-responsive translocation of FGD1. Indeed, there is a faciogenital dysplasia patient who has a missense mutation in proline-rich domain of FGD1, by which the serine residue at position 205 is substituted with isoleucine. When expressed in cells, the mutant FGD1 with S(205)/I substitution fails to translocate to the membrane in response to the mitogenic stimuli. Thus we present a novel mechanism by which the activity of FGD1, a GEF for Cdc42, is temporally and spatially regulated in cells.
Glycogen synthase kinase-3 (GSK3) plays important roles in numerous signaling pathways that regulate a variety of cellular processes including cell proliferation, differentiation, apoptosis and embryonic development. In the canonical Wnt signaling pathway, GSK3 phosphorylation mediates proteasomal targeting and degradation of beta-catenin via the destruction complex. We recently reported a biochemical screen that discovered multiple additional protein substrates whose stability is regulated by Wnt signaling and/or GSK3 and these have important implications for Wnt/GSK3 regulation of different cellular processes.(1) In this article, we also present a bio-informatics based screen for proteins whose stability may be controlled by GSK3 and beta-Trcp, the SCF E3 ubiquitin ligase that is responsible for beta-catenin degradation in the Wnt signaling pathway. Furthermore, we review various GSK3 regulated proteolysis substrates described in the literature. We propose that GSK3 phosphorylation dependent proteolysis is a widespread mechanism that the cell employs to regulate a variety of cell processes in response to signals.
In the current study, a novel coreceptor-specific cell-cell fusion (CCF) assay system is reported. The system possesses the following features: dual CCR5-dependent and CXCR4-dependent CCF assays, all stable cell lines, inducible expression of gp160 to minimize cytotoxicity, robust luciferase reporter, and 384-well format. These assays have been validated using various known HIV entry inhibitors targeting various stages of the HIV entry/fusion process, including fusion inhibitors, gp120 inhibitors, CCR5 antagonists, CCR5 antibodies, and CXCR4 antagonists. IC50 data generated from this assay system were well correlated to that from the antiviral assays. The effects of DMSO on this assay system were assessed, and a 2- to 3-fold increase in luciferase activity was observed in the presence of 0.05% to 2% DMSO. Although cell-cell fusion efficiency was enhanced, no changes in drug response kinetics for entry inhibitors were found in the presence of 0.1% or 0.5% DMSO. This assay system has been successfully used for the identification and characterization of thousands of CCR5 inhibitors.
The dbl proto-oncogene product is a prototype of a growing family of guanine nucleotide exchange factors (GEFs) that stimulate the
activation of small GTP-binding proteins from the Rho family. Mutations that result in the loss of proto-Dbl's amino terminus
produce a variant with constitutive GEF activity and high oncogenic potential. Here, we show that proto-Dbl is a short-lived
protein that is kept at low levels in cells by efficient ubiquitination and degradation. The cellular fate of proto-Dbl is
regulated by interactions with the chaperones Hsc70 and Hsp90 and the protein-ubiquitin ligase CHIP, and these interactions
are mediated by the spectrin domain of proto-Dbl. We show that CHIP is the E3 ligase responsible for ubiquitination and proteasomal
degradation of proto-Dbl, while Hsp90 functions to stabilize the protein. Onco-Dbl, lacking the spectrin homology domain,
cannot bind these regulators and therefore accumulates in cells at high levels, leading to persistent stimulation of its downstream
signaling pathways.
Smurf1, a member of HECT-type E3 ubiquitin ligases, regulates cell polarity and protrusive activity by inducing ubiquitination and subsequent proteasomal degradation of the small GTPase RhoA. We report here that hPEM-2, a guanine nucleotide exchange factor for the small GTPase Cdc42, is a novel target of Smurf1. Pulse-chase labeling and a ubiquitination experiment using MG132, a proteasomal inhibitor, indicate that Smurf1 induces proteasomal degradation of hPEM-2 in cells. GST pull-down assays with heterologously expressed firefly luciferase-fusion proteins that include partial sequences of hPEM-2 reveal that part of the PH domain (residues 318-343) of hPEM-2 is sufficient for binding to Smurf1. In contrast, the hPEM-2 binding domain in Smurf1 was mapped to the C2 domain. Although it has been reported that the binding activities of some C2 domains to target proteins are regulated by Ca2+, Smurf1 interacts with hPEM-2 in a Ca2+-independent manner. Our discovery that hPEM-2 is, in addition to RhoA, a target protein of Smurf1 suggests that Smurf1 plays a crucial role in the spatiotemporal regulation of Rho GTPase family members.
Evolutionary conserved members of the Ras superfamily of small GTP-binding proteins function as binary molecular switches to control diverse biological processes. In the context of cellular signaling, these include functions in exocytic and endocytic trafficking, as well as roles in signal relay downstream of various cell surface receptors. We previously reviewed roles played by the large family of GTPase, activating proteins in these processes. In this companion review, we highlight recent findings relating to the regulation of another major class of Ras superfamily regulatory proteins, the guanine nucleotide exchange factors.
We previously demonstrated that both Tiam1, an activator of Rac, and constitutively active V12Rac promote E-cadherin-mediated cell-cell adhesion in epithelial Madin Darby canine kidney (MDCK) cells. Moreover, Tiam1 and V12Rac inhibit invasion of Ras-transformed, fibroblastoid MDCK-f3 cells by restoring E-cadherin-mediated cell-cell adhesion. Here we show that the Tiam1/Rac-induced cellular response is dependent on the cell substrate. On fibronectin and laminin 1, Tiam1/Rac signaling inhibits migration of MDCK-f3 cells by restoring E-cadherin-mediated cell- cell adhesion. On different collagens, however, expression of Tiam1 and V12Rac promotes motile behavior, under conditions that prevent formation of E-cadherin adhesions. In nonmotile cells, Tiam1 is present in adherens junctions, whereas Tiam1 localizes to lamellae of migrating cells. The level of Rac activation by Tiam1, as determined by binding to a glutathione-S-transferase- PAK protein, is similar on fibronectin or collagen I, suggesting that rather the localization of the Tiam1/Rac signaling complex determines the substrate-dependent cellular responses. Rac activation by Tiam1 requires PI3-kinase activity. Moreover, Tiam1- but not V12Rac-induced migration as well as E-cadherin-mediated cell- cell adhesion are dependent on PI3-kinase, indicating that PI3-kinase acts upstream of Tiam1 and Rac.
Chemotaxis-competent cells respond to a variety of ligands by activating second messenger pathways leading to changes in the actin/myosin cytoskeleton and directed cell movement. We demonstrate that Dictyostelium Akt/PKB, a homologue of mammalian Akt/PKB, is very rapidly and transiently activated by the chemoattractant cAMP. This activation takes place through G protein-coupled chemoattractant receptors via a pathway that requires homologues of mammalian p110 phosphoinositide-3 kinase. pkbA null cells exhibit aggregation-stage defects that include aberrant chemotaxis, a failure to polarize properly in a chemoattractant gradient and aggregation at low densities. Mechanistically, we demonstrate that the PH domain of Akt/PKB fused to GFP transiently translocates to the plasma membrane in response to cAMP with kinetics similar to those of Akt/PKB kinase activation and is localized to the leading edge of chemotaxing cells in vivo. Our results indicate Akt/PKB is part of the regulatory network required for sensing and responding to the chemoattractant gradient that mediates chemotaxis and aggregation.
Morphologic polarity is necessary for chemotaxis of mammalian cells. As a probe of intracellular signals responsible for this
asymmetry, the pleckstrin homology domain of the AKT protein kinase (or protein kinase B), tagged with the green fluorescent
protein (PHAKT-GFP), was expressed in neutrophils. Upon exposure of cells to chemoattractant, PHAKT-GFP is recruited selectively
to membrane at the cell's leading edge, indicating an internal signaling gradient that is much steeper than that of the chemoattractant.
Translocation of PHAKT-GFP is inhibited by toxin-B from Clostridium difficile, indicating that it requires activity of one or more Rho guanosine triphosphatases.
The directed movement of fibroblasts towards locally released platelet-derived growth factor (PDGF) is a critical event in wound healing. Although recent studies have implicated polarized activation of phosphoinositide (PI) 3-kinase in G protein-mediated chemotaxis, the role of 3' PI lipids in tyrosine kinase-triggered chemotaxis is not well understood. Using evanescent wave microscopy and green fluorescent protein-tagged Akt pleckstrin homology domain (GFP-AktPH) as a molecular sensor, we show that application of a shallow PDGF gradient triggers a markedly steeper gradient in 3' PI lipids in the adhesion zone of fibroblasts. Polar GFP-AktPH gradients, as well as a new type of radial gradient, were measured from front to rear and from the periphery to the center of the adhesion zone, respectively. A strong spatial correlation between polarized 3' PI production and rapid membrane spreading implicates 3' PI lipids as a direct mediator of polarized migration. Analysis of the temporal changes of 3' PI gradients in the adhesion zone revealed a fast diffusion coefficient (0.5 microm(2)/s) and short lifetime of 3' PIs of <1 min. Together, this study suggests that the tyrosine kinase-coupled directional movement of fibroblasts and their radial membrane activity are controlled by local generation and rapid degradation of 3' PI second messengers.
FGD1, the gene responsible for the inherited disease faciogenital dysplasia, encodes a guanine nucleotide exchange factor (GEF) that specifically activates the p21 GTPase Cdc42. In order, FGD1 is composed of a proline-rich N-terminal region, adjacent GEF and pleckstrin homology (PH) domains, a FYVE-finger domain and a second C-terminal PH domain (PH2), structural motifs involved in signaling and subcellular localization. Fgd1, the mouse FGD1 ortholog, is expressed in regions of active bone formation within osteoblasts and in the osteoblast-like cell line MC3T3-E1, a finding consistent with its role in skeletal formation. Here, we use subcellular fractionation studies to show that endogenous Fgd1 protein is localized in the cytosolic and Golgi and plasma membrane fractions of mouse calvarial cells. Immunocytochemical studies performed with osteoblast-like MC3T3-E1 cells and other mammalian cell lines confirm the localization of Fgd1 and show that the proline-rich N-terminal region is necessary and sufficient for Fgd1 subcellular localization to the plasma membrane and Golgi complex. In contrast, the FYVE-finger and PH2 domains do not appear to direct the localization of Fgd1 or the activation of Cdc42. In addition, microinjection studies indicate that the N-terminal Fgd1 domain inhibits filopodia formation, suggesting that this region down-regulates GEF function. These results characterize the function of the Fgd1 domains for both protein localization and Cdc42 activation and indicate that the Fgd1 Cdc42GEF protein is involved in the regulation of Cdc42 activity at the subcortical actin cytoskeleton and Golgi complex.
Cell polarity is a fundamental property of all cells. In higher eukaryotes, the small GTPase Cdc42, acting through a Par6-atypical protein kinase C (aPKC) complex, is required to establish cellular asymmetry during epithelial morphogenesis, asymmetric cell division and directed cell migration. However, little is known about what lies downstream of this complex. Here we show, through the use of primary rat astrocytes in a cell migration assay, that Par6-PKCzeta interacts directly with and regulates glycogen synthase kinase-3beta (GSK-3beta) to promote polarization of the centrosome and to control the direction of cell protrusion. Cdc42-dependent phosphorylation of GSK-3beta occurs specifically at the leading edge of migrating cells, and induces the interaction of adenomatous polyposis coli (Apc) protein with the plus ends of microtubules. The association of Apc with microtubules is essential for cell polarization. We conclude that Cdc42 regulates cell polarity through the spatial regulation of GSK-3beta and Apc. This role for Apc may contribute to its tumour-suppressor activity.
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 discs large (hDlg) tumor suppressor is intimately involved in the control of cell contact, polarity, and proliferation by interacting with several components of the epithelial junctional complex and with the APC tumor suppressor protein. In epithelial cells, hDlg protein stability is regulated through the ubiquitin-proteasome pathway: hDlg is actively degraded in isolated cells, whereas it accumulates upon cell-cell contact. During neoplastic transformation of epithelial cells, loss of the differentiated morphology and progression toward a metastatic phenotype correlate with down-regulation of hDlg levels and loss of contact-dependent stabilization. Here we show that upon hyperphosphorylation, hDlg interacts with the beta-TrCP ubiquitin ligase receptor through a DSGLPS motif within its Src homology 3 domain. As a consequence, overexpression of beta-TrCP enhances ubiquitination of Dlg protein and decreases its stability, whereas a dominant negative beta-TrCP mutant inhibits this process. Furthermore, a mutant Dlg protein that is unable to bind beta-TrCP displays a higher protein stability and is insensitive to beta-TrCP. Using RNA interference, we also demonstrate that endogenous beta-TrCP regulates hDlg protein levels in epithelial cells. Finally, we show that beta-TrCP selectively induces the degradation of the membrane-cytoplasmic pool, without affecting the nuclear pool of hDlg.
FGD1, the gene responsible for the inherited disease faciogenital dysplasia, encodes a guanine nucleotide exchange factor (GEF) that specifically activates the p21 GTPase Cdc42. In order, FGD1 is composed of a proline-rich N-terminal region, adjacent GEF and pleckstrin homology (PH) domains, a FYVE-finger domain and a second C-terminal PH domain (PH2), structural motifs involved in signaling and subcellular localization. Fgd1, the mouse FGD1 ortholog, is expressed in regions of active bone formation within osteoblasts and in the osteoblast-like cell line MC3T3-E1, a finding consistent with its role in skeletal formation. Here, we use subcellular fractionation studies to show that endogenous Fgd1 protein is localized in the cytosolic and Golgi and plasma membrane fractions of mouse calvarial cells. Immunocytochemical studies performed with osteoblast-like MC3T3-E1 cells and other mammalian cell lines confirm the localization of Fgd1 and show that the proline-rich N-terminal region is necessary and sufficient for Fgd1 subcellular localization to the plasma membrane and Golgi complex. In contrast, the FYVE-finger and PH2 domains do not appear to direct the localization of Fgd1 or the activation of Cdc42. In addition, microinjection studies indicate that the N-terminal Fgd1 domain inhibits filopodia formation, suggesting that this region down-regulates GEF function. These results characterize the function of the Fgd1 domains for both protein localization and Cdc42 activation and indicate that the Fgd1 Cdc42GEF protein is involved in the regulation of Cdc42 activity at the subcortical actin cytoskeleton and Golgi complex.
HIV-1 Vpu interacts with CD4 in the endoplasmic reticulum and triggers CD4 degradation, presumably by proteasomes. Human βTrCP identified by interaction with Vpu connects CD4 to this proteolytic machinery, and CD4–Vpu–βTrCP ternary complexes have been detected by coimmunoprecipitation. βTrCP binding to Vpu and its recruitment to membranes require two phosphoserine residues in Vpu essential for CD4 degradation. In βTrCP, WD repeats at the C terminus mediate binding to Vpu, and an F box near the N terminus is involved in interaction with Skp1p, a targeting factor for ubiquitin-mediated proteolysis. An F-box deletion mutant of βTrCP had a dominant-negative effect on Vpu-mediated CD4 degradation. These data suggest that βTrCP and Skp1p represent components of a novel ER-associated protein degradation pathway that mediates CD4 proteolysis.
β-catenin is a pivotal player in the signaling pathway initiated by Wnt proteins, mediators of several developmental processes. β-catenin activity is controlled by a large number of binding partners that affect the stability and the localization of β-catenin and is thereby able to participate in such varying processes as gene expression and cell adhesion. Activating mutations in β-catenin and in components regulating its stability can contribute to the formation of certain tumors.
Beta-catenin is a pivotal player in the signaling pathway initiated by Wnt proteins, mediators of several developmental processes. beta-catenin activity is controlled by a large number of binding partners that affect the stability and the localization of beta-catenin and is thereby able to participate in such varying processes as gene expression and cell adhesion. Activating mutations in beta-catenin and in components regulating its stability can contribute to the formation of certain tumors.
Faciogenital dysplasia (FGDY), also known as Aarskog-Scott syndrome, is an X-linked developmental disorder characterized by disproportionately short stature and by facial, skeletal, and urogenital anomalies. Molecular genetic analyses mapped FGDY to chromosome Xp11.21. To clone this gene, YAC clones spanning an FGDY-specific translocation breakpoint were isolated. An isolated cDNA, FGD1, is disrupted by the breakpoint, and FGD1 mutations cosegregate with the disease. FGD1 codes for a 961 amino acid protein that has strong homology to Rho/Rac guanine nucleotide exchange factors (GEFs), contains a cysteine-rich zinc finger-like region, and, like the RasGEF mSos, contains two potential SH3-binding sites. These results provide compelling evidence that FGD1 is responsible for FGDY and suggest that FGD1 is a Rho/RacGEF involved in mammalian development. Peer Reviewed http://deepblue.lib.umich.edu/bitstream/2027.42/31188/1/0000089.pdf
THE superfamily of low molecular mass GTP-binding proteins, for which the ras proteins are prototypes, has been implicated in the regulation of diverse biological activities including protein trafficking, secretion, and cell growth and differentiation. One member of this family, CDC42Hs (originally referred to as Gp or G25K), seems to be the human homologue of the Saccharomyces cerevisiae cell-division-cycle protein, CDC42Sc. A second S. cerevisiae protein, CDC24, which is known from complementation studies to act with CDC42Sc to regulate the development of normal cell shape and the selection of nonrandom budding sites in yeast, contains a region with sequence similarity to the dbl oncogene product. Here we show that dbl specifically catalyses the dissociation of GDP from CDC42Hs and thereby qualifies as a highly selective guanine nucleotide exchange factor for the GTP-binding protein. Although guanine nucleotide exchange activities have been previously described for other members of the Ras-related GTP-binding protein family, this is the first demonstration, to our knowledge, of the involvement of a human oncogenic protein in catalysing exchange activity.
Utilizing DNA transfection analysis with the continuous NIH 3T3 cell line as assay cell, we and other have observed that as many as 10-50% of human haematopoietic tumours contain oncogenes, the vast majority of which are members of the ras proto-oncogene family. In addition, Cooper and co-workers have reported the detection and isolation of specific oncogenes, B-lym and T-lym, which appear to be activated in human and rodent tumours of certain B and T lymphoid cells, respectively. In surveying human haematopoietic malignancies, we observed that DNA of a primary human diffuse B-cell lymphoma induced an unusual transformed focus on transfection of NIH 3T3 cells. Here, we report the molecular cloning and physical characterization of this human oncogene, whose transforming activity was shown to reside within a human DNA sequence of 45 kilobases (kb) cloned in a cosmid vector. Its properties distinguish it from previously reported retroviral or nonretroviral oncogenes.
The adenomatous polyposis coli gene (APC) is mutated in familial adenomatous polyposis and in sporadic colorectal tumors, and its product binds to the adherens junction protein beta-catenin. Overexpression of APC blocks cell cycle progression. The APC-beta-catenin complex was shown to bind to DLG, the human homolog of the Drosophila discs large tumor suppressor protein. This interaction required the carboxyl-terminal region of APC and the DLG homology repeat region of DLG. APC colocalized with DLG at the lateral cytoplasm in rat colon epithelial cells and at the synapse in cultured hippocampal neurons. These results suggest that the APC-DLG complex may participate in regulation of both cell cycle progression and neuronal function.
The Rho GTPases form a subgroup of the Ras superfamily of 20- to 30-kD GTP-binding proteins that have been shown to regulate a wide spectrum of cellular functions. These proteins are ubiquitously expressed across the species, from yeast to man. The mammalian Rho-like GTPases comprise at least 10 distinct proteins: RhoA, B, C, D, and E; Rac1 and 2; RacE; Cdc42Hs, and TC10. A comparison of the amino acid sequences of the Rho proteins from various species has revealed that they are conserved in primary structure and are 50%–55% homologous to each other. Like all members of the Ras superfamily, the Rho GTPases function as molecular switches, cycling between an inactive GDP-bound state and an active GTP-bound state. Until recently, members of the Rho subfamily were believed to be involved primarily in the regulation of cytoskeletal organization in response to extracellular growth factors. However, research from a number of laboratories over the past few years has revealed that the Rho GTPases play crucial roles in diverse cellular events such as membrane trafficking, transcriptional regulation, cell growth control, and development. Consequently, a major challenge has been to unravel the underlying molecular mechanisms by which the Rho GTPases mediate these various activities. Many targets of the Rho GTPases have now been identified and further characterization of some of them has provided major insights toward our understanding of Rho GTPase function at the molecular level. This review aims to summarize the general established principles about the Rho GTPases and some of the more recent exciting findings, hinting at novel, unanticipated functions of the Rho GTPases.
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.
NF-kappaB (nuclear factor-kappaB) is a collective name for inducible dimeric transcription factors composed of members of the Rel family of DNA-binding proteins that recognize a common sequence motif. NF-kappaB is found in essentially all cell types and is involved in activation of an exceptionally large number of genes in response to infections, inflammation, and other stressful situations requiring rapid reprogramming of gene expression. NF-kappaB is normally sequestered in the cytoplasm of nonstimulated cells and consequently must be translocated into the nucleus to function. The subcellular location of NF-kappaB is controlled by a family of inhibitory proteins, IkappaBs, which bind NF-kappaB and mask its nuclear localization signal, thereby preventing nuclear uptake. Exposure of cells to a variety of extracellular stimuli leads to the rapid phosphorylation, ubiquitination, and ultimately proteolytic degradation of IkappaB, which frees NF-kappaB to translocate to the nucleus where it regulates gene transcription. NF-kappaB activation represents a paradigm for controlling the function of a regulatory protein via ubiquitination-dependent proteolysis, as an integral part of a phosphorylationbased signaling cascade. Recently, considerable progress has been made in understanding the details of the signaling pathways that regulate NF-kappaB activity, particularly those responding to the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-1. The multisubunit IkappaB kinase (IKK) responsible for inducible IkappaB phosphorylation is the point of convergence for most NF-kappaB-activating stimuli. IKK contains two catalytic subunits, IKKalpha and IKKbeta, both of which are able to correctly phosphorylate IkappaB. Gene knockout studies have shed light on the very different physiological functions of IKKalpha and IKKbeta. After phosphorylation, the IKK phosphoacceptor sites on IkappaB serve as an essential part of a specific recognition site for E3RS(IkappaB/beta-TrCP), an SCF-type E3 ubiquitin ligase, thereby explaining how IKK controls IkappaB ubiquitination and degradation. A variety of other signaling events, including phosphorylation of NF-kappaB, hyperphosphorylation of IKK, induction of IkappaB synthesis, and the processing of NF-kappaB precursors, provide additional mechanisms that modulate the level and duration of NF-kappaB activity.
The conjugation of ubiquitin to other cellular proteins regulates a broad range of eukaryotic cell functions. The high efficiency and exquisite selectivity of ubiquitination reactions reflect the properties of enzymes known as ubiquitin-protein ligases or E3s. An E3 recognizes its substrates based on the presence of a specific ubiquitination signal, and catalyzes the formation of an isopeptide bond between a substrate (or ubiquitin) lysine residue and the C terminus of ubiquitin. Although a great deal is known about the molecular basis of E3 specificity, much less is known about molecular mechanisms of catalysis by E3s. Recent findings reveal that all known E3s utilize one of just two catalytic domains--a HECT domain or a RING finger--and crystal structures have provided the first detailed views of an active site of each type. The new findings shed light on many aspects of E3 structure, function, and mechanism, but also emphasize that key features of E3 catalysis remain to be elucidated.
The Rho GTPases are members of the Ras superfamily and include several isoforms of Cdc42, Rac, and Rho. The Rho subfamily regulates a variety of signal transduction pathways in eukaryotic cells, including cell adhesion and migration, by modulating cytoskeletal dynamics. Cdc42 induces formation of filopodia, whereas Rac regulates actin polymerization at the plasma membrane where ruffles are formed. Both induce the formation of cytoskeletal/signaling aggregates known as focal complexes. Rho controls the assembly of focal adhesions and the reorganization of actin into stress fibers. More recently, it has been recognized that Rho GTPases also initiate signaling pathways that impact on gene expression and cell growth regulation, and that each of these GTPases is essential for Ras to induce malignant transformation. A specific role for Rac has been identified in phagocytic cells, such as neutrophils or monocytes, where it activates the NADPH Oxidase enzymatic complex. This chapter describes an assay developed for Rac and Cdc42 activation using the GTPase/p21-binding region of p21-activated kinase 1 (PAK1). Similar strategies developed as assays for Rho GTP are discussed.
The Dlg tumour suppressor protein is intimately involved in the control of cell contact and polarity. Previous studies have shown that hDlg is a target for a number of viral transforming proteins. In particular, the high risk human papillomavirus (HPV) E6 proteins target hDlg for proteasome-mediated degradation, an activity that appears to contribute to HPV-induced malignancy. However, little information is available concerning the normal regulation of hDlg. In this study we have investigated the role of the proteasome in the regulation of endogenous hDlg protein levels in epithelial cell lines. We demonstrate that hDlg is, indeed, degraded via the proteasome both in the presence and absence of HPV, in a fashion that is dependent on the ability of the cells to form cell junctions. By western blot and immunofluorescence analysis we show that hDlg is efficiently degraded in isolated cells; however, upon cell-cell contact, hDlg is both hyper-phosphorylated and stabilised. Strikingly, in both transformed rodent cells and undifferentiated cervical cancer cells, this ability to stabilise Dlg upon increased cell density is lost. These results demonstrate a complex pattern of hDlg regulation by phosphorylation and proteasome degradation in response to cell contact. Loss of this regulation probably represents a significant step in the development of malignancy.
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?
It has been postulated that reactive oxygen species (ROS) may act as second messengers leading to nuclear factor (NF)-kappaB activation. This hypothesis is mainly based on the findings that N-acetyl-L-cysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), compounds recognized as potential antioxidants, can inhibit NF-kappaB activation in a wide variety of cell types. Here we reveal that both NAC and PDTC inhibit NF-kappaB activation independently of antioxidative function. NAC selectively blocks tumor necrosis factor (TNF)-induced signaling by lowering the affinity of receptor to TNF. PDTC inhibits the IkappaB-ubiquitin ligase activity in the cell-free system where extracellular stimuli-regulated ROS production does not occur. Furthermore, we present evidence that endogenous ROS produced through Rac/NADPH oxidase do not mediate NF-kappaB signaling, but instead lower the magnitude of its activation.
Mechanisms underlying ubiquitination
- Pickart
Rho-GTPases and cancer
- E Sahai
- C J Marshall
Sahai, E. & Marshall, C.J. (2002) Rho-GTPases and cancer.
Nature Rev. Cancer 2, 133 -142.
Cdc42 regulates GSK-3β and adenomatous polyposis coli to cotrol cell polarity
- Etienne-Manneville