No full-text available
To read the full-text of this research,
you can request a copy directly from the authors.
Feedback Regulation of Ras Signaling by Rabex-5-Mediated Ubiquitination
Abstract
Ras proteins play a central role in transducing signals that control cell proliferation, differentiation, motility, and survival. The location-specific signaling activity of Ras has been previously shown to be regulated by ubiquitination [1]. However, the molecular machinery that controls Ras ubiquitination has not been defined. Here we demonstrate through biochemical and functional analyses that Rabex-5 (also known as RabGEF1) [2, 3] functions as an E3 ligase for Ras. Rabex-5-mediated Ras ubiquitination promotes Ras endosomal localization and leads to the suppression of ERK activation. Moreover, the Ras effector RIN1 [4, 5] is required for Rabex-5-dependent Ras ubiquitination, suggesting a feedback mechanism by which Ras activation can be coupled to ubiquitination. These findings define new elements in the regulatory circuitry that link Ras compartmentalization to signaling output.
... Although these modifications do not affect the binding of RAF proteins, they diminish RAF activation by shortening the dwell time of RAF at the PM and its exposure to activating events [71]. Interestingly, this modification is mediated by the E3 ubiquitin ligase Rabex-5 in a RIN1 dependent manner [72]. As RIN1 is a RAS effector, this circuitry may constitute a negative feedback loop that limits RAS activation. ...
... Interestingly, tyrosine phosphorylation of RAS proteins by ABL on tyrosine 137 has the opposite effect-it enhances RAF kinase binding to HRAS and downstream signaling [84] ( Figure 5F). The ABL kinase is activated by HRAS in a RIN1 dependent manner [84], but RIN1 also stimulates HRAS sequestration in endosomes [72]. Thus, RIN1 may exert a complex regulation on RAS signaling acting as accelerator and brake-likely in this temporal sequence. ...
... HRAS had only 6 isoform specific interactors (out of 153), NRAS had 28 (out of 232). Interestingly, the two KRAS splicing isoforms KRAS4A and KRAS4B had more specific interactors combined (72), but they also featured splice form specific interactions with KRAS4A specifically binding to 15 proteins and KRAS4B to 29 proteins. These two proteins have a very high degree of sequence homology and only differ in the sequence of their HVR, which must play a role in the regulation of selective interactions. ...
RAS oncogenes are among the most commonly mutated proteins in human cancers. They regulate a wide range of effector pathways that control cell proliferation, survival, differentiation, migration and metabolic status. Including aberrations in these pathways, RAS-dependent signaling is altered in more than half of human cancers. Targeting mutant RAS proteins and their downstream oncogenic signaling pathways has been elusive. However, recent results comprising detailed molecular studies, large scale omics studies and computational modeling have painted a new and more comprehensive portrait of RAS signaling that helps us to understand the intricacies of RAS, how its physiological and pathophysiological functions are regulated, and how we can target them. Here, we review these efforts particularly trying to relate the detailed mechanistic studies with global functional studies. We highlight the importance of computational modeling and data integration to derive an actionable understanding of RAS signaling that will allow us to design new mechanism-based therapies for RAS mutated cancers.
... This is not a degradative posttranslational modification but rather stabilizes these Ras isoforms in endosomes and inhibits their recycling to plasma membrane (Jura et al. 2006) (see [7] in Fig. 3.1). The HVR of these Ras proteins is not an obligatory acceptor for ubiquitin but participates in ubiquitination by the E3 ubiquitin ligase Rabex5 (Xu et al. 2010). Interestingly, activated Ras induces the recruitment of Rabex-5 to endosomes through a RAS and RAB interactor 1 (RIN1)-containing machinery. ...
... impairs interaction of H-and NRas with Raf1. Consequently, MAPK activation is reduced, yet PI3K/Akt activation is not affected (Xu et al. 2010;Yan et al. 2010;Jura et al. 2006). Hence, inhibition or overexpression of the ubiquitin ligase Rabex5, which is responsible for H-and NRas ubiquitination on endosomes, resulted in increased or attenuated Raf1/MAPK activation, respectively (Xu et al. 2010;Yan et al. 2010). ...
... Consequently, MAPK activation is reduced, yet PI3K/Akt activation is not affected (Xu et al. 2010;Yan et al. 2010;Jura et al. 2006). Hence, inhibition or overexpression of the ubiquitin ligase Rabex5, which is responsible for H-and NRas ubiquitination on endosomes, resulted in increased or attenuated Raf1/MAPK activation, respectively (Xu et al. 2010;Yan et al. 2010). In a much more complex scenario, Rin1, the GEF for Rab5, can bind HRas on endosomes and simultaneously stimulates Rab5-dependent endocytosis Tall et al. 2001). ...
The endocytic compartment is not only the functional continuity of the plasma membrane but consists of a diverse collection of intracellular heterogeneous complex structures that transport, amplify, sustain, and/or sort signaling molecules. Over the years, it has become evident that early, late, and recycling endosomes represent an interconnected vesicular-tubular network able to form signaling platforms that dynamically and efficiently translate extracellular signals into biological outcome. Cell activation, differentiation, migration, death, and survival are some of the endpoints of endosomal signaling. Hence, to understand the role of the endosomal system in signal transduction in space and time, it is therefore necessary to dissect and identify the plethora of decoders that are operational in the different steps along the endocytic pathway. In this chapter, we focus on the regulation of spatiotemporal signaling in cells, considering endosomes as central platforms, in which several small GTPases proteins of the Ras superfamily, in particular Ras and Rac1, actively participate to control cellular processes like proliferation and cell mobility.
... Extensive research in the past has also revealed that RAS proteins are subjected to other posttranslational modifications including phosphorylation, ubiquitination, acetylation, and S-nitrosylation [8,[11][12][13][14][15]. For example, KRAS4B is phosphorylated on serine 181 by protein kinase C and the phosphorylation is involved in the negative regulation of its association with the plasma membrane. ...
... For example, KRAS4B is phosphorylated on serine 181 by protein kinase C and the phosphorylation is involved in the negative regulation of its association with the plasma membrane. RAS proteins are also modified by monoubiquitination and bi-ubiquitination [11][12][13]. Lys117, Lys147 and Lys170 are potential sites of ubiquitination [1]. The E3 ligase specific for RAS ubiquitination is RABEX5 [13]. ...
... Lys117, Lys147 and Lys170 are potential sites of ubiquitination [1]. The E3 ligase specific for RAS ubiquitination is RABEX5 [13]. Cys118, a highly conserved site in RAS isoforms and orthologues, can be modified by nitrosylation [16]. ...
RAS proteins are GTPases that participate in multiple signal cascades, regulating crucial cellular processes including cell survival, proliferation, differentiation, and autophagy. Mutations or deregulated activities of RAS are frequently the driving force for oncogenic transformation and tumorigenesis. Given the important roles of the small ubiquitin-related modifier (SUMO) pathway in controlling the stability, activity, or subcellular localization of key cellular regulators, we investigated here whether RAS proteins are posttranslationally modified (i.e. SUMOylated) by the SUMO pathway. We observed that all three RAS protein isoforms (HRAS, KRAS, and NRAS) were modified by the SUMO3 protein. SUMOylation of KRAS protein, either endogenous or ectopically expressed, was observed in multiple cell lines. The SUMO3 modification of KRAS proteins could be removed by SUMO1/sentrin-specific peptidase 1 (SENP1) and SENP2, but not by SENP6, indicating that RAS SUMOylation is a reversible process. A conserved residue in RAS, Lys-42, was a site that mediates SUMOylation. Results from biochemical and molecular studies indicated that the SUMO-E3 ligase PIASγ specifically interacts with RAS and promotes its SUMOylation. Moreover, SUMOylation of RAS appeared to be associated with its activation. In summary, our study reveals a new posttranslational modification for RAS proteins. Since we found that HRAS, KRAS, and NRAS can all be SUMOylated, we propose that SUMOylation might represent a mechanism by which RAS activities are controlled.
... RAS stability is controlled by the E3 ubiquitin ligases, b-TrCP1, and Nedd4-1, that directly polyubiquitinate RAS proteins triggering degradation (Shukla et al, 2014;Zeng et al, 2014). In addition, we and others have recently demonstrated that RAS family members can undergo reversible mono-and di-ubiquitination (Jura et al, 2006;Xu et al, 2010;Sasaki et al, 2011;Baker et al, 2013a;Simicek et al, 2013). However, how reversible ubiquitination affects RAS activity and its tumorigenic properties remains very much controversial. ...
... Earlier studies reported that reversible ubiquitination restricts the activity of HRAS and NRAS, but not that of KRAS, whereas more recent reports demonstrated that KRAS can also undergo mono-and di-ubiquitination (Jura et al, 2006). Xu et al (2010) demonstrated that di-ubiquitination of HRAS and NRAS by the E3 ubiquitin ligase RABEX5 (RABGEF1) induces their re-localization to the endomembranes, leading to a decrease in RAS activity and downstream signaling. On the other hand, two other groups demonstrated that monoubiquitination of HRAS at Lys117 accelerates intrinsic nucleotide exchange and promotes GTP loading, whereas monoubiquitination of KRAS at Lys147 impaired NF1-mediated GTP hydrolysis Baker et al, 2013a,b). ...
... OTUB1 triggers activation of the MAPK pathway by inhibiting RAS ubiquitination. Consistently with these observations, previous reports demonstrated that ubiquitination of HRAS and NRAS modulates their ability to activate the MAPK pathway (Xu et al, 2010;Yan et al, 2010). The RAS-specific E3 ligase RABEX5 may act as a tumor suppressor by regulating MAPK cascade activation. ...
Activation of the RAS oncogenic pathway, frequently ensuing from mutations in RAS genes, is a common event in human cancer. Recent reports demonstrate that reversible ubiquitination of RAS GTPases dramatically affects their activity, suggesting that enzymes involved in regulating RAS ubiquitination may contribute to malignant transformation. Here, we identified the de‐ubiquitinase OTUB1 as a negative regulator of RAS mono‐ and di‐ubiquitination. OTUB1 inhibits RAS ubiquitination independently of its catalytic activity resulting in sequestration of RAS on the plasma membrane. OTUB1 promotes RAS activation and tumorigenesis in wild‐type RAS cells. An increase of OTUB1 expression is commonly observed in non‐small‐cell lung carcinomas harboring wild‐type KRAS and is associated with increased levels of ERK1/2 phosphorylation, high Ki67 score, and poorer patient survival. Our results strongly indicate that dysregulation of RAS ubiquitination represents an alternative mechanism of RAS activation during lung cancer development.
... Although these modifications do not affect the binding of RAF proteins, they diminish RAF activation by shortening the dwell time of RAF at the PM and its exposure to activating events [69]. Interestingly, this modification is mediated by the E3 ubiquitin ligase Rabex-5 in a RIN1 dependent manner [70]. As RIN1 is a RAS effector this circuitry may constitute a negative feedback loop that limits RAS activation. ...
... Interestingly, tyrosine phosphorylation of RAS proteins by ABL on tyrosine 137 has the opposite effect -it enhances RAF kinase binding to HRAS and downstream signaling [82] (Fig. 5F). The ABL kinase is activated by HRAS in a RIN1 dependent manner [82], but RIN1 also stimulates HRAS sequestration in endosomes [70]. Thus, RIN1 may exert a complex regulation on RAS signaling acting as accelerator and brake -likely in this temporal sequence. ...
RAS oncogenes are amongst the most commonly mutated proteins in human cancers. They regulate a wide range of effector pathways that control cell proliferation, survival, differentiation, migration and metabolic status. Including aberrations in these pathways, RAS dependent signaling is altered in more than half of human cancers. Targeting mutant RAS proteins and their downstream oncogenic signaling pathways has been elusive. However, recent results comprising detailed molecular studies, large scale omics studies and computational modeling have painted a new and more comprehensive portrait of RAS signaling that helps us to understand the intricacies of RAS, how its physiological and pathophysiological functions are regulated, and how we can target them. Here, we review these efforts particularly trying to relate the detailed mechanistic studies with global functional studies. We highlight the importance of computational modeling and data integration to derive an actionable understanding of RAS signaling that will allow us to design new mechanism based therapies for RAS mutated cancers.
... Furthermore, Ras ubiquitination and the resulting endosomal retention negatively regulates Ras-dependent recruitment of Raf to the plasma membrane and Erk activation (Jura et al., 2006). RabGEF1 mediates Ras ubiquitination and promotes Ras endosomal localization, leading to the suppression of Erk activation (Xu et al., 2010). This effect is independent of the GEF activation of RabGEF1 (Xu et al., 2010). ...
... RabGEF1 mediates Ras ubiquitination and promotes Ras endosomal localization, leading to the suppression of Erk activation (Xu et al., 2010). This effect is independent of the GEF activation of RabGEF1 (Xu et al., 2010). Then, Shioda et al. report that D2LR elicits Erk activation and dendritic spine formation through RabGEF1/ PDGFRβ-mediated endocytosis in mouse striatum (Shioda et al., 2017). ...
Background
RabGEF1 is a guanine-nucleotide exchange factor for RAB-5, which plays an oncogenic role in certain human cancers. However, the function of RabGEF1 in glioma has not been studied. Here, we report that the down-regulation of RabGEF1 inhibits the proliferation and metastasis, and induces autophagy of U251 glioblastoma cells.
Methods
The expression of RabGEF1 in glioma and normal tissues were measured by immunohistochemistry. Four siRNAs targeting different sites of RabGEF1 were conducted and the interference efficiencies were verified by qRT-PCR assay. Western blot was used to detect the expression of interest proteins. Cell proliferation was detected using CCK-8 and clone formation assay. Cell migration and invasion were analyzed by scratch assay and transwell assay, respectively. Flow cytometry was used to detect cell cycle distribution and apoptosis.
Results
RabGEF1 was significantly up-regulated in human glioma tissues. RabGEF1 knockdown reduced cell viability, induced cell cycle arrest and apoptosis in U251 cells. Cell migration and invasion were also inhibited when RabGEF1 silencing. Mechanism studies showed that Cyclin D1 and CDK4/6 were significantly down-regulated when RabGEF1 silencing. p53 and caspase mediated apoptotic pathway was activated by down-regulation of RabGEF1. Moreover, RabGEF1 knockdown also induced autophagy in glioma cells. The investigation of AKT and Erk pathways suggested that phosphorylated AKT, p70S6K and phosphorylated Erk were all decreased when RabGEF1 silencing.
Conclusion
In conclusion, our data suggest that RabGEF1 is up-regulated in human glioma and down-regulation of RabGEF1 inhibited cell proliferation and metastasis, and induced autophagy of U251 glioblastoma cells, which might be mediated by inactivation of AKT and Erk signaling pathways.
... In contrast to the molecular mechanisms that regulate F box protein function, upstream cellular signaling for most F box proteins in SCF complex formation and E3 ligase activation remains largely unclear. Studies have indicated a role of post-translational modifications (PTMs), including sumoylation and particularly phosphorylation, in altering the subcellular localization and E3 ligase activity of F box proteins (Fuchs et al., 2002;Gao et al., 2009;Jura et al., 2006;Lin et al., 2009;Wang et al., 2018;Xu et al., 2010). How FBXL21 activity is regulated upstream is largely unknown. ...
... Compared with target substrate identification, upstream regulatory mechanisms governing E3 ligases are often not as well understood. Consistent with the growing evidence highlighting PTMs as key regulatory events for E3 ligases (Fuchs et al., 2002;Gao et al., 2009;Jura et al., 2006;Lin et al., 2009;Wang et al., 2018;Xu et al., 2010), we report that GSK-3b regulates FBXL21-CULLIN1 complex formation as a critical step of E3 ligase activation. TCAP phosphorylation is known to regulate myofibrillogenesis, and PKD has been found to interact with cardiac TCAP and function in the maintenance of transverse tubule organization and intracellular Ca 2+ transients in myocytes (Candasamy et al., 2014;Mayans et al., 1998). ...
FBXL21 is a clock-controlled E3 ligase modulating circadian periodicity via subcellular-specific CRYPTOCHROME degradation. How FBXL21 regulates tissue-specific circadian physiology and what mechanism operates upstream is poorly understood. Here we report the sarcomere component TCAP as a cytoplasmic substrate of FBXL21. FBXL21 interacts with TCAP in a circadian manner antiphasic to TCAP accumulation in skeletal muscle, and circadian TCAP oscillation is disrupted in Psttm mice with an Fbxl21 hypomorph mutation. GSK-3β phosphorylates FBXL21 and TCAP to activate FBXL21-mediated, phosphodegron-dependent TCAP degradation. GSK-3β inhibition or knockdown diminishes FBXL21-Cul1 complex formation and delays FBXL21-mediated TCAP degradation. Finally, Psttm mice show significant skeletal muscle defects, including impaired fiber size, exercise tolerance, grip strength, and response to glucocorticoid-induced atrophy, in conjunction with cardiac dysfunction. These data highlight a circadian regulatory pathway where a GSK-3β-FBXL21 functional axis controls TCAP degradation via SCF complex formation and regulates skeletal muscle function.
... Rabex-5 (also called RabGEF1), an A20-like E3 ubiquitin ligase, promotes inhibitory mono-and di-ubiquitination of Drosophila Ras and mammalian H-Ras and N-Ras to restrict signaling to downstream effectors [13][14][15][16]. Rabex-5 inhibits both wild-type Ras and also the constitutively active oncogenic mutant Ras G12V (also referred to as RasV12 in the literature) [13][14][15][16]. ...
... Rabex-5 (also called RabGEF1), an A20-like E3 ubiquitin ligase, promotes inhibitory mono-and di-ubiquitination of Drosophila Ras and mammalian H-Ras and N-Ras to restrict signaling to downstream effectors [13][14][15][16]. Rabex-5 inhibits both wild-type Ras and also the constitutively active oncogenic mutant Ras G12V (also referred to as RasV12 in the literature) [13][14][15][16]. ...
Dysregulation of the Ras oncogene in development causes developmental disorders, “Rasopathies,” whereas mutational activation or amplification of Ras in differentiated tissues causes cancer. Rabex-5 (also called RabGEF1) inhibits Ras by promoting Ras mono- and di-ubiquitination. We report here that Rabex-5-mediated Ras ubiquitination requires Ras Tyrosine 4 (Y4), a site of known phosphorylation. Ras substitution mutants insensitive to Y4 phosphorylation did not undergo Rabex-5-mediated ubiquitination in cells and exhibited Ras gain-of-function phenotypes in vivo. Ras Y4 phosphomimic substitution increased Rabex-5-mediated ubiquitination in cells. Y4 phosphomimic substitution in oncogenic Ras blocked the morphological phenotypes associated with oncogenic Ras in vivo dependent on the presence of Rabex-5. We developed polyclonal antibodies raised against an N-terminal Ras peptide phosphorylated at Y4. These anti-phospho-Y4 antibodies showed dramatic recognition of recombinant wild-type Ras and RasG12V proteins when incubated with JAK2 or SRC kinases but not of RasY4F or RasY4F,G12V recombinant proteins suggesting that JAK2 and SRC could promote phosphorylation of Ras proteins at Y4 in vitro. Anti-phospho-Y4 antibodies also showed recognition of RasG12V protein, but not wild-type Ras, when incubated with EGFR. A role for JAK2, SRC, and EGFR (kinases with well-known roles to activate signaling through Ras), to promote Ras Y4 phosphorylation could represent a feedback mechanism to limit Ras activation and thus establish Ras homeostasis. Notably, rare variants of Ras at Y4 have been found in cerebellar glioblastomas. Therefore, our work identifies a physiologically relevant Ras ubiquitination signal and highlights a requirement for Y4 for Ras inhibition by Rabex-5 to maintain Ras pathway homeostasis and to prevent tissue transformation.
... Rab5 GDP/GTP exchange factor (RABGEF1) acts as an E3 ligase for HRAS, and HRAS monoubiquitination promotes its localization to the endosome, thereby decreasing ERK signaling [32]. RABGEF1 possesses a zinc finger (ZnF) domain similar to that of A20 with E3 ligase activity [33][34][35], and Xu et al. [32] suggested that the interaction between RABGEF1 and HRAS occurs via this domain. ...
... Rab5 GDP/GTP exchange factor (RABGEF1) acts as an E3 ligase for HRAS, and HRAS monoubiquitination promotes its localization to the endosome, thereby decreasing ERK signaling [32]. RABGEF1 possesses a zinc finger (ZnF) domain similar to that of A20 with E3 ligase activity [33][34][35], and Xu et al. [32] suggested that the interaction between RABGEF1 and HRAS occurs via this domain. A mutation in the ZnF domain was reported to obstruct RABGEF1's ability to ubiquitinate RAS, providing support for ZnF-mediated RAS binding. ...
Background
RAS protein interactions have predominantly been studied in the context of the RAF and PI3kinase oncogenic pathways. Structural modeling and X-ray crystallography have demonstrated that RAS isoforms bind to canonical downstream effector proteins in these pathways using the highly conserved switch I and II regions. Other non-canonical RAS protein interactions have been experimentally identified, however it is not clear whether these proteins also interact with RAS via the switch regions.
Results
To address this question we constructed a RAS isoform-specific protein-protein interaction network and predicted 3D complexes involving RAS isoforms and interaction partners to identify the most probable interaction interfaces. The resulting models correctly captured the binding interfaces for well-studied effectors, and additionally implicated residues in the allosteric and hyper-variable regions of RAS proteins as the predominant binding site for non-canonical effectors. Several partners binding to this new interface (SRC, LGALS1, RABGEF1, CALM and RARRES3) have been implicated as important regulators of oncogenic RAS signaling. We further used these models to investigate competitive binding and multi-protein complexes compatible with RAS surface occupancy and the putative effects of somatic mutations on RAS protein interactions.
Conclusions
We discuss our findings in the context of RAS localization to the plasma membrane versus within the cytoplasm and provide a list of RAS protein interactions with possible cancer-related consequences, which could help guide future therapeutic strategies to target RAS proteins.
Electronic supplementary material
The online version of this article (doi:10.1186/s13628-017-0037-6) contains supplementary material, which is available to authorized users.
... Mechanistically, Rabex-5 is reported to regulate immune responses via the negative regulation of RasGTPase activated pathways (4,25,26). Previous studies have focused on the function D). Error bars show the SD of the mean of two replicate wells (two separate aliquots of cells treated with the same stimulations; n = 2). ...
... of Rabex-5 in mast cells, suggesting that the phenotype of Rabex-5-deficient mice might reflect, at least in part, defective mast cell function (4,25,26). Here, we have reported that Rabex-5 is a critical negative regulator of type 1 IFN production. Based on the strength of our observations in vitro, we suggest that excessive type 1 IFN production may be the driving factor of the spontaneous inflammatory disorder exhibited by Rabex-5-deficient mice. ...
Significance
The identification of the Lon protease Cereblon as the thalidomide receptor has led to significant progress in our understanding of immunomodulatory drugs (IMiDs). IMiD binding alters the surface of Cereblon to facilitate the recruitment of proteins including Ikaros and CK1α. The recruitment of these proteins results in their ubiquitin-mediated proteosomal degradation. How Cereblon mediates the efficacy of these compounds in the treatment of inflammatory skin conditions remains unclear. Significantly, this study has identified Rabex-5 as a Cereblon-interacting protein that is affected by IMiD binding. Furthermore, knockdown studies in a human macrophage cell line reveals a function of Rabex-5 in regulating Toll-like receptor–induced type 1 IFN production. These findings may help explain the antiinflammatory properties of IMiDs.
... H-RasC186S mutant in HVR region abrogates the ubiquitination, indicating this ubiquitination is HVR dependent. The endosomal E3 ligase, Rabex-5/RabGEF1, the mammalian ortholog of yeast Vps9p, and a GEF for Rab5, is responsible for mono-and di-ubiquitination of H-Ras and N-Ras [103]. Ubiquitination of Ras restricts endosomal Ras anchoring and suppresses downstream ERK activation [103]. ...
... The endosomal E3 ligase, Rabex-5/RabGEF1, the mammalian ortholog of yeast Vps9p, and a GEF for Rab5, is responsible for mono-and di-ubiquitination of H-Ras and N-Ras [103]. Ubiquitination of Ras restricts endosomal Ras anchoring and suppresses downstream ERK activation [103]. It was also shown that Ras ubiquitination by Rabex-5 restricts Ras signaling in Drosophila in vivo to establish proper organ size, wing vein pattern, and eye versus antennal fate [104]. ...
Ras proteins are molecular switches cycling between the GTP-bound state and the GDP-bound state to transduce signals from outside of the cell to the interior. They all have the CAAX motifs at the C-terminus. Post-translational modifications of the CAAX motifs determine the subcellular localization and correct biological function of Ras proteins. Here we have described how prenylation of the CAAX motifs affects membrane association of Ras and how this modification can be targeted to block Ras-transduced signaling. In addition, other post-translational modifications of Ras, such as palmitoylation, phosphorylation, acetylation, ubiquitination, and S-nitrosylation, are discussed. Because oncogenic Ras mutants are drivers for tumorigenesis, understanding how post-translational modifications of Ras mutants influence their activities will help us design therapeutic drugs to treat patients with cancer.
... 12 Mono-and/or bi-ubiquitination of H/N-Ras are location-specific and mediated by the E3-ligase Rabex-5, eventually promoting Ras endosomal localization and suppression of ERK activation. 13 This modification has important functional consequences since inhibition of H/N-Ras ubiquitination results in their hyperactivity in vitro 12,13 and in overgrowth phenotypes in vivo. 14 Accordingly, targeting Ras post-translational modification and/or subcellular localization are currently explored as possible anticancer strategies. ...
... 12 Mono-and/or bi-ubiquitination of H/N-Ras are location-specific and mediated by the E3-ligase Rabex-5, eventually promoting Ras endosomal localization and suppression of ERK activation. 13 This modification has important functional consequences since inhibition of H/N-Ras ubiquitination results in their hyperactivity in vitro 12,13 and in overgrowth phenotypes in vivo. 14 Accordingly, targeting Ras post-translational modification and/or subcellular localization are currently explored as possible anticancer strategies. ...
The small GTPases of the Ras family play a pivotal role in the regulation of cell proliferation and motility, both in normal and transformed cells. In particular, the three genes encoding for the N-, H- and K-Ras are frequently mutated in human cancer and their inappropriate regulation, expression and subcellular localization can drive tumor onset and progression. Activation of the Ras-MAPK pathway directly signals on the cell cycle machinery by regulating the expression and/or localization of two key cell cycle player, Cyclin D1 and p27(Kip1). We recently reported that in normal fibroblasts, following mitogenic stimuli, p27(Kip1) translocates to the cytoplasm where it regulates H-Ras localization and activity. This regulatory mechanism ensures that cells pass beyond the restriction point of the cell cycle only when the proper level of stimulation is reached. Here, we comment on this new evidence that possibly represents one of the ways that cells have developed during evolution to ensure that the decision to divide is taken only at the right time.
... In particular, Ras localization to Rab-5-positive endosomes mediates its mono-and bi-ubiquitination, leading to dampening of ERK activation. 7 Recent studies in neurons demonstrated that p27 controls the trafficking of Rab5-containing vesicles via regulation of MT organization. 8 In line with these observations, our work provides evidence that cytoplasmic p27, through its interaction with Stathmin, affects Ras activity, favoring its accumulation in Rab-5-positive vesicles and its mono-/bi-ubiquitination. 7 Using different approaches, we demonstrated that the increase in MAPK pathway activity observed in p27 null cells is, at least in part, dependent on MTdependent transport. ...
... 7 Recent studies in neurons demonstrated that p27 controls the trafficking of Rab5-containing vesicles via regulation of MT organization. 8 In line with these observations, our work provides evidence that cytoplasmic p27, through its interaction with Stathmin, affects Ras activity, favoring its accumulation in Rab-5-positive vesicles and its mono-/bi-ubiquitination. 7 Using different approaches, we demonstrated that the increase in MAPK pathway activity observed in p27 null cells is, at least in part, dependent on MTdependent transport. When p27 expression levels are low or lost, as frequently observed in tumors, Stathmin activity is released, mono-/bi-ubiquitination of Ras is diminished and, as a consequence, MAPK signaling pathway activation is augmented (Fig. 1, right panel). ...
Our recent study has uncovered an additional way by which the cell cycle inhibitor p27kip1 controls cell proliferation. Acting on the activity of the microtubule destabilizing protein Stathmin, p27kip1 modulates full H-Ras activation and, as a consequence, the MAPK signaling cascade. This regulatory mechanism influences cell cycle in vitro and mice growth in vivo and, when unbalanced, may lead to uncontrolled proliferation and tumor onset.
... Ras is represented by one gene in Drosophila and by HRas, NRas, and KRas in mammals. Drosophila Ras and HRas and NRas are negatively regulated by inhibitory ubiquitination [Jura et al., 2006;Yan et al., 2009] by the E3 Rabex-5 [Yan et al. 2010, Xu et al 2010. We previously showed that Rabex-5 requires Tyrosine 4 (Y4) to promote Ras ubiquitination in Drosophila [Washington et al., 2020]; Y4F mutations cause Ras to evade ubiquitination by Rabex-5 in vitro and behave as gain-of-function Ras mutations in vivo [Washington et al. 2020]. ...
Ras signaling plays a highly conserved role from flies to mammals in establishing proper development, and its dysregulation can lead to cancer. In Drosophila , we demonstrated that Ras Tyrosine 4 (Y4) was required for inhibitory ubiquitination by Rabex-5. In humans, rare histidine substitution mutations at Y4 are found in HRas in cerebellar glioblastomas (cGBMs). We report here that analogous Y4H mutations in Drosophila Ras make it less sensitive to Rabex-5-mediated ubiquitination in cells and show increased frequency of vein phenotypes per wing compared to wild-type Ras, which would be consistent with Ras gain-of-function and with their appearance in human cGBMs.
... Constitutive activation of RAS due to point mutation is seen in different cancer types including lung cancer and pancreatic adenocarcinoma (Moore et al., 2020). RAS activates the downstream MAPK pathway at the plasma membrane, which is inhibited by RAS monoubiquitination (Xu et al., 2010). To identify potential DUBs regulating RAS, Baietti et al. performed a protein-protein interaction screening (Baietti et al., 2016). ...
Protein ubiquitination plays a pivotal role in protein homeostasis. Ubiquitination may regulate the stability, activity, protein–protein interaction, and localization of a protein. Ubiquitination is subject to regulation by two groups of counteracting enzymes, the E3 ubiquitin ligases and deubiquitinases. Consistently, deubiquitinases are involved in essentially all biological processes. OTUB1, an OTU-family deubiquitinase, is a critical regulator of development, cancer, DNA damage response, and immune response. OTUB1 antagonizes the ubiquitination of a wide-spectrum of proteins through at least two different mechanisms. Besides direct deubiquitination, OTUB1 can also inhibit ubiquitination by non-canonically blocking ubiquitin transfer from certain ubiquitin-conjugases (E2). In this review, we start with a general background of protein ubiquitination and deubiquitination. Next, we introduce the basic characteristics of OTUB1 and then elaborate on the updated biological functions of OTUB1. Afterwards, we discuss potential mechanisms underlying the versatility and specificity of OTUB1 functions. In the end, we discuss the perspective that OTUB1 can be a potential therapeutic target for cancer.
... The KRas, HRas, and NRas genes in mammals are represented by a single Ras gene in Drosophila (referred to in the literature as Ras1, Ras85D, and here referred to as Ras). The E3 ubiquitin ligase Rabex-5 inhibits Drosophila Ras and mammalian HRas and NRas by promoting their mono-and di-ubiquitination (Jura et al. 2006;Yan et al. 2009Yan et al. , 2010Xu et al. 2010;Washington et al. 2020). The Cul3-Lztr1 ubiquitin ligase also inhibits Ras by ubiquitination in both flies and mammals (Steklov et al. 2018;Bigenzahn et al. 2018). ...
Ras signaling plays an important role in growth, proliferation, and developmental patterning. Maintaining appropriate levels of Ras signaling is important to establish patterning in development and to prevent diseases such as cancer in mature organisms. The Ras protein is represented by Ras85D in Drosophila and by HRas, NRas, and KRas in mammals. In the past dozen years, multiple reports have characterized both inhibitory and activating ubiquitination events regulating Ras proteins. Inhibitory Ras ubiquitination mediated by Rabex-5 or Lztr1 is highly conserved between flies and mammals. Activating ubiquitination events at K117 and K147 have been reported in mammalian HRas, NRas, and KRas, but it is unclear if these activating roles of K117 and K147 are conserved in flies. Addressing a potential conserved role for these lysines in Drosophila Ras activation requires phenotypes strong enough to assess suppression. Therefore, we utilized oncogenic Ras, RasG12V, which biases Ras to the GTP-loaded active conformation. We created double mutants RasG12V,K117R, RasG12V,K147R, and triple mutant RasG12V,K117R,K147R, to prevent lysine-specific post-translational modification of K117, K147, or both respectively. We compared their phenotypes to RasG12V in the wing to reveal the roles of these lysines. Although RasG12V,K147R did not show compelling or quantifiable differences from RasG12V, RasG12V,K117R showed visible and quantifiable suppression compared to RasG12V, and triple mutant RasG12V,K117R,K147R showed dramatic suppression compared to RasG12V and increased suppression compared to RasG12V,K117R. These data are consistent with highly conserved roles for K117 and K147 in Ras activation from flies to mammals.
... Rabex5 is the canonical Rab5GEF with clearly established roles in endosomal fusion and sorting into EEA1-positive endosomes (Horiuchi et al., 1997). Rin1 has a Ras binding domain and mediates Rabex5-dependent ubiquitination of endosomal Ras leading to its attenuation (Xu et al., 2010). It is striking however that loss of Rme-6 is sufficient to cause profound effects on proliferation and cell survival indicating that there are important non-redundant functions for individual Rab5GEFs, even in the transport of a single cargo. ...
Epidermal growth factor receptor (EGFR) signalling results in a variety of cell behaviours, including cell proliferation, migration and apoptosis, which depend on cell context. Here we have explored how the Rab5GEF, Rme-6, regulates EGFR signalling by modulating endocytic flux. We demonstrate that Rme-6, which acts early in the endocytic pathway, regulates EGFR trafficking through an endocytic compartment that is competent for ERK1/2 signalling. While overexpression of Rme-6 results in enhanced ERK1/2 nuclear localisation and c-Fos activation, loss of Rme-6 results in aberrant ERK1/2 signalling with increased cytoplasmic ERK1/2 phosphorylation (Thr202/Tyr204) but decreased ERK1/2 nuclear translocation and c-Fos activation, the latter leading to decreased cell proliferation. Phosphorylation of ERK1/2 by protein kinase 2 (CK2) is required for its nuclear translocation and our data support a model whereby Rme-6 provides a scaffold for a population of CK2 which is required for efficient nuclear translocation of ERK1/2. Rme-6 is itself a substrate for CK2 on Thr642 and Ser996 and phosphorylation on these sites can activate its Rab5GEF activity and endocytic trafficking of EGFR. Together our results indicate that Rme-6 co-ordinates EGFR trafficking and signalling to regulate the assembly and disassembly of an ERK1/2 signalosome.
Summary statement
Here we demonstrate how Rme-6, a Rab5GEF, co-ordinates trafficking and signalling of EGFR on the early endocytic pathway to ensure appropriate regulation of downstream ERK1/2 signalling.
... Washington et al. identified that the phosphorylation of Tyr4 is indispensable for the ubiquitination of Ras and Phe substitution at this site, rendering Ras insensitive to Rabex5 ubiquitination [50]. Rabex5/RabGEF1 promotes mono-and diubiquitination of H-Ras and N-Ras [51]. H-Ras is modified at Lys63, which stabilizes its association with the endosome and modulates downstream signaling potential. ...
Ras, a GTP-GDP binary switch protein, transduces signals from diverse receptors to regulate various signaling networks. Three Ras genes encode for protein isoforms, namely, Harvey Ras (H-Ras), Kirsten Ras (K-Ras, with two splice variants, K-Ras4A and K-Ras4B), and Neuroblastoma Ras (N-Ras). The isoforms undergo a series of post-translational modifications that enable their membrane attachment and biological activity. The activation of Ras isoforms is tightly regulated, and any dysregulation affects cellular processes, such as cell division, apoptosis, differentiation, cell migration, etc. The Ras gene is highly prone to mutation, and ~30% of cancers carry somatic mutations in Ras, whereas germline mutations clinically manifest as various rasopathies. In addition to regulation by the Guanine nucleotide exchange factors and the GTPase activation proteins, Ras signaling, and localization are also regulated by phosphorylation-dephosphorylation, ubiquitination, nitrosylation, and acetylation. Herein, we review the regulation of Ras signaling and localization by various regulatory enzymes in depth and assess the current status of Ras drug discovery targeting these regulatory enzymes.
... HRAS can activate ABL via RIN1, a direct RAS effector [74]. However, RIN1 also stimulates HRAS ubiquitination and sequestration in endosomes [75]. Thus, RIN1 could co-ordinate HRAS activation with subsequent inhibition. ...
RAS proteins regulate most aspects of cellular physiology. They are mutated in 30% of human cancers and 4% of developmental disorders termed Rasopathies. They cycle between active GTP-bound and inactive GDP-bound states. When active, they can interact with a wide range of effectors that control fundamental biochemical and biological processes. Emerging evidence suggests that RAS proteins are not simple on/off switches but sophisticated information processing devices that compute cell fate decisions by integrating external and internal cues. A critical component of this compute function is the dynamic regulation of RAS activation and downstream signaling that allows RAS to produce a rich and nuanced spectrum of biological outputs. We discuss recent findings how the dynamics of RAS and its downstream signaling is regulated. Starting from the structural and biochemical properties of wild-type and mutant RAS proteins and their activation cycle, we examine higher molecular assemblies, effector interactions and downstream signaling outputs, all under the aspect of dynamic regulation. We also consider how computational and mathematical modeling approaches contribute to analyze and understand the pleiotropic functions of RAS in health and disease.
... RAB5 exchange factor (Rabex-5) is a key player embedding both ubiquitin binding and E3 ligase domains for NRAS and HRAS but not KRAS modification. Rabex-5 is able to promote both mono-and di-ubiquitination and downregulates RAS function through directing non-degradational ubiquitin-mediated relocalization at the endosomes [109,110]. Beside influencing protein amount and localization, KRAS monoubiquitination was found to alter RAS interactions with regulatory proteins and effectors, thus upregulating its activity by enhancing GTP loading. In general, it was reported that, in the cell, the ubiquitinated KRAS forms are in the activated GTP-bound state and show enhanced binding to the downstream effectors as rapidly accelerated fibrosarcoma (RAF), phosphatidylinositol 3-kinase (PI3K), and RAS-like guanine nucleotide exchange factors (RalGEF) [82,83]. ...
A cancer outcome is a multifactorial event that comes from both exogenous injuries and an endogenous predisposing background. The healthy state is guaranteed by the fine-tuning of genes controlling cell proliferation, differentiation, and development, whose alteration induces cellular behavioral changes finally leading to cancer. The function of proteins in cells and tissues is controlled at both the transcriptional and translational level, and the mechanism allowing them to carry out their functions is not only a matter of level. A major challenge to the cell is to guarantee that proteins are made, folded, assembled and delivered to function properly, like and even more than other proteins when referring to oncogenes and onco-suppressors products. Over genetic, epigenetic, transcriptional, and translational control, protein synthesis depends on additional steps of regulation. Post-translational modifications are reversible and dynamic processes that allow the cell to rapidly modulate protein amounts and function. Among them, ubiquitination and ubiquitin-like modifications modulate the stability and control the activity of most of the proteins that manage cell cycle, immune responses, apoptosis, and senescence. The crosstalk between ubiquitination and ubiquitin-like modifications and post-translational modifications is a keystone to quickly update the activation state of many proteins responsible for the orchestration of cell metabolism. In this light, the correct activity of post-translational machinery is essential to prevent the development of cancer. Here we summarize the main post-translational modifications engaged in controlling the activity of the principal oncogenes and tumor suppressors genes involved in the development of most human cancers.
... By literature search, we confirmed that 27% of the proposed feedback cases are already known (Additional file 5: Table S4). Some well-documented examples include the RIN1-HRAS-loop (RIN1 is required for Rabex-5-dependent Ras ubiquitination; [38]), RAF1-PAK1-loop (PAK phosphorylates and activates RAF1; [39]), GRB7-ERBB2loop (ERBB2 can phosphorylate and activate Grb7; [40]) and RASSF1-TP53-loop (TP53 binding to the RASSF1A promoter down-regulated RASSF1A expression; [41]). ...
Background -
Ras is a key cellular signaling hub that controls numerous cell fates via multiple downstream effector pathways. While pathways downstream of effectors such as Raf, PI3K and RalGDS are extensively described in the literature, how other effectors signal downstream of Ras is often still enigmatic.
Methods -
A comprehensive and unbiased Ras-effector network was reconstructed downstream of 43 effector proteins (converging onto 12 effector classes) using public pathway and protein–protein interaction (PPI) databases. The output is an oriented graph of pairwise interactions defining a 3-layer signaling network downstream of Ras. The 2290 proteins comprising the network were studied for their implication in signaling crosstalk and feedbacks, their subcellular localizations, and their cellular functions.
Results -
The final Ras-effector network consists of 2290 proteins that are connected via 19,080 binary PPIs, increasingly distributed across the downstream layers, with 441 PPIs in layer 1, 1660 in layer 2, and 16,979 in layer 3. We identified a high level of crosstalk among proteins of the 12 effector classes. A class-specific Ras sub-network was generated in CellDesigner (.xml file) and a functional enrichment analysis thereof shows that 58% of the processes have previously been associated to a respective effector pathway, with the remaining providing insights into novel and unexplored functions of specific effector pathways.
Conclusions -
Our large-scale and cell general Ras-effector network is a crucial steppingstone towards defining the network boundaries. It constitutes a ‘reference interactome’ and can be contextualized for specific conditions, e.g. different cell types or biopsy material obtained from cancer patients. Further, it can serve as a basis for elucidating systems properties, such as input–output relationships, crosstalk, and pathway redundancy.
... H-Ras and N-Ras have been reported to be diubiquitinated in a lysine residue, while K-Ras resists these modifications (Jura et al., 2006). Ubiquitination of Ras inhibits endosomal Ras and prevents activation of ERK (Xu et al., 2010;Yan et al., 2010). Ras can also be activated via ubiquitination (Sasaki et al., 2011). ...
Mutation in RAS gene is one of the most common genetic alterations, which seems to be seen in one third of human cancers.
Ras, as a molecular switch, has been considered in wide variety of signaling pathways such as cell division and apoptosis. Ras
proteins have a binary function to transmit diferent extracellular messages into intracellular signaling network. It has been
proved that Ras proteins associate with diferent plasma membranes. Although all Ras isoforms have been found at plasma
membrane, H-Ras and N-Ras are located in Golgi, and K-Ras at ER and mitochondria outer membrane. There have been a
lot of eforts to inhibit Ras signaling that can be a pivotal approach to treat Ras-induced tumors. Efect of RalA and RalB on
the growth of embryonal tumors, at downstream region of Ras, has been studied in a number of studies, which showed that
inhibition of these signaling pathways can provide a strong therapeutic approach to cancer. Also, post translational modifca-
tions (PTMs) in proteins interfere extremely with cell signaling pathways in cells that can react to external signals. In this
review, the role of Ral signaling in cancer and PTM of Ras proteins has been reviewed.
... Lysine deacetylases, HDAC6 and SIRT2, are suggested to negatively regulate K-RAS acetylation in cancer cells (Yang et al., 2013;Knyphausen et al., 2016). RABEX5, an E3 Ubiquitin ligase, catalyzes monoand di-ubiquitylation of H-and N-RAS, but not K-RAS, which downregulates RAS activity (Xu et al., 2010;Yan et al., 2010;Washington et al., 2020). The ubiquitylation site(s) by RABEX5 remains unclear. ...
RAS is a founding member of the RAS superfamily of GTPases. These small 21 kDa proteins function as molecular switches to initialize signaling cascades involved in various cellular processes, including gene expression, cell growth, and differentiation. RAS is activated by GTP loading and deactivated upon GTP hydrolysis to GDP. Guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) accelerate GTP loading and hydrolysis, respectively. These accessory proteins play a fundamental role in regulating activities of RAS superfamily small GTPase via a conserved guanine binding (G)-domain, which consists of five G motifs. The Switch regions lie within or proximal to the G2 and G3 motifs, and undergo dynamic conformational changes between the GDP-bound “OFF” state and GTP-bound “ON” state. They play an important role in the recognition of regulatory factors (GEFs and GAPs) and effectors. The G4 and G5 motifs are the focus of the present work and lie outside Switch regions. These motifs are responsible for the recognition of the guanine moiety in GTP and GDP, and contain residues that undergo post-translational modifications that underlie new mechanisms of RAS regulation. Post-translational modification within the G4 and G5 motifs activates RAS by populating the GTP-bound “ON” state, either through enhancement of intrinsic guanine nucleotide exchange or impairing GAP-mediated down-regulation. Here, we provide a comprehensive review of post-translational modifications in the RAS G4 and G5 motifs, and describe the role of these modifications in RAS activation as well as potential applications for cancer therapy.
... As mentioned above, RAS is regulated by ubiquitin-mediated degradation. A number of E3 ubiquitin ligases have been implicated in negatively regulating RAS including Rabex-5 [57,58], leucine zipperlike transcription regulator 1 (LZTR1) [59,60], and β-TrCP [44]. In human cells, ubiquitylation of RAS by Rabex-5 targeted RAS to the endosome and reduced ERK-MAPK activation. ...
RAS GTPases are important mediators of oncogenesis in humans. However, pharmacologic inhibition of RAS has proved challenging. We have taken a novel approach to discover vulnerabilities in RAS that can be exploited to inhibit RAS signaling and tumorigenesis. Monobodies are single-domain synthetic binding proteins that achieve levels of affinity and selectivity similar to antibodies but are insensitive to the redox potential of their environment. We have developed a panel of monobodies that target distinct vulnerabilities in RAS. We recently described the activity of the NS1 monobody at inhibiting RAS signaling. NS1 binds to the α4-α5 allosteric lobe of RAS to prevent RAS dimerization and nanoclustering. When introduced into cells as a genetically encoded reagent, NS1 inhibits RAS signaling and oncogenic transformation in vitro through blocking the ability of RAS to self-associate and stimulate the dimerization and activation of RAF. Using a chemically regulated NS1 expression system, we demonstrate that targeting the α4-α5 dimerization interface with NS1 inhibits KRAS-driven tumors in vivo. In addition to NS1, we will discuss our results with monobodies targeting additional aspects of RAS biochemistry. Our results establish the importance of RAS dimerization through the α4-α5 region in mediating RAS signaling and oncogenic transformation of cells both in vitro and in vivo and reveal additional vulnerabilities in RAS that may be targeted to inhibit RAS-driven tumors.
Citation Format: Imran Khan, Russell Spencer-Smith, Kevin Teng, Akiko Koide, Shohei Koide, John P. O'Bryan. Inhibition of RAS signaling and tumorigenesis through targeting vulnerabilities in RAS biochemistry [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr B01.
... In support of the nuclear location of Tc964, we predicted the presence of two nuclear location motifs in the Tc964 protein, i.e., a non-classical Nuclear Localization Signal composed by the PRVRY and NPYTTRP motifs and a Nuclear Export Signal rich in Leucine (L) [45,46]. Regarding the NLS sequence, in the PRVFY motif, the Proline (P), the first Arginine (R), Valine (V), and Tyrosine (Y) residues, are identical (amino acids 105-109) ( Figure S2). ...
The Tc964 protein was initially identified by its presence in the interactome associated with the LYT1 mRNAs, which code for a virulence factor of Trypanosoma cruzi. Tc964 is annotated in the T. cruzi genome as a hypothetical protein. According to phylogenetic analysis, the protein is conserved in the different genera of the Trypanosomatidae family; however, recognizable orthologues were not identified in other groups of organisms. Therefore, as a first step, an in-depth molecular characterization of the Tc946 protein was carried out. Based on structural predictions and molecular dynamics studies, the Tc964 protein would belong to a particular class of GTPases. Subcellular fractionation analysis indicated that Tc964 is a nucleocytoplasmic protein. Additionally, the protein was expressed as a recombinant protein in order to analyze its antigenicity with sera from Chagas disease (CD) patients. Tc964 was found to be antigenic, and B-cell epitopes were mapped by the use of synthetic peptides. In parallel, the Leishmania major homologue (Lm964) was also expressed as recombinant protein and used for a preliminary evaluation of antigen cross-reactivity in CD patients. Interestingly, Tc964 was recognized by sera from Chronic CD (CCD) patients at different stages of disease severity, but no reactivity against this protein was observed when sera from Colombian patients with cutaneous leishmaniasis were analyzed. Therefore, Tc964 would be adequate for CD diagnosis in areas where both infections (CD and leishmaniasis) coexist, even though additional assays using larger collections of sera are needed in order to confirm its usefulness for differential serodiagnosis.
... All three Ras isoforms, H-Ras, K-Ras (two splice variants, K-Ras4A and K-Ras4B), and N-Ras reside in the plasma membrane and switch on/off for the downstream signal transduction [74], whereas the ubiquitination of Ras has been shown to control Ras protein turnover as well as its subcellular localization [50]. Rabex-5 (also known as RabGEF1) functions as an E3 ligase for mediating Ras (H-Ras and N-Ras, but not K-Ras) ubiquitination to promote Ras endosomal localization, and further leads to the suppression of ERK activation [75]. Meanwhile NEDD4-1 can regulate Ras-GDP level of all three forms and subsequently drives PTEN degradation, leading to tumor processes [76] (Fig. 1). ...
Abstract The ubiquitin system, known as a common feature in eukaryotes, participates in multiple cellular processes, such as signal transduction, cell-cycle progression, receptor trafficking and endocytosis, and even the immune response. In lung cancer, evidence has revealed that aberrant events in ubiquitin-mediated processes can cause a variety of pathological outcomes including tumorigenesis and metastasis. Likewise, ubiquitination on the core components contributing to the activity of cell signaling controls bio-signal turnover and cell final destination. Given this, inhibitors targeting the ubiquitin system have been developed for lung cancer therapies and have shown great prospects for clinical application. However, the exact biological effects and physiological role of the drugs used in lung cancer therapies are still not clearly elucidated, which might seriously impede the progress of treatment. In this work, we summarize current research advances in cell signal regulation processes mediated through the ubiquitin system during the development of lung cancer, with the hope of improving the therapeutic effects by means of aiming at efficient targets.
... As mentioned above, RAS is regulated by ubiquitin-mediated degradation. A number of E3 ubiquitin ligases have been implicated in negatively regulating RAS including Rabex-5 [57,58], leucine zipperlike transcription regulator 1 (LZTR1) [59,60], and β-TrCP [44]. In human cells, ubiquitylation of RAS by Rabex-5 targeted RAS to the endosome and reduced ERK-MAPK activation. ...
RAS is the most frequently mutated oncogene in cancer and a critical driver of oncogenesis. Therapeutic targeting of RAS has been a goal of cancer research for >30 years due to its essential role in tumor formation and maintenance. Yet the quest to inhibit this challenging foe has been elusive. Although once considered "undruggable", the struggle to directly inhibit RAS has seen recent success with the development of pharmacological agents that specifically target the KRAS(G12C) mutant protein, which include the first direct RAS inhibitor to gain entry to clinical trials. However, the limited applicability of these inhibitors to G12C-mutant tumors demands further efforts to identify more broadly efficacious RAS inhibitors. Understanding allosteric influences on RAS may open new avenues to inhibit RAS. Here, we provide a brief overview of RAS biology and biochemistry, discuss the allosteric regulation of RAS, and summarize the various approaches to develop RAS inhibitors.
... The earliest studies examining the effects of ubiquitination on RAS were conducted in mammalian and Drosophila cells. In both studies, Rabex-5 was identified as a key regulator of RAS function (69,70). Rabex-5 is a multi-domain protein that contains an exchange factor domain that promotes activation of RAB5 GTPases during endocytosis. ...
Many sensory and chemical signal inputs are transmitted by intracellular GTP-binding (G) proteins. G proteins make up two major subfamilies: "large" G proteins comprising three subunits and "small" G proteins, such as the proto-oncogene product RAS, which contains a single subunit. Members of both subfamilies are regulated by post-translational modifications, including lipidation, proteolysis and carboxyl methylation. Emerging studies have shown that these proteins are also modified by ubiquitination. Much of our current understanding of this post-translational modification comes from investigations of the large G protein α subunit from yeast (Gpa1) and the three RAS isotypes in humans, NRAS, KRAS and HRAS. Gα undergoes both mono- and polyubiquitination, and these modifications have distinct consequences for determining the sites and mechanisms of its degradation. Genetic and biochemical reconstitution studies have revealed the enzymes and binding partners required for addition and removal of ubiquitin, as well as the delivery and destruction of both the mono- and polyubiquitinated forms of the G protein. Complementary studies of RAS have identified multiple ubiquitination sites, each having distinct consequences for binding to regulatory proteins, shuttling to and from the plasma membrane and degradation. Here, we review what is currently known about these two well-studied examples, Gpa1 and the human RAS proteins, which have revealed additional mechanisms of signal regulation and dysregulation relevant to human physiology. We also compare and contrast the effects of G protein ubiquitination with other post-translational modifications of these proteins.
... Ubiquitination plays a relevant role in the modulation of RAS-MAPK signaling dynamics (33), and its defective function associated with dominantly acting inactivating germline mutations in CBL, which encodes a multivalent adaptor protein with E3 ubiquitin ligase activity controlling cell surface receptor endocytosis and degradation, has been documented in RASopathies (34)(35)(36)(37). Mono-and di-ubiquitination of HRAS and NRAS has been demonstrated to drive endosomal targeting of both the GTPases and attenuate RAS-MAPK signaling (38), and an E3 ligase, RABGEF1 (also known as RABEX5), has been identified to mediate RAS ubiquitination and promote suppression of MAPK activation (39,40). On the other hand, KRAS monoubiquitination has been demonstrated to enhance its activation and facilitates its binding to downstream effectors (41). ...
Noonan syndrome (NS), the most common RASopathy, is caused by mutations affecting signaling through RAS and the MAPK cascade. Recently, genome scanning has discovered novel genes implicated in NS, whose function in RAS-MAPK signaling remains obscure, suggesting the existence of unrecognized circuits contributing to signal modulation in this pathway. Among these genes, LZTR1 encodes a functionally poorly characterized member of the BTB/POZ protein superfamily. Two classes of germline LZTR1 mutations underlie dominant and recessive forms of NS, while constitutional monoallelic, mostly inactivating, mutations in the same gene cause schwannomatosis, a cancer-prone disorder clinically distinct from NS. Here we show that dominant NS-causing LZTR1 mutations do not affect significantly protein stability and subcellular localization. We provide the first evidence that these mutations, but not the missense changes occurring as biallelic mutations in recessive NS, enhance stimulus-dependent RAS-MAPK signaling, which is triggered, at least in part, by an increased RAS protein pool. Moreover, we document that dominant NS-causing mutations do not perturb binding of LZTR1 to CUL3, a scaffold coordinating the assembly of a multimeric complex catalyzing protein ubiquitination, but are predicted to affect the surface of the Kelch domain mediating substrate binding to the complex. Collectively, our data suggest a model in which LZTR1 contributes to the ubiquitination of protein(s) functioning as positive modulator(s) of the RAS-MAPK signaling pathway. In this model, LZTR1 mutations are predicted to variably impair binding of these substrates to the multi-component ligase complex and their efficient ubiquitination and degradation, resulting in MAPK signaling upregulation.
... S12E), Thus, LZTR1 regulates RAS by a nondegradative mechanism. Ubiquitination of RAS also induces its relocalization to endomembranes (19,20). However, LZTR1 overexpression increased the endomembrane fraction of both wt-RAS and the HRAS-K170R mutant ( fig. ...
Regulation of RAS by ubiquitination
The protein LZTR1 is mutated in human cancers and developmental diseases. Work from two groups now converges to implicate the protein in regulating signaling by the small guanosine triphosphatase RAS. Steklov et al. showed that mice haploinsufficient for LZTR1 recapitulated aspects of the human disease Noonan syndrome. Their biochemical studies showed that LZTR1 associated with RAS. LZTR1 appears to function as an adaptor that promotes ubiquitination of RAS, thus inhibiting its signaling functions. Bigenzahn et al. found LZTR1 in a screen for proteins whose absence led to resistance to the tyrosine kinase inhibitors used to treat cancers caused by the BCR-ABL oncogene product. Their biochemical studies and genetic studies in fruitflies also showed that loss of LZTR1 led to increased activity of RAS and signaling through the mitogen-activated protein kinase pathway.
Science , this issue p. 1177 , p. 1171
... Their activities, subcellular localizations, and stabilities are therefore tightly controlled in normal cells; deregulation of any of these features often causes malignant transformation (1). Ras proteins are regulated by a wide range of post-translational modifications, including farnesylation, carboxylmethylation, palmitoylation, phosphorylation, ubiquitination, and acetylation (1,7-10) (8,(11)(12)(13)(14)(15). The RAS/RAF/MEK/ERK signaling pathway plays a central role in mediating cell survival and proliferation, and its dysregulation was detected in more than 50% of human tumors (16). ...
Ras proteins participate in multiple signal cascades, regulating crucial cellular processes including cell survival, proliferation, and differentiation. We have previously reported that Ras proteins are modified by sumoylation and that lysine-42 (K42) plays an important role in mediating the modification. In the current study, we further investigated the role of K42 in regulating cellular activities of K-Ras. Inducible expression of K-RasV12 led to the activation of downstream components including c-RAF, MEK1, and ERKs whereas expression of K-RasV12/R42 mutant compromised the activation of the RAF/MEK/ERK signaling axis. Expression of K-RasV12/R42 also led to reduced phosphorylation of several other protein kinases including JNK, Chk2, and FAK. Significantly, K-RasV12/R42 expression inhibited cellular migration and invasion in vitro in multiple cell lines including transformed pancreatic cells. Given K-Ras plays a crucial role in mediating oncogenesis in pancreas, we treated transformed pancreatic cells of both BxPC-3 and MiaPaCa-2 with 2-D08, an SUMO E2 inhibitor. Treatment with the compound inhibited cell migration in a concentration-dependent manner, which was correlated with a reduced level of K-Ras sumoylation. Moreover, 2-D08 suppressed expression of ZEB1 (a mesenchymal cell marker) with concomitant induction of ZO-1 (an epithelial cell marker). Combined, our studies strongly suggest that post-translational modification(s) including sumoylation mediated by K42 plays a crucial role in K-Ras activities in vivo.
... Regarding activity modulation, ubiquitination is known to regulate the activity of both oncogenes and tumor suppressors in cancer. For example, the oncogenic GTPase K-RAS was previously shown to be ubiquitinated by Rabex-5 to promote its endosomal localization by beta-TrCP (TrCP), a key member of the SkpI-Cdc53-F-box E3, to mediate its proteasomal degradation (Xu et al., 2010). However, ubiquitination also impairs K-Ras response to GTPase-activating proteins (GAPs). ...
Dynamic modulation and posttranslational modification of proteins are tightly controlled biological processes that occur in response to physiological cues. One such dynamic modulation is ubiquitination, which marks proteins for degradation via the proteasome, altering their localization, affecting their activity, and promoting or interfering with protein interactions. Hence, ubiquitination is crucial for a plethora of physiological processes, including cell survival, differentiation and innate and adaptive immunity. Similar to kinases, components of the ubiquitination system are often deregulated, leading to a variety of diseases, such as cancer and neurodegenerative disorders. In a context-dependent manner, ubiquitination can regulate both tumor-suppressing and tumor-promoting pathways in cancer. This review outlines how components of the ubiquitination systems (e.g. E3 ligases and deubiquitinases) act as oncogenes or tumor suppressors according to the nature of their substrates. Furthermore, I interrogate how the current knowledge of the differential roles of ubiquitination in cancer lead to technical advances to inhibit or reactivate the components of the ubiquitination system accordingly.
... These modifications were first reported on NRAS and HRAS and modification of the latter promoted association with endosomes. The E3 ligase for HRAS was identified as RABEX5 (Xu et al. 2010), a protein that also has exchange activity for RAB5. Interestingly, the RAS effector RIN1 was required for the ubiquitination reaction. ...
The three human RAS genes encode four proteins that play central roles in oncogenesis by acting as binary molecular switches that regulate signaling pathways for growth and differentiation. Each is subject to a set of posttranslational modifications (PTMs) that modify their activity or are required for membrane targeting. The enzymes that catalyze the various PTMs are potential targets for anti-RAS drug discovery. The PTMs of RAS proteins are the focus of this review. Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved.
... The endosomally localized Rabex5 (also known as RabGEF1) functions partly as an E3 ubiquitin ligase and promotes mono-and di-ubiquitination of H-RAS and N-RAS, leading to the anchoring of RAS at the endosomes and reducing downstream signalling [150,151]. It has been shown that Rabex-5 contains an A20 like Zing finger ubiquitin ligase domain (ZnF) which mediates the interaction of Rabex-5 with H-RAS and N-RAS. ...
Article Methylation of KRAS by SETD7 promotes KRAS degradation in non-small cell lung cancer Graphical abstract Highlights d SETD7 interacts with KRAS and methylates KRAS d SETD7-mediated methylation of KRAS leads to KRAS degradation d RABGEF1 induces KRAS poly-ubiquitination in a methylation-dependent manner d SETD7 is inversely correlated with KRAS at the protein level in clinical NSCLC tissues In brief Post-translational methylation and its role in modifying KRAS activity remain largely unclear. Chiang et al. identify that SETD7 interacts with KRAS and methylates KRAS at lysines 182 and 184. Then, E3 ligase RABGEF1 is recruited in a methylation-dependent manner and promotes polyubiquitination and degradation of KRAS. SUMMARY Oncogenic KRAS mutations are a key driver for initiation and progression in non-small cell lung cancer (NSCLC). However, how post-translational modifications (PTMs) of KRAS, especially methylation, modify KRAS activity remain largely unclear. Here, we show that SET domain containing histone lysine methyltrans-ferase 7 (SETD7) interacts with KRAS and methylates KRAS at lysines 182 and 184. SETD7-mediated methyl-ation of KRAS leads to degradation of KRAS and attenuation of the RAS/MEK/ERK signaling cascade, endowing SETD7 with a potent tumor-suppressive role in NSCLC, both in vitro and in vivo. Mechanistically, RABGEF1, a ubiquitin E3 ligase of KRAS, is recruited and promotes KRAS degradation in a K182/K184 methylation-dependent manner. Notably, SETD7 is inversely correlated with KRAS at the protein level in clinical NSCLC tissues. Low SETD7 or RABGEF1 expression is associated with poor prognosis in lung adenocar-cinoma patients. Altogether, our results define a tumor-suppressive function of SETD7 that operates via modulating KRAS methylation and degradation.
Mutations of RAS genes drive cancer more frequently than any other oncogene. RAS proteins integrate signals from a wide array of receptors and initiate downstream signaling through pathways that control cellular growth. RAS proteins are fundamentally binary molecular switches in which the off/on state is determined by the binding of GDP or GTP, respectively. As such, the intrinsic and regulated nucleotide-binding and hydrolytic properties of the RAS GTPase were historically believed to account for the entirety of the regulation of RAS signaling. However, it is increasingly clear that RAS proteins are also regulated by a vast array of post-translational modifications (PTMs). The current challenge is to understand what are the functional consequences of these modifications and which are physiologically relevant. Because PTMs are catalyzed by enzymes that may offer targets for drug discovery, the study of RAS PTMs has been a high priority for RAS biologists.
Ras proteins are small GTPases that participate in multiple signal cascades, regulating crucial cellular processes including cell survival, proliferation, and differentiation. Mutations or deregulated activities of Ras are frequently the driving force for oncogenic transformation and tumorigenesis. Posttranslational modifications play a crucial role in mediating the stability, activity, or subcellular localization/trafficking of numerous cellular regulators including Ras proteins. A series of recent studies reveal that Ras proteins are also regulated by sumoylation. All three Ras protein isoforms (HRas, KRas, and NRas) are modified by SUMO3. The conserved lysine42 appears to be the primary site for mediating sumoylation. Expression of KRasV12/R42 mutants compromised the activation of the Raf/MEK/ERK signaling axis, leading to a reduced rate of cell migration and invasion in vitro in multiple cell lines. Moreover, treatment of transformed pancreatic cells with a SUMO E2 inhibitor blocks cell migration in a concentration-dependent manner, which is associated with a reduced level of both KRas sumoylation and expression of mesenchymal cell markers. Furthermore, mouse xenograft experiments reveal that expression of a SUMO-resistant mutant appears to suppress tumor development in vivo. Combined, these studies indicate that sumoylation functions as an important mechanism in mediating the roles of Ras in cell proliferation, differentiation, and malignant transformation and that the SUMO-modification system of Ras oncoproteins can be explored as a new druggable target for various human malignancies.
RAS, a member of the small GTPase family, functions as a binary switch by shifting between inactive GDP-loaded and active GTP-loaded state. RAS gain-of-function mutations are one of the leading causes in human oncogenesis, accounting for ∼19% of the global cancer burden. As a well-recognized target in malignancy, RAS has been intensively studied in the past decades. Despite the sustained efforts, many failures occurred in the earlier exploration and resulted in an ‘undruggable’ feature of RAS proteins. Phosphorylation at several residues has been recently determined as regulators for wild-type and mutated RAS proteins. Therefore, the development of RAS inhibitors directly targeting the RAS mutants or towards upstream regulatory kinases supplies a novel direction for tackling the anti-RAS difficulties. A better understanding of RAS phosphorylation can contribute to future therapeutic strategies. In this review, we comprehensively summarized the current advances in RAS phosphorylation and provided mechanistic insights into the signaling transduction of associated pathways. Importantly, the preclinical and clinical success in developing anti-RAS drugs targeting the upstream kinases and potential directions of harnessing allostery to target RAS phosphorylation sites were also discussed.
RAS was identified as a human oncogene in the early 1980s and subsequently found to be mutated in nearly 30% of all human cancers. More importantly, RAS plays a central role in driving tumor development and maintenance. Despite decades of effort, there remain no FDA approved drugs that directly inhibit RAS. The prevalence of RAS mutations in cancer and the lack of effective anti-RAS therapies stem from RAS’ core role in growth factor signaling, unique structural features, and biochemistry. However, recent advances have brought promising new drugs to clinical trials and shone a ray of hope in the field. Here, we will exposit the details of RAS biology that illustrate its key role in cell signaling and shed light on the difficulties in therapeutically targeting RAS. Furthermore, past and current efforts to develop RAS inhibitors will be discussed in depth.
Ubiquitin ligases (E3) play a crucial role in the regulation of different cellular processes such as proliferation and differentiation via recognition, interaction, and ubiquitination of key cellular proteins in a spatial and temporal regulated manner. The type of ubiquitin chain formed determines the fate of the substrates. The ubiquitinated substrates can be degraded by the proteasome, display altered subcellular localization, or can suffer modifications on their interaction with functional protein complexes. Deregulation of E3 activities is frequently found in various human pathologies, including cancer. The illegitimated or accelerated degradation of oncosuppressive proteins or, inversely, the abnormally high accumulation of oncoproteins, contributes to cell proliferation and transformation. Anomalies in protein abundance may be related to mutations that alter the direct or indirect recognition of proteins by the E3 enzymes or alterations in the level of expression or activity of ubiquitin ligases. Through a few examples, we illustrate here the complexity and diversity of the molecular mechanisms related to protein ubiquitination involved in cell cycle regulation. We will discuss the role of ubiquitin-dependent degradation mediated by the proteasome, the role of non-proteolytic ubiquitination during cell cycle progression, and the consequences of this deregulation on cellular transformation. Finally, we will highlight the novel opportunities that arise from these studies for therapeutic intervention.
H-Ras is a unique isoform of the Ras GTPase family, one of the most prominently mutated oncogene families across the cancer landscape. Relative to other isoforms, though, mutations of H-Ras account for the smallest proportion of mutant Ras cancers. Yet, in recent years, there have been renewed efforts to study this isoform, especially as certain H-Ras–driven cancers, like those of the head and neck, have become more prominent. Important advances have therefore been made not only in the understanding of H-Ras structural biology but also in approaches designed to inhibit and impair its signaling activity. In this review, we outline historic and present initiatives to elucidate the mechanisms of H-Ras–dependent tumorigenesis as well as highlight ongoing developments in the quest to target this critical oncogene.
The three RAS genes — HRAS, NRAS and KRAS — are collectively mutated in one-third of human cancers, where they act as prototypic oncogenes. Interestingly, there are rather distinct patterns to RAS mutations; the isoform mutated as well as the position and type of substitution vary between different cancers. As RAS genes are among the earliest, if not the first, genes mutated in a variety of cancers, understanding how these mutation patterns arise could inform on not only how cancer begins but also the factors influencing this event, which has implications for cancer prevention. To this end, we suggest that there is a narrow window or ‘sweet spot’ by which oncogenic RAS signalling can promote tumour initiation in normal cells. As a consequence, RAS mutation patterns in each normal cell are a product of the specific RAS isoform mutated, as well as the position of the mutation and type of substitution to achieve an ideal level of signalling.
The ubiquitin system regulates diverse biological processes, many involved in cancer pathogenesis, by altering the ubiquitination state of protein substrates. This is accomplished by ubiquitin ligases and deubiquitinases (DUBs), which respectively add or remove ubiquitin from substrates to alter their stability, activity, localization, and interactions. While lack of catalytic activity makes therapeutic targeting of ubiquitin ligases difficult, DUB inhibitors represent an active area of research and the identification of cancer-associated DUBs may lead to the development of novel therapeutics. A growing body of literature demonstrates that the DUB Otubain 1 (OTUB1) regulates many cancer-associated signaling pathways including MAPK, ERa, EMT, RHOa, mTORC1, FOXM1, and P53 to promote tumor cell survival, proliferation, invasiveness, and therapeutic resistance. In addition, clinical studies have associated elevated OTUB1 expression with high grade, invasiveness, and metastasis in several tumor types including lung, breast, ovarian, glioma, colon, and gastric. Interestingly, in addition to catalytic DUB activity, OTUB1 displays a catalytic-independent, non-canonical activity where it inhibits the transfer of ubiquitin onto protein substrates by sequestration of E2 ubiquitin conjugating enzymes. The aim of this review is to describe the canonical and non-canonical activities of OTUB1, summarize roles for OTUB1 in cancer-associated pathways, and discuss its potential therapeutic targeting.
The cellular response to external stress signals and DNA damage depends on the activity of ubiquitin ligases (E3s), which regulate numerous cellular processes, including homeostasis, metabolism and cell cycle progression. E3s recognize, interact with and ubiquitylate protein substrates in a temporally and spatially regulated manner. The topology of the ubiquitin chains dictates the fate of the substrates, marking them for recognition and degradation by the proteasome or altering their subcellular localization or assembly into functional complexes. Both genetic and epigenetic alterations account for the deregulation of E3s in cancer. Consequently, the stability and/or activity of E3 substrates are also altered, in some cases leading to downregulation of tumour-suppressor activities and upregulation of oncogenic activities. A better understanding of the mechanisms underlying E3 regulation and function in tumorigenesis is expected to identify novel prognostic markers and to enable the development of the next generation of anticancer therapies. This Review summarizes the oncogenic and tumour-suppressor roles of selected E3s and highlights novel opportunities for therapeutic intervention.
The endosomal sorting complexes required for transport (ESCRTs) machinery consists of four protein assemblies (ESCRT-0 to −III subcomplexes) which mediate various processes of membrane remodeling in the cell. In the endocytic pathway, ESCRTs sort cargo destined for degradation into intraluminal vesicles (ILVs) of endosomes. Cargos targeted by ESCRTs include various signaling molecules, mainly internalized cell-surface receptors but also some cytosolic proteins. It is therefore expected that aberrant trafficking caused by ESCRT dysfunction affects different signaling pathways. Here we review how perturbation of ESCRT activity alters intracellular transport of membrane receptors, causing their accumulation on endocytic compartments, decreased degradation and/or altered recycling to the plasma membrane. We further describe how perturbed trafficking of receptors impacts the activity of their downstream signaling pathways, with or without changes in transcriptional responses. Finally, we present evidence that ESCRT components can also control activity and intracellular distribution of cytosolic signaling proteins (kinases, other effectors and soluble receptors). The underlying mechanisms involve sequestration of such proteins in ILVs, their sorting for degradation or towards non-lysosomal destinations, and regulating their availability in various cellular compartments. All these ESCRT-mediated processes can modulate final outputs of multiple signaling pathways.
Identification of a novel post-translational modification (PTM) for a target protein, defining its physiologic role, and studying its potential crosstalk with other PTMs is a challenging process. A set of highly sensitive tools termed Signal-Seeker kits was developed, which enables rapid and simple detection of post-translational modifications on any target protein. The methodology for these tools utilizes affinity purification of modified proteins from a cell or tissue lysate and immunoblot analysis. These tools utilize a single lysis system that is effective at identifying endogenous, dynamic PTM changes, as well as the potential crosstalk between PTMs. As a proof-of-concept experiment, the acetylation, tyrosine phosphorylation, SUMOylation 2/3, and ubiquitination profiles of the EGFR - Ras - c-Fos axis were examined in response to EGF stimulation. All 10 previously identified PTMs of this signaling axis were confirmed using these tools, and it also identified acetylation as a novel modification of c-Fos. This axis in the EGF/EGFR signaling pathway was chosen because it is a well-established signaling pathway with proteins localized in the membrane, cytoplasmic, and nuclear compartments that ranged in abundance from 4.18x10(8) (EGFR) to 1.35x10(4) (c-Fos) molecules per A431 cell. These tools enabled the identification of low abundance PTMs, such as c-Fos Ac, at 17 molecules per cell. These studies highlight how pervasive PTMs are, and how stimulants like EGF induce multiple PTM changes on downstream signaling axis. Identification of endogenous changes and potential crosstalk between multiple PTMs for a target protein or signaling axis will provide regulatory mechanistic insight to investigators.
Nearly 30% of human tumours harbour mutations in RAS family members. Post-translational modifications and the localisation of RAS within subcellular compartments affect RAS interactions with regulator, effector and scaffolding proteins. New insights into the control of spatiotemporal RAS signalling reveal that activation kinetics and subcellular compartmentalisation are tightly coupled to the generation of specific biological outcomes. Computational modelling can help utilising these insights for the identification of new targets and design of new therapeutic approaches.
The adaptation to changing environmental conditions often requires a comprehensive reconstruction of the proteome. These rearrangements are initiated by extra- or intracellular signals that activate appropriate cellular programs. They include the removal of proteins that are not useful anymore or detrimental in the new condition, expression of new proteins or the relocalization of existing ones, and activation or inactivation of kinases and other enzymes. The modification of proteins with ubiquitin (ubiquitination) plays crucial roles in the coordination of these dynamic processes.
Human RIN1 was first characterized as a RAS binding protein based on the properties of its carboxyl-terminal domain. We now
show that full-length RIN1 interacts with activated RAS in mammalian cells and defines a minimum region of 434 aa required
for efficient RAS binding. RIN1 interacts with the “effector domain” of RAS and employs some RAS determinants that are common
to, and others that are distinct from, those required for the binding of RAF1, a known RAS effector. The same domain of RIN1
that binds RAS also interacts with 14-3-3 proteins, extending the similarity between RIN1 and other RAS effectors. When expressed
in mammalian cells, the RAS binding domain of RIN1 can act as a dominant negative signal transduction blocker. The amino-terminal
domain of RIN1 contains a proline-rich sequence similar to consensus Src homology 3 (SH3) binding regions. This RIN1 sequence
shows preferential binding to the ABL–SH3 domain in vitro. Moreover, the amino-terminal domain of RIN1 directly associates with, and is tyrosine phosphorylated by, c-ABL. In addition,
RIN1 encodes a functional SH2 domain that has the potential to activate downstream signals. These data suggest that RIN1 is
able to mediate multiple signals. A differential pattern of expression and alternate splicing indicate several levels of RIN1
regulation.
Rab GTPases are central elements of the vesicular transport machinery. An emerging view is that downstream effectors of these GTPases are multiprotein complexes that include nucleotide exchange factors to ensure coupling between GTPase activation and effector function. We have previously shown that Rab5, which regulates various steps of transport along the early endocytic pathway, is activated by a complex consisting of Rabex-5, a Rab5 nucleotide exchange factor, and the effector Rabaptin-5. We postulated that the physical association of these two proteins is necessary for their activity in Rab5-dependent endocytic membrane transport. To evaluate the functional implications of such complex formation, we have reconstituted it with the use of recombinant proteins and characterized its properties. First, we show that Rabaptin-5 increases the exchange activity of Rabex-5 on Rab5. Second, Rab5-dependent recruitment of Rabaptin-5 to early endosomes is completely dependent on its physical association with Rabex-5. Third, complex formation between Rabaptin-5 and Rabex-5 is essential for early endosome homotypic fusion. These results reveal a functional synergy between Rabaptin-5 and Rabex-5 in the complex and have implications for the function of analogous complexes for Rab and Rho GTPases.
Current models evoke the plasma membrane (PM) as the exclusive platform from which Ras regulates signalling. We developed a fluorescent probe that reports where and when Ras is activated in living cells. We show that oncogenic H-Ras and N-Ras engage Raf-1 on the Golgi and that endogenous Ras and unpalmitoylated H-Ras are activated in response to mitogens on the Golgi and endoplasmic reticulum (ER), respectively. We also demonstrate that H-Ras that is restricted to the ER can activate the Erk pathway and transform fibroblasts, and that Ras localized on different membrane compartments differentially engages various signalling pathways. Thus, Ras signalling is not limited to the PM, but also proceeds on the endomembrane.
We show that the specific subcellular distribution of H- and Nras guanosine triphosphate–binding proteins is generated by
a constitutive de/reacylation cycle that operates on palmitoylated proteins, driving their rapid exchange between the plasma
membrane (PM) and the Golgi apparatus. Depalmitoylation redistributes farnesylated Ras in all membranes, followed by repalmitoylation
and trapping of Ras at the Golgi, from where it is redirected to the PM via the secretory pathway. This continuous cycle prevents
Ras from nonspecific residence on endomembranes, thereby maintaining the specific intracellular compartmentalization. The
de/reacylation cycle also initiates Ras activation at the Golgi by transport of PM-localized Ras guanosine triphosphate. Different
de/repalmitoylation kinetics account for isoform-specific activation responses to growth factors.
Palmitoylation is postulated to regulate Ras signaling by modulating its intracellular trafficking and membrane microenvironment. The mechanisms by which palmitoylation contributes to these events are poorly understood. Here, we show that dynamic turnover of palmitate regulates the intracellular trafficking of HRas and NRas to and from the Golgi complex by shifting the protein between vesicular and nonvesicular modes of transport. A combination of time-lapse microscopy and photobleaching techniques reveal that in the absence of palmitoylation, GFP-tagged HRas and NRas undergo rapid exchange between the cytosol and ER/Golgi membranes, and that wild-type GFP-HRas and GFP-NRas are recycled to the Golgi complex by a nonvesicular mechanism. Our findings support a model where palmitoylation kinetically traps Ras on membranes, enabling the protein to undergo vesicular transport. We propose that a cycle of depalmitoylation and repalmitoylation regulates the time course and sites of Ras signaling by allowing the protein to be released from the cell surface and rapidly redistributed to intracellular membranes.
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.
Rabex-5, the mammalian orthologue of yeast Vps9p, is a guanine nucleotide exchange factor for Rab5. Rabex-5 forms a tight complex with Rabaptin-5, a multivalent adaptor protein that also binds to Rab4, Rab5, and to domains present in gamma-adaptins and the Golgi-localized, gamma-ear-containing, ARF-binding proteins (GGAs). Rabaptin-5 augments the Rabex-5 exchange activity, thus generating GTP-bound, membrane-associated Rab5 that, in turn, binds Rabaptin-5 and stabilizes the Rabex-5.Rabaptin-5 complex on endosomes. Although the Rabex-5.Rabaptin-5 complex is critical to the regulation of endosomal fusion, the structural determinants of this interaction are unknown. Likewise, the possible binding and covalent attachment of ubiquitin to Rabex-5, two modifications that are critical to the function of yeast Vps9p in endosomal transport, have not been studied. In this study, we identify the 401-462 and 551-661 coiled-coils as the regions in Rabex-5 and Rabaptin-5, respectively, that interact with one another. We also demonstrate that Rabex-5 undergoes ubiquitination and binds ubiquitin, though not via its proposed C-terminal CUE-like domain. Instead, the N-terminal region of Rabex-5 (residues 1-76), comprising an A20-like Cys2/Cys2 zinc finger and an adjacent alpha-helix, is important for ubiquitin binding and ubiquitination. Importantly, we demonstrate that the Rabex-5 zinc finger displays ubiquitin ligase (E3) activity. These observations extend our understanding of the regulation of Rabex-5 by Rabaptin-5. Moreover, the demonstration that Rabex-5 is a ubiquitin ligase that binds ubiquitin and undergoes ubiquitination indicates that its role in endosome fusion may be subject to additional regulation by ubiquitin-dependent modifications.
Rabex-5 is an exchange factor for Rab5, a master regulator of endosomal trafficking. Rabex-5 binds monoubiquitin, undergoes covalent ubiquitination and contains an intrinsic ubiquitin ligase activity, all of which require an N-terminal A20 zinc finger followed immediately by a helix. The structure of the N-terminal portion of Rabex-5 bound to ubiquitin at 2.5-A resolution shows that Rabex-5-ubiquitin interactions occur at two sites. The first site is a new type of ubiquitin-binding domain, an inverted ubiquitin-interacting motif, which binds with approximately 29-microM affinity to the canonical Ile44 hydrophobic patch on ubiquitin. The second is a diaromatic patch on the A20 zinc finger, which binds with approximately 22-microM affinity to a polar region centered on Asp58 of ubiquitin. The A20 zinc-finger diaromatic patch mediates ubiquitin-ligase activity by directly recruiting a ubiquitin-loaded ubiquitin-conjugating enzyme.
A healthy individual can mount an immune response to exogenous pathogens while avoiding an autoimmune attack on normal tissues. The ability to distinguish between self and non-self is called 'immunological tolerance' and, for T lymphocytes, involves the generation of a diverse pool of functional T cells through positive selection and the removal of overtly self-reactive thymocytes by negative selection during T-cell ontogeny. To elucidate how thymocytes arrive at these cell fate decisions, here we have identified ligands that define an extremely narrow gap spanning the threshold that distinguishes positive from negative selection. We show that, at the selection threshold, a small increase in ligand affinity for the T-cell antigen receptor leads to a marked change in the activation and subcellular localization of Ras and mitogen-activated protein kinase (MAPK) signalling intermediates and the induction of negative selection. The ability to compartmentalize signalling molecules differentially in the cell endows the thymocyte with the ability to convert a small change in analogue input (affinity) into a digital output (positive versus negative selection) and provides the basis for establishing central tolerance.
Rabex-5 is a guanine nucleotide exchange factor (GEF) for Rab5. Here, we report the identification of a novel functional domain of Rabex-5 that is essential for its membrane targeting and Rab5 GEF activity in vivo. The data show that full-length Rabex-5 efficiently activates Rab5 in the cell. However, the GEF domain itself (residues 135-399) is inactive in this respect, despite its activity in vitro. Generation and characterization of a series of Rabex-5 constructs reveal that the GEF domain is unable to target to early endosomes and that a sequence N-terminal to the GEF domain can restore its early endosomal targeting and its ability to activate Rab5 in the cell. This region (residues 81-135) is termed membrane-binding motif, which together with the downstream helical bundle domain (residues 135-230) forms an early endosomal targeting (EET) domain necessary and sufficient for association with early endosomes. Furthermore, several active Rabex-5 constructs do not contain the Rabaptin-5-binding domain in the C-terminal region. Thus, Rabex-5 can target to early endosomes via the EET domain and activate Rab5 in a Rabaptin-5-independent manner in vivo. We discuss a model to reconcile these in vivo data with previous in vitro results on Rabex-5 function and its interaction with Rabaptin-5.
Transforming growth factor β (TGF-β) receptor (TβR) signaling contributes to normal development as well as tumorigenesis.
Here we report that RIN1, a RAB5 guanine nucleotide exchange factor (GEF) and down regulator of receptor tyrosine kinases
(RTKs), promotes TβR signaling through enhanced endocytosis. TβR activation induces SNAI1 (Snail), a transcription repressor
that reduces RIN1 expression, providing a negative feedback mechanism to control TβR trafficking and downstream signaling.
Persistent RAS signaling disrupts this equilibrium by stabilizing SNAI1 protein, resulting in strong silencing of RIN1 and
stabilization of RTKs. TGF-β-induced RIN1 silencing in breast cancer cells prolonged sensitivity to hepatocyte growth factor,
a ligand for the MET-type RTK, and enhanced growth factor-directed cell motility. We conclude that in some tumor cells TβR
and RAS signals are integrated through the silencing of RIN1, leading to a reduction in RAB5-mediated endocytosis. These findings
shed new light on the basis for distinct interpretations of TGF-β signaling by normal versus transformed cells.
The Ras signaling pathway allows cells to translate external cues into diverse biological responses. Depending on context and the threshold reached, Ras signaling can promote growth, proliferation, differentiation, or cell survival. Failure to maintain precise control of Ras can have adverse physiological consequences. Indeed, excess Ras signaling disrupts developmental patterning and causes developmental disorders [1, 2], and in mature tissues, it can lead to cancer [3-5]. We identify Rabex-5 as a new component of Ras signaling crucial for achieving proper pathway outputs in multiple contexts in vivo. We show that Drosophila Rabex-5 restricts Ras signaling to establish organism size, wing vein pattern, and eye versus antennal fate. Rabex-5 has both Rab5 guanine nucleotide exchange factor (GEF) activity that regulates endocytic trafficking [6] and ubiquitin ligase activity [7, 8]. Surprisingly, overexpression studies demonstrate that Rabex-5 ubiquitin ligase activity, not its Rab5 GEF activity, is required to restrict wing vein specification and to suppress the eye phenotypes of oncogenic Ras expression. Furthermore, genetic interaction experiments indicate that Rabex-5 acts at the step of Ras, and tissue culture studies show that Rabex-5 promotes Ras ubiquitination. Together, these findings reveal a new mechanism for attenuating Ras signaling in vivo and suggest an important role for Rabex-5-mediated Ras ubiquitination in pathway homeostasis.
Ras signaling can promote proliferation, cell survival and differentiation. Mutations in components of the Ras pathway are found in many solid tumors and are associated with developmental disorders. We demonstrate here that Drosophila tissues containing hypomorphic mutations in E1, the most upstream enzyme in the ubiquitin pathway, display cell-autonomous upregulation of Ras-ERK activity and Ras-dependent ectopic proliferation. Ubiquitylation is widely accepted to regulate receptor tyrosine kinase (RTK) endocytosis upstream of Ras. However, although the ectopic proliferation of E1 hypomorphs is dramatically suppressed by removing one copy of Ras, removal of the more upstream components Egfr, Grb2 or sos shows no suppression. Thus, decreased ubiquitylation may lead to growth-relevant Ras-ERK activation by failing to regulate a step downstream of RTK endocytosis. We further demonstrate that Drosophila Ras is ubiquitylated. Our findings suggest that Ras ubiquitylation restricts growth and proliferation in vivo. We also report our intriguing observation that complete inactivation of E1 causes non-autonomous activation of Ras-ERK in adjacent tissue, mimicking oncogenic Ras overexpression. We demonstrate that maintaining sufficient E1 function is required both cell autonomously and non-cell autonomously to prevent inappropriate Ras-ERK-dependent growth and proliferation in vivo and may implicate loss of Ras ubiquitylation in developmental disorders and cancer.
The overexpression of some human proteins can cause interference with the Ras signal transduction pathway in the yeast Saccharomyces
cerevisiae. The functional block is located at the level of the effector itself, since these proteins do not suppress activating
mutations further downstream in the same pathway. We now demonstrate, with in vivo and in vitro experiments, that the protein
encoded by one human cDNA (clone 99) can interact directly with yeast Ras2p and with human H-Ras protein, and we have named
this gene rin1 (Ras interaction/interference). The interaction between Ras and Rin1 is enhanced when Ras is bound to GTP.
Rin1 is not able to interact with either an effector mutant or a dominant negative mutant of H-Ras. Thus, Rin1 displays a
human H-Ras interaction profile that is the same as that seen for Raf1 and yeast adenylyl cyclase, two known effectors of
Ras. Moreover, Raf1 directly competes with Rin1 for binding to H-Ras in vitro. Unlike Raf1, however, the Rin1 protein resides
primarily at the plasma membrane, where H-Ras is localized. These data are consistent with Rin1 functioning in mammalian cells
as an effector or regulator of H-Ras.
The small GTPase Rab5 plays an essential role in endocytic traffic. Rab GDP dissociation inhibitor delivers Rab5 to the membrane, where a nucleotide exchange activity allows recruitment of an effector protein, Rabaptin-5. Here we uncovered a novel 60 kDa Rab5-binding protein, Rabex-5. Rabex-5 forms a tight physical complex with Rabaptin-5, and this complex is essential for endocytic membrane fusion. Sequencing of mammalian Rabex-5 by nanoelectrospray mass spectrometry and cloning revealed striking homology to Vps9p, a yeast protein implicated in endocytic traffic. Rabex-5 displays GDP/GTP exchange activity on Rab5 upon delivery of the GTPase to the membrane. This demonstrates that a soluble exchange factor coupled to a Rab effector translocates from cytosol to the membrane, where the complex stabilizes the GTPase in the active state.
S. typhimurium stimulates signaling pathways leading to membrane ruffling, actin cytoskeleton rearrangements, and nuclear responses. The stimulation requires a protein secretion system (type III) that translocates bacterial proteins into the host cell. We show that SopE, a substrate of this secretion system, stimulates cytoskeletal reorganization and JNK activation in a CDC42- and Rac-1-dependent manner. A lambda gt11 cDNA library screen for proteins that interact with SopE identified Rac-1 and CDC42. Furthermore, purified SopE was shown to stimulate GDP/GTP nucleotide exchange in several Rho GTPases in vitro, including Rac-1 and CDC42. These findings establish a paradigm for microbial stimulation of cellular responses in which the pathogen induces signaling events by directly engaging the signaling machinery within the host cell.
The activities of three Rab-specific factors with GDP/GTP exchange activity, Vps9p, Rabex-5 and DSS4, with their cognate GTPases, Ypt51p, Rab5 and Ypt1p, have been analysed quantitatively. In contrast to other exchange factors examined and to DSS4, Vps9p, and by analogy probably Rabex-5, have considerably lower affinity than GDP to the respective GTPases. In keeping with this, they are relatively weak exchangers, with a maximal rate constant for GDP release from the ternary complex between exchange factor, GTPase and GDP of ca 0.01 s(-1), which is several orders of magnitude lower than for other exchange factors examined. If interaction with these proteins is a mandatory aspect of the Rab cycle, this suggests that the overall rate of cycling might be controlled at this point of the cycle. Surprisingly, DSS4, which has the thermodynamic potential to displace GDP effectively from Ypt1p, also does this very slowly, again with a maximal rate constant of ca 0.01 s(-1). An additional, and based on present knowledge, unique, feature of the Ypt1p.DSS4 complex, is that the association of GTP (or GDP) is more than 10(3)-fold slower than to Ypt1p, thus leading to a long life-time of the binary complex between the two proteins, even at the high nucleotide concentrations that prevail in the cell. This leads to the conclusion that the protein-protein complex is likely to have an important biological significance in addition to its probable role in GTP/GDP exchange.
RIN1 was originally identified by its ability to inhibit activated Ras and likely participates in multiple signaling pathways because it binds c-ABL and 14-3-3 proteins, in addition to Ras. RIN1 also contains a region homologous to the catalytic domain of Vps9p-like Rab guanine nucleotide exchange factors (GEFs). Here, we show that this region is necessary and sufficient for RIN1 interaction with the GDP-bound Rabs, Vps21p, and Rab5A. RIN1 is also shown to stimulate Rab5 guanine nucleotide exchange, Rab5A-dependent endosome fusion, and EGF receptor-mediated endocytosis. The stimulatory effect of RIN1 on all three of these processes is potentiated by activated Ras. We conclude that Ras-activated endocytosis is facilitated, in part, by the ability of Ras to directly regulate the Rab5 nucleotide exchange activity of RIN1.
Ras signalling has classically been thought to occur exclusively at the inner surface of a relatively uniform plasma membrane. Recent studies have shown that Ras proteins interact dynamically with specific microdomains of the plasma membrane as well as with other internal cell membranes. These different membrane microenvironments modulate Ras signal output and highlight the complex interplay between Ras location and function.
Mast cell activation induced by aggregation of Fc epsilon RI receptors with immunoglobulin E and antigen is mediated through the activation of multiple protein kinase cascades. Here we report that the regulatory protein RabGEF1 bound to Ras and negatively regulated Ras activation and its 'downstream' effector pathways in Fc epsilon RI-dependent mast cell activation. RabGEF1-deficient mast cells showed enhanced degranulation and release of lipid mediators and cytokines in response to Fc epsilon RI aggregation. RabGEF1-deficient mice developed severe skin inflammation and had increased numbers of mast cells. Thus, RabGEF1 is a negative regulator of Fc epsilon RI-dependent mast cell activation, and a lack of RabGEF1 results in the development of skin inflammation in vivo.
The Rab5 GTPase, an essential regulator of endocytosis and endosome biogenesis, is activated by guanine-nucleotide exchange factors (GEFs) that contain a Vps9 domain. Here, we show that the catalytic core of the Rab GEF Rabex-5 has a tandem architecture consisting of a Vps9 domain stabilized by an indispensable helical bundle. A family-wide analysis of Rab specificity demonstrates high selectivity for Rab5 subfamily GTPases. Conserved exchange determinants map to a common surface of the Vps9 domain, which recognizes invariant aromatic residues in the switch regions of Rab GTPases and selects for the Rab5 subfamily by requiring a small nonacidic residue preceding a critical phenylalanine in the switch I region. These and other observations reveal unexpected similarity with the Arf exchange site in the Sec7 domain.
Protein ubiquitylation is a recognized signal for protein degradation. However, it is increasingly realized that ubiquitin conjugation to proteins can be used for many other purposes. Furthermore, there are many ubiquitin-like proteins that control the activities of proteins. The central structural element of these post-translational modifications is the ubiquitin superfold. A common ancestor based on this superfold has evolved to give various proteins that are involved in diverse activities in the cell.
Endocytosis of cell surface receptors plays an important role in regulating cell signaling cascades. In some cases, internalization of an activated receptor attenuates the signaling process, while in other cases the clustering of activated receptors on early endosomal structures has been proposed to be essential for fully activating signaling cascades. Regulating the movement of receptors and other signaling proteins through the endocytic pathway, therefore, has a direct impact on cellular homeostasis. The small GTPase Rab5 is a crucial regulatory component of the endocytic pathway. Activation of Rab5 is mediated by GDP-GTP exchange factors (GEFs) that generate the Rab5-GTP complex. A large number of proteins have been identified that contain a specific, highly conserved domain (Vps9) that catalyzes nucleotide exchange on Rab5, linking the regulation of cell signaling cascades with intracellular receptor trafficking through the endocytic pathway.
K-Ras associates with the plasma membrane (PM) through farnesylation that functions in conjunction with an adjacent polybasic sequence. We show that phosphorylation by protein kinase C (PKC) of S181 within the polybasic region promotes rapid dissociation of K-Ras from the PM and association with intracellular membranes, including the outer membrane of mitochondria where phospho-K-Ras interacts with Bcl-XL. PKC agonists promote apoptosis of cells transformed with oncogenic K-Ras in a S181-dependent manner. K-Ras with a phosphomimetic residue at position 181 induces apoptosis via a pathway that requires Bcl-XL. The PKC agonist bryostatin-1 inhibited the growth in vitro and in vivo of cells transformed with oncogenic K-Ras in a S181-dependent fashion. These data demonstrate that the location and function of K-Ras are regulated directly by PKC and suggest an approach to therapy of K-Ras-dependent tumors with agents that stimulate phosphorylation of S181.
The interaction between ubiquitinated proteins and intracellular proteins harboring ubiquitin binding domains (UBDs) is critical to a multitude of cellular processes. Here, we report that Rabex-5, a guanine nucleotide exchange factor for Rab5, binds to Ub through two independent UBDs. These UBDs determine a number of properties of Rabex-5, including its coupled monoubiquitination and interaction in vivo with ubiquitinated EGFRs. Structural and biochemical characterization of the UBDs of Rabex-5 revealed that one of them (MIU, motif interacting with ubiquitin) binds to Ub with modes superimposable to those of the UIM (ubiquitin-interacting motif):Ub interaction, although in the opposite orientation. The other UBD, RUZ (Rabex-5 ubiquitin binding zinc finger) binds to a surface of Ub centered on Asp58(Ub) and distinct from the "canonical" Ile44(Ub)-based surface. The two binding surfaces allow Ub to interact simultaneously with different UBDs, thus opening new perspectives in Ub-mediated signaling.
Ras proteins are essential components of signal transduction pathways that control cell proliferation, differentiation, and survival. It is well recognized that the functional versatility of Ras proteins is accomplished through their differential compartmentalization, but the mechanisms that control their spatial segregation are not fully understood. Here we show that HRas is subject to ubiquitin conjugation, whereas KRas is refractory to this modification. The membrane-anchoring domain of HRas is necessary and sufficient to direct the mono- and diubiquitination of HRas. Ubiquitin attachment to HRas stabilizes its association with endosomes and modulates its ability to activate the Raf/MAPK signaling pathway. Therefore, differential ubiquitination of Ras proteins may control their location-specific signaling activities.
Signal transduction down the Ras/MAPK pathway, including that critical to T cell activation, proliferation, and differentiation, has been generally considered to occur at the plasma membrane. It is now clear that the plasma membrane does not represent the only platform for Ras/MAPK signaling. Moreover, the plasma membrane itself is no longer considered a uniform structure but rather a patchwork of microdomains that can compartmentalize signaling. Signaling on internal membranes was first recognized on endosomes. Genetically encoded fluorescent probes for signaling events such as GTP/GDP exchange on Ras have revealed signaling on a variety of intracellular membranes, including the Golgi apparatus. In fibroblasts, Ras is activated on the plasma membrane and Golgi with distinct kinetics. The pathway by which Golgi-associated Ras becomes activated involves PLCgamma and RasGRP1 and may also require retrograde trafficking of Ras from the plasma membrane to the Golgi as a consequence of depalmitoylation. Thus, the Ras/MAPK pathway represents a clear example of compartmentalized signaling.
RabGEF1/Rabex-5, a guanine nucleotide exchange factor (GEF) for the endocytic pathway regulator, Rab5, contains a Vps9 domain, an A20-like zinc finger (ZnF) domain, and a coiled coil domain. To investigate the importance of these domains in regulating receptor internalization and cell activation, we lentivirally delivered RabGEF1 mutants into RabGEF1-deficient (-/-) mast cells and examined Fc epsilon RI-dependent responses. Wild-type RabGEF1 expression corrected phenotypic abnormalities in -/- mast cells, including decreased basal Fc epsilon RI expression, slowed Fc epsilon RI internalization, elevated IgE + Ag-induced degranulation and IL-6 production, and the decreased ability of -/- cytosol to support endosome fusion. We showed that RabGEF1's ZnF domain has ubiquitin ligase activity. Moreover, the coiled coil domain of RabGEF1 is required for Rabaptin-5 binding and for maintaining basal levels of Rabaptin-5 and surface Fc epsilon RI. However, mutants lacking either of these domains normalized phenotypic abnormalities in IgE + antigen-activated -/- mast cells. By contrast, correction of these -/- phenotypes required a functional Vps9 domain. Thus, Fc epsilon RI-mediated mast cell functional activation is dependent on RabGEF1's GEF activity.
Author manuscript; available in PMC
- Page
Page 6 Curr Biol. Author manuscript; available in PMC 2012 September 07
Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway
- Goodwin
- Js
- Drake
- Rogers C Kr
- Wright L J Lippincott-Schwartz
- Philips Mr Kenworthy
Goodwin JS, Drake KR, Rogers C, Wright L, Lippincott-Schwartz J, Philips MR, Kenworthy AK. Depalmitoylated Ras traffics to and from the Golgi complex via a nonvesicular pathway. J Cell Biol. 2005; 170:261–272.
In this and all other figures, levels of endogenous or ectopically expressed proteins were determined by immunoblotting of whole cell lysates (WCL)
- See
- Figure
- S
See also Figure S1. In this and all other figures, levels of endogenous or ectopically expressed proteins were determined by immunoblotting of whole cell lysates (WCL);
Ubiquitin and ubiquitin-like proteins as multifunctional signals
- Gordon C Welchman Rl
- Mayer
Welchman RL, Gordon C, Mayer RJ. Ubiquitin and ubiquitin-like proteins as multifunctional signals. Nat Rev Mol Cell Biol. 2005; 6:599–609.
Author manuscript; available in PMC NIH-PA Author Manuscript NIH-PA Author Manuscript (A) HEK 293T cells were co-transfected with expression vectors for His 6 -tagged ubiquitin (His-Ub), HA-tagged Rabex-5 wild type (WT) and T7-tagged HRas WT as indicated
- Xu
[PubMed: 11703925] Xu et al. Page 7 Curr Biol. Author manuscript; available in PMC 2012 September 07. NIH-PA Author Manuscript NIH-PA Author Manuscript (A) HEK 293T cells were co-transfected with expression vectors for His 6 -tagged ubiquitin (His-Ub), HA-tagged Rabex-5 wild type (WT) and T7-tagged HRas WT as indicated. Ub conjugates were isolated from the transfected cells by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography and HRas polypeptides were detected by immunoblotting (IB).
PKC regulates a farnesyl-electrostatic switch on K-Ras that promotes its association with Bcl-XL on mitochondria and induces apoptosis
- Bivona Tg
- Quatela
- Se
- Bo Bodemann
- Ahearn Im
- Soskis Mj
- A Mor
- J Miura
- Wiener
- Wright L Hh
- Saba
- Sg
Bivona TG, Quatela SE, Bodemann BO, Ahearn IM, Soskis MJ, Mor A, Miura J, Wiener HH, Wright L, Saba SG, et al. PKC regulates a farnesyl-electrostatic switch on K-Ras that promotes its association with Bcl-XL on mitochondria and induces apoptosis. Mol Cell. 2006; 21:481–493.
Ubiquitin and ubiquitin-like proteins as multifunctional signals
- Welchman