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Rabex-5 Ubiquitin Ligase Activity Restricts Ras Signaling to Establish Pathway Homeostasis in Drosophila
Abstract
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 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]. ...
... We isolated Ras ubiquitin conjugates from cells using previous protocols [Yan et al., 2009;Yan et al., 2010;Washington et al., 2020]. S2 cells were transfected with Ras constructs and HA-UB plasmid with vector control or Rabex-5, and Ras was pulled down on nickel beads and then analyzed by Western. ...
... Schneider S2 cell extracts were prepared and used to isolate tagged Ras in Figure 1A as described previously [Washington et al., Yan et al. 2009;Yan et al 2010]. Extracts were prepared from harvested cells or from frozen cell pellets in lysis buffer, 1X NP40 buffer (50 mM HEPES pH 7.4, 1% NP40, 1mM EDTA, 150 mM NaCl), 8M Urea, 5 mM betamercaptoethanol, 10 mM imidazole supplemented with Roche protease inhibitor cocktail and 1mM each of PMSF and Sodium Orthovanadate) and used in pull down assays described above or analyzed by Western. ...
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.
... 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]. ...
... To elucidate the molecular mechanism of Ras inhibition by Rabex-5 and to advance our understanding of the A20 family of E3 ubiquitin ligases, we mapped a signal in Drosophila Ras responsible for Rabex-5 mediated ubiquitination with a deletion strategy (S1A Fig). In our previous work, we used a double FLAG-His6 tag on full length Ras [13,16]. Deletion constructs were tagged with a triple tag of GFP-FLAG-His6 so that smaller constructs (corresponding to larger deletions) would be large enough to eliminate concerns of peptide instability. ...
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.
... Rabex-5 (also called RABGEF1) negatively regulates Ras by promoting Ras ubiquitylation causing its relocalization to an endosomal compartment (Xu et al., 2010;Yan et al., 2010). We demonstrate here that loss of Rabex-5 affects both hematopoietic waves and results in a number of hematopoietic abnormalities including increased hemocyte numbers, increased size of the larval lymph gland, lamellocyte differentiation and formation of melanotic masses. ...
... We previously reported melanotic mass formation (Fig. 1A), and larval and pupal lethality in Drosophila that lack the neoplastic tumor suppressor Rabex-5 (Yan et al., 2010). At least one melanotic mass was found in 3.8% of larvae homozygous for the deletion allele Rabex-5 ex42 (referred to as Rabex-5-null larvae) 6 days after egg laying (AEL). ...
... The size of Rabex-5-null lymph glands increased drastically (Fig. 2B,C and Fig. S2A); lymph glands became so overgrown that they dissociated from the dorsal vessel upon dissection, were morphologically unrecognizable, and/or physically indistinguishable from other overgrown tissues. This is consistent with overgrowth seen previously for Rabex-5-mutant epithelial tissues (Yan et al., 2010;Thomas and Strutt, 2014). Srp-gal4 directed dap expression did not affect the lymph gland area in control larvae (srp>dap) but restored the lymph glands of Rabex-5-null larvae to wild-type size (Rabex-5 ex42/ex42 ; srp>dap, Fig. 2C). ...
Hematopoietic homeostasis requires the maintenance of a reservoir of undifferentiated blood cell progenitors and the ability to replace or expand differentiated blood cell lineages when necessary. Multiple signaling pathways function in these processes, but how their spatiotemporal control is established and their activity is coordinated in the context of the entire hematopoietic network are still poorly understood. We report here that loss of the gene Rabex-5 in Drosophila causes several hematopoietic abnormalities including blood cell (hemocyte) overproliferation, increased size of the hematopoietic organ (the lymph gland), lamellocyte differentiation, and melanotic mass formation. Hemocyte-specific Rabex-5 knockdown was sufficient to increase hemocyte populations, increase lymph gland size, and induce melanotic masses. Rabex-5 negatively regulates Ras, and we show Ras activity is responsible for specific Rabex-5 hematopoietic phenotypes. Surprisingly, Ras-independent Notch protein accumulation and transcriptional activity in the lymph gland underlie multiple distinct hematopoietic phenotypes of Rabex-5 loss. Thus, Rabex-5 plays an important role in Drosophila hematopoiesis and may serve as an axis coordinating Ras and Notch signaling in the lymph gland.
... stat92E F is a hypomorph (Baksa et al., 2002). Rabex-5 ex42 is a hypomorph (a gift from Cathie Pfleger, Yan et al., 2010). Transgenes used were UAS-Rabex-5:Myc and UAS-Rabex-5 DPYT :Myc (gifts from Cathie Pfleger and Yan et al., 2010), UAS-YFP-Rab5 S43N (Bloomington and Zhang et al., 2007). ...
... Rabex-5 ex42 is a hypomorph (a gift from Cathie Pfleger, Yan et al., 2010). Transgenes used were UAS-Rabex-5:Myc and UAS-Rabex-5 DPYT :Myc (gifts from Cathie Pfleger and Yan et al., 2010), UAS-YFP-Rab5 S43N (Bloomington and Zhang et al., 2007). UAS-Rbpn-5 was constructed by insertion of the Rbpn-5 coding region from the cDNA clone LD23155 (BDGP) into pUASt (Brand and Perrimon, 1993). ...
... In vitro assays have shown that Rabaptin-5 requires Rabex-5 both for its recruitment to early endosomes and its ability to promote early endosome fusion, whereas Rabex-5 alone can stimulate excessive early endosome fusion if expressed at high enough levels Lippe et al., 2001). We obtained RNAi and UAS lines to examine the effect of Rabex-5 on early endosome dynamics in the Drosophila wing (VDRC and Yan et al., 2010). As with RNAi-Rbpn-5, apical Rab5 puncta are significantly reduced in RNAi-Rabex-5 expressing tissue (Fig. 4B). ...
Endocytosis plays an important role in the regulation of tumour growth and metastasis. In Drosophila, a
number of endocytic neoplastic tumour suppressor genes have been identified that when mutated cause
epithelial disruption and over-proliferation. Here we characterise the Drosophila homologue of the Rab5
effector Rabaptin-5, and show that it is a novel neoplastic tumour suppressor. Its ability to bind Rab5 and
modulate early endosomal dynamics is conserved in Drosophila, as is its interaction with the Rab5 GEF
Rabex5, for which we also demonstrate neoplastic tumour suppressor characteristics. Surprisingly, we do
not observe disruption of apico-basal polarity in Rabaptin-5 and Rabex-5 mutant tissues; instead the
tumour phenotype is associated with upregulation of Jun N-terminal Kinase (JNK) and Janus Kinase
(JAK)/Signal Transducer and Activator of Transcription (STAT) signalling.
... Evidence for the latter was provided by the discovery that H-and N-Ras proteins are both mono-and di-ubiquitylated via a K63-linkage, a mode of ubiquitylation that is often required for endocytosis as well as protein sorting at the endosome. 13 When Ras proteins are deficient in ubiquitylation by either mutating the lysine residues in Ras 13 or by silencing the Ras E3 ligase Rabex-5/RabGEF1, 14,15 the Ras signaling outputs are enhanced. Conversely, when Rabex-5 is overexpressed, Ras signaling is inhibited. ...
... Conversely, when Rabex-5 is overexpressed, Ras signaling is inhibited. 14,15 These results suggest that a key outcome of Ras internalization is to attenuate growth factor signaling. Despite Instead of palmitoylation, K-Ras-4B associates with the plasma membrane via electrostatic interactions with a lysine rich region in the C-terminus. ...
... 14 One consequence of endosomal internalization is suppression of the signaling output from the Ras pathway (I). [13][14][15] A fraction of the GTP-bound and ubiquitylated Ras can also stimulate ESCRT-III components CHMP6 and VPS4A to enable sorting and recycling of components from the Ras pathway, such as EGFR (and possibly Ras itself) back to the plasma membrane. This pathway apparently enhances Ras signaling (II). ...
Ras proteins are best known to function on the plasma membrane to mediate growth factor signaling. Controlling the length of time that Ras proteins stay on the plasma membrane is an effective way to properly modulate the intensity and duration of growth factor signaling. It has been shown previously that H- and N-Ras proteins in the GTP-bound state can be ubiquitylated via a K-63 linkage, which leads to endosome internalization and results in a negative-feedback loop for efficient signal attenuation. In a more recent study, two new Ras effectors have been isolated, CHMP6 and VPS4A, which are components of the ESCRT-III complex, best known for mediating protein sorting in the endosomes. Surprisingly, these molecules are required for efficient Ras-induced transformation. They apparently do so by controlling recycling of components of the Ras pathway back to the plasma membrane, thus creating a positive-feedback loop to enhance growth factor signaling. These results suggest the fates of endosomal Ras proteins are complex and dynamic - they can be either stored and/or destroyed or recycled. Further work is needed to decipher how the fate of these endosomal Ras proteins is determined.
... 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). ...
... Crosses were performed at the indicated temperatures on standard Drosophila medium as in our previous work (Yan et al. 2009;Yan et al. 2010;Washington et al. 2020). In each experimental trial, crosses were set up on food from the same batch, and all vials were kept in close proximity in the same box and experienced the same environment; if there were any environmental variables (such as variations in batches of food or slight fluctuations in temperature when incubator doors were opened), all vials in each trial experienced them simultaneously. ...
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.
... 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.
... The abilities of RabGEF1 to bind ubiquitin, undergo monoubiquitination, and express E3 ubiquitin ligase activity can contribute to its functions to regulate endosome trafficking via sorting of ubiquitinated cargoes as well as Rab5-dependent endosome fusion. Furthermore, recent studies have shown that RabGEF1 can bind to active Ras to induce Ras ubiquitination and thereby inhibit Ras-dependent ERK activation [32][33][34]. ...
... Taken together, we hypothesize that the exact mechanisms by which RabGEF1 interacts with Ras, or other signaling molecules in particular, are dependent on the cellular context in which the functional activation is induced. Recent studies have shown that RabGEF1 can promote Ras ubiquitination, leading to a reduced ERK activation [32][33][34]. In the present study, we showed that RabGEF1 can bind to the activated form of Rac1 and negatively regulate the activation of Rac1 and ERK in response to the interaction of a single ligand with its cognate receptor (Figs 5B and 7). ...
Nerve growth factor (NGF) binds to its cognate receptor TrkA and induces neuronal differentiation by activating distinct downstream signal transduction events. RabGEF1 (also known as Rabex-5) is a guanine nucleotide exchange factor for Rab5, which regulates early endosome fusion and vesicular trafficking in endocytic pathways. Here, we used the antisense (AS) expression approach to induce an NGF-dependent sustained knockdown of RabGEF1 protein expression in stable PC12 transfectants. We show that RabGEF1 is a negative regulator of NGF-induced neurite outgrowth and modulates other cellular and signaling processes that are activated by the interaction of NGF with TrkA receptors, such as cell cycle progression, cessation of proliferation, and activation of NGF-mediated downstream signaling responses. Moreover, RabGEF1 can bind to Rac1, and the activation of Rac1 upon NGF treatment is significantly enhanced in AS transfectants, suggesting that RabGEF1 is a negative regulator of NGF-induced Rac1 activation in PC12 cells. Furthermore, we show that RabGEF1 can also interact with NMDA receptors by binding to the NR2B subunit and its associated binding partner SynGAP, and negatively regulates activation of nitric oxide synthase activity induced by NMDA receptor stimulation in NGF-differentiated PC12 cells. Our data suggest that RabGEF1 is a negative regulator of TrkA-dependent neuronal differentiation and of NMDA receptor-mediated signaling activation in NGF-differentiated PC12 cells.
... Biochemical and genetic studies simultaneously led to the identification of the E3 involved in H-Ras ubiquitylation [67,267]. Xu et al. focused on Rabex-5, the guanine nucleotide exchange factor (GEF) for Rab5, because it was previously shown to interact with H-Ras [268]. ...
... Overexpression of Rabex-5 triggered an increase in the endosomal pool of H-Ras and inhibition of H-Ras-mediated signaling [67]. In complete agreement with these observations, Drosophila Rabex-5 was identified as a negative regulator of Ras and was shown to mediate drosophila Ras ubiquitylation with the same characteristic di-ubiquitylated form [267]. ...
Modification by Lys63-linked ubiquitin (UbK63) chains is the second most
abundant form of ubiquitylation. In addition to their role in DNA repair or kinase activation,
UbK63 chains interfere with multiple steps of intracellular trafficking. UbK63 chains
decorate many plasma membrane proteins, providing a signal that is often, but not always,
required for their internalization. In yeast, plants, worms and mammals, this same
modification appears to be critical for efficient sorting to multivesicular bodies and
subsequent lysosomal degradation. UbK63 chains are also one of the modifications involved
in various forms of autophagy (mitophagy, xenophagy, or aggrephagy). Here, in the context
of trafficking, we report recent structural studies investigating UbK63 chains assembly by
various E2/E3 pairs, disassembly by deubiquitylases, and specifically recognition as sorting
signals by receptors carrying Ub-binding domains, often acting in tandem. In addition, we
address emerging and unanticipated roles of UbK63 chains in various recycling pathways
that function by activating nucleators required for actin polymerization, as well as in the
transient recruitment of signaling molecules at the plasma or ER membrane. In this review,
we describe recent advances that converge to elucidate the mechanisms underlying the
wealth of trafficking functions of UbK63 chains.
... A20_ZF domain possesses an E3 ubiquitin ligase activity in combination with UbcH5C [14,15]. Ras proteins have been shown to be substrates for the E3 ligase activity of A20_ZF [16,17] . Rabex-5 uses its tandem UBDs (A20_ZF-MIU) for binding EGFR [18]. ...
... In parallel with that A20_ZF may be utilized for functions other than polyubiquitin binding in vivo, we observed that monoubiquitinated L1 neural adhesion molecule is endocytosed in a strictly MIU-dependent manner in cultured cells (unpublished data). Considering that A20_ZF of Rabex-5 possesses an E3 ubiquitin ligase activity [14,15,18] with its substrate specificity toward Ras proteins [16,17] , the ubiquitin binding capability of A20_ZF may be for functions other than simple ubiquitin recognition per se. A few cases have been reported for biochemical characterization of tandem ubiquitin binding domains in polyubiquitin recognition . ...
... 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.
... However, little is known about how the cellular levels of KRAS are regulated at the protein level. Although it was previously reported that RAS proteins can be controlled by protein kinase Cδ (PKCδ) 54 , glycogen synthase kinase 3β (GSK3β) 55 , leucine zipper-like transcriptional regulator 1 (LZTR1) 56 , and Rab5 guanine nucleotide exchange factor (Rabex-5) 57 , these processes do not distinguish KRAS from other isoforms, and the exact mechanism is not yet fully understood. Here, we report a mechanism whereby KRAS stability is specifically controlled by another oncogenic factor, DX2. ...
Recent development of the chemical inhibitors specific to oncogenic KRAS (Kirsten Rat Sarcoma 2 Viral Oncogene Homolog) mutants revives much interest to control KRAS-driven cancers. Here, we report that AIMP2-DX2, a variant of the tumor suppressor AIMP2 (aminoacyl-tRNA synthetase-interacting multi-functional protein 2), acts as a cancer-specific regulator of KRAS stability, augmenting KRAS-driven tumorigenesis. AIMP2-DX2 specifically binds to the hypervariable region and G-domain of KRAS in the cytosol prior to farnesylation. Then, AIMP2-DX2 competitively blocks the access of Smurf2 (SMAD Ubiquitination Regulatory Factor 2) to KRAS, thus preventing ubiquitin-mediated degradation. Moreover, AIMP2-DX2 levels are positively correlated with KRAS levels in colon and lung cancer cell lines and tissues. We also identified a small molecule that specifically bound to the KRAS-binding region of AIMP2-DX2 and inhibited the interaction between these two factors. Treatment with this compound reduces the cellular levels of KRAS, leading to the suppression of KRAS-dependent cancer cell growth in vitro and in vivo. These results suggest the interface of AIMP2-DX2 and KRAS as a route to control KRAS-driven cancers. Direct targeting of oncogenic KRAS activity is a challenge. Here the authors report that a splice variant of AIMP2, AIMP2-DX2, enhances KRAS stability by blocking ubiquitin-mediated degradation of KRAS via the E3 ligase, Smurf2, and identify a chemical that can hinder AIMP2-DX2 from interacting with KRAS.
... 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.
... The most significant group (red) contains kinesin-like protein (KIF) family, intraflagellar transport protein (IFT), pericentrin (PCNT), and some gammatubulin complex components that are known as primary cilia proteins [17] . The second group (blue) contains RAB guanine nucleotide exchange factor 1 (RABGEF1), vacuolar protein sorting-associated protein 16 homolog (VPS16), and vacuolar protein sorting-associated protein 45 (VPS45) that is associated with endocytosis, ciliary membrane homeostasis, and autophagy process [18][19][20] . The third, the most minor, group (green) is autophagy related proteins including autophagy protein 5, and 7 (ATG5, ATG7), rapamycin-insensitive companion of mTOR (RICTOR), and regulatoryassociated protein of mTOR (RPTOR) [21] . ...
Primary cilium is a microtubule-based organelle that projects from the surfaces of most mammalian cell types and protrudes into the extracellular milieu as an antenna-like sensor to senses extracellular physical and biochemical signals, and then transmits signals into cytoplasm or nucleus to regulate numerous physical and developmental processes. Therefore, loss of primary cilia is associated to multiple cancer progression, including skin, breast, pancreas, ovarian, prostate, and kidney cancers. Our previous studies demonstrate that high prevalent loss of DAB2 Interacting Protein (DAB2IP) is associated with renal cell carcinoma, and we found a kinesin-like protein, kinesin family member 3A (KIF3a), was significantly increased in DAB2IP-interacting protein fraction. KIF3 is one of the most abundant kinesin-2 family proteins expressed in cells, and it is necessary for ciliogenesis. In this study, we observed that loss of DAB2IP in normal kidney epithelial cell significantly impair primary cilia formation. We unveiled a new mechanism of primary cilia stability via DAB2IP and KIF3a physical interaction at DAB2IP-PH domain. Furthermore, we found that KIF3a also act as a tumor suppressor in renal cell carcinoma, affect tumor development and patient survival.
... 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.
... 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.
... Midguts stained for phosphorylated histone 3 (p-H3) ( Fig. 3A) revealed condensed chromatin in ISCs, which is consistent with ISCs being the major mitotic cells in adult Drosophila midgut (14,22). UAS-driven Rab11 RNAi resulted in a pronounced increase in the number of mitotic enterocytes ( Taking advantage of the Drosophila midgut genetics model, we examined the impact of Rab11 endosome function on tumorigenesis driven by oncogenic activity via EGFR-activating (EGFR A887T ) mutation, Ras-activating (Ras V12 ) mutation, APC loss-of-function (LOF, APC RNAi), and Misshapen loss of function (msn RNAi) (24). In Drosophila, the RasV12 mutant is widely used as a cancer model in various fly tissue (25,26). ...
The effects of polarized membrane trafficking in mature epithelial tissue on cell growth and cancer progression have not been fully explored in vivo. A majority of colorectal cancers have reduced and mislocalized Rab11, a small GTPase dedicated to trafficking of recycling endosomes. Patients with low Rab11 protein expression have poor survival rates. Using genetic models across species, we show that intact recycling endosome function restrains aberrant epithelial growth elicited by APC or RAS mutations. Loss of Rab11 protein led to epithelial dysplasia in early animal development and synergized with oncogenic pathways to accelerate tumor progression initiated by carcinogen, genetic mutation, or aging. Transcriptomic analysis uncovered an immediate expansion of the intestinal stem cell pool along with cell-autonomous Yki/Yap activation following disruption of Rab11a-mediated recycling endosomes. Intestinal tumors lacking Rab11a traffic exhibited marked elevation of nuclear Yap, upd3/IL6-Stat3, and amphiregulin-MAPK signaling, whereas suppression of Yki/Yap or upd3/IL6 reduced gut epithelial dysplasia and hyperplasia. Examination of Rab11a function in enteroids or cultured cell lines suggested that this endosome unit is required for suppression of the Yap pathway by Hippo kinases. Thus, recycling endosomes in mature epithelia constitute key tumor suppressors, loss of which accelerates carcinogenesis.
Significance
Recycling endosome traffic in mature epithelia constitutes a novel tumor suppressing mechanism.
... 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.
... We tested whether LZTR1 affected ubiquitination of RAS proteins by acting as a substrate adaptor for the CUL3 E3 ligase complex (11,20). Ubiquitination of RAS proteins is known, Bigenzahn Europe PMC Funders Author Manuscripts but little is known of its consequences (21)(22)(23)(24)(25). Co-expression of HA-ubiquitin with either one of the four RAS isoforms alone only resulted in a basal state of ubiquitination, but addition of MYC-tagged CUL3 and V5-tagged LZTR1 led to increased ubiquitination of RAS proteins (Fig. S16A-D). ...
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
... 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
... 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. ...
... 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]. It is uncertain which specific lysine site is involved although the authors generated an H-Ras8KR mutant in which all eight lysine sites were mutated to arginine and found this mutant was enriched in the Golgi compartment. ...
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.
... Rabaptin-5 that possesses GEF activity toward RAB-5 and related GTPases [19]. Upregulation of RABEX-5 has been detected in various cancer types, including breast cancer, prostate cancer, colorectal cancer, gastric cancer, and lung cancer [20]. RABEX-5 acts as an oncogene and due to is its involvement in the formation and development of malignant tumors, it might influence tumor biological behavior. ...
Objective:
This study aimed to investigate mRNA and protein expression levels of RABEX-5 and matrix metalloproteinase-9 (MMP-9), their mutual correlation, and biological behavior in gastric cancer (GC) patients.
Methods:
The expression levels of RABEX-5 and MMP-9 were determined by real-time quantitative PCR and Western blotting in cell lines, GC tissues, and adjacent normal tissues. In addition, RABEX-5 and MMP-9 expression was analyzed by immunohistochemistry in formalin-fixed tissues from 113 GC patients.
Results:
The mRNA and protein expression levels of RABEX-5 and MMP-9 in GC cell lines and GC tissues were higher than those in normal gastric mucosa cell line and adjacent normal tissues. RABEX-5 expression and MMP-9 expression in GC tissues were significantly and positively correlated. In addition, the size of tumor (p<0.001), Lauren's classification (p=0.009), and N stage (p<0.001) were identified as the relative factors of RABEX-5 expression, whereas the expression of MMP-9 was correlated with N stage (p=0.003). The results of the multivariate analysis revealed that the independent predictive factors of overall survival were T stage (hazard ratio (HR)=2.382; p=0.028), N stage (HR=1.755; p<0.001), RABEX-5 expression (HR=0.452; p=0.004), and MMP-9 expression (HR=0.561; p=0.032).
Conclusions:
RABEX-5 and MMP-9 expression levels were elevated in GC tissues and were associated with tumor invasion, metastasis, and prognosis. Therefore, they may be promising prognostic indicators of survival in GC patients.
... This observation was confirmed by confocal microscopy showing increased plasma membrane localization (SI Appendix, Fig. S9A) and a decreased amount of GFP-H-Ras in Rab-5-positive vesicles in p27KO cells, compared with WT or DKO cells (Fig. 5B). Rab-5 is a small GTPase regulator of vesicular trafficking, and it is involved in the mono-or biubiquitination of H-Ras (31), a process resulting in decreased H-Ras activity (31,32). We thus tested whether H-Ras was differently mono-or biubiquitinated in the presence/absence of p27 and stathmin. ...
Significance
Different functions have been ascribed to p27 kip1 , originally identified as a universal cyclin-dependent kinase (CDK) inhibitor, fundamental for the control of cell proliferation and tumor progression. Yet, not all p27 functions can be explained by its ability to bind and inhibit CDKs. Here, we demonstrate that p27 kip1 controls cell cycle entry also through a CDK-independent function, by regulating microtubule stability. Following growth factor stimulation, p27 kip1 prevents full activation of H-Ras, acting on its subcellular compartmentalization, eventually restraining the activation of the MAPK pathway. Our work provides additional understanding of the mechanisms regulating the cell cycle and anticipates potential implications in diseases characterized by deregulated proliferation, such as cancer.
... A seductive observation is that most UBDs make the proteins they reside in susceptible to ubiquitination, affording them a vast potential for different modes of regulation. For instance, the GTP exchange factor for Rab5, Rabex5, has proposed roles as a Ub ligase against itself and other targets in the endocytic pathway and as a Ub-binding protein that engages Ub cargo as well as a ubiquitinated form of itself-all of which may impinge on the ability of Rabex-5 to activate Rab5 (Mattera et al. 2006;Mattera and Bonifacino 2008;Xu et al. 2010;Yan et al. 2010;Aikawa et al. 2012). Such discoveries promise a rich and interesting future. ...
When ubiquitin (Ub) is attached to membrane proteins on the plasma membrane, it directs them through a series of sorting steps that culminate in their delivery to the lumen of the lysosome where they undergo complete proteolysis. Ubiquitin is recognized by a series of complexes that operate at a number of vesicle transport steps. Ubiquitin serves as a sorting signal for internalization at the plasma membrane and is the major signal for incorporation into intraluminal vesicles of multivesicular late endosomes. The sorting machineries that catalyze these steps can bind Ub via a variety of Ub-binding domains. At the same time, many of these complexes are themselves ubiquitinated, thus providing a plethora of potential mechanisms to regulate their activity. Here we provide an overview of how membrane proteins are selected for ubiquitination and deubiquitination within the endocytic pathway and how that ubiquitin signal is interpreted by endocytic sorting machineries.
... While many Ras proteins enter the endosomes, the complex fates of these internalized Ras proteins are just beginning to resolve. When the lysine residues in the Ras proteins are mutated (Jura et al., 2006;Zheng, Cheng, et al., 2012) or when an ubiquitin ligase responsible for Ras ubiquitylation (Rabex-5) is repressed (Xu, Lubkov, Taylor, & Bar-Sagi, 2010;Yan, Jahanshahi, Horvath, Liu, & Pfleger, 2010), Ras signaling outputs are apparently enhanced. Conversely, when Rabex-5 is overexpressed, the Ras signaling outputs are inhibited. ...
While Ras GTPases are best known for mediating growth factor signaling on the plasma membrane, these proteins also have surprisingly complex activities in the endosome. Assisted by a method called bimolecular fluorescent complementation (BiFC), which can detect weak and transient protein-protein interactions and reveal where the binding takes place in live cells, we have identified three effectors, Cdc42, CHMP6, and VPS4A that interact with Ras proteins in endosomes. These effectors are all necessary for Ras-induced transformation, suggesting that for Ras proteins to efficiently induce tumor formation, they must also activate effectors in cytoplasm, such as those in endosomes. Here, we describe how BiFC can be used to detect and screen for Ras effectors and for readily revealing where in the cell the binding occurs.
... One such example is that Ub employs the Asp-58 surface residue to associate with the A20-ZnF domain and the Ile-44 patch to bind to the MIU domain (13,14). The A20-ZnF domain possesses Ub-ligase activity (13)(14)(15)(16) and promotes Ras ubiquitination to attenuate the Ras signaling pathway (17). To exert Ub-ligase activity, the A20-ZnF domain binds Ub-charged Ubc5 (an E2 enzyme) in a manner that depends upon the residues involved in Ub binding. ...
Ubiquitin (Ub)-dependent endocytosis of membrane proteins requires precise molecular recognition of ubiquitinated cargo by Ub-binding proteins (UBPs). Many UBPs are often themselves monoubiquitinated, a mechanism referred to as coupled monoubiquitination, which prevents them from binding in trans to the ubiquitinated cargo. However, the spatiotemporal regulatory mechanism underlying the interaction of UBPs with the ubiquitinated cargo, via their Ub-binding domains (UBDs) remains unclear. Previously, we reported the interaction of Rabex-5, a UBP and guanine nucleotide exchange factor (GEF) for Rab5, with ubiquitinated neural cell adhesion molecule L1, via its motif interacting with Ub (MIU) domain. This interaction is critical for the internalization and sorting of the ubiquitinated L1 into endosomal/lysosomal compartments. The present study demonstrated that the interaction of Rabex-5 with Rab5 depends specifically on interaction of the MIU domain with the ubiquitinated L1 to drive its internalization. Notably, impaired GEF mutants and the Rabex-5E213A mutant increased the flexibility of the hinge region in the HB-VPS9 tandem domain, which significantly affected their interactions with the ubiquitinated L1. In addition, GEF mutants increased the catalytic efficiency, which resulted in a reduced interaction with the ubiquitinated L1. Furthermore, the coupled monoubiquitination status of Rabex-5 was found to be significantly associated with interaction of Rabex-5 and the ubiquitinated L1. Collectively, our study reveals a novel mechanism, wherein the GEF activity of Rabex-5 acts as an intramolecular switch orchestrating ubiquitinated cargo-binding activity and coupled monoubiquitination to permit the spatiotemporal dynamic exchange of the ubiquitinated cargos.
... This is consistent with RIN1 and RABGEF1 both contributing to EGFR downregulation in response to EGF stimulation. RABGEF1 silenced cells also had an increase in EGF-induced ERK1/2 phosphorylation (Fig. 1B), which is in line with the established role of RABGEF1 in RASRRAFRMEKRERK pathway repression through RAS ubiquitylation (Xu et al., 2010;Yan et al., 2010). The human RAB5 GEF domain, also called VPS9 domain, family has ten members including RIN1-3 and RABGEF1 (http://www.ensembl. ...
Epidermal growth factor receptor (EGFR) stimulation initiates RAS signaling simultaneously with EGFR internalization. Endocytosed EGFR is then either recycled or degraded. EGFR fate is determined in part by the RAS effector RIN1, a guanine nucleotide exchange factor (GEF) for RAB5 GTPases. EGFR degradation was slowed by RIN1 silencing, enhanced by RIN1 over-expression and accelerated by RIN1 localization to the plasma membrane. RIN1 also directly activates ABL tyrosine kinases, which regulate actin remodeling, a function not previously connected to endocytosis. We report that RIN1-RAB5 signaling favors EGFR down regulation over EGFR recycling, while RIN1-ABL signaling stabilizes EGFR and inhibits macropinocytosis. RIN1(QM), a mutant that blocks ABL activation, caused EGF-stimulated membrane ruffling, actin remodeling, dextran uptake and EGFR degradation. An ABL kinase inhibitor phenocopied these effects in cells over-expressing RIN1. EGFR activation also promotes RIN1 interaction with BIN1, a membrane bending protein. These findings suggest that RIN1 orchestrates RAB5 activation, ABL kinase activation and BIN1-recruitment to determine EGFR fate.
... 16 -18). It is noteworthy that the proto-oncogenes H-Ras and N-Ras are ubiquitinated (18,19), and this ubiquitination markedly disrupts the balance between plasma and endomembrane localization of Ras, favoring the latter and disfavoring signal transduction toward ERK1/2 (18,20,21). We show here that the endogenous RalA and RalB GTPases are ubiquitinated, which contributes to selective membrane localization. ...
Ras GTPases signal by orchestrating a balance among several effector pathways, of which those driven by the GTPases RalA and
RalB are essential to Ras oncogenic functions. RalA and RalB share the same effectors but support different aspects of oncogenesis.
One example is the importance of active RalA in anchorage-independent growth and membrane raft trafficking. This study has
shown a new post-translational modification of Ral GTPases: nondegradative ubiquitination. RalA (but not RalB) ubiquitination
increases in anchorage-independent conditions in a caveolin-dependent manner and when lipid rafts are endocytosed. Forcing
RalA mono-ubiquitination (by expressing a protein fusion consisting of ubiquitin fused N-terminally to RalA) leads to RalA
enrichment at the plasma membrane and increases raft exposure. This study suggests the existence of an ubiquitination/de-ubiquitination
cycle superimposed on the GDP/GTP cycle of RalA, involved in the regulation of RalA activity as well as in membrane raft trafficking.
... Therefore, phosphorylation by aPKC appears to be at the nexus of regulation of ubiquitination of small Rho GTPases, promoting degradation in Rho1/A and preventing it in CDC42. Finally, two of the small Rab GTPase GEFs, Rabex5 and Rabring7 (Xu, et al., 2010;Yan, et al., 2010) are known to have ubiquitin E3 ligase activity (Sakane, et al., 2007), and Rabex5 cellular localisation is regulated by its ability to bind a ubiquitin signal (Mattera, et al., 2006). As yet there are no known Rab proteins that are themselves ubiquitinated. ...
... Considering high sequence homology between AtSAPs in plants and Rabex-5 and A20 in animals, one can envisage that AtSAPs may function in plant immune system. The E3 ubiquitin ligase activity of AtSAP5 [17] adds one more reason to conjecture potential roles of AtSAP5 in plants since the same E3 ubiquitin ligase activity is known to be crucial in the function of Rabex-5 [22,23] and A20 [11]. It remains to be uncovered what the specific physiological functions of AtSAP5 and other A20_ZF-containing AtSAPs in plants are. ...
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.
The increasing levels of pesticide resistance in agricultural pests and disease vectors represents a threat to both food security and global health. As insecticide resistance intensity strengthens and spreads, the likelihood of a pest encountering a sub-lethal dose of pesticide dramatically increases. Here, we apply dynamic Bayesian networks to a transcriptome time-course generated using sub-lethal pyrethroid exposure on a highly resistant Anopheles coluzzii population. The model accounts for circadian rhythm and ageing effects allowing high confidence identification of transcription factors with key roles in pesticide response. The associations generated by this model show high concordance with lab-based validation and identifies 44 transcription factors putatively regulating insecticide-responsive transcripts. We identify six key regulators, with each displaying differing enrichment terms, demonstrating the complexity of pesticide response. The considerable overlap of resistance mechanisms in agricultural pests and disease vectors strongly suggests that these findings are relevant in a wide variety of pest species.
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.
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.
The increasing levels of pesticide resistance in agricultural pests and disease vectors represents a threat to both food security and global health. As insecticide resistance intensity strengthens and spreads, the likelihood of a pest encountering a sub-lethal dose of pesticide dramatically increases. Here, we apply dynamic Bayesian networks to a transcriptome time-course generated using sub-lethal pyrethroid exposure on a highly resistant Anopheles coluzzii population. The model accounts for circadian rhythm and ageing effects allowing high confidence identification of transcription factors with key roles in pesticide response. The associations generated by this model show high concordance with lab-based validation and identifies 44 transcription factors regulating insecticide-responsive transcripts. We identify six key regulators, with each displaying differing enrichment terms, demonstrating the complexity of pesticide response. The considerable overlap of resistance mechanisms in agricultural pests and disease vectors strongly suggests that these findings are relevant in a wide variety of pest species.
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.
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.
Growth factor receptors play a variety of roles during embryonic development and in adult homeostasis. These receptors are activated repeatedly in different cellular contexts and with different cellular outcomes. This begs the question as to how cells in a particular developmental, spatial and temporal context, or in adult tissue, interpret signalling by growth factor receptors in order to deliver qualitatively different signalling outputs. One mechanism by which this could occur is via endocytic regulation. The original paradigm for the role of endocytosis in growth factor receptor signalling was that receptor uptake has a quantitative role in signalling by reducing the number of cell surface receptors available for activation and targeting activated receptors for degradation. However, a range of studies over the last several years, in many different experimental systems, has demonstrated an additional qualitative role for endocytic trafficking in receptor signalling, with specific outcomes depending on the location of the signalling complex. Confinement of receptors within endosomes can spatially regulate signalling, facilitating specific protein interactions or post-translational modifications that alter throughout the trafficking process. Therefore, endocytosis does not simply regulate cell surface expression, but tightly controls protein interactions and function to produce distinct outcomes.
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.
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.
A few signaling pathways are used repeatedly in the Drosophila larval eye disc to form the ordered array of ommatidia seen in the adult eye. Disruption of signaling by a change in either the levels or timing of expression of signaling pathway components, often leads to dramatic effects: the lack of eye tissue altogether, tumor growth, misdetermination of cell types, altered patterning, or degeneration. The level and timing of signaling in the eye are efficiently controlled by the proteostasis network; a collection of mechanisms responsible for the folding, modification, trafficking, and degradation of proteins. We review evidence from the Notch, EGFR, and rhodopsin pathways that the proteostasis network plays a unique and substantial role in enabling the iterative use of signaling pathways in the confines of the developing Drosophila eye and in adult vision. © 2013 Springer Science+Business Media New York. All rights are reserved.
Endosomes play key roles in the control and execution of such diverse and spatially restricted processes as cell signaling, epithelial to mesenchymal transitions as well as cell adhesion and migration. The endocytosis of growth factor receptors and adhesion molecules, such as cadherins and integrins, is coming into focus as a major mechanism in the regulation of cellular processes that govern cell growth, differentiation, survival, and motility. Subversion of these pathways accompanies disease progression, especially cancer. We suggest that endosomes are multifunctional dynamic platforms on which unique sets of molecular components are assembled and sorted to adapt to different environmental and cellular cues. A better understanding of how endosomes can function as conduits for the acquisition of oncogenic phenotypes will lead to more specific therapeutic approaches to combat cancer progression. © 2013 Springer Science+Business Media New York. All rights are reserved.
The GTPase Ras can either promote or inhibit cell survival. Inactivating mutations in RasGAP (vap), a Ras GTPase-activating protein, lead to age-related brain degeneration in Drosophila. Genetic interactions implicate the epidermal growth factor receptor (EGFR)-Ras pathway in promoting neurodegeneration but the mechanism is not known. Here we show that the Src homology 2 (SH2) domains of RasGAP are essential for its neuroprotective function. By using affinity purification and mass spectrometry, we identify a complex containing RasGAP together with Sprint, a Ras effector and putative activator of the endocytic GTPase Rab5. Formation of the RasGAP-Sprint complex requires the SH2 domains of RasGAP and tyrosine phosphorylation of Sprint. RasGAP and Sprint co-localize with Rab5-positive early endosomes but not with Rab7-positive late endosomes. We demonstrate a key role for this interaction in neurodegeneration: mutation of Sprint (or Rab5) suppresses neuronal cell death caused by the loss of RasGAP. These results indicate that the long-term survival of adult neurons in Drosophila is critically dependent on the activities of two GTPases, Ras and Rab5, regulated by the interplay of RasGAP and Sprint.
RAS genes are among the most frequently mutated proto-oncogenes in cancer. However, how Ras stability is regulated remains largely unknown. Here, we report a regulatory loop involving the E3 ligase Nedd4-1, Ras, and PTEN. We found that Ras signaling stimulates the expression of Nedd4-1, which in turn acts as an E3 ubiquitin ligase that regulates Ras levels. Importantly, Ras activation, either by oncogenic mutations or by epidermal growth factor (EGF) signaling, prevents Nedd4-1-mediated Ras ubiquitination. This leads to Ras-induced Nedd4-1 overexpression, and subsequent degradation of the tumor suppressor PTEN in both human cancer samples and cancer cells. Our study thus unravels the molecular mechanisms underlying the interplay of Ras, Nedd4-1, and PTEN and suggests a basis for the high prevalence of Ras-activating mutations and EGF hypersignaling in cancer.
The endosomal pathway provides a major platform for ubiquitin-modifying enzymes, which act upon membrane-associated proteins in transit. Ubiquitylated cargo proteins are recognized by ubiquitin-binding domains inherent to key adaptor proteins at the plasma membrane and sorting endosome. A balance between ubiquitylation and deubiquitylation activities may govern the efficiency of recycling from endosomes to the plasma membrane versus lysosomal sorting through the multivesicular body pathway. We discuss the current knowledge of the properties of adaptors and ubiquitin-modifying proteins and their effects upon the trafficking and signaling of receptors and ligands associated with pathways fundamental to development.
Ubiquitination of integral membrane proteins is a common posttranslational modification used to mediate endocytosis and endocytic
sorting of cell surface proteins in eukaryotic cells. Ubiquitin (Ub)-binding proteins (UBPs) regulate the stability, function,
and localization of ubiquitinated cell surface proteins in the endocytic pathway. Here, I report that the immunoglobulin superfamily
cell adhesion molecule L1 undergoes ubiquitination and dephosphorylation on the plasma membrane upon L1 antibody-induced clustering,
which mimics L1-L1 homophilic binding, and that these modifications are critical for obtaining the maximal rate of internalization
and trafficking to the lysosome, but not to the proteasome. Notably, L1 antibody-induced clustering leads to the association
of ubiquitinated L1 with Rabex-5, a UBP and guanine nucleotide exchange factor for Rab5, via interaction with the motif interacting
with Ub (MIU) domain, but not the A20-type zinc finger domain. This interaction specifically depends on the presence of an
Ub moiety on lysine residues in L1. Rabex-5 expression accelerates the internalization rates of L1WT and L1Y1176A, a tyrosine-based motif mutant, but not L1K11R, an ubiquitination-deficient mutant, leading to the accumulation of ubiquitinated L1 on endosomes. In contrast, RNA interference-mediated
knockdown of Rabex-5 impairs the internalizations of L1WT and L1Y1176A, but not L1K11R from the plasma membrane. Overall, these results provide a novel mechanistic insight into how Rabex-5 regulates internalization
and postendocytic trafficking of ubiquitinated L1 destined for lysosomal degradation.
Cullin/RING ubiquitin ligases (CRL) comprise the largest subfamily of ubiquitin ligases. CRLs are involved in cell cycle regulation, DNA replication, DNA damage response (DDR), development, immune response, transcriptional regulation, circadian rhythm, viral infection, and protein quality control. One of the main functions of CRLs is to regulate the DDR, a fundamental signaling cascade that maintains genome integrity. In this review, we will discuss the regulation of CRL ubiquitin ligases and their roles in control of the DDR.
ESCRT proteins were initially isolated in yeast as a single functional set of conserved components controlling endosomal cargo sorting and multivesicular body (MVB) biogenesis. Recent work has suggested that metazoan ESCRT proteins might have more functionally diverse roles, but the limited availability of ESCRT mutants in species other than yeast has hampered a thorough analysis. Here, we used a genetic screening strategy based on both cell-autonomous and non-autonomous growth-promotion phenotypes to isolate null mutations in nearly half of the ESCRT-encoding genes of Drosophila, including components of ESCRT-I, ESCRT-II and ESCRT-III complexes. All ESCRT components are required for trafficking of ubiquitylated proteins and are required to prevent excess Notch and EGFR signaling. However, cells lacking certain ESCRT-III components accumulate fewer ubiquitylated molecules in endosomes and display reduced degrees of cell proliferation compared with those lacking components of ESCRT-I and ESCRT-II. Moreover, although we find by ultrastructural analysis that MVB formation is impaired in ESCRT-I and ESCRT-II mutant cells, MVB biogenesis still occurs to some degree in ESCRT-III mutant cells. This work highlights the multiple cell biological and developmental roles of ESCRT proteins in Drosophila, suggests that the metazoan ESCRT-I, ESCRT-II and ESCRT-III complexes do not serve identical functions, and provides the basis for an extensive analysis of metazoan ESCRT function.
Src family kinases regulate multiple cellular processes including proliferation and oncogenesis. C-terminal Src kinase (Csk)
encodes a critical negative regulator of Src family kinases. We demonstrate that the Drosophila melanogaster Csk ortholog, dCsk, functions as a tumor suppressor: dCsk mutants display organ overgrowth and excess cellular proliferation. Genetic analysis indicates that the dCsk−/− overgrowth phenotype results from activation of Src, Jun kinase, and STAT signal transduction pathways. In particular, blockade
of STAT function in dCsk mutants severely reduced Src-dependent overgrowth and activated apoptosis of mutant tissue. Our data provide in vivo evidence
that Src activity requires JNK and STAT function.
An attack by a parasitic wasp activates a vigorous cellular immune response in Drosophila larvae. This response is manifested by an increased number of circulating cells, the hemocytes, and by the appearance of a specialized class of hemocyte, the lamellocytes, which participate in the encapsulation and killing of the parasite. To study the molecular mechanisms of this response, we have overexpressed different genes in the hemocytes, by using the GAL4-upstream activating sequence system and a hemocyte-specific Hemese-GAL4 driver. Multiple transgenes were tested, representing several important signaling pathways. We found that the proliferation response and the activation of lamellocyte formation are independent phenomena. A drastic increase in the number of circulating hemocytes is caused by receptor tyrosine kinases, such as Egfr, Pvr, and Alk, as well as by the downstream signaling components Ras85D and pointed, supporting the notion that the Ras-mitogen-activated protein kinase pathway regulates hemocyte numbers. In the case of Pvr and Alk, this phenotype also is accompanied by lamellocyte formation. By contrast, constitutively active hopscotch and hemipterous give massive activation of lamellocyte formation with little or no increase in total hemocyte numbers. This finding indicates that both the Jak/Stat and the Jun kinase pathways affect lamellocyte formation. Still other signals, mediated by aop(ACT), Toll(10b), and Rac1 expression, cause a simultaneous increase in lamellocyte and total cell numbers, and the same effect is seen when WNT signaling is suppressed. We conclude that the activation of a cellular response is complex and affected by multiple signaling pathways.
Mutations in the PTPN11 gene, which encodes the protein tyrosine phosphatase SHP-2, causes Noonan syndrome (NS), an autosomal dominant disorder with
pleomorphic developmental abnormalities. Certain germline and somatic PTPN11 mutations cause leukemias. Mutations have gain-of-function (GOF) effects with the commonest NS allele, N308D, being weaker
than the leukemia-causing mutations. To study the effects of disease-associated PTPN11 alleles, we generated transgenic fruitflies with GAL4-inducible expression of wild-type or mutant csw, the Drosophila orthologue of PTPN11. All three transgenic mutant CSWs rescued a hypomorphic csw allele's eye phenotype, documenting activity. Ubiquitous expression of two strong csw mutant alleles were lethal, but did not perturb development from some CSW-dependent receptor tyrosine kinase pathways. Ubiquitous
expression of the weaker N308D allele caused ectopic wing veins, identical to the EGFR GOF phenotype. Epistatic analyses established
that cswN308D's ectopic wing vein phenotype required intact EGF ligand and receptor, and that this transgene interacted genetically with
Notch, DPP and JAK/STAT signaling. Expression of the mutant csw transgenes increased RAS-MAP kinase activation, which was necessary but not sufficient for transducing their phenotypes.
The findings from these fly models provided hypotheses testable in mammalian models, in which these signaling cassettes are
largely conserved. In addition, these fly models can be used for sensitized screens to identify novel interacting genes as
well as for high-throughput screening of therapeutic compounds for NS and PTPN11-related cancers.
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.
We recently reported that RabGEF1 is a negative regulator of high-affinity Fc receptor for IgE (FcεRI)-dependent mast cell activation and that mice lacking RabGEF1 develop severe skin inflammation and increased numbers of dermal mast cells. To better understand how RabGEF1 can regulate signaling events and biological responses in mast cells, we examined the responses of bone marrow-derived cultured mast cells (BMCMCs) from wild-type (+/+) and Rabgef1 knockout (−/−) mice after stimulation with the c-Kit ligand, stem cell factor (SCF), an important regulator of mast cell development, survival, proliferation, and activation. We found that RabGEF1-deficient mast cells exhibited enhanced and prolonged activation of Ras and extracellular regulated kinase, and significantly elevated IL-6 secretion, after stimulation with SCF. SCF-induced activation of c-Jun N-terminal kinase was increased in Rabgef1 −/− BMCMCs, but without corresponding significant increases in SCF-induced migration or adhesion. SCF-mediated activation of the survival-enhancing kinase, Akt, also was increased in Rabgef1 −/− BMCMCs, and these cells had a survival advantage over their +/+ counterparts in vitro. Despite enhanced Ras activation in the absence of RabGEF1, SCF-induced proliferation was lower in Rabgef1 −/− BMCMCs compared with their +/+ counterparts. Finally, we found that c-Kit internalization was delayed in the absence of RabGEF1, probably reflecting a positive role for RabGEF1 in the regulation of endocytic events, and that infection of Rabgef1 −/− BMCMCs with a wild-type RabGEF1 lentiviral construct normalized c-Kit internalization to the levels seen in +/+ BMCMCs. Thus, RabGEF1 plays a critical role in the regulation of SCF/c-Kit-mediated signaling events and biological responses in mast cells.
• endocytosis
• proliferation
• Rab5
• Rabex-5
• survival
Noonan syndrome is a relatively common, genetically heterogeneous Mendelian trait with a pleiomorphic phenotype. Prior to the period covered in this review, missense mutations in PTPN11 had been found to account for nearly 50% of Noonan syndrome cases. That gene encodes SHP-2, a protein tyrosine kinase that plays diverse roles in signal transduction including signaling via the RAS-mitogen activated protein kinase (MAPK) pathway. Noonan syndrome-associated PTPN11 mutations are gain-of-function, with most disrupting SHP-2's activation-inactivation mechanism. Here, we review recent information that has elucidated further the types and effects of PTPN11 defects in Noonan syndrome and compare them to the related, but specific, missense PTPN11 mutations causing other diseases including LEOPARD syndrome and leukemias. These new data derive from biochemical and cell biological studies as well as animal modeling with fruit flies and chick embryos. The discovery of KRAS missense mutation as a minor cause of Noonan syndrome and the pathogenetic mechanisms of those mutants is discussed. Finally, the elucidation of gene defects underlying two phenotypically related disorders, Costello and cardio-facio-cutaneous syndromes is also reviewed. As these genes also encode proteins relevant for RAS-MAPK signal transduction, all of the syndromes discussed in this article now can be understood to constitute a class of disorders caused by dysregulated RAS-MAPK signaling.
Forward genetic screens in model organisms have provided important insights into numerous aspects of development, physiology and pathology. With the availability of complete genome sequences and the introduction of RNA-mediated gene interference (RNAi), systematic reverse genetic screens are now also possible. Until now, such genome-wide RNAi screens have mostly been restricted to cultured cells and ubiquitous gene inactivation in Caenorhabditis elegans. This powerful approach has not yet been applied in a tissue-specific manner. Here we report the generation and validation of a genome-wide library of Drosophila melanogaster RNAi transgenes, enabling the conditional inactivation of gene function in specific tissues of the intact organism. Our RNAi transgenes consist of short gene fragments cloned as inverted repeats and expressed using the binary GAL4/UAS system. We generated 22,270 transgenic lines, covering 88% of the predicted protein-coding genes in the Drosophila genome. Molecular and phenotypic assays indicate that the majority of these transgenes are functional. Our transgenic RNAi library thus opens up the prospect of systematically analysing gene functions in any tissue and at any stage of the Drosophila lifespan.
The Drosophila compound eye is specified by the concerted action of seven nuclear factors that include Eyeless/Pax6. These factors have been called "master control" proteins because loss-of-function mutants lack eyes and ectopic expression can direct ectopic eye development. However, inactivation of these genes does not cause the presumptive eye to change identity. Surprisingly, we find that several of these eye specification genes are not coexpressed in the same embryonic cells-or even in the presumptive eye. We demonstrate that the EGF Receptor and Notch signaling pathways have homeotic functions that are genetically upstream of the eye specification genes, and show that specification occurs much later than previously thought-not during embryonic development but in the second larval stage.
even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic devel- opment. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction path- way. SUMMARY
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.
More than 25 years have passed since activating mutations in Ras genes were identified in DNA from human tumors. In this time, it has been established beyond doubt that these mutations play a direct role in causing cancer, and do so in collaboration with a number of other oncogenes and tumor suppressors. Oncogenic mutant Ras proteins are resistant to downregulation by GAP-mediated hydrolysis of bound GTP, and therefore signal persistently. Efforts to develop therapies that block Ras oncoprotein function directly have failed. The high affinity of Ras proteins for GTP has discouraged attempts to identify GTP-analogs. Ras processing enzymes have been targeted, but unfortunately, K-Ras, the Ras protein that plays the major role in human cancer, has proven refractory to these approaches. Further progress has been made with drugs that block downstream signaling: the approved drug Sorafenib inhibits Raf kinase, and its clinical benefits in liver cancer are greatest in patients in which the mitogen activated protein kinase (MAPK) signaling pathway is hyperactive. Other Raf kinase inhibitors, as well as drugs that block mitogen-activated protein kinase / extracellular signal-regulated kinase kinase (MEK) and various steps in the PI 3' kinase pathway, are under development. Here we will discuss the complexities of Ras signaling and their effects on targeting the Ras pathway in the future.
The Ras/mitogen activated protein kinase (MAPK) pathway is essential in the regulation of the cell cycle, differentiation, growth and cell senescence, all of which are critical to normal development. It is therefore not surprising that its dysregulation has profound effects on development. A class of developmental syndromes, the 'RASopathies', is caused by germline mutations in genes that encode protein components of the Ras/MAPK pathway. The vast majority of these mutations result in increased signal transduction down the Ras/MAPK pathway, but usually to a lesser extent than somatic mutations associated with oncogenesis. Each syndrome exhibits unique phenotypic features, however, since they all cause dysregulation of the Ras/MAPK pathway, there are numerous overlapping phenotypic features between the syndromes, including characteristic facial features, cardiac defects, cutaneous abnormalities, neurocognitive delay and a predisposition to malignancies. Here we review the clinical and underlying molecular basis for each of these syndromes.
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 clinical success of selective kinase inhibitors, such as imatinib and erlotinib, as therapeutic agents for several human cancers has prompted substantial interest in the further development and clinical testing of such inhibitors for a wide variety of malignancies. While much of this effort has been focused on the receptor tyrosine kinases, including EGFR, HER2, PDGF receptor, c-KIT, and MET, inhibitors of serine/threonine kinases are also beginning to emerge within discovery pipelines. Among these kinases, the RAF and MEK kinases have received substantial attention, owing largely to the relatively high frequency of activating mutations of RAS ( approximately 20% of all human cancers), an upstream activator of the well established RAF-MEK-ERK signaling cascade, as well as frequent activating mutations in the BRAF kinase ( approximately 7% of all human cancers). Here, we summarize the current state of development of kinase inhibitors directed at this signaling pathway, a few of which have already demonstrating favorable toxicity profiles as well as promising activity in early phase clinical studies.
In Drosophila, as in mammalian cells, the Raf serine/threonine kinase appears to act as a common transducer of signals from several different receptor tyrosine kinases. We describe a new role for Raf in Drosophila development, showing that Raf acts in the somatic follicle cells to specify the dorsoventral polarity of the egg. Targeted expression of activated Raf (Rafgof) within follicle cells is sufficient to dorsalize both the eggshell and the embryo, whereas reduced Raf activity ventralizes the eggshell. We show that Raf functions downstream of the EGF receptor to instruct the dorsal follicle cell fate. In this assay, human and Drosophila Rafgof are functionally similar, in that either can induce ventral follicle cells to assume a dorsal fate.
The Drosophila argos gene, which encodes a secreted protein with an EGF motif, is involved in several developmental processes regulating cell-cell interactions such as eye morphogenesis. Loss-of-function mutations in the argos gene cause an increase in the number of photoreceptor cells and cone cells, impaired retinal projections to the optic lobe, and the formation of extra veins. We show here that ubiquitously expressed argos product restored all these loss-of-function phenotypes. Overexpression of argos in the wild-type background resulted in the reduced number of photoreceptor cells, cone cells, and pigment cells, which are phenotypes opposite to those of the loss-of-function mutants. The argos gene is expressed in developing wing veins. Ubiquitous argos expression caused loss of veins in a dose-dependent manner. This phenotype was enhanced by the loss-of-function rhomboid mutation, implying the possibility that argos and rhomboid play key roles in a common pathway for normal wing vein formation. We propose that argos acts as an inhibitory signal for cellular differentiation in the developing eye and wing.
In the Drosophila eye, activation of the sevenless (sev) receptor tyrosine kinase is required for the specification of the R7 photoreceptor cell fate. In a genetic screen for mutations that result in the activation of the sev signaling pathway in the absence of the inducing signal, we identified a gain-of-function mutation in rolled (rlSevenmaker [rlSem]), which encodes a homolog of mitogen-activated protein (MAP) kinase. In addition to the sev pathway, this mutation activates the pathways controlled by torso and the epidermal growth factor receptor homology. The rlSem mutation results in the substitution of a single conserved amino acid in the kinase domain. Activation of MAP kinase by the rlSem mutation is both necessary and sufficient to activate multiple signaling pathways controlled by receptor tyrosine kinases.
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.
The Drosophila Ras1 gene is required for proper cell fate specification throughout development, and the loss-of-function phenotype of Ras1 suggests an additional role in cell proliferation or survival. A direct role for RAS1 in promoting cell proliferation, however, has not been established. We show that expression of an activated form of RAS1 (RAS1V12) during Drosophila imaginal disc development is sufficient to drive ectopic cell proliferation and hyperplastic tissue growth. In addition, expression of RAS1V12 induces widespread cell death in the imaginal discs, including cells not expressing the transgene, which results in ablation of adult structures. Loss-of-function mutations in the genes encoding RAF, MEK, MAPK and KSR dominantly suppress RAS1V12-induced cell proliferation. Furthermore, two RAS effector loop mutations (E37G and Y40C) that block the RAS-RAF interaction, also suppress RAS1V12-induced proliferation, consistent with a requirement for the MAPK cascade during the RAS1 mitogenic response. These two RAS effector loop mutants, however, retain some activity and can act synergistically with a MAPK gain-of-function mutation, suggesting that RAS1 may also act through signaling pathway(s) distinct from the MAPK cascade.
The Drosophila compound eye is specified by the concerted action of seven nuclear factors that include Eyeless/Pax6. These factors have been called "master control" proteins because loss-of-function mutants lack eyes and ectopic expression can direct ectopic eye development. However, inactivation of these genes does not cause the presumptive eye to change identity. Surprisingly, we find that several of these eye specification genes are not coexpressed in the same embryonic cells-or even in the presumptive eye. We demonstrate that the EGF Receptor and Notch signaling pathways have homeotic functions that are genetically upstream of the eye specification genes, and show that specification occurs much later than previously thought-not during embryonic development but in the second larval stage.
Activating point mutations of the small GTPase Ras are present in about 30% of all human tumors. Constitutively active Ras induces growth factor independent cell proliferation and cell survival. Oncogenic Ras appears to be essential for tumor progression and maintenance. Several therapeutic agents have been developed to inhibit Ras, such as FTIs and antisense oligonucleotides. A new tool for blocking oncogenes in cancer cells has emerged with the discovery that RNA interference can specifically silence expression of endogenous human genes. The therapeutic potential of a RNAi-mediating vector was recently demonstrated by the stable suppression of oncogenic K-Ras in tumor cells.
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.
Intracellular protein transport is a key factor in epithelial cell polarity. Here we report that mutations in two core components of the vesicle trafficking machinery - a syntaxin and a Rab protein - cause an expansion of the apical membrane domain of Drosophila melanogaster epithelia; this polarity defect is coupled with overproliferation to form neoplastic tumours. Surprisingly, these proteins are associated with the endocytic, and not the exocytic, pathway. The syntaxin Avalanche (Avl) localizes to early endosomes, and loss of avl results in the cellular accumulation of specific membrane proteins, including the Notch signalling receptor and the polarity determinant Crumbs (Crb). Protein accumulation results from a failure in endosomal entry and progression towards lysosomal degradation; these and other avl phenotypes are also detected in Rab5 null mutant cells. Overexpression of Crb alone is sufficient to induce overproliferation of wild-type imaginal tissue, suggesting that polarity alterations in avl and Rab5 mutants directly contribute to tumour formation. Our findings reveal a critical and specific role for endocytic traffic in the control of both apico-basal polarity and cell proliferation.
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.
Inactivating mutations in the Drosophila tumor-suppressor genes result in tissue overgrowth. This can occur because the mutant tissue either grows faster than wild-type tissue and/or continues to grow beyond a time when wild-type tissue stops growing. There are three general classes of tumor-suppressor genes that regulate the growth of imaginal disc epithelia. Mutations in the hyperplastic tumor-suppressor genes result in increased cell proliferation but do not disrupt normal tissue architecture. These genes include pten, Tsc1, Tsc2, and components of the hippo/salvador/warts pathway. Mutations in a second class of genes, the neoplastic tumor-suppressor genes, disrupt proteins that function either as scaffolds at cell-cell junctions (scribble, discs large, lgl) or as components of the endocytic pathway (avalanche, rab5, ESCRT components). For the third group, the nonautonomous tumor-suppressor genes, mutant cells stimulate the proliferation of adjacent wild-type cells. Understanding the interactions between these three classes of genes will improve our understanding of how cell and tissue growth are coordinated during organismal development and perturbed in disease states such as cancer.
A gain-of-function mutation in Drosophila MAP kinase (A) Control engal4/+ wing. (B and C) Expressing Rabex-5 WT (B) or Rabex-5 DPYT (C) using engal4 re
- D Brunner
- N Oellers
- J Szabad
- W H Biggs
- rd
- S L Zipursky
- E Hafen
Brunner, D., Oellers, N., Szabad, J., Biggs, W.H., 3rd, Zipursky, S.L., and Hafen, E. (1994). A gain-of-function mutation in Drosophila MAP kinase (A) Control engal4/+ wing. (B and C) Expressing Rabex-5 WT (B) or Rabex-5 DPYT (C) using engal4 re-sulted in crossvein loss (arrows).