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RAS proteins conduct signaling from surface receptors to cytoplasmic effectors, and RAS gain-of-function mutations are pervasive in cancer. A new mechanism for RAS signal attenuation with implications for receptor trafficking has been uncovered.
RAS GTPases (HRAS, KRAS and NRAS) function at the inner surface of the plasma membrane to translate receptor activation into cytoplasmic signaling . Following activation by guanine nucleotide exchange factors (GEFs), GTP-bound RAS proteins transduce signals through multiple downstream effector proteins with the capacity to influence virtually every aspect of cell biology. GTPase-activating proteins (GAPs) return RAS to the quiescent, GDP-bound, state. Regulation of RAS signaling is essential during development, and loss-of-function mutations in RAS, RAS-specific GEFs/GAPs, or RAS effectors are associated with a variety of abnormalities. Cells in adult organisms require tight control of the same signaling pathways, and gain-of-function mutations in RAS and its effectors are pervasive in cancer.
In this issue of Current Biology, two groups, using distinctly different approaches, report a new pathway for attenuation of RAS signaling [2,3]. They show that RABGEF1 (Rabex-5), named for its role as a GEF and activator of RAB5 GTPases during endocytosis, functions as an E3 ubiquitin ligase that targets RAS proteins to reduce downstream signaling [2,3].
Mammalian HRAS and NRAS proteins are post-translationally palmitoylated and farnesylated , promoting their localization to the plasma membrane; the predominant isoform of KRAS is farnesylated but not palmitoylated, and its membrane localization is regulated by a polybasic motif and conditional phosphorylation ). From their position on the plasma membrane, RAS proteins effectively transduce signals from receptor tyrosine kinases, such as epidermal growth factor receptor (EGFR), to downstream effectors such as the RAF–MEK–ERK kinase cascade and other pathways. Mono- and di-ubiquitination of HRAS and NRAS is associated with their translocation to endosomal membranes and a concomitant reduction in ERK activation, which is measured by dual phosphorylation of ERK1 and ERK2 . By contrast, ubiquitinated KRAS continues signaling through the RAF–MEK–ERK pathway, whereas ubiquitinated EGFR is internalized and traffics to the late endosome .
In one of the new studies, the Bar-Sagi group  considered RABGEF1 (Rabex5), a known binding partner of RAS proteins , to be a strong candidate for promoting HRAS and NRAS ubiquitination. RABGEF1 contains an A20-type zinc finger (ZnF-A20, IPR002653) domain with E3 ubiquitin ligase activity and an adjacent α helix with ubiquitin-binding activity [8,9]. Indeed, ectopic expression of RABGEF1 enhanced the ubiquitination of HRAS and NRAS in cultured mammalian cells, while RABGEF1 silencing diminished HRAS and NRAS ubiquitination . Importantly, the capacity of RABGEF1 to promote Ras ubiquitination was blocked by a mutation in the ZnF domain but not by a mutation in the GEF domain. RAS ubiquitination by wild-type RABGEF1, but not the ZnF mutant, was recapitulated in a cell-free system to confirm target specificity.
The Pfleger group  began with the observation that a Drosophila Rabex5 (RABGEF1 ortholog) loss-of-function mutant has dramatic phenotypic alterations, including ectopic wing veins, melanotic tumors and eye/antenna fate switching, phenotypes that are strikingly similar to those caused by a gain-of-function mutation in Ras (Drosophila has a single ortholog of mammalian HRAS, NRAS and KRAS). They also showed that phenotypes resulting from Rabex-5 silencing were suppressed by a Ras loss-of-function mutation . These inverse relationships suggested that Rabex5 normally inhibits Ras signaling in this system. Further, the phenotypes of a Rabex5 loss-of-function mutant were rescued by ectopic expression of a wild-type or GEF domain mutant of Rabex5, but not by a ZnF mutant of Rabex5. This demonstrated that the Rabex5 ubiquitin ligase function, but not the guanine nucleotide exchange function, was responsible for Rabex5-mediated regulation of Ras activity. In addition, Drosophila Rabex5 was shown to ubiquitinate Ras in an insect cell culture system.
A RABGEF1/Rabex5 gain-of-function mutation resulted in a reduction in downstream signaling by HRAS and NRAS in mammalian cells and by Drosophila Ras, as judged by lower levels of dual phosphorylated ERK proteins. Reinforcing this interpretation, phenotypes caused by a constitutively active Egfr (which acts upstream of Ras) were exacerbated by loss of Rabex5 in the Drosophila system. These findings shed light on a previous report , which showed that silencing or loss of mouse RABGEF1 increased RAS activity and ERK phosphorylation following stimulation of mast cell immunoglobulin E receptors (FcεRI). The new findings suggest a mechanism for reducing ERK signaling after receptor internalization, but leave unresolved whether ubiquitinated and endosome-localized RAS proteins continue to signal through alternative downstream effector pathways.
RAS ubiquitination is associated with translocation to endosome membranes  and, consistent with this, RABGEF1 overexpression correlated with higher levels of GFP-tagged HRAS on EEA1-positive vesicles, while RABGEF1 silencing diminished endosomal localization of GFP–HRAS . It remains uncertain, however, whether ubiquitination drives the movement of HRAS and NRAS to endosomes or whether this modification is simply required to retain RAS proteins that have already been incorporated into endosomes during receptor endocytosis.
Much about the choreography of RAB5-driven endocytosis remains unresolved. RAB5 family members (RAB5A–C and the related proteins RAB17, RAB21, RAB22, RAB31) participate in the early stages of endosome trafficking and fusion. These GTPases depend on activation by GEFs of the VPS9 family (RABGEF1, GAPVD1, RIN1–3 and the related proteins RINL, ALS2, ALS2CL, ANKRD27, C16orf7). The functional interaction of RAB5 proteins with regulatory GEFs and effectors has received much attention (reviewed in ), and a defined-component RAB5 endosome fusion system using RABGEF1 has been described . Much less is understood about precisely where and when RAB5 proteins are activated (i.e. GTP loaded), or how this leads to engagement of RAB5 effector proteins (RABEP1–2, ZFYVE20, ANKFY1, EEA1 and MON1A–B).
The work of Xu and colleagues  implicates a second exchange factor in the pathway leading to RAS ubiquitination. RIN1 is a RAS effector [12,13] and a RAB5-directed GEF  implicated in endocytosis. Overexpression of RIN1 enhanced HRAS ubiquitination, but this effect was dependent on RABGEF1 and therefore likely to reflect an event occurring upstream of RABGEF1. And, unlike RABGEF1, the GEF function of RIN1 is required for enhancement of HRAS ubiquitination. Of note, RIN1 may also participate in endocytosis through a Src homology 2 (SH2) domain that binds activated receptor tyrosine kinases  and a domain that activates ABL tyrosine kinase, promoting actin cytoskeleton remodeling .
These findings suggest a possible sequence of events following RAS activation (for example, following receptor tyrosine kinase stimulation; Figure 1). Signaling through the downstream effector RAF triggers MEK and ERK activation, while the RAS effector RIN1 converts GDP-bound (inactive) RAB5 to GTP-bound (active) RAB5. Once activated, RAB5 can then engage its own effectors including RABEP (rabaptin) proteins. RABGEF1 may be recruited to the growing complex by its early endosome targeting domain  and through associations with RABEP  and RAB22 . Following recruitment to the growing complex, RABGEF1 acts through its ZnF-A20 domain as an E3 ligase for RAS. This leaves RABGEF1 well positioned to function through a feedback loop  to maintain elevated levels of GTP-bound RAB5 proteins on the nascent endosome.
Characterizing the role of RABGEF1 in RAS ubiquitination and endosome localization represents a significant advance in understanding RAS signal regulation following receptor stimulation that will invigorate research in this area. Among the questions raised by this work are the timing of RAS ubiquitination relative to endosome trafficking and the identity of other ubiquitination targets of RABGEF1. Also unclear is the fate of ubiquitinated RAS: is it de-ubiquitinated for recycling to the plasma membrane or sent to a degradation pathway?