Downloaded from http://perspectivesinmedicine.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press MRAS: A Close but Understudied Member of the RAS Family Lucy C. Young1 and Pablo Rodriguez-Viciana2 1UCSF Helen Diller Family Comprehensive Cancer Center, San Francisco, California 94158 2UCL Cancer Institute, Paul O’Gorman Building, University College London, London WC1E 6BT, United Kingdom Correspondence: [email protected] MRAS is the closest relative to the classical RAS oncoproteins and shares most regulatory and effector interactions. However, it also has unique functions, including its ability to function as a phosphatase regulatory subunit when in complex with SHOC2 and protein phosphatase 1 (PP1). This phosphatase complex regulates a crucial step in the activation cycle of RAF kinases and provides a key coordinate input required for efficient ERK pathway activation and trans- formation by RAS. MRAS mutations rarely occur in cancer but deregulated expression may play a role in tumorigenesis in some settings. Activating mutations in MRAS (as well as SHOC2 and PP1) do occur in the RASopathy Noonan syndrome, underscoring a key role for MRAS within the RAS-ERK pathway. MRAS also has unique roles in cell migration and differentiation and has properties consistent with a key role in the regulation of cell polarity. Further inves- tigations should shed light on what remains a relatively understudied RAS family member. he RRAS subgroup (RRAS, TC21/RRAS2, SEQUENCE FEATURES OF MRAS Tand MRAS/RRAS3) of the RAS family GTPases (RFGs) are the closest relatives to the The RRAS subgroup lies within a distinct classical RAS oncogenes (H/N/KRAS, hereafter branch of the tree of all small GTPases with referred to collectively as RAS). These GTPases classical RAS and ERAS (Fig. 1A). MRAS is share many regulatory and effector proteins with highly conserved between vertebrates and has RAS as well as transforming abilities (Chan et al. considerable similarity to the ras-2 gene in Cae- www.perspectivesinmedicine.org 1994; Saez et al. 1994; Kimmelman et al. 1997; norhabditis elegans (Fig. 1B). Interestingly, al- Rodriguez-Viciana et al. 2006). Members of this though classical RAS orthologs exist in fly, group have distinct functions, and MRAS has fish, and nematode, they are absent in ascidian, been shown to play a number of roles in cellular which does have orthologs to MRAS and RRAS processes such as differentiation, cytoskeletal re- (Fig. 1B). Therefore, MRAS evolved indepen- modeling, and cell migration. Uniquely among dently of RRAS in metazoans and has been sug- RFGs, MRAS is part of a phosphatase complex gested to compensate for the lack of classical that positively regulates RAF kinase activation RAS in ascidian (Keduka et al. 2009). and is required to cooperate with RAS proteins The G domain of MRAS is similar in se- for efficient ERK pathway activation. quence to the classical RAS proteins (it shares Editors: Linda VanAelst, Julian Downward, and Frank McCormick Additional Perspectives on Ras and Cancer in the 21st Century available at www.perspectivesinmedicine.org Copyright © 2018 Cold Spring Harbor Laboratory Press; all rights reserved Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a033621 1 Downloaded from http://perspectivesinmedicine.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press L.C. Young and P. Rodriguez-Viciana ABERAS Hs RAS-2 Ce ERAS Hs MRAS Ci MRAS MRAS Hs MRAS Hs RRAS Hs Mras Dr TC21 Hs RAS-1 Ce NRAS Hs RRAS Hs HRAS Hs Rras Dr KRAS4A Hs Ras64B Dm RRAS/TC21 KRAS4B Hs TC21 Hs Rras2 Dr RRAS Ci LET-60 Ce Ras85D Dm KRAS4A Hs KRAS4B Hs Classical HRAS Hs RAS Hrasa Dr Hrasb Dr Nras Dr NRAS Hs Figure 1. MRAS phylogeny. (A) Phylogenetic relationship of human RAS family proteins. (B) Phylogenetic analysis of RAS family proteins in human (Hs), zebrafish (Dr), fruitfly (Dm), nematode (Ce), and ascidian Ciona intestinalis (Ci). Sequence alignment was performed using MUSCLE (see ebi.ac.uk/Tools/msa/muscle) and tree generation with iTOL (see itol.embl.de). 53% amino acid identity with KRAS), and like (Mitin et al. 2005). Although some of these pro- RRAS and TC21, it has a short amino-terminal teins also regulate RRAS/TC21, full analysis of extension. The terminal residue of MRAS is ly- this regulation links MRAS closely to classical sine, which differs from the terminal residues of RAS in terms of GEF/GAP specificity (Ohba RAS (Fig. 2A), and consequently rather than et al. 2000). This implies that most physiological being farnesylated, MRAS is predicted to be signals that activate RAS will also activate MRAS posttranslationally modified instead by gera- simultaneously and is consistent with MRAS nylgeranylation (Zhang and Casey 1996). The functioning together with RAS to provide coor- hypervariable region (HVR) of MRAS has sim- dinate inputs for efficient RAF activation (see ilarities to KRAS4B in that carboxy-terminal below). cysteines capable of palmytoylation are absent Given its sequence similarity and identical www.perspectivesinmedicine.org and instead a polybasic region is found (Fig. effector domain (Fig. 2B), it is unsurprising that 2A). Like KRAS4B, MRAS is found in disor- MRAS can bind many of the same effectors as dered membrane domains rather than orga- RAS such as A-, B-, and CRAF, AFDN/AF6, nized lipid rafts (Ehrhardt et al. 2002), which RASSF5, RalGEFs, and PI3K (Quilliam et al. suggests these proteins may signal in similar 2001; Ortiz-Vega et al. 2002; Rodriguez-Vicia- pathways and/or be similarly regulated. na et al. 2004). Through binding RGL2/RLF, MRAS activates RAL and ELK1 in MCF-7 cells in an ERK-independent manner (Ehrhardt et al. MRAS REGULATION AND CONTROL 2001; Castro et al. 2012). MRAS also controls OF DOWNSTREAM PATHWAYS activation of RAP activity through binding MRAS shares many regulatory proteins with MR-GEF/RAPGEF5 (Rebhun et al. 2000) and other RFGs; it can be activated by SOS1, RA-GEF2/RAPGEF6 (Gao et al. 2001), the latter RASGRF1, RASGRP1, and RASGRP3 guanine being specifically linked to control of cell adhe- nucleotide exchange factors (GEFs) and inacti- sion through tumor necrosis factor (TNF)-α- vated by p120 RASGAP, neurofibromin, and triggered integrin activation in haematopoietic GAP1m GTPase-activating proteins (GAPs) cells (Yoshikawa et al. 2007). 2 Advanced Online Article. Cite this article as Cold Spring Harb Perspect Med doi: 10.1101/cshperspect.a033621 Downloaded from http://perspectivesinmedicine.cshlp.org/ on September 25, 2021 - Published by Cold Spring Harbor Laboratory Press MRAS A CAAX NRAS 165 QYRMKKLNSS–––––––DDGTQGCMGLPCVVM 189 165 QHKLRKLNPP–––––––DESGPGCMSCKCVLS 189 HRAS Palmitoylated KRAS4A 165 QYRLKKISKE–––––––EKTPGCVKIKKCIIM 189 KRAS4B 165 KHK-EKMSKD-------GKKKKKKSKTKCVIM 188 Phosphorylated MRAS 177 QQIPEKSQKKKKKTKWRGDRATGTHKLQCVIL 208 Basic RRAS 193 KYQEQELPPSP-----PSAPRKKGGGCPCVLL 224 TC21 177 KFQEQECPPSPE----PTRKEKDKKGCHCVIF 204 B Switch I Switch II MRAS (21-93) DGGVGKSALTIQFFQKIFVPDYDPTIEDSYLKHTEIDNQWAILDVLDTAGQEEFSAMREQYMRTGDGFLIVYS RRAS (37-109) G------------I-SY--SD---------T-ICSV-GIP-R--I---------G--------A-H-----FA TC21 (22-92) G------------I-SY--TD---------T-QCVI-DRA-R--I---------G--------T-E-----FS H/N/KRAS (11-83) A-----------LI-NH--DE---------R-QVVI-GETCL--I--------YS---D----T-E---C-FA Effector domain Figure 2. Comparison of MRAS amino acid sequence with other RAS family GTPases (RFGs). (A) Alignment of RAS protein hypervariable regions with features shaded according to property. (B) Sequence alignment (of Switch I and II containing regions) of MRAS with H/N/KRAS, RRAS, and TC21. MRAS is a weak activator of the ERK path- tified in C. elegans as a positive modulator of the way compared with RAS (Kimmelman et al. ERK pathway (Selfors et al. 1998; Sieburth et al. 1997; Rodriguez-Viciana et al. 2004), which, at 1998). Active MRAS forms a ternary complex least in part, could be attributed to the lower with SHOC2 and PP1 to form a phosphatase affinity of MRAS for RAF compared with RAS. holoenzyme that specifically dephosphorylates a One reason for this may be the differences in conserved inhibitory site in RAF kinases (S259 in sequence of MRAS, particularly in the Switch I CRAF, S365 in BRAF, and S214 in ARAF, hereby region (Fig. 2B). Both the GppNHp- and gua- referred to as S259) that functions as a 14-3-3 nosine diphosphate (GDP)-bound forms of binding site and plays a key role in the RAF-acti- MRAS have an open Switch I conformation, vation cycle (Rodriguez-Viciana et al. 2006). and mutation of these residues to those of The consensus model of RAF activation RAS result in a higher proportion of MRAS in stipulates that RAF is maintained in an auto- the closed state. In addition, these mutations inhibited inactive state in the cytosol by an increase the affinity of MRAS for the RAS-bind- intramolecular interaction between the amino- www.perspectivesinmedicine.org ing domain (RBD) of RAF (Ye et al. 2005), which terminal region and the catalytic domain, which implies that there are regions unique to MRAS is in part mediated by a 14-3-3 dimer bound (with others yet to be identified) that govern its to two phosphorylated residues (S259 and S621 affinity and specificity for various effectors. in CRAF) (Fig. 3A) (Tzivion et al. 1998; Matallanas et al. 2011; Lavoie and Therrien 2015). RAS-guanosine triphosphate (GTP) MRAS REGULATION OF RAF ACTIVITY binding to the RBD of RAF results in RAF THROUGH SHOC2/PROTEIN translocation to the plasma membrane in which PHOSPHATASE 1 (PP1) other activating steps then take place. Chief In addition to sharing interactions with many of among these is the dephoshphorylation of the RAS effectors, MRAS also has its own unique “S259” site, which leads to 14-3-3 displacement effector interactions with RAPGEFs (Gao et al. from this site, destabilizes the closed conforma- 2001) as well as SHOC2 and PP1 (Rodriguez- tion of RAF, allows the cysteine-rich domain Viciana et al.