RAS subfamily members show high conservation within the G1, G3, G4, and G5 boxes (). Most proteins in this group are relatively small (183 to 340 amino acids in length) and show no prominent functional motifs outside of those defining their RAS relatedness.
Fig 2 Alignment of human RAS subfamily members. G box consensus residues are highlighted in blue. N- and C-terminal region cysteines, some of which are substrates for prenylation or fatty acid modification, are highlighted in green. N-terminal glycines in positions (more ...)
Most of the RAS subfamily proteins localize predominantly to the plasma membrane. Membrane localization results in part from C-terminal prenylation. Prenylation signals mostly conform to the Caax (a = aliphatic, x = terminal amino acid) motif that directs cysteine farnesylation (except when x = L or F, which instructs geranylgeranylation as occurs on RRAS proteins and some RAP proteins). The prenylation reaction is followed by proteolysis of the three C-terminal residues (aax) and methylation of the lipid-modified cysteine [reviewed in (23
)]. The later two posttranslational processing steps take place in the endoplasmic reticulum (ER) before transport to the plasma membrane (53
). RAL proteins contain the geranylgeranylation signal CCaa. Some RAS subfamily members lack either type of isoprenylation motif and are not subject to any known lipid modification.
Some RAS subfamily proteins contain fatty acid acylation signals. Notably, HRAS, NRAS, ERAS, RRAS1 and RAP2A, RAP2B, and RAP2C have palmitoylated cysteine residues proximal to their C-terminal prenylated cysteines. This modification requires transit through the Golgi compartment (54
). Endomembrane localization of RAS proteins may be more than just a posttranslational modification detour, however. Several lines of evidence suggest that RAS proteins are functional signal transducers in the ER-Golgi complex (55
N-terminal lipidations may contribute to the localization of other RAS subfamily members such as ARHI (Ras homolog member I), which has a potential myristoylation site (MetGly) at its N terminus, and NKIRAS1 and NKIRAS2 (NF-κB inhibitor–interacting Ras-like 1 and 2) proteins, which have putative myristoylation or palmitoylation modification signals (MGxxCxxxxC) .
An additional factor in RAS protein trafficking and localization is the presence of a C-terminal polybasic region, as seen on the predominant KRAS splice variant KRAS2B. This protein lacks a palmitoylation site but has a strong polybasic region immediately upstream of the C-terminal farnesylation site. In contrast, HRAS and NRAS have palmitoylation sites but no polybasic regions. These differences are believed to underlie the distinct membrane localization characteristics (58
) and signaling properties (59
) of these otherwise close paralogs. In the 2A isoform of KRAS (N terminus = KTPGCVKIKKCIIM), the polybasic sequence is replaced with a palmitoylation site. As a result, the KRAS2A isoform may be more similar to HRAS and NRAS in its subcellular localization. Interestingly, RAP1 (prenylation + polybasic) and RAP2 (prenylation + fatty acylation) proteins appear to have a relationship similar to that of KRAS2B and HRAS. The RIT1 and RIT2 proteins encode C-terminal polybasic sequences (6 of 10 residues are R or K) but lack both prenylation and fatty acylation signals. RERG (Ras-related and estrogen-regulated growth inhibitor) appears to be devoid of all standard lipid membrane localization signals and displays cytosolic localization, suggesting that it functions outside the context of cellular membranes (60
Other posttranslational modifications have been described for RAS subfamily proteins. These include serine phosphorylation (61
) and nitrosylation (62
) of HRAS and tyrosine phosphorylation of RRAS1 (65
); the functions of these modifications are still under investigation.
Variation at the level of alternative splicing has been described for some RAS genes. For KRAS (67
) and HRAS (69
), the primary function of alternate splicing may be to generate isoforms with distinct subcellular localizations.
A comparison of RAS subfamily sequences from Homo sapiens, Drosophila Melanogaster, and Caenorhabditis elegans () shows strong conservation through evolution, with most branches of the dendrogram containing representatives from each species. This analysis also illustrates a notable expansion of RAS subfamily proteins (human = 35, fly = 14, worm = 12) and suggests 12 structural or functional branches.
Fig. 3 Dendrogram of RAS subfamily members from H. sapiens, D. melanogaster (Dm), and C. elegans (Ce). Human protein names are in uppercase letters. Branch lengths are directly proportional to the number of differences between sequences compared. See (more ...)
RAS oncoprotein branch (HRAS, KRAS, and NRAS)
HRAS, KRAS, and NRAS (H, K, NRAS) proteins are perhaps best known for their mitogenic properties. As discussed above, mutationally activated forms of these proteins can efficiently transform cells in vitro and in vivo, and such mutations are common in a broad spectrum of human tumors. There is also strong evidence from cell culture experiments (70
) and model organisms (71
) that H, K, NRAS proteins contribute to cell differentiation and organ development. These same proteins have more recently been implicated in neuronal plasticity in the central nervous system (73
The protein kinase RAF1 (also called c-Raf) was the first identified RAS effector (79
) and, together with the closely related ARAF (also called A-Raf) and BRAF (also called BRaf), has been the most intensively studied [reviewed in (84
)]. Activated RAS binds with high affinity to the “Raf-like Ras–binding domain” (Interpro IPR003116), as well as an adjacent cysteine-rich domain, and leads to activation of the kinase activity of RAF and initiation of the MEK-ERK mitogen-activated protein kinase cascade, which affects transcription and other cellular functions. The precise mechanism of RAS-mediated activation is complex and not yet fully elucidated, but seems to involve enhanced membrane association, as well as allosteric derepression (deletion of the RAS binding domain results in constitutive kinase activity) (85
) and promotion of RAF phosphorylation by serine-threonine and tyrosine kinases. Hyperactivation of the RAF effector pathway alone can transform immortalized rodent fibroblast cells, but appears to be insufficient for transformation of some other cell types (86
). The frequent occurrence of dominant BRAF mutations in some human cancers (88
) further suggests that this effector pathway has a major role in tumorigenesis. Other human proteins with “Raf-like Ras–binding domains” include TIAM1, which functions as a RAS-controlled GEF-type activator of RAC (a member of the Rho subfamily) (89
). Mice deficient in TIAM1 function develop normally but are impaired in carcinogen-induced, RAS-mediated, tumorigenesis (90
), consistent with a role for this effector in RAS-mediated growth regulation.
The catalytic subunits of phosphotidylinositol 4,5 bisphosphate 3-kinase (PI3K) constitute another well-established class of RAS effectors (91
). RAS binds to a consensus “phosphoinositide 3-kinase Ras binding domain” (Interpro IPR000341) found in seven distinct human proteins (PIK3CG, PIK3C2A, PIK3C2G, PIK3CB, PIK3CA, PIK3C2B, and PIK3CD). This interaction promotes PI3K catalytic activity (92
), resulting in increased production of membrane-associated PIP3
(phosphatidylinositol 3,4,5-trisphosphate) and the subsequent plasma membrane recruitment of PIP3
-binding PH domain proteins such as the protein kinases AKT1 and PDPK1 (3-phosphoinositide–dependent protein kinase 1, also called PDK1). RAS-mediated activation of PI3K is also an important component of cell transformation (8
Several GEFs for RAL proteins are RAS effectors (93
). RALGDS, RGL1 (Ral GDP dissociation stimulator–like; also called ARHGAP9), RGL2 (also called Rab2L), and RGL3 each encode a Ras association (RA) domain (Interpro IPR000159), a third type of RAS-effector interaction motif. RAS proteins stimulate the nucleotide-exchange activity of RALGDS (98
), and this appears to have a critical role in human cell transformation (99
RIN1 is another RA domain–containing RAS effector protein (101
). The RIN1 protein functions as a RAS-responsive GEF for RAB5 (103
) and also stimulates the catalytic activity of the ABL tyrosine kinase (104
). RIN1 has a restricted expression pattern (78
) and, because of its high-affinity binding to RAS proteins (101
), may function in part as a physiological competitor of other effectors. The related proteins RIN2 and RIN3 have discernable RA domains but have not been functionally connected to any RAS protein. Another RAS effector, NORE1 (novel Ras effector 1; also called RASSF5 and RapL), is a positive regulator of cell death through association with the proapoptotic kinase STK4 (106
). NORE1 is itself part of a family of related proteins (RASSF1 through RASSF6) that all contain RA domains but have not all been functionally connected to RAS. The RA domain–containing enzyme phospholipase C epsilon (PLCE1; also called PLCε) has also been described as a RAS effector (107
). However, RA domains show affinity for RAP as well as RAS proteins. In the case of the RA protein MLLT4 (also called AF6), Rap1 proteins may be the preferred physiological binding partners (108
). Finally, another RA domain–containing protein, RASIP1 (Ras-interacting protein 1, also called RAIN), is an effector of RAS and RAP (109
). Systematic analysis of RAS family GTPases and multiple effectors has demonstrated binding specificity that often correlates with biochemical and biological activation (110
BRAP (also called IMP, impedes mitogenic signal propagation) is another protein that binds specifically to activated RAS (111
), although BRAP has no RA or other recognizable RAS-interaction domain. BRAP appears to function as a dedicated inhibitor of signaling between RAF and MEK.
RRAS (Related to RAS) branch
RRAS1, RRAS2 (also called TC21), and MRAS (also called RRas3) appear to be involved in control of mitogenesis and the cytoskeleton. RRAS1 localizes to focal adhesions where it promotes cell adhesion and activates integrins (112
). Activating (GTPase-defective) mutants of all the RRAS proteins can transform cultured fibroblast cells, with RRAS2 being the most potently transforming (114
). Activating mutations and overexpression of RRAS2 are found in some human tumors (118
). Effectors implicated in the function of RRAS family members include PI3K (121
), RALGDS and related proteins (97
), and RAF kinases (124
), but RRAS1 appears to work primarily through PI3K (121
). This overlap with effectors of the H, K, NRAS family likely reflects the complete conservation of G2 box (switch 1) sequences among members of both branches. The differences between the physiological consequences of RRAS activation versus that of H, K, NRAS activation may reflect quantitative differences in effector engagement, as well as the contribution of some unique effectors for each protein.
RAP (Ras-Proximal) branch
RAP proteins are activated by mitogenic stimuli and function as regulators of integrin-mediated cell adhesion and cell spreading (126
). In cultured cells, RAP proteins do not show transforming activity. Rather, overexpression of RAP1A inhibits RAS-mediated transformation (128
). However, RAP1A has been reported to bind and activate BRAF (129
), suggesting that it has the capacity to promote mitogenesis and perhaps transformation in some contexts but not others (130
). Two observations suggest contributions of RAP proteins in tumorigenesis, but with possible tissue-type specificity. Activation of a RAP-directed GEF (131
) or inactivation of a RAP-directed GAP (132
) promotes hematopoietic tumor formation. Conversely, the loss of an activator of RAP1 proteins has been found in a mouse osteosarcoma and in several nonhematopoietic human cancer cell lines (133
RAP proteins may function through activation of RALGDS and related proteins, but not in the same way that RAS does (134
), and through associations with PLCE1 (135
). In lymphoid cells, RAP1 proteins promote integrin activation through NORE1 (136
RAL (RAS-Like) branch
RALA and RALB have been implicated in a broad spectrum of functions including mitogenic responses, differentiation, protein trafficking, and cytoskeleton dynamics [reviewed in (137
)]. As discussed above, H, K, NRAS, RRAS2, MRAS, and RAP proteins all appear to work in part through RALGDS-type effectors that are expected to stimulate RAL functions. Although mutationally activated RAL proteins are not themselves oncogenic, they can enhance transformation of cultured cells by RAS and EGFR (epidermal growth factor receptor) (98
). The two RAL proteins appear to have distinct and complementary roles in cell transformation; RALB is required for tumor cell survival, whereas RALA promotes anchorage-independent cell proliferation (139
). Each RAL also has a distinct role in epithelial cell polarization (140
Several RAL effectors have been identified but, to date, these do not include members of the RAF-PI3K-RALGDS triumvirate. This may seem surprising because RAL proteins show high overall relatedness to H, K, N-RAS proteins. However, the two subfamilies diverge appreciably in their Switch 1 regions (). The sequence YDPTIED is completely conserved in H, K, N-RAS proteins as well as in RRAS1 RRAS2, MRAS, and all RAP proteins, all of which share many effectors. In RALA and RALB the equivalent sequence is YEPTKAD.
The RAL effector RALBP1 (also called RLIP), which has a RAC- and CDC42-directed GAP domain (141
), regulates endocytosis (144
). RAL is also a component of the exocyst complex. RAL directly binds to both SEC5L1 (also called Sec5) and EXOC8 (also called EXO84), promoting exocyst complex assembly and membrane trafficking (147
RIT (RAS-like Protein in All Tissues) branch
RIT1 and RIT2 (also called Rin) are positive factors for neuronal cell survival as well as for the initiation, elongation, and branching of neu-rites in culture (150
). The enhanced expression of RIT1 and RIT2 in developing and mature neurons (153
) supports the biological relevance of these properties. RIT2 includes a Ca2+
-calmodulin binding site (153
), which appears to be required for its neurite outgrowth function (150
). Although an activated (GTPase-deficient) mutant of RIT1 can transform a fibroblast cell line (154
), there is no evidence that either RIT gene functions in tumorigenesis.
On the basis of protein interaction experiments, RALGDS (and related proteins) and AF6 are potential effectors of RIT1 and RIT2 (156
), but no RIT-specific effectors have been characterized. Several lines of evidence indicate that RAF and PI3K are not direct effectors of RIT proteins (150
ERAS (Embyonic Stem Cell–Expressed Ras) branch
ERAS is an unusual subfamily member in several respects. As indicated in , ERAS occupies a branch with no human paralogs and no fly or worm orthologs. ERAS expression is restricted to undifferentiated embryonic stem (ES) cells (157
Ectopic expression of wild-type ERAS transforms cultured fibroblast cells (157
). This unusual property likely reflects the effect of sequence differences at residues that regulate the GTP/GDP binding equilibrium in other RAS proteins (that is, the amino acid corresponding to Gly12
in H, K, NRAS). ERAS may be an important factor in the propensity of ES cells to form teratomas. A strong candidate effector of ERAS is PI3K (157
DIRAS (Distinct Subgroup of RAS) and ARHI branches
The DIRAS1 (also called Rig) and DIRAS2 proteins, like RHEBs, show reduced GTPase activity compared to that of most RAS superfamily GTPases, and DIRAS proteins remain predominantly in the GTP-bound state (158
). DIRAS and ARHI proteins may have tumor suppressor functions. Overexpression of DIRAS1 antagonizes Ras-mediated signaling and transformation, and DIRAS1 is silenced or down-regulated in many neural tumors and tumor-derived cell lines (159
). The ARHI (also called Noey2) protein has been implicated as a tumor suppressor in breast and ovarian cancer (160
Ectopic expression of DIRAS1 or DIRAS2 can induce the formation of large vacuolar structures (158
), but downstream effectors have not been identified for these proteins.
RASD (Ras Induced by Dexamethasone) branch
RASD1. (also called dexRas) was identified as a transcript that shows strong, rapid, and transient induction after treatment of cells with dexamethasone (162
), and RASD2 (also called RHES, for Ras homolog enriched in striatum) was identified as a protein expressed in pancreatic beta cells in response to efaroxan, an imidazoline that functions as an α2
-adrenergic receptor antagonist and insulin secretagogue (163
). There is no evidence to support involvement of RASD1 or RASD2 in transformation or tumorigenesis. RASD1 appears to function as a negative regulator of peptide hormone secretion (164
) and as a cell growth suppressor (165
). RASD2 has the capacity to activate PI3K and may interfere with G protein–coupled receptor signaling (166
The uncharacterized gene products RASL10A and RASL10B are the closest related proteins to RASD1 and RASD2.
NKIRAS (NFKB Inhibitor–interacting RAS-like, also called kB-Ras) branch
NKIRAS1 and NKIRAS2 were discovered as proteins that interact with NFKBI (usually called IκB), an inhibitor of the transcription factor NFKB (usually called NF-κB) (167
). Binding of NKIRAS to NFKBI-NFKB complexes prevents nuclear translocation of the complex in resting cells, suggesting that NKIRAS proteins participate in the negative regulation of NFKB (168
REM (Rad and Gem–related) branch
REM1, REM2, RRAD (also called Rad), and GEM (also called Kir) were identified primarily on the basis of their restrictive and regulated expression patterns (169
). They share a conserved C-terminal cysteine (position –7), but this is not within a context recognized for lipid modification. REM subfamily proteins show no transforming or tumorigenic properties. REM1, RRAD, and GEM function in part as negative regulators of calcium currents through a direct interaction with the β subunit of a voltage-gated Ca2+
). Overexpression of GEM produces cytoskeletal changes marked by cellular processes. These changes may result from a direct interaction of GEM with the kinesin-like protein KIF9 (175
) and RHOA inactivation [reviewed in (176
)], perhaps through GMIP (Gem interacting protein), a RHOGAP (177
RERG (RAS-related and Estrogen-Regulated Growth inhibitor) branch
RERG was identified during a search for genes whose expression in breast tumors correlates with prolonged survival (60
). As its name implies, transcription of the RERG gene is increased in response to estrogen, perhaps through direct estrogen receptor binding to the RERG gene promoter. RERG shows no binding to H, K, NRAS effectors tested (RAF, RALGDS, PI3K, and RIN1), and RERG neither transformed cultured fibroblasts nor enhanced HRAS-mediated transformation (60
). Ectopic expression of RERG actually blocked transformation and tumorigenesis in a breast tumor cell line (60
Three gene products
RASL11A, RASL11B, and RASL12—show relatedness to REM. Abundance of RASL11A
transcripts is decreased in some prostate tumors (178
), but its function is uncharacterized. The RASL11A, RASL11B, and RASL12 gene products have no lipid modification signals, suggesting functions that are not restricted to membrane surfaces. Further analysis of these proteins will determine if they are best considered as a separate branch of RAS proteins.
RHEB (Ras Homolog Enriched in Brain) branch
RHEB proteins are involved in the control of cell cycle and cell growth (179
). Although early studies found that RHEB proteins block MAPK (mitogen-activated protein kinase) signaling and inhibit RAS-mediated transformation of cultured fibroblasts (180
), it is not yet clear whether these observations represent physiological activities.
RHEB proteins have low intrinsic GTPase activity and exist predominantly in the GTP-bound form. RHEBs are subject to negative regulation, however, by the GAP activity of a TSC1-TSC2 complex. The best-characterized downstream effector of RHEB is the Ser-Thr kinase FRAP1 (also called mTOR, target of rapamycin) (179
), which in turn regulates translation through its substrates RPS6K (ribosomal protein S6 kinase 1) and EIF4EBP1 (eukaryotic initiation factor 4E–binding protein). Loss-of-function mutations in TSC genes are associated with tuberous sclerosis complex, a benign tumor syndrome, suggesting that RHEB may have tumor promoter functions in vivo.