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KRAS , HRAS and NRAS proto-oncogenes belong to a family of 40 highly homologous genes, which in turn are a subset of a superfamily of >160 genes encoding small GTPases. RAS proteins consist of a globular G-domain (aa1-166) and a 22-23 aa unstructured hypervariable region (HVR) that mediates membrane targeting. The evolutionary origins of the RAS isoforms, their HVRs and alternative splicing of the KRAS locus has not been explored. We found that KRAS is basal to the RAS proto-oncogene family and its duplication generated HRAS in the common ancestor of vertebrates. In a second round of duplication HRAS generated NRAS and KRAS generated an additional RAS gene we have designated KRASBL , absent in mammals and birds. KRAS4A arose through a duplication and insertion of the 4 th exon of NRAS into the 3 rd intron of KRAS . We found evolutionary conservation of a short polybasic region (PBR1) in HRAS, NRAS and KRAS4A, a second polybasic region (PBR2) in KRAS4A, two neutralized basic residues (NB) and a serine in KRAS4B and KRASBL, and a modification of the CaaX motif in vertebrates with farnesyl rather than geranylgeranyl polyisoprene lipids, suggesting that a less hydrophobic membrane anchor is critical to RAS protein function. The persistence of four RAS isoforms through >400 million years of evolution argues strongly for differential function.

Key Points RAS proteins are molecular switches that regulate a wide range of signalling pathways by engaging effectors on cellular membranes. They are themselves regulated by various post-translational modifications (PTMs). RAS proteins associate with membranes by virtue of a series of constitutive PTMs of their carboxy-terminal CAAX sequence. These PTMs include prenylation, proteolysis and carboxyl methylation. Membrane association and trafficking of all RAS isoforms other than KRAS4B are also regulated by the reversible palmitoylation of Cys residues in the C-terminal hypervariable regions of the proteins. Cis – trans isomerization of a peptidyl-prolyl bond adjacent to a palmitate in HRAS acts as a molecular timer that regulates depalmitoylation and retrograde trafficking. Phosphorylation of KRAS4B in its polybasic region allows this protein to dissociate from the plasma membrane through a mechanism known as the farnesyl–electrostatic switch. Monoubiquitylation and diubiquitylation of HRAS regulate its association with endosomes, and monoubiquitylation of KRAS4B enhances its activation. S -nitrosylation of Cys118 of RAS promotes guanine nucleotide exchange. Toxins produced by Pseudomonas aeruginosa and Clostridium sordelli ADP-ribosylate and monglucosylate RAS, respectively, leading to diminished signalling. RAS proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. Their trafficking and activity are regulated by constitutive post-translational modifications (PTMs), including farnesylation, methylation and palmitoylation, as well as conditional PTMs, such as phosphorylation, peptidyl-proly isomerization, ubiquitylation, nitrosylation, ADP ribosylation and glucosylation. RAS proteins are monomeric GTPases that act as binary molecular switches to regulate a wide range of cellular processes. The exchange of GTP for GDP on RAS is regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs), which regulate the activation state of RAS without covalently modifying it. By contrast, post-translational modifications (PTMs) of RAS proteins direct them to various cellular membranes and, in some cases, modulate GTP–GDP exchange. Important RAS PTMs include the constitutive and irreversible remodelling of its carboxy-terminal CAAX motif by farnesylation, proteolysis and methylation, reversible palmitoylation, and conditional modifications, including phosphorylation, peptidyl-prolyl isomerisation, monoubiquitylation, diubiquitylation, nitrosylation, ADP ribosylation and glucosylation.

The three mammalian RAS genes ( HRAS , NRAS and KRAS ) encode four proteins that play central roles in cancer biology. Among them, KRAS is mutated more frequently in human cancer than any other oncogene. The pre-mRNA of KRAS is alternatively spliced to give rise to two products, KRAS4A and KRAS4B, which differ in the membrane targeting sequences at their respective C-termini. Notably, both KRAS4A and KRAS4B are oncogenic when KRAS is constitutively activated by mutation in exon 2 or 3. Whereas KRAS4B is the most studied oncoprotein, KRAS4A is understudied and until recently considered relatively unimportant. Emerging work has confirmed expression of KRAS4A in cancer and found non-overlapping functions of the splice variants. The most clearly demonstrated of these is direct regulation of hexokinase 1 by KRAS4A, suggesting that the metabolic vulnerabilities of KRAS -mutant tumors may be determined in part by the relative expression of the splice variants. The aim of this review is to address the most relevant characteristics and differential functions of the KRAS splice variants as they relate to cancer onset and progression.

The two products of the KRAS locus, K-Ras4A and K-Ras4B, are encoded by alternative fourth exons and therefore, possess distinct membrane-targeting sequences. The common activating mutations occur in exons 1 or 2 and therefore, render both splice variants oncogenic. K-Ras4A has been understudied, because it has been considered a minor splice variant. By priming off of the splice junction, we developed a quantitative RT-PCR assay for K-Ras4A and K-Ras4B message capable of measuring absolute amounts of the two transcripts. We found that K-Ras4A was widely expressed in 30 of 30 human cancer cell lines and amounts equal to K-Ras4B in 17 human colorectal tumors. Using splice variant-specific antibodies, we detected K-Ras4A protein in several tumor cell lines at a level equal to or greater than that of K-Ras4B. In addition to the CAAX motif, the C terminus of K-Ras4A contains a site of palmitoylation as well as a bipartite polybasic region. Although both were required for maximal efficiency, each of these could independently deliver K-Ras4A to the plasma membrane. Thus, among four Ras proteins, K-Ras4A is unique in possessing a dual membrane-targeting motif. We also found that, unlike K-Ras4B, K-Ras4A does not bind to the cytosolic chaperone δ-subunit of cGMP phosphodiesterase type 6 (PDE6δ). We conclude that efforts to develop anti–K-Ras drugs that interfere with membrane trafficking will have to take into account the distinct modes of targeting of the two K-Ras splice variants. Significance The KRAS oncogene is mutated more frequently in human cancer than any other. The KRAS transcript is alternatively spliced to give rise to two products, K-Ras4A and K-Ras4B, both of which are oncogenic when KRAS is mutated. We detected significant amounts of each transcript in human tumor cells and colorectal carcinomas. We found that K-Ras4A is targeted to the plasma membrane by dual targeting motifs distinct from those of K-Ras4B. Because interfering with membrane association of Ras proteins remains one of the most attractive approaches to anti-Ras therapy, efforts in this direction will have to disrupt both the K-Ras4A and the K-Ras4B membrane-targeting pathways.

RAS mutations are among the most prevalent oncogenic drivers in cancers. RAS proteins propagate signals only when associated with cellular membranes as a consequence of lipid modifications that impact their trafficking. Here, we discovered that RAB27B, a RAB family small GTPase, controlled NRAS palmitoylation and trafficking to the plasma membrane, a localization required for activation. Our proteomic studies revealed RAB27B upregulation in CBL- or JAK2-mutated myeloid malignancies, and its expression correlated with poor prognosis in acute myeloid leukemias (AMLs). RAB27B depletion inhibited the growth of CBL-deficient or NRAS-mutant cell lines. Strikingly, Rab27b deficiency in mice abrogated mutant but not WT NRAS-mediated progenitor cell growth, ERK signaling, and NRAS palmitoylation. Further, Rab27b deficiency significantly reduced myelomonocytic leukemia development in vivo. Mechanistically, RAB27B interacted with ZDHHC9, a palmitoyl acyltransferase that modifies NRAS. By regulating palmitoylation, RAB27B controlled c-RAF/MEK/ERK signaling and affected leukemia development. Importantly, RAB27B depletion in primary human AMLs inhibited oncogenic NRAS signaling and leukemic growth. We further revealed a significant correlation between RAB27B expression and sensitivity to MEK inhibitors in AMLs. Thus, our studies presented a link between RAB proteins and fundamental aspects of RAS posttranslational modification and trafficking, highlighting future therapeutic strategies for RAS-driven cancers.

α-ketoglutarate (KG), also referred to as 2-oxoglutarate, is a key intermediate of cellular metabolism with pleiotropic functions. Cell-permeable esterified analogs are widely used to study how KG fuels bioenergetic and amino acid metabolism and DNA, RNA, and protein hydroxylation reactions, as cellular membranes are thought to be impermeable to KG. Here we show that esterified KG analogs rapidly hydrolyze in aqueous media, yielding KG that, in contrast to prevailing assumptions, imports into many cell lines. Esterified KG analogs exhibit spurious KG-independent effects on cellular metabolism, including extracellular acidification, arising from rapid hydrolysis and de-protonation of α-ketoesters, and significant analog-specific inhibitory effects on glycolysis or mitochondrial respiration. We observe that imported KG decarboxylates to succinate in the cytosol and contributes minimally to mitochondrial metabolism in many cell lines cultured in normal conditions. These findings demonstrate that nuclear and cytosolic KG-dependent reactions may derive KG from functionally distinct subcellular pools and sources. Analogues of α-ketoglutarate are used in many cellular studies but assumptions are made about cellular uptake. Here, the authors show that esterified analogues rapidly hydrolyse in aqueous medium resulting in an analogue which can be quickly taken up by many cell lines, contrary to prevailing assumptions.

K-Ras4B is targeted to the plasma membrane by a farnesyl modification that operates in conjunction with a polybasic domain. We characterized a farnesyl-electrostatic switch whereby protein kinase C phosphorylates K-Ras4B on serine 181 in the polybasic region and thereby induces translocation from the plasma membrane to internal membranes that include the endoplasmic reticulum (ER) and outer mitochondrial membrane. This translocation is associated with cell death. Here we have explored the mechanism of phospho–K-Ras4B toxicity and found that GTP-bound, phosphorylated K-Ras4B associates with inositol trisphosphate receptors on the ER in a Bcl-xL–dependent fashion and, in so doing, blocks the ability of Bcl-xL to potentiate the InsP ₃ regulated flux of calcium from ER to mitochondria that is required for efficient respiration, inhibition of autophagy, and cell survival. Thus, we have identified inositol trisphosphate receptors as unique effectors of K-Ras4B that antagonize the prosurvival signals of other K-Ras effectors.

Members of the RAS small GTPase family regulate cellular responses to extracellular stimuli by mediating the flux through downstream signal transduction cascades. RAS activity is strongly dependent on its subcellular localization and its nucleotide-binding status, both of which are modulated by posttranslational modification. We have determined that RAS is posttranslationally acetylated on lysine 104. Molecular dynamics simulations suggested that this modification affects the conformational stability of the Switch II domain, which is critical for the ability of RAS to interact with guanine nucleotide exchange factors. Consistent with this model, an acetylation-mimetic mutation in K-RAS4B suppressed guanine nucleotide exchange factor-induced nucleotide exchange and inhibited in vitro transforming activity. These data suggest that lysine acetylation is a negative regulatory modification on RAS. Because mutations in RAS family members are extremely common in cancer, modulation of RAS acetylation may constitute a therapeutic approach.

A farnesylated and methylated form of prelamin A called progerin causes Hutchinson-Gilford progeria syndrome (HGPS). Inhibiting progerin methylation by inactivating the isoprenylcysteine carboxylmethyltransferase (ICMT) gene stimulates proliferation of HGPS cells and improves survival of Zmpste24 -deficient mice. However, we don't know whether Icmt inactivation improves phenotypes in an authentic HGPS mouse model. Moreover, it is unknown whether pharmacologic targeting of ICMT would be tolerated by cells and produce similar cellular effects as genetic inactivation. Here, we show that knockout of Icmt improves survival of HGPS mice and restores vascular smooth muscle cell numbers in the aorta. We also synthesized a potent ICMT inhibitor called C75 and found that it delays senescence and stimulates proliferation of late-passage HGPS cells and Zmpste24 -deficient mouse fibroblasts. Importantly, C75 did not influence proliferation of wild-type human cells or Zmpste24 -deficient mouse cells lacking Icmt , indicating drug specificity. These results raise hopes that ICMT inhibitors could be useful for treating children with HGPS.

Members of the Rho family of small GTPases have been shown to be involved in tumorigenesis and metastasis. Currently, most of the available information on the function of Rho proteins in malignant transformation is based on the use of dominant-negative mutants of these GTPases. The specificity of these dominant-negative mutants is limited however. In this study, we used small interfering RNA directed against either Rac1 or Rac3 to reduce their expression specifically. In line with observations using dominant-negative Rac1 in other cell types, we show that RNA interference-mediated depletion of Rac1 strongly inhibits lamellipodia formation, cell migration and invasion in SNB19 glioblastoma cells. Surprisingly however, Rac1 depletion has a much smaller inhibitory effect on SNB19 cell proliferation and survival. Interestingly, whereas depletion of Rac3 strongly inhibits SNB19 cell invasion, it does not affect lamellipodia formation and has only minor effects on cell migration and proliferation. Similar results were obtained in BT549 breast carcinoma cells. Thus, functional analysis of Rac1 and Rac3 using RNA interference reveals a critical role for these GTPases in the invasive behavior of glioma and breast carcinoma cells.