Ras oncogene at 85D

EVOLUTIONARY HOMOLOGS
Table of contents

Ras attachment to membranes

Attachment of Ras protein to the membrane, which requires farnesylation at the Ras C-terminus, is essential for its biological activity. A promising pharmacological approach of antagonizing oncogenic Ras activity is to develop inhibitors of farnesyltransferase. C. elegans vulval differentiation, which is controlled by a Ras-mediated signal transduction pathway, is used as a model system to test previously identified farnesyltransferase inhibitors. Two farnesyltransferase inhibitors, manumycin and gliotoxin, suppress the Multivulva phenotype resulting from an activated let-60 ras mutation, but not the Multivulva phenotype resulting from mutations in the lin-1 gene or the lin-15 gene (respectively), which act downstream (lin-1) and upstream (lin15) of let-60 ras in the signaling pathway. These results are consistent with the idea that the suppression of the Multivulva phenotype of let-60 ras by the two inhibitors is specific for Ras protein and that the mutant Ras protein might be more sensitive than wild-type Ras to the farnesyltransferase inhibitors. This work suggests that C. elegans vulval development could be a simple and effective in vivo system for evaluation of farnesyltransferase inhibitors against Ras-activated tumors (Hara, 1995).

This paper proposes a novel mechanism for the regulation of Ras processing and a new function for Ras is demonstrated, one involved with the regulation of the expression of cardiac autonomic receptors and their associated G proteins. Induction of endogenous cholesterol synthesis in cultured cardiac myocytes results in a coordinated increase in the expression of muscarinic receptors, the G protein alpha-subunit, G-alphai2, and the inward rectifying K+ channel, GIRK1. These changes in gene expression are associated with a marked increase in the response of heart cells to parasympathetic stimulation. The induction of the cholesterol metabolic pathway regulates Ras processing and Ras regulates expression of G-alphai2. In primary cultured myocytes most of the RAS is localized to the cytoplasm in an unfarnesylated form. Induction of the cholesterol metabolic pathway results in increased farnesylation and membrane association of RAS. Studies of Ras mutants expressed in cultured heart cells demonstrate that activation of Ras by induction of the cholesterol metabolic pathway results in increased expression of G-alphai2 mRNA. Hence, farnesylation of Ras is a regulatable process that plays a novel role in the control of second messenger pathways (Gadbut, 1997).

The Nras oncogene is transiently localized in the Golgi prior being localized to the plasma membrane (PM). Moreover, green fluorescent protein (GFP)-tagged Nras illuminates motile, peri-Golgi vesicles, and prolongs BFA treatment blocks PM expression. GFP-Hras colocalizes with GFP-Nras, but GFP-Kras4B reveals less Golgi and no vesicular fluorescence. Whereas a secondary membrane targeting signal is required for PM expression, the CAAX motif alone is necessary and sufficient to target proteins to the endomembrane where they are methylated, a modification required for efficient membrane association. Thus, prenylated CAAX proteins do not associate directly with the PM but instead associate with the endomembrane and are subsequently transported to the PM, a process that requires a secondary targeting motif (Choy, 1999).

Yeast Ras regulates the complex that catalyzes the first step in GPI-anchor biosynthesis at the ER

The yeast ERI1 gene encodes a small ER-localized protein that associates in vivo with GTP bound Ras2 in an effector loop-dependent manner. Loss of Eri1 function results in hyperactive Ras phenotypes. This study demonstrates that Eri1 is a component of the GPI-GlcNAc transferase (GPI-GnT) complex in the ER, which catalyzes transfer of GlcNAc from UDP-GlcNAc to an acceptor phosphatidylinositol, the first step in the production of GPI-anchors for cell surface proteins. GTP bound Ras2 associates with the GPI-GnT complex in vivo and inhibits its activity, indicating that yeast Ras uses the ER as a signaling platform from which to negatively regulate the GPI-GnT. It is proposed that diminished GPI-anchor protein production contributes to hyperactive Ras phenotypes (Sobering, 2004).

The results suggest that inhibition of GPI-GnT activity is a pivotal function of Ras signaling that acts in parallel with stimulation of adenylyl cyclase, the established effector of Ras in yeast. The hyperactive Ras phenotypes of mutants in the GPI-GnT are suppressed by loss of Ras2 function. This suppression may result either from a compensatory increase in GPI-GnT activity, or from diminished adenylyl cyclase activity. The latter explanation is favored, because the failure of ras2Δ to suppress the high-temperature growth defects of GPI-GnT mutants, which result from a deficiency in GPI-protein production, argues against a compensatory increase in GPI-GnT activity (Sobering, 2004).

The significance of GPI-GnT regulation by Ras is not yet clear. However, the dimorphic shift from the yeast form to the filamentous form undoubtedly requires alterations in the composition of the cell wall. Little is known about the ways in which the Saccharomyces cell wall is modified during filamentous/invasive growth aside from an increase in chitin content. It is proposed that Ras regulates changes in cell wall architecture through inhibition of the GPI-GnT, which would have the dual effect of decreasing GPI-anchor proteins at the cell surface and increasing chitin by diverting the pool of UDP-GlcNAc from anchor production to chitin biosynthesis (Sobering, 2004).

Ras and KSR

Genetic screens in Drosophila melanogaster and Caenorhabditis elegans have identified the kinase suppressor of Ras, Ksr, as a new component in the Ras intracellular signaling pathway. In these organisms, mutations in Ksr results in attenuation of Ras-mediated signaling. Homologs of Ksr have also been isolated from mice and humans; their precise role in Ras signaling is not yet well defined. Interactions between the murine form of Ksr (mKsr-1) and other components of the Ras pathway have now been studied. To gain insight into the biological function of Ksr, a yeast two-hybrid screen was used and an interaction between the carboxy-terminal region of mKsr-1 and mitogen-activated protein (MAP) kinase kinase 1 (MAPKK-1 or MEK-1) was found. An interaction is also detected between MAP kinase (also called extracellular signal-regulated kinase, or ERK), and the amino-terminal region of mKsr-1. These interactions are recapitulated in COS-7 cells. When COS-7 cells are transfected with either full-length mKsr-1 or just its carboxy-terminal region, an inhibition of serum-stimulated MAP kinase activation is observed. Microinjection of full-length mKsr-1 or its carboxy-terminal, but not its amino-terminal region, blocks serum-induced DNA synthesis in rat embryo fibroblasts. Co-injection of mKsr-1 with MEK-1 reverses the blockage. Together with the data from genetic analyses, these findings lead to the proposal that mKsr-1 may control MAP kinase signaling by serving as a scaffold protein that links MEK and its substrate ERK (Yu, 1998).

By screening for mutations that suppress the vulval defects caused by a constitutively active let-60 ras gene, six loss-of-function alleles of ksr-1, a novel C. elegans gene, were identified. ksr-1 positively mediates Ras signaling and functions downstream of or in parallel to let-60. In the absence of ksr-1 function, normal Ras signaling is impaired only slightly, suggesting ksr-1 may act to modulate the main signaling pathway, or in a branch that diverges from it. The predicted KSR-1 protein has a protein kinase domain and is most similar to a recently identified Drosophila protein involved in Ras signaling. It is proposed that the function of ksr-1 is evolutionarily conserved (Kornfeld, 1995).

Vulval induction in C. elegans is controlled by a highly conserved signaling pathway similar to the RTK-Ras-MAPK cascade in mammals. By screening for suppressors of the Multivulva phenotype caused by an activated let-60 ras allele, mutations in the ksr-1 gene were isolated that act as positive modifiers of vulval induction and are required for at least two other let-60 ras-mediated processes. Although ksr-1 mutations do not perturb vulval induction in an otherwise wild-type background, they have very strong effects on vulval induction in genetic backgrounds where Ras pathway activity is constitutively activated or compromised, suggesting that ksr-1 activity is required for maximal stimulation of vulval fates by the Ras pathway. Genetic epistasis analysis suggests that ksr-1 acts downstream of or in parallel to let-60 ras. ksr-1 encodes a novel putative protein kinase related to the Raf family of Ser/Thr kinases (Sundaramm, 1995).

A regulatory B subunit of protein phosphatase 2A (PP2A) positively regulates an RTK-Ras-MAP kinase signaling cascade during Caenorhabditis elegans vulval induction. Although reduction of sur-6 PP2A-B function causes few vulval induction defects in an otherwise wild-type background, sur-6 PP2A-B mutations suppress the Multivulva phenotype of an activated ras mutation and enhance the Vulvaless phenotype of mutations in lin-45 raf, sur-8, or mpk-1. Double mutant analysis suggests that sur-6 PP2A-B acts downstream or in parallel to ras, but likely upstream of raf, and functions with ksr-1 in a common pathway to positively regulate Ras signaling (Sieburth, 1999).

KSR proteins positively regulate Ras signaling in C. elegans and Drosophila, as well as in Xenopus oocytes and certain mammalian cells. Murine KSR associates with several proteins in vivo, including Raf, MEK, and MAP kinase, and has been proposed to function as a scaffold protein involved in signal propagation through the Raf/MEK/MAP kinase cascade. Murine KSR is a phosphoprotein; although the role of phosphorylation in KSR regulation is unclear. Thus, KSR-1 is a potential target for regulation by PP2A during vulval induction. Alternatively, KSR-1 may act to regulate PP2A-B function. Another potential sur-6 PP2A-B-dependent PP2A target is LIN-45 Raf. The mechanism of Raf activation is still poorly understood, but there is evidence for both inhibitory and activating phosphates on Raf. Whereas in vitro studies suggest that PP2A can dephosphorylate Raf, it is probably not the major phosphatase to remove activating phosphates. However, a role for PP2A in removing inhibitory phosphates has not been ruled out. The placement of a B regulatory subunit of PP2A as a positive regulator of the Ras pathway, and the unexpected finding that it acts together with KSR-1, should lead to a better understanding of PP2A regulation and its physiological substrates (Sieburth, 1999).

A proline-directed serine/threonine ceramide-activated protein (CAP) kinase mediates transmembrane signaling through the sphingomyelin pathway. CAP kinase reportedly initiates proinflammatory TNF alpha action by phosphorylating and activating Raf-1. The present studies delineate kinase suppressor of Ras (KSR), identified genetically in C. elegans and Drosophila, as CAP kinase. Mouse KSR, like CAP kinase, renatures and autophosphorylates as a 100-kDa membrane-bound polypeptide. KSR overexpression constitutively activates Raf-1. TNF alpha or ceramide analogs markedly enhance KSR autophosphorylation and its ability to complex with, phosphorylate, and activate Raf-1. In vitro, low nanomolar concentrations of natural ceramide stimulate KSR to autophosphorylate, and transactivate Raf-1. Other lipid second messengers are ineffective. Thr269, the Raf-1 site phosphorylated by CAP kinase, is also recognized by KSR. Thus, by previously established criteria, KSR appears to be CAP kinase (Zhang, 1997).

Kinase suppressor of Ras (KSR) is an evolutionarily conserved component associated with Ras-dependent signaling pathways. Murine KSR (mKSR1) translocates from the cytoplasm to the plasma membrane in the presence of activated Ras. At the membrane, mKSR1 modulates Ras signaling by enhancing Raf-1 activity in a kinase-independent manner. The activation of Raf-1 is mediated by the mKSR1 cysteine-rich CA3 domain and involves a detergent labile cofactor that is not ceramide. These findings reveal another point of regulation for Ras-mediated signal transduction and further define a noncatalytic role for mKSR1 in the multistep process of Raf-1 activation (Michaud, 1997).

Kinase suppressor of Ras (KSR) is a loss-of-function allele that suppresses the rough eye phenotype of activated Ras in Drosophila and the multivulval phenotype of activated Ras in Caenorhabditis elegans. Genetic and biochemical studies suggest that KSR is a positive regulator of Ras signaling that functions between Ras and Raf or in a pathway parallel to Raf. The effect of mammalian KSR expression was examined on the activation of extracellular ligand-regulated (ERK) mitogen-activated protein (MAP) kinase in fibroblasts. Ectopic expression of KSR inhibits the activation of ERK MAP kinase by insulin, phorbol ester, or activated alleles of Ras, Raf, and mitogen and extracellular-regulated kinase. Expression of deletion mutants of KSR demonstrates that the KSR kinase domain is necessary and sufficient for the inhibitory effect of KSR on ERK MAP kinase activity. KSR inhibits cell transformation by activated RasVal-12 but has no effect on the ability of RasVal-12 to induce membrane ruffling. These data indicate that KSR is a potent modulator of a signaling pathway essential to normal and oncogenic cell growth and development (Joneson, 1998).

Genetic and biochemical studies have identified kinase suppressor of Ras (KSR) to be a conserved component of Ras-dependent signaling pathways. To better understand the role of KSR in signal transduction, studies investigating the effect of phosphorylation and protein interactions on KSR function have been initiated. Five in vivo phosphorylation sites of KSR have been identified. In serum-starved cells, KSR contains two constitutive sites of phosphorylation (Ser297 and Ser392), which mediate the binding of KSR to the 14-3-3 family of proteins. In the presence of activated Ras, KSR contains three additional sites of phosphorylation (Thr260, Thr274, and Ser443), all of which match the consensus motif (Px[S/T]P) for phosphorylation by mitogen-activated protein kinase (MAPK). Treatment of cells with the MEK inhibitor PD98059 blocks phosphorylation of the Ras-inducible sites and activated MAPK associates with KSR in a Ras-dependent manner. Together, these findings indicate that KSR is an in vivo substrate of MAPK. Mutation of the identified phosphorylation sites does not alter the ability of KSR to facilitate Ras signaling in Xenopus oocytes, suggesting that phosphorylation at these sites may serve other functional roles, such as regulating catalytic activity. Interestingly, during the course of this study, it was found that the biological effect of KSR varies dramatically with the level of KSR protein expressed. In Xenopus oocytes, KSR functions as a positive regulator of Ras signaling when expressed at low levels, whereas at high levels of expression, KSR blocks Ras-dependent signal transduction. Likewise, overexpression of Drosophila KSR blocks R7 photoreceptor formation in the Drosophila eye. Therefore, the biological function of KSR as a positive effector of Ras-dependent signaling appears to be dependent on maintaining KSR protein expression at low or near-physiological levels (Cacace, 1999).

Kinase suppressor of Ras (KSR) is an evolutionarily conserved component of Ras-dependent signaling pathways. The identification of B-KSR1, a novel splice variant of murine KSR1 that is highly expressed in brain-derived tissues, is reported. B-KSR1 protein is detectable in mouse brain throughout embryogenesis, is most abundant in adult forebrain neurons, and is complexed with activated mitogen-activated protein kinase (MAPK) and MEK in brain tissues. Expression of B-KSR1 in PC12 cells results in accelerated nerve growth factor (NGF)-induced neuronal differentiation and detectable epidermal growth factor (EGF)-induced neurite outgrowth. Sustained MAPK activity is observed in cells stimulated with either NGF or EGF, and all effects on neurite outgrowth can be blocked by the MEK inhibitor PD98059. In B-KSR1-expressing cells, the MAPK-B-KSR1 interaction is inducible and correlates with MAPK activation, while the MEK-B-KSR1 interaction is constitutive. Further examination of the MEK-B-KSR1 interaction reveals that all genetically identified loss-of-function mutations in the catalytic domain severely diminish MEK binding. Moreover, B-KSR1 mutants defective in MEK binding are unable to augment neurite outgrowth. Together, these findings demonstrate the functional importance of MEK binding and indicate that B-KSR1 may function to transduce Ras-dependent signals that are required for neuronal differentiation or that are involved in the normal functioning of the mature central nervous system (Muller, 2000).

In Drosophila and C. elegans, kinase suppressor of Ras (KSR) functions as a positive modulator of Ras-dependent signaling either upstream of or parallel to Raf. Attempts to characterize the biochemical and biological properties of mammalian KSR, however, have had limited success. Although some studies have demonstrated a requirement of KSR kinase activity for its action, others indicate the kinase function of KSR is dispensable and suggest that KSR acts primarily as a scaffold protein. Interpretations of KSR function are further hampered by the lack of a standardized assay for its kinase activity in vitro. To address this issue, a two-stage in vitro kinase assay was established in which KSR never comes in contact with any recombinant kinases other than c-Raf-1. Using this assay, KSR has been shown to be inactive when immunoprecipitated from quiescent COS-7 cells that overexpress Flag-tagged KSR, but KSR activity is rapidly and markedly induced upon epidermal growth factor treatment. Moreover, KSR-reconstituted mitogen-activated protein kinase activation is detected in KSR immunoprecipitates depleted of all contaminating kinases (c-Raf-1, MEK1, ERK2) by multiple high salt washes. Only full-length kinase-active KSR is capable of signaling c-Raf-1-dependent activity, since kinase inactive and C- and N-terminal deletion mutants are without effect. Furthermore, endogenous KSR isolated from A431 cells, which contain high levels of activated EGF receptor, displays constitutively enhanced kinase activity. Hence, KSR kinase activity is not an artifact of overexpression but a property intrinsic to this protein. The recognition of EGF as a potent activator of KSR kinase activity and the availability of a well defined in vitro kinase assay should facilitate the definition of the function of KSR as a Ras-effector molecule (Xing, 2000).

A two-stage in vitro assay for KSR kinase activity has been established in which KSR never comes in contact with any recombinant kinase other than c-Raf-1, and EGF has been defined as a potent activator of KSR kinase activity. However, these studies do not address the mechanism of c-Raf-1 stimulation by activated KSR. Here it is shown that phosphorylation of c-Raf-1 on Thr269 by KSR is necessary for optimal activation in response to EGF stimulation. In vitro, KSR specifically phosphorylates c-Raf-1 on threonine residues during the first stage of the two-stage kinase assay. Using purified wild type and mutant c-Raf-1 proteins, it has been demonstrated that Thr269 is the major c-Raf-1 site phosphorylated by KSR in vitro, and that phosphorylation of this site is essential for c-Raf-1 activation by KSR. KSR acts via transphosphorylation, not by increasing c-Raf-1 autophosphorylation, because kinase inactive c-Raf-1^K375M serves as an equally effective KSR substrate. In vivo, low physiologic doses of EGF (0.001-0.1 ng/ml) stimulate KSR activation, and induce Thr269 phosphorylation and activation of c-Raf-1. Low dose EGF does not induce serine or tyrosine phosphorylation of c-Raf-1. High dose EGF (10-100 ng/ml) induces no additional Thr269 phosphorylation but rather increases c-Raf-1 phosphorylation on serine residues and tyrosines340/341. A Raf-1 mutant with valine substituted for Thr269 is unresponsive to low dose EGF, but is serine and Tyr340/341 phosphorylated and partially activated at high EGF doses. These studies show that Thr269 is the major c-Raf-1 site phosphorylated by KSR. Further, phosphorylation of this site is essential for c-Raf-1 activation by KSR in vitro and for optimal c-Raf-1 activation in response to physiologic EGF stimulation in vivo (Xing, 20001).

Kinase suppressor of Ras (KSR) is a conserved component of the Ras pathway that interacts directly with MEK and MAPK. KSR1 translocates from the cytoplasm to the cell surface in response to growth factor treatment and this process is regulated by Cdc25C-associated kinase 1 (C-TAK1). C-TAK1 constitutively associates with mammalian KSR1 and phosphorylates serine 392 to confer 14-3-3 binding and cytoplasmic sequestration of KSR1 in unstimulated cells. In response to signal activation, the phosphorylation state of S392 is reduced, allowing the KSR1 complex to colocalize with activated Ras and Raf-1 at the plasma membrane, thereby facilitating the phosphorylation reactions required for the activation of MEK and MAPK (Muller, 2001).

Kinase Suppressor of Ras (KSR) is a conserved component of the Ras pathway that acts as a molecular scaffold to facilitate signal transmission through the MAPK cascade. Although recruitment of KSR1 from the cytosol to the plasma membrane is required for its scaffolding function, the precise mechanism(s) regulating the translocation of KSR1 have not been fully elucidated. Using mass spectrometry to analyze the KSR1-scaffolding complex, the serine/threonine protein phosphatase PP2A has been identified as a KSR1-associated protein; PP2A is a critical regulator of KSR1 activity. The enzymatic core subunits of PP2A (PR65A and catalytic C) constitutively associate with the N-terminal domain of KSR1, whereas binding of the regulatory PR55B subunit is induced by growth factor treatment. Specific inhibition of PP2A activity prevents the growth factor-induced dephosphorylation event involved in the membrane recruitment of KSR1 and blocks the activation of KSR1-associated MEK and ERK. Moreover, PP2A activity is required for activation of the Raf-1 kinase and that both Raf and KSR1 must be dephosphorylated by PP2A on critical regulatory 14-3-3 binding sites for KSR1 to promote MAPK pathway activation. These findings identify KSR1 as novel substrate of PP2A and demonstrate the inducible dephosphorylation of KSR1 in response to Ras pathway activation. Further, these results elucidate a common regulatory mechanism for KSR1 and Raf-1 whereby their localization and activity are modulated by the PP2A-mediated dephosphorylation of critical 14-3-3 binding sites (Ory, 2003).

Kinase Suppressor of Ras (KSR) is a molecular scaffold that interacts with the core kinase components of the ERK cascade, Raf, MEK, and ERK and provides spatial and temporal regulation of Ras-dependent ERK cascade signaling. In this report, the heterotetrameric protein kinase, casein kinase 2 (CK2), has been identified as a new KSR1-binding partner. Moreover, the KSR1/CK2 interaction is required for KSR1 to maximally facilitate ERK cascade signaling and contributes to the regulation of Raf kinase activity. Binding of the CK2 holoenzyme is constitutive and requires the basic surface region of the KSR1 atypical C1 domain. Loss of CK2 binding does not alter the membrane translocation of KSR1 or its interaction with ERK cascade components; however, disruption of the KSR1/CK2 interaction or inhibition of CK2 activity significantly reduces the growth-factor-induced phosphorylation of C-Raf and B-Raf on the activating serine site in the negative-charge regulatory region (N-region). This decrease in Raf N-region phosphorylation further correlates with impaired Raf, MEK, and ERK activation. These findings identify CK2 as a novel component of the KSR1 scaffolding complex that facilitates ERK cascade signaling by functioning as a Raf family N-Region kinase (Ritt, 2006).

Ions regulation of Ras-mediated signaling

C. elegans cdf-1 was identified in a genetic screen for regulators of Ras-mediated signaling. CDF-1 is a cation diffusion facilitator protein that is structurally and functionally similar to vertebrate ZnT-1. These proteins have an evolutionarily conserved function as positive regulators of the Ras pathway, and the Ras pathway has an evolutionarily conserved ability to respond to CDF proteins. CDF proteins regulate Ras-mediated signaling by promoting Zn²⁺ efflux and reducing the concentration of cytosolic Zn²⁺, and cytosolic Zn²⁺ negatively regulates Ras-mediated signaling. Physiological concentrations of Zn²⁺ cause a significant inhibition of Ras-mediated signaling. These findings suggest that Zn²⁺ negatively regulates a conserved element of the signaling pathway and that Zn²⁺ regulation is important for maintaining the inactive state of the Ras pathway (Bruinsma, 2002).

SIRT6 regulates Ras-related protein R-Ras2 by lysine defatty-acylation

The Ras family of GTPases are important in cell signaling and frequently mutated in human tumors. Understanding their regulation is thus important for studying biology and human diseases. This study reports that a novel posttranslational mechanism, reversible lysine fatty acylation, regulates R-Ras2, a member of the Ras family. SIRT6 (see Drosophila Sirt6), a sirtuin with established tumor suppressor function, regulates the lysine fatty acylation of R-Ras2. In mouse embryonic fibroblasts (MEFs), Sirt6 knockout (KO) increases R-Ras2 lysine fatty acylation. Lysine fatty acylation promotes the plasma membrane localization of R-Ras2 and its interaction with phosphatidylinositol 3-kinase PI3K, leading to activated Akt (see Drosophila Akt1) and increased cell proliferation. These data establish lysine fatty acylation as a previously unknown mechanism that regulates the Ras family of GTPases and provides an important mechanism by which SIRT6 functions as a tumor suppressor (Zhang, 2017).

Evolutionary homologs: Table of contents

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