Ras oncogene at 85D


Table of contents

Ras and cell cycle

The retinoblastoma tumour-suppressor protein, a regulator of G1 exit, functionally links Ras to passage through the G1 phase (See Drosophila Retinoblastoma-family protein). Inactivation of Ras in cycling cells causes a decline in cyclin D1 protein levels, accumulation of the hypophosphorylated, growth-suppressive form of Rb and G1 arrest. When Rb is disrupted either genetically or biochemically, cells fail to arrest in G1 following Ras inactivation. In contrast, inactivation of Ras in quiescent cells prevents growth-factor induction of both immediate-early gene transcription and exit from G0 in an Rb-independent manner. It is suggested that the Ras pathway regulates the expression of cyclin D1 protein, which in turn targets Rb, resulting in Rb phosphorylation and consequently in Rb inactivation, thereby provoking exit from G1 (Peeper, 1997).

Stable IIC9 cell lines, Goa1 and Goa2, were generated that overexpress full-length antisense Goalpha RNA. Expression of antisense Goalpha RNA ablates the alpha subunit of the heterotrimeric G protein, Go, resulting in growth in the absence of mitogen. To better understand this change in IIC9 phenotype, the signaling pathway and cell cycle events previously shown to be important in control of IIC9 G1/S phase progression were characterized. Ablation of Goalpha results in growth, constitutively active Ras/ERK, elevated expression of cyclin D1, and constitutively active cyclin D1-CDK complexes, all in the absence of mitogen. These characteristics are abolished by the transient overexpression of the transducin heterotrimeric G protein alpha subunit, strongly suggesting the transformation of Goalpha-ablated cells involves Gobetagamma subunits. This is the first study to implicate a heterotrimeric G protein in tumor suppression (Weber, 1997a).

In Chinese hamster embryo fibroblasts (IIC9 cells), platelet-derived growth factor (PDGF) stimulates mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAP kinase/ERK) activity, but not that of c-jun N-terminal kinase (JNK), and induces G1 phase progression. ERK1 activation is biphasic and is sustained throughout the G1 phase of the cell cycle. PDGF induces cyclin D1 protein and mRNA levels in a time-dependent manner. Inhibition of PDGF-induced ERK1 activity by the addition of a selective inhibitor of MEK1 (MAP kinase kinase/ERK kinase 1) activation, PD98059, or transfection with a dominant-negative ERK1 (dnERK-) is correlated with growth arrest. In contrast, growth is unaffected by expression of dominant-negative JNK (dnJNK-). Interestingly, addition of PD98059 or dnERK-, but not dnJNK-, results in a dramatic decrease in cyclin D1 protein and mRNA levels, concomitant with a decrease in cyclin D1-cyclin-dependent kinase activity. To investigate the importance of sustained ERK1 activation, ERK1 activity was blocked by the addition of PD98059 throughout G1. Addition of PD98059 up to 4 h after PDGF treatment decreases ERK1 activity to the levels found in growth-arrested IIC9 cells. Loss of cyclin D1 mRNA and protein expression is observed within 1 h after inhibition of the second sustained phase of ERK1 activity. Disruption of sustained ERK1 activity also results in G1 growth arrest. These data provide evidence of a role for sustained ERK activity in controlling G1 progression through positive regulation of the continued expression of cyclin D1, a protein known to positively regulate G1 progression (Weber, 1997b).

The persistent activation of p42/p44(MAPK) is required to pass the G1 restriction point in fibroblasts and it has been postulated that MAPKs control the activation of G1 cyclin-dependent complexes. This study examined the mitogen-dependent induction of cyclin D1 expression, one of the earliest cell cycle-related events to occur during the G0/G1 to S-phase transition, as a potential target of MAPK regulation. Effects exerted either by the p42/p44(MAPK) or the p38/HOGMAPK cascade on the regulation of cyclin D1 promoter activity or cyclin D1 expression have been compared in CCL39 cells, using a co-transfection procedure. Inhibition of the p42/p44(MAPK) signaling by expression of dominant-negative forms of either mitogen-activated protein kinase kinase 1 (MKK1) or p44(MAPK), or by expression of the MAP kinase phosphatase, MKP-1, strongly inhibits expression of a reporter gene driven by the human cyclin D1 promoter as well as the endogenous cyclin D1 protein. Conversely, activation of this signaling pathway by expression of a constitutively active MKK1 mutant dramatically increases cyclin D1 promoter activity and cyclin D1 protein expression, in a growth factor-independent manner. Moreover, the use of a CCL39-derived cell line that stably expresses an inducible chimera of the estrogen receptor fused to a constitutively active Raf-1 mutant (DeltaRaf-1:ER) reveals that in the absence of growth factors, activation of the Raf > MKK1 > p42/p44MAPK cascade is sufficient to fully induce cyclin D1. In marked contrast, the p38(MAPK) cascade shows an opposite effect on the regulation of cyclin D1 expression. In cells co-expressing high levels of the p38(MAPK) kinase (MKK3), together with the p38(MAPK), a significant inhibition of mitogen-induced cyclin D1 expression is observed. Furthermore, inhibition of p38(MAPK) activity with the specific inhibitor, SB203580, enhances cyclin D1 transcription and protein level. Altogether, these results support the notion that MAPK cascades drive specific cell cycle responses to extracellular stimuli, at least in part, through the modulation of cyclin D1 expression and associated cdk activities (Lavoie, 1996).

In the germline of C. elegans hermaphrodites, meiotic cell cycle progression occurs in spatially restricted regions. Immediately after leaving the distal mitotic region, germ cells enter meiosis and thereafter remain in the pachytene stage of first meiotic prophase for an extended period. At the dorsoventral gonadal flexure, germ cells exit pachytene and subsequently become arrested in diakinesis. Exit from pachytene is dependent on the function of three members of the MAP kinase signaling cascade. One of these genes, mek-2, is a newly identified C. elegans MEK (MAP kinase kinase). The other two genes, mpk-1/sur-1 (MAP kinase) and let-60 ras, have been previously identified based on their roles in vulval induction; in this study they act in combination with mek-2 to permit exit from pachytene. The expression of mpk-1/sur-1 is required within the germline to permit exit from pachytene (Church, 1995).

Transformation of primary cells by ras requires either a cooperating oncogene or the inactivation of tumor suppressors, such as p53 or p16. Expression of oncogenic ras in primary human or rodent cells results in a permanent G1 arrest. The arrest induced by ras is accompanied by accumulation of p53 and p16, and is phenotypically indistinguishable from cellular senescence. The features of ras arrested cells are remarkably similar to cells that have surpassed their proliferative capacity and become senescent. For example, senescent cells adopt a flat enlarged morphology and cease proliferation at subconfluent densities despite the presence of serum. Senescent cells arrest with a G1 DNA content, and express increased levels and/or activity of p53, p21 and p16. Inactivation of either p53 or p16 prevents ras-induced arrest in rodent cells. These observations suggest that the onset of cellular senescence does not simple reflect the accumulation of cell divisions, but can be prematurely activated in response to an oncogenic stimulus. Negation of ras-induced senescence may be relevant during multistep tumorigenesis (Serrano, 1997).

The Ras and Raf1 proto-oncogenes transduce extracellular signals that promote cell growth. Cdc25 phosphatases activate the cell division cycle by dephosphorylation of critical threonine and tyrosine residues within the cyclin-dependent kinases. Cdc25 phosphatase associates with raf1 in somatic mammalian cells and in meiotic frog oocytes. Furthermore, Cdc25 phosphatase can be activated in vitro in a Raf1-dependent manner. Activation of the cell cycle by the Ras/Raf1 pathways might be mediated in part by Cdc25 (Galaktionov, 1995).

Anchorage-independent growth is a hallmark of transformed cells, but little is known of the molecular mechanisms that underlie this phenomenon. Control, nontransformed cultured cells cannot proliferate in semisolid medium. Three important cell cycle events are dependent on adhesion of these cells to a substratum: (1) phosphorylation of the retinoblastoma protein, pRB; (2) cyclin E-dependent kinase activity, and (3) cyclin A expression. Cells that express ras proliferate in nonadherent cultures, and each of these three events occurs in the absence of adhesion in PKC3-F4/ras cells. Thus, ras can override the adhesion requirement of cellular functions that are necessary for cell cycle progression. Cells that express ras possess hyperphosphorylated forms of pRB and cyclin E-dependent kinase activity (See Drosophila Cyclin E)in the absence of adhesion but remain adhesion dependent for expression of cyclin A (See Drosophila Cyclin A). The adhesion dependence of pRB phosphorylation and cyclin E-dependent kinase activity is therefore dissociable from the adhesion dependence of cyclin A expression. Furthermore, ectopic expression of cyclin A is sufficient to rescue anchorage-independent growth of ras+ cells but does not induce anchorage-independent growth. However, like pRB phosphorylation and cyclin E-dependent kinase activity, the kinase activity associated with ectopically expressed cyclin A is dependent on cell adhesion, and this dependence is overcome by ras. Thus, the induction of anchorage-independent growth by ras may involve multiple signals that lead to both expression of cyclin A and activation of G1 cyclin-dependent kinase activities in the absence of cell adhesion (Kang, 1996).

The Ras-GTPase-activating protein (RasGAP) is an important modulator of p21ras - dependent signal transduction in Xenopus oocytes and in mammalian cells. The role of the RasGAP SH3 domain was investigated in signal transduction with a monoclonal antibody against the SH3 domain of RasGaP. This antibody prevents the activation of the maturation-promoting factor complex (cyclin B-p34cdc2) by oncogenic Ras. The antibody appears to be specific because as little as 5 ng injected per oocyte reduces the level of Cdc2 activation by 50% whereas 100 ng of nonspecific immunoglobulin G does not affect Cdc2 activation. The antibody blocks the Cdc2 activation induced by oncogenic Ras but not that induced by progesterone, which acts independently of Ras. A peptide corresponding to positions 317 to 326 of a sequence in the SH3 domain of human RasGAP blocks Cdc2 activation, whereas a peptide corresponding to positions 273 to 305 of a sequence in the N-terminal moiety of the SH3 domain of RasGAP has no effect. The antibody does not block the mitogen-activated protein (MAP) kinase cascade (activation of MAPK/ERK kinase [MEK], MAP kinase, and S6 kinase p90rsk). Surprisingly, injection of the negative MAP kinase mutant protein ERK2 K52R (containing a K-to-R mutation at position 52) blocks the Cdc2 activation induced by oncogenic Ras as well as blocking the activation of MAP kinase. Thus, MAP kinase is also implicated in the regulation of Cdc2 activity. The regulation of the synthesis of the c-mos oncogene product, which is necessary for the activation of Cdc2 was investigated. These results suggest that oncogenic Ras activates two signaling mechanisms: the MAP kinase cascade and a signaling pathway implicating the SH3 domain of RasGAP. These mechanisms might control Mos protein expression implicated in Cdc2 activation (Pomerance, 1996).

The involvement of Ras in the activation of multiple early signaling pathways is well understood, but it is less clear how the various Ras effectors interact with the cell cycle machinery to cause G(1) progression. Ras-mediated activation of extracellular-regulated kinase/mitogen-activated protein kinase has been implicated in cyclin D(1) up-regulation, but there is little extracellular-regulated kinase activity during the later stages of G(1), when cyclin D(1) expression becomes maximal, implying that other effector pathways may also be important in cyclin D1 induction. The involvement of Ras effectors from the phosphatidylinositol (PI) 3-kinase and Ral-GDS families in G1 progression has been addressed and this involvement is compared to that of the Raf/mitogen-activated protein kinase pathway. PI 3-kinase activity is required for the expression of endogenous cyclin D1 and for S phase entry following serum stimulation of quiescent NIH 3T3 fibroblasts. Activated PI 3-kinase induces cyclin D1 transcription and E2F activity, at least in part mediated by the serine/threonine kinase Akt/PKB, and to a lesser extent the Rho family GTPase Rac. In addition, both activated Ral-GDS-like factor and Raf stimulate cyclin D1 transcription and E2F activity and act in synergy with PI 3-kinase. Therefore, multiple cooperating pathways mediate the effects of Ras on progression through the cell cycle (Gille, 1999).

The lethal toxin (LT) from Clostridium sordellii is a glucosyltransferase that modifies and inhibits small G proteins of the Ras family, Ras and Rap, as well as Rac proteins. LT induces cdc2 kinase activation and germinal vesicle breakdown (GVBD) when microinjected into full-grown Xenopus oocytes. Toxin B from Clostridium difficile, that glucosylates and inactivates Rac proteins, does not induce cdc2 activation, indicating that proteins of the Ras family, Ras and/or Rap, negatively regulate cdc2 kinase activation in Xenopus oocyte. In oocyte extracts, LT catalyzes the incorporation of [14C]glucose into a group of proteins of 23 kDa and into one protein of 27 kDa. The 23-kDa proteins are recognized by anti-Rap1 and anti-Rap2 antibodies, whereas the 27-kDa protein is recognized by several anti-Ras antibodies and probably corresponds to K-Ras. Microinjection of LT into oocytes together with UDP-[14C]glucose results in a glucosylation pattern similar to that found in in vitro glucosylation, indicating that the 23- and 27-kDa proteins are in vivo substrates of LT. In vivo time-course analysis reveals that the 27-kDa protein glucosylation is completed within 2 h, well before cdc2 kinase activation, whereas the 23-kDa proteins are partially glucosylated at GVBD. This observation suggests that the 27-kDa Ras protein could be the in vivo target of LT, allowing cdc2 kinase activation. Interestingly, inactivation of Ras proteins does not prevent the phosphorylation of c-Raf1 and the activation of MAP kinase that occurs normally around GVBD (Rime, 1998).

Conditional expression of an activated ras mutant in Balb/c-3T3 fibroblasts fails to stimulate S phase entry in the absence of plasma-derived progression factors, but does shorten the G1 interval from 12 to 6 h and abrogate the normal proliferative requirement for platelet-derived growth factor. Ras-dependent alteration of the 3T3 cell cycle is accompanied by a dramatic increase in the expression of the G1 regulatory protein, cyclin D1, while expression of cyclin E and cyclin A proteins are only weakly induced. Cyclin/cdk complexes assembled in response to ectopic ras expression in the absence of growth factor stimulation bind the cdk inhibitory factor, Kip1 (See Drosophila Dacapo), and are inactive. However, plasma-stimulated regulatory pathways function co-operatively with the oncogenic ras molecule to decrease Kip1 levels, induce the kinase activities associated with cyclins D, E and A, and trigger the initiation of DNA replication. These results suggest that a ras-activated signal transduction pathway may link environmental mitogenic stimuli to the cell cycle machinery via modulation of G1 cyclin expression (Winston, 1996).

Small GTPases act as molecular switches in intracellular signal-transduction pathways. In the case of the Ras family of GTPases, one of their most important roles is as regulators of cell proliferation: the mitogenic response to a variety of growth factors and oncogenes can be blocked by inhibiting Ras function. But in certain situations, activation of Ras signaling pathways arrests the cell cycle rather than causing cell proliferation. Extracellular signals may trigger different cellular responses by activating Ras-dependent signaling pathways to varying degrees. Other signaling pathways could also influence the consequences of Ras signaling. When signaling through the Ras-related GTPase Rho is inhibited, constitutively active Ras induces the cyclin-dependent-kinase inhibitor p21Waf1/Cip1 and entry into the DNA-synthesis phase of the cell cycle is blocked. When Rho is active, induction of p21Waf1/Cip1 by Ras is suppressed and Ras induces DNA synthesis. Therefore, Rho helps Ras to drive cells into S phase. Cells that lack p21Waf1/Cip1 do not require Rho signaling for the induction of DNA synthesis by activated Ras, indicating that, once Ras has become activated, the primary requirement for Rho signaling is the suppression of p21Waf1/Cip1 induction (Olson, 1998).

In cellular transformation, activated forms of the small GTPases Ras and RhoA can cooperate to drive cells through the G1 phase of the cell cycle. A similar but substrate-regulated mechanism is involved in the anchorage-dependent proliferation of untransformed NIH-3T3 cells. Among several extracellular matrix components tested, only fibronectin supports growth factor-induced, E2F-dependent S phase entry. Although all substrates support the mitogen-activated protein kinase (MAPK) response to growth factors, RhoA activity is specifically enhanced on fibronectin. Moreover, induction of cyclin D1 and suppression of p21(Cip/Waf) occurs specifically, in a Rho-dependent fashion, in cells attached to fibronectin. This ability of fibronectin to stimulate both Ras/MAPK- and RhoA-dependent signaling can explain its potent cooperation with growth factors in the stimulation of cell cycle progression (Danen, 2000).

While most untransformed cells require substrate attachment for growth (anchorage dependence), the oncogenically transformed cells lack this requirement (anchorage independence) and are often tumorigenic. However, the mechanism of loss of anchorage dependence is not fully understood. When normal rat fibroblasts are cultured in suspension without substrate attachment, the cell cycle arrests in G1 phase and the cyclin-dependent kinase inhibitor p27Kip1 protein and its mRNA accumulate. Conditional expression of oncogenic Ras induces the G1-S transition of the cell cycle and significantly shortens the half-life of p27Kip1 protein without altering its mRNA level. Inhibition of the activation of mitogen-activated protein (MAP) kinase by cyclic AMP-elevating agents and a MEK inhibitor prevents the oncogenic Ras-induced degradation of p27Kip1 (See Drosophila Dacapo). These results suggest that the loss of substrate attachment induces cell cycle arrest through the up-regulation of p27Kip1 mRNA, but the oncogenic Ras confers anchorage independence by accelerating p27Kip1 degradation through the activation of the MAP kinase signaling pathway. It appears that p27Kip1 is phosphorylated by MAP kinase in vitro and the phosphorylated p27Kip1 cannot bind to and inhibit cdk2 (Kawada, 1997).

Several different oncogenes and growth factors promote G1 phase progression. Cyclin D1, the regulatory subunit of several cyclin-dependent kinases, is required for, and capable of shortening, the G1 phase of the cell cycle. The present study demonstrates that transforming mutants of p21ras (Ras Val-12, Ras Leu-61) induce the cyclin D1 promoter. Site-directed mutagenesis of AP-1-like sequences at -954 abolish p21ras-dependent activation of cyclin D1 expression. The AP-1-like sequences are also required for activation of the cyclin D1 promoter by c-Jun. Several AP-1 proteins (c-Jun, JunB, JunD, and c-Fos) bind the cyclin D1 -954 region. Cyclin D1 promoter activity is stimulated by overexpression of mitogen-activated protein kinase (p41MAPK) or c-Ets-2 through the proximal 22 base pairs. Expression of plasmids encoding either dominant negative MAPK (p41MAPKi) or dominant negatives of ETS activation (Ets-LacZ), antagonize MAPK-dependent induction of cyclin D1 promoter activity. Epidermal growth factor induction of cyclin D1 transcription, through the proximal promoter region, is antagonized by either p41MAPKi or Ets-LacZ, suggesting that ETS functions downstream of epidermal growth factor and MAPK in the context of the cyclin D1 promoter. The activation of cyclin D1 transcription by p21ras provides evidence for cross-talk between the p21ras and cell cycle regulatory pathways (Albanese, 1995).

Ectopic overexpression of v-H-Ras protein in NIH 3T3 cells results in cellular transformation and an acceleration of G1 progression in these cells. A shortened G1 phase is found to be associated with an increased level of cyclin D1 but not cyclin E protein. Reduced synthesis of cyclin D1 in v-H-Ras transformants results in a slower G1 progression rate in these cells. Although constitutive overexpression of cyclin D1 in NIH 3T3 cells accelerates G1 progression, cells remain untransformed. Inhibition of cyclin D1 synthesis greatly impairs the soft-agar cloning efficiency of v-H-Ras transformants. These results suggest that increased expression of cyclin D1 is necessary but not sufficient for the transforming activity of v-H-Ras. A similar effect on cell cycle progression is also observed in Raf-transformed cells. In addition to cyclin D1, cyclin E protein is found to be elevated in Src transformants. This may account for the further shortening of the G1 phase in these cells. Activation of an additional Ras-independent pathway is thought to be responsible for the further acceleration of the G1 phase in Src transformants (Liu, 1995).

The cyclin-dependent kinase 4 (CDK4) regulates progression through the G1 phase of the cell cycle. The activity of CDK4 is controlled by the opposing effects of the D-type cyclin, an activating subunit, and p16INK4, an inhibitory subunit. Ectopic expression of p16INK4 blocks entry into S phase of the cell cycle induced by oncogenic Ha-Ras, and this block is relieved by coexpression of a catalytically inactive CDK4 mutant. Expression of p16INK4 suppresses cellular transformation of primary rat embryo fibroblasts by oncogenic Ha-Ras and Myc, but not by Ha-Ras and E1a. Together, these observations provide direct evidence that p16INK4 can inhibit cell growth (Serrano, 1995).

Considerable evidence points to a role for G1 cyclin-dependent kinase (CDK) in allowing the accumulation of E2F transcription factor activity and induction of the S phase of the cell cycle. Numerous experiments have also demonstrated a critical role for both Myc and Ras activities in allowing cell-cycle progression. Inhibition of Ras activity blocks the normal growth-dependent activation of G1 CDK, prevents activation of the target genes of E2F, and results in cell-cycle arrest in G1. Ras is essential for entry into the S phase in Rb+/+ fibroblasts but not in Rb-/- fibroblasts, establishing a link between Ras and the G1 CDK/Rb/E2F pathway. However, although expression of Ras alone will not induce G1 CDK activity or S phase, coexpression of Ras with Myc allows the generation of cyclin E-dependent kinase activity and the induction of S phase, coincident with the loss of the p27 cyclin-dependent kinase inhibitor (CKI). These results suggest that Ras, along with the activation of additional pathways, is required for the generation of G1 CDK activity, and that activation of cyclin E-dependent kinase in particular depends on the cooperative action of Ras and Myc (Leone, 1997).

It is well documented that Ras functions as a molecular switch for reentry into the cell cycle at the border between G0 and G1 by transducing extracellular growth stimuli into early G1 mitogenic signals. The role of Ras was investigated during the late stage of the G1 phase by using NIH 3T3 (M17) fibroblasts in which the expression of a dominant negative Ras mutant [Ha-Ras(Asn17)] was induced in response to dexamethasone treatment. Delaying the expression of Ras(Asn17) until late in the G1 phase by introducing dexamethasone 3 h after the addition of epidermal growth factor (EGF) abolishes the downregulation of the p27kip1 cyclin-dependent kinase (CDK) inhibitor that normally occurs during this period, with resultant suppression of cyclin Ds/CDK4 and cyclin E/CDK2 and G1 arrest. The immunodepletion of p27kip1 completely eliminates the CDK inhibitor activity from EGF-stimulated, dexamethasone-treated cell lysate. The failure of p27kip1 downregulation and G1 arrest is also observed in cells in which Ras(Asn17) is induced after growth stimulation with either a phorbol ester or alpha-thrombin and is mimicked by the addition of inhibitors for phosphatidylinositol-3-kinase late in the G1 phase. Ras-mediated downregulation of p27kip1 involves both the suppression of synthesis and the stimulation of the degradation of the protein. Unlike the earlier expression of Ras(Asn17) at the border between G0 and G1, its delayed expression does not compromise the EGF-stimulated transient activation of extracellular signal-regulated kinases or inhibit the stimulated expression of a principal D-type cyclin, cyclin D1, until close to the border between G1 and S. It is concluded that Ras plays temporally distinct, phase-specific roles throughout the G1 phase and that Ras function late in G1 is required for p27kip1 downregulation and passage through the restriction point, a prerequisite for entry into the S phase (Takuwa, 1997).

v-Abl is an oncogenic form of the c-Abl nonreceptor tyrosine kinase. v-Abl induces transcription of c-myc, and c-Myc function is a necessary but not sufficient component of the v-Abl transformation program. The E2F site in the c-myc promoter is a v-Abl response element and v-Abl appears to induce c-myc by initiating a phosphorylation cascade that ultimately activates E2F-binding proteins. The Ras GTPase and Raf1 serine/threonine kinase are required in this pathway. However, in contrast to other aspects of v-Abl signaling, induction of c-myc transcription is independent of the Rac GTPase. These results also establish a requirement for activated cyclin-dependent kinases (cdks), as v-Abl-dependent induction of c-myc transcription is blocked by cdk inhibitor p21 and induction of c-myc is accompanied by activation of cdk2 and cdk4. v-Abl-dependent induction of c-myc is accompanied by hyperphosphorylation of pRb, p107, and p130 (Zou, 1997).

BCR/ABL is a chimaeric oncogene generated by translocation of sequences from the c-ABL protein-tyrosine kinase gene on chromosome 9 (See Drosophila Abl Oncogene) into the BCR (breakpoint cluster region) gene on chromosome 22. Alternative chimeric proteins, p210(BCR/ABL) and p190(BCR/ABL), are produced that are characteristic of chronic myelogenous leukemia and acute lymphoblastic leukemia, respectively. Their role in the etiology of human leukemia remains to be defined. The tumorigenic effect of BCR/ABL oncogenes is mediated by Bcl-2 (Drosophila homolog: death executioner Bcl-2 homologue). Bcl-2 is a protein essential for transformation by BCR/ABL. However, it is not known how Bcl-2 and Ras fit together in cell transformation by BCR/ABL. The data presented here establish that Bcl-2 is a downstream target gene of the Ras signaling pathway in cells transformed by BCR/ABL, and that constitutive Ras activation results in constitutive expression of the gene. Conversely, a truncated form of the BCR/ABL, which lacks a critical BCR region required for activation of the Ras signaling pathway, fails to induce Bcl-2 expression. These results indicate that BCR/ABL prevents apoptosis by inducing Bcl-2 through a signaling pathway involving Ras and links constitutive Ras activation and Bcl-2 gene regulation. Hence, these results further imply that Ras is involved in both mitogenic signals and survival signals (Sanchez-Garcia, 1997).

Activation of the Ras/Raf/ERK pathway extends the half-life of the Myc protein and thus enhances the accumulation of Myc activity. Investigated were two N-terminal phosphorylation sites in Myc, Thr 58 and Ser 62, known to be regulated by mitogen stimulation. Phosphorylation of these two residues is critical for determining the stability of Myc. Phosphorylation of Ser 62 is required for Ras-induced stabilization of Myc, likely mediated through the action of ERK. Conversely, phosphorylation of Thr 58, likely mediated by GSK-3 but dependent on the prior phosphorylation of Ser 62, is associated with degradation of Myc. Further analysis demonstrates that the Ras-dependent PI-3K pathway is also critical for controlling Myc protein accumulation, likely through the control of GSK-3 activity. These observations thus define a synergistic role for multiple Ras-mediated phosphorylation pathways in the control of Myc protein accumulation during the initial stage of cell proliferation (Sears, 2000).

The amino acid sequence surrounding Ser 62 represents a consensus ERK recognition sequence, and evidence has been presented that ERK can mediate the phosphorylation of Myc at Ser 62. Mutation of Ser 62 prevents mitogen- and Ras-induced stabilization of Myc. Moreover, phosphorylation at Ser 62 is enhanced under conditions where Myc is stabilized. The importance of Ser 62 in the control of Myc stability is seen in the strict requirement for the stabilization of Myc by Ras, but seen from work that has demonstrated an impaired transforming function when Ser 62 is altered. In contrast, phosphorylation at Thr 58 coincides with a decreased stability of Myc and mutations that prevent Thr 58 phosphorylation lead to stable Myc protein. Once again, this coincides with work that has shown that alteration of Thr 58 enhances the transforming activity of Myc and that mutations at this site are common in Myc proteins derived from tumors. Various lines of work suggest that the GSK-3 protein kinase is most likely responsible for the phosphorylation of Myc at Thr 58. Thr 58 lies within an established consensus, and GSK-3 has been shown to phosphorylate Thr 58 in Myc in vitro. However, unlike ERK, which is tightly regulated by cell growth, the level of GSK-3 protein is constant and does not fluctuate with cell growth. Nevertheless, despite the continual presence of GSK-3 protein, the activity of the kinase is regulated during the initial phase of cell proliferation. In particular, GSK-3 activity is inhibited through the action of PI-3K/AKT. Thus, as Ras initiates the PI-3K/AKT pathway, GSK-3 activity is held in check, preventing the phosphorylation of Thr 58. Only when AKT activity declines would GSK-3 then have the capacity to phosphorylate Thr 58 to induce the degradation of Myc. Thus, Ras activation elicits two responses within the cell that can cooperate to enhance Myc stability: a direct effect of ERK and an indirect effect of AKT (Sears, 2000 and references therein).

Ras in the regulation of redox signals

Ras p21 signaling is involved in multiple aspects of growth, differentiation, and stress response. There is evidence pointing to superoxides as relays of Ras signaling messages. Chemicals with antioxidant activity suppress Ras-induced DNA synthesis. The inhibition of Ras significantly reduces the production of superoxides by the NADPH-oxidase complex. Kirsten and Harvey are nonallelic Ras cellular genes that share a high degree of structural and functional homology. The sequences of Ki- and Ha-Ras proteins are almost identical. They diverge only in the 20-amino acid hypervariable domain at the COOH termini. To date, their functions remain indistinguishable. Ki- and Ha-Ras genes are shown in this study to differently regulate the redox state of the cell. Ha-Ras-expressing cells produce high levels of reactive oxygen species (ROS) by inducing the NADPH-oxidase system. Ki-Ras, on the other hand, stimulates the scavenging of ROS by activating posttranscriptionally the mitochondrial antioxidant enzyme, Mn-superoxide dismutase (Mn-SOD), via an ERK1/2-dependent pathway. Glutamic acid substitution of the four lysine residues in the polybasic stretch at the COOH terminus of Ki-Ras completely abolishes the activation of Mn-SOD, although it does not inhibit ERK1/2-induced transcription. In contrast, an alanine substitution of the cysteine of the CAAX box has very little effect on Mn-SOD activity but eliminates ERK1/2- dependent transcription (Santillo, 2001).

Ras and photoperiod response in Neurospora

band, an allele enabling clear visualization of circadianly regulated spore formation (conidial banding), has remained an integral tool in the study of circadian rhythms for 40 years. bd was mapped using single-nucleotide polymorphisms (SNPs), cloned, and determined to be a T79I point mutation in ras-1. Alterations in light-regulated gene expression in the ras-1bd mutant suggests that the Neurospora photoreceptor WHITE COLLAR-1 is a target of RAS signaling, and increases in transcription of both wc-1 and fluffy show that regulators of conidiation are elevated in ras-1bd. Comparison of ras-1bd with dominant active and dominant-negative ras-1 mutants and biochemical assays of RAS function indicate that RAS-1bd displays a modest enhancement of GDP/GTP exchange and no change in GTPase activity. Because the circadian clock in ras-1bd appears to be normal, ras-1bd apparently acts to amplify a subtle endogenous clock output signal under standard assay conditions. Reactive oxygen species (ROS), which can affect and be affected by RAS signaling, increase conidiation, suggesting a link between generation of ROS and RAS-1 signaling; surprisingly, however, ROS levels are not elevated in ras-1bd. The data suggest that interconnected RAS- and ROS-responsive signaling pathways regulate the amplitude of circadian- and light-regulated gene expression in Neurospora (Belden, 2007).

Ras and directional sensing in Dictyostelium

Cells' ability to detect and orient themselves in chemoattractant gradients has been the subject of numerous studies, but the underlying molecular mechanisms remain largely unknown. Ras activation is the earliest polarized response to chemoattractant gradients downstream from heterotrimeric G proteins in Dictyostelium, and inhibition of Ras signaling results in directional migration defects. Activated Ras is enriched at the leading edge, promoting the localized activation of key chemotactic effectors, such as PI3K and TORC2. To investigate the role of Ras in directional sensing, the effect of its misregulation was studied by using cells with disrupted RasGAP activity. An ortholog of mammalian NF1, DdNF1, was identified as a major regulator of Ras activity in Dictyostelium. Disruption of nfaA leads to spatially and temporally unregulated Ras activity, causing cytokinesis and chemotaxis defects. By using unpolarized, latrunculin-treated cells, it was shown that tight regulation of Ras is important for gradient sensing. Together, these findings suggest that Ras is part of the cell's compass and that the RasGAP-mediated regulation of Ras activity affects directional sensing (Zhang, 2008).

Evolutionary homologs: Table of contents

Ras85D: Biological Overview | Regulation | Protein Interactions | Effects of Mutation | Ras as Oncogene | References

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