Ras oncogene at 85D

EVOLUTIONARY HOMOLOGS
Table of contents

Targets of Ras signaling: Cytoplasmic targets

Activation of Ras stimulates cell surface membrane ruffling and pinocytosis. Although seen as coupled events, it is clear that membrane ruffling and pinocytosis are regulated by distinct Ras signal transduction pathways. Ras controls membrane ruffling via the small GTPase Rac (see Drosophila Rac). In BHK-21 cells, expression of the constitutively active Rac1(G12V) mutant results in a dramatic stimulation of membrane ruffling without affecting pinocytosis. Expression of Ha-Ras(G12V), an activated Ras mutant, stimulates both membrane ruffling and pinocytosis. The Ha-Ras(G12V)-stimulated pinocytosis but not the membrane ruffling is abolished by either wortmannin or co-expression with a dominant negative mutant of Rab5, Rab5(S34N). Expression of the activated Rab5(Q79L) mutant mimics the stimulatory effect of Ha-Ras(G12V) on pinocytosis but not on membrane ruffling. These results indicate that Ha-Ras(G12V) separately activates Rab5-dependent pinocytosis and Rac1-dependent membrane ruffling (Li, 1997).

Ras plays a key role in regulating cellular proliferation, differentiation, and transformation. Raf is the major effector of Ras in the Ras > Raf > Mek > extracellular signal-activated kinase (ERK) cascade. A second effector is phosphoinositide 3-OH kinase (PI 3-kinase) that, in turn, activates the small G protein Rac. Rac also has multiple effectors, one of which is the serine threonine kinase Pak [p65(Pak)]. Ras, but not Raf, activates Pak1 in cotransfection assays of Rat-1 cells but not NIH 3T3 cells. Agents that activate or block specific components downstream of Ras were tested and a Ras > PI 3-kinase > Rac/Cdc42 > Pak signal has been demonstrated. Although these studies suggest that the signal from Ras through PI 3-kinase is sufficient to activate Pak, additional studies suggest that other effectors contribute to Pak activation. RasV12S35 and RasV12G37, two effector mutant proteins that fail to activate PI 3-kinase, do not activate Pak when tested alone but activate Pak when they are cotransfected. Similarly, RacV12H40, an effector mutant that does not bind Pak, and Rho both cooperate with Raf to activate Pak. A dominant negative Rho mutant also inhibits Ras activation of Pak. All combinations of Rac/Raf and Ras/Raf and Rho/Raf effector mutants that transform cells cooperatively stimulate ERK. Cooperation is Pak dependent, since all combinations are inhibited by kinase-deficient Pak mutants in both transformation assays and ERK activation assays. These data suggest that other Ras effectors can collaborate with PI 3-kinase and with each other to activate Pak. Furthermore, the strong correlation between Pak activation and cooperative transformation suggests that Pak activation is necessary, although not sufficient, for cooperative transformation of Rat-1 fibroblasts by Ras, Rac, and Rho (Tang, 1999).

Ras-mediated transformation of mammalian cells has been shown to activate multiple signaling pathways, including those involving mitogen-activated protein kinases and the small GTPase Rho. Members of the Rho family affect cell morphology by controlling the formation of actin-dependent structures: specifically, filopodia are induced by Cdc42Hs, lamellipodia and ruffles by Rac, and stress fibers by RhoA. In addition, Rho GTPases are involved in progression through the G1 phase of the cell cycle, and Rac1 and RhoA are implicated in the morphogenic and mitogenic responses to transformation by oncogenic Ras. In order to examine the cross-talk between Ras and Rho proteins, the effects on focus-forming activity and cell growth of the Rho-family members Cdc42Hs, Rac1 and RhoG were examined by expressing constitutively active or dominant-negative forms in NIH3T3 cells. Expression of Rac1 or RhoG modulates the saturation density to which the cells grew, probably by affecting the level of contact inhibition. Although all three GTPases are required for cell transformation mediated by Ras but not by constitutively active Raf, the selective activation of each GTPase is not sufficient to induce the formation of foci. The coordinated activation of Cdc42Hs, RhoG and Rac1, however, elicit a high focus-forming activity, independent of the mitogen-activated ERK and JNK protein kinase pathways. It is concluded that Ras-mediated transformation induces extensive changes in cell morphology that require the activity of members of the Rho family of GTPases. These data show that the pattern of coordinated Rho family activation, which elicits a focus-forming activity in NIH3T3 cells, is distinct from the regulatory cascade that has been proposed for the control of actin-dependent structures in Swiss 3T3 cells (Roux, 1997).

RIN1 was originally identified by its ability to inhibit activated Ras and likely participates in multiple signaling pathways because it binds c-ABL and 14-3-3 proteins, in addition to Ras. RIN1 also contains a region homologous to the catalytic domain of Vps9p-like Rab guanine nucleotide exchange factors (GEFs). This region is necessary and sufficient for RIN1 interaction with the GDP-bound Rabs, Vps21p, and Rab5A. RIN1 has also been shown to stimulate Rab5 guanine nucleotide exchange, Rab5A-dependent endosome fusion, and EGF receptor-mediated endocytosis. The stimulatory effect of RIN1 on all three of these processes is potentiated by activated Ras. It is concluded that Ras-activated endocytosis is facilitated, in part, by the ability of Ras to directly regulate the Rab5 nucleotide exchange activity of RIN1 (Tall, 2001).

With the discovery of a pathway that links Ras to endocytosis via RIN1, the question arises, what is the purpose of this pathway? At least two models can be envisioned. In the first, internalization and degradation of cell surface receptors is a mechanism used to attenuate receptor based signaling. Because Ras activation is an early response to receptor activation, it is likely that Ras functions in negative feedback pathways to attenuate signals it receives by potentiating flux through the endocytic pathway and therefore increasing the rate at which activated cell surface receptors are cleared. One established pathway originates from within the activated MAP kinase cascade and serves to disrupt the Shc-Grb2-Sos Ras nucleotide exchange complex. However, this feedback pathway is specific for attenuating Ras signaling (downstream of activated receptors) and does not explain Ras-mediated attenuation of active cell surface receptors. The rapid kinetics of receptor internalization upon ligand binding suggests that receptor-mediated endocytosis is subject to extremely tight temporal control mechanisms. Ras-mediated activation of RIN1, which stimulates Rab5A-dependent receptor internalization, represents a direct link between Ras and negative feedback control of cell surface receptor signaling (Tall, 2001).

In a second model of RIN1 function, feed-forward mechanisms stemming from Ras activation could actually result in the facilitation of specific types of receptor signaling. Receptor-mediated Ras activation on the inner surface of the plasma membrane could initiate the proximal RIN1-mediated endocytic pathway to stimulate internalization of receptors. In certain signaling pathways, receptor internalization is a prerequisite for signaling. Activation of RIN1-mediated receptor endocytosis by Ras could serve to initiate internal membrane derived signaling pathways in a feed forward style mechanism by stimulating internalization of active receptors to their physiological signaling compartment. However, initial analysis indicates that signaling is attenuated by RIN1. Further experimentation will be required to determine the precise impact RIN1 activation has on downstream cell signaling events (Tall, 2001).

Targets of Ras signaling: Nuclear targets

The mechanism has been examined by which growth factor-mediated induction of the Ras pathway interferes with signaling via the second messenger cAMP. Activation of cellular Ras with insulin or NGF stimulates recruitment of the S6 kinase pp90RSK to the signal-dependent coactivator CBP. Formation of the pp90RSK-CBP complex occurs with high stoichiometry and persists for 6-8 hr following growth factor addition. pp90RSK specifically recognizes the E1A-binding domain of the coactivator CBP. In addition, like E1A, binding of pp90RSK to CBP is sufficient to repress transcription of cAMP-responsive genes via the cAMP-inducible factor CREB (see Drosophila CREB). By contrast with its effects on the cAMP pathway, formation of the pp90RSK-CBP complex is required for induction of Ras-responsive genes. These results provide a demonstration of cross-coupling between two signaling pathways that occurs at the level of a signal-dependent coactivator (Nakajima, 1996).

The c-jun proto-oncogene encodes a transcription factor that is activated by mitogens both transcriptionally and as a result of phosphorylation by Jun N-terminal kinase (JNK). The cellular signaling pathways involved in epidermal growth factor (EGF) induction of the c-jun promoter have been investigated. Two sequence elements that bind ATF1 (a leucine zipper DNA binding protein) and MEF2D transcription factors are required in HeLa cells, although these elements are not sufficient for maximal induction. Activated forms of Ras, RacI, Cdc42Hs, and MEKK increase expression of the c-jun promoter, while dominant negative forms of Ras, RacI, and MEK kinase (MEKK) inhibit EGF induction. These results suggest that EGF activates the c-jun promoter by a Ras-to-Rac-to-MEKK pathway. No change is found in protein binding to the jun ATF1 site in EGF-treated cells. A potential mechanism for regulation of ATF1 and CREB is phosphorylation (Clarke, 1997).

The beta-amyloid protein, the major component of the vascular and plaque amyloid deposits that characterize Alzheimer's disease, derives from a larger beta-amyloid precursor protein (APP) that is expressed in both neural and nonneural cells. An increased expression of APP might actively contribute to the development of the pathology; however, the mechanisms involved in the regulation of APP gene expression are not yet well understood. In PC12 cells, a rat pheochromocytoma cell line, nerve growth factor (NGF) induces the APP gene expression and increases APP mRNA levels in the presence of 0.5 or 15% serum. Expression of activated ras in the PC12 cell subline UR61 also leads to a significant increase in content of APP transcripts; a dominant negative mutant of ras blocks the NGF-induced response. Other ligands of tyrosine kinase receptors, such as fibroblast growth factor, which causes morphological differentiation, or epidermal growth factor, which induces cell growth, also increase APP mRNA levels in PC12 cells. These results suggest that ras mediates the induction of APP gene expression by NGF and other ligands of tyrosine kinase receptors (Cosgaya, 1996).

Cell transformation by the Ras oncogene is mediated by members of the ets gene family. To analyse the mechanisms of regulation, activation of several ets factors by Ras expression have been studied. Expression of Ha-Ras strongly activates the Ets1 p68 and p54 isoforms and Ets2 in F9 EC cells. The Ras responsive elements of Ets1 p68 were mapped to two domains: RI+II and RIII. Mutation of threonine 82 to alanine in RI+II abolishes both Ras activation and phosphorylation by MAP kinase. Threonine 82 is part of a sequence that is conserved in Drosophila Pointed P2, an ets protein that has been shown both genetically and biochemically to mediate Ras signaling in Drosophila cells. Pointed P2 is activated by Ras in mammalian cells and mutation of the homologous threonine abolishes activation. Pointed P2 resembles Ets1, in that it has conserved sequences in a similar position adjacent to the ets DNA binding domain that negatively auto-regulates DNA binding. These results indicate that the Drosophila Pointed and vertebrate Ets1 are most probably evolutionarily related proteins that have remarkably conserved Ras regulatory mechanisms downstream from MAP kinase (Wasylyk, 1997).

Despite extensive evidence implicating Ras in cardiac muscle hypertrophy, the mechanisms involved are unclear. Ras, through an effector-like function of Ras GTPase-activating protein (GAP) in neonatal cardiac myocytes, can up-regulate expression from a comprehensive set of promoters, including both cardiac cell-specific and constitutive ones. To investigate the mechanism(s) underlying these findings, recombinant adenoviruses were used that harbored either a dominant negative Ras (17N Ras) allele or the N-terminal domain of GAP (nGAP), which is responsible for the Ras-like effector function. Inhibition of endogenous Ras reduces basal levels of [3H]uridine and [3H]phenylalanine incorporation into total RNA, mRNA, and protein, with parallel changes in apparent cell size. In addition, 17N Ras markedly inhibits phosphorylation of the C-terminal domain (CTD) of RNA polymerase II (pol II), known to regulate transcript elongation, accompanied by down-regulation of its principal kinase, cyclin-dependent kinase 7 (Cdk7). In contrast, nGAP elicits the opposite effects on each of these parameters. Furthermore, cotransfection of constitutively active Ras (12R Ras) with wild-type pol II, rather than a truncated mutant lacking the CTD, demonstrates that Ras activation of transcription is dependent on the pol II CTD. Consistent with a potential role for this pathway in the development of cardiac myocyte hypertrophy, alpha1-adrenergic stimulation similarly enhances pol II phosphorylation and Cdk7 expression, where both effects are inhibited by dominant negative Ras, while pressure overload hypertrophy leads to an increase in both hyperphosphorylated and hypophosphorylated pol II in addition to Cdk7 (Abdellatif, 1998).

During B cell development, rearrangement and expression of Ig heavy chain (HC) genes promotes development and expansion of pre-B cells accompanied by the onset of Ig light chain (LC) variable region gene assembly. To elucidate the signaling pathways that control these events, the ability of activated Ras expression to promote B cell differentiation to the stage of LC gene rearrangement in the absence of Ig HC gene expression was tested. For this purpose, an activated Ras expression construct was introduced into JH-deleted embryonic stem cells that lack the ability to assemble HC variable region genes. Differentiation potential was assayed by recombination activating gene (RAG) 2-deficient blastocyst complementation. Activated Ras expression induces the progression of B lineage cells beyond the developmental checkpoint ordinarily controlled by mu HC. Such Ras/JH-deleted B cells accumulate in the periphery but continue to express markers associated with precursor B cells, including RAG gene products. These peripheral Ras/JH-deleted B cell populations show extensive Ig LC gene rearrangement but maintain an extent of kappa LC gene rearrangement and a preference for kappa over lambda LC gene rearrangement similar to that of wild-type B cells. These findings are discussed in the context of potential mechanisms that may regulate Ig LC gene rearrangement (Shaw, 1999).

TGFbeta can override the proliferative effects of EGF and other Ras-activating mitogens in normal epithelial cells. However, epithelial cells harboring oncogenic Ras mutations often show a loss of TGFbeta antimitogenic responses. Oncogenic Ras inhibits TGFbeta signaling in mammary and lung epithelial cells by negatively regulating the TGFbeta mediators Smad2 and Smad3. Oncogenically activated Ras inhibits the TGFbeta-induced nuclear accumulation of Smad2 and Smad3 and Smad-dependent transcription. Ras acting via Erk MAP kinases causes phosphorylation of Smad2 and Smad3 at specific sites in the region linking the DNA-binding domain and the transcriptional activation domain. These sites are separate from the TGFbeta receptor phosphorylation sites that activate Smad nuclear translocation. Mutation of these MAP kinase sites in Smad3 yields a Ras-resistant form that can rescue the growth inhibitory response to TGFbeta in Ras-transformed cells. EGF, which is weaker than oncogenic mutations at activating Ras, induces a less extensive phosphorylation and cytoplasmic retention of Smad2 and Smad3. These results suggest a mechanism for the counterbalanced regulation of Smad2/Smad3 by TGFbeta and Ras signals in normal cells, and for the silencing of antimitogenic TGFbeta functions by hyperactive Ras in cancer cells (Kretzschmar, 1999).

Smad transcription factors mediate the actions of TGF-ß cytokines during development and tissue homeostasis. TGF-ß receptor-activated Smad2 regulates gene expression by associating with transcriptional co-activators or co-repressors. The Smad co-repressor TGIF competes with the co-activator p300 for Smad2 association, such that TGIF abundance helps determine the outcome of a TGF-ß response. Small alterations in the physiological levels of TGIF can have profound effects on human development, as shown by the devastating brain and craniofacial developmental defects in heterozygotes carrying a hypomorphic TGIF mutant allele. TGIF levels modulate sensitivity to TGF-ß-mediated growth inhibition, TGIF is a short-lived protein and epidermal growth factor (EGF) signaling via the Ras-Mek pathway causes the phosphorylation of TGIF at two Erk MAP kinase sites, leading to TGIF stabilization and favoring the formation of Smad2-TGIF co-repressor complexes in response to TGF-ß. These results identify the first mechanism for regulating TGIF levels and suggest a potential link for Smad and Ras pathway convergence at the transcriptional level (Lo, 2001).

TGIF acts at the intersection of Ras and Smad pathways. Expression of oncogenic Ha-Ras inhibits G1 cell cycle arrest by saturating concentrations of TGF-ß. In this context activation of the Mek pathway, whether by EGF stimulation, expression of a constitutively active Ras, or expression of an activated Mek, leads to a rapid increase in the level of the TGIF protein, whereas pharmacological inhibition of activated Mek blocks the EGF-induced increase in TGIF level. This enhancement in TGIF level occurs by accumulation of a phosphatase-sensitive, hyperphosphorylated TGIF form, which has a retarded electrophoretic mobility. The increase in phosphorylation of this upper form of TGIF in response to EGF requires a pair of Erk MAP kinase consensus sites near the C-terminus of TGIF. In addition, this upper TGIF form has a longer metabolic half-life than the lower TGIF form, leading to an overall build-up in the steady-state level of TGIF itself and hence its increased assembly with activated Smad and HDAC, forming co-repressor complexes. Thus, the effect of the Ras-Mek pathway on TGIF protein stability described here suggests a novel mechanism for modulating TGF-ß signaling at the transcriptional level (Lo, 2001).

The interplay between the TGF-ß and EGF/Ras signal transduction pathways occurs at other levels as well. These include Ras inhibition of TGF-ß receptor expression and of Smad accumulation in the nucleus. EGF stimulation via Ras activation has been shown to diminish nuclear accumulation of TGF-ß-activated Smad proteins. However, at high levels of TGF-ß signaling, EGF addition or transformation by an oncogenic H-ras allele is unable to prevent Smad entry into the nucleus, even though it can profoundly alter the cellular response to TGF-ß. The subcellular distribution of Smad in the cell is a function of its interactions with protein partners in the cytoplasm and nucleus. Smad proteins have intrinsic nuclear import activity that in the basal state is negated by contacts with SARA (Smad anchor for receptor activation). Likewise, overexpression of a nuclear partner of Smad, namely the Smad DNA binding co-factor FAST1, leads to Smad2 nuclear accumulation in the absence of receptor activation. Receptor-mediated Smad phosphorylation diminishes the affinity of Smad for SARA, which results in Smad movement to the nucleus and association with various protein partners. In light of these insights, attenuation of Smad nuclear accumulation by Ras-Mek signaling could result not only from direct effects on Smad nuclear import and/or export machinery, but also from effects of Ras-Mek signaling on Smad interactions with protein partners (Lo, 2001).

Ras signaling has long been known to act as a modifier of cellular responsiveness to TGF-ß. During embryo development, many processes are cooperatively stimulated by TGF-ß and Ras signaling. In principle, this cooperativity could be achieved by Ras modulating gene activation or repression by Activin, Nodal and other TGF-ß-like signals. Smad complexes activated by these factors can associate with either general co-activators, such as p300/CBP, or co-repressors like TGIF that specifically target nuclear Smad proteins. Regulation of co-activator activity by mitogenic signals, such as EGF, may result in general transcriptional upregulation. Increased TGIF activity in response to the same signals provides a mechanism to repress a specific subset of gene responses. Hence, regulation of TGIF levels by Ras signaling allows an effective and selective way to adjust the level of Smad-activated transcription in vivo. TGIF thus provides a potential link within the nucleus between signals that activate the Ras pathway and TGF-ß morphogens that exert different effects on gene expression at different levels of signal (Lo, 2001).

Likewise, during tumorigenesis, transformation by disregulation of Ras or EGFR and related tyrosine kinases in various types of epithelial cells modifies their responsiveness to TGF-ß by conferring resistance to growth inhibition by TGF-ß, while allowing other responses to TGF-ß, including extracellular matrix production, cellular motility and stimulation of angiogenesis. In fact, TGF-ß collaborates with oncogenic Ras to bring about metastatic and invasive phenotypic alterations in Ras-transformed mammary epithelial cells. Thus, oncogenic Ras signaling can attenuate certain TGF-ß responses while allowing or even enabling others. These results suggest that stabilization of TGIF provides a mechanism for the modification of Smad responses by Ras-Mek signaling. In this context, it is noteworthy that a recently identified form of human TGIF, TGIF2, has been found to be amplified and overexpressed in a third of ovarian cancer cell lines. TGIF and TGIF2 are highly conserved in the C-terminus containing the EGF-inducible phosphorylation sites (Lo, 2001).

The E-cadherin-based adherens junction (AJ) is essential for organogenesis of epithelial tissues including the liver, although the regulatory mechanism of AJ formation during development remains unknown. Using a primary culture system of fetal hepatocytes in which oncostatin M (OSM) induces differentiation, it has been shown that OSM induces AJ formation by altering the subcellular localization of AJ components, including E-cadherin and catenins. By retroviral expression of dominant-negative forms of signaling molecules, Ras was shown to be required for the OSM-induced AJ formation. Fetal hepatocytes derived from K-Ras knockout (K-Ras-/-) mice fail to form AJs in response to OSM, whereas AJ formation is induced normally by OSM in mutant hepatocytes lacking both H-Ras and N-Ras. Moreover, the defective phenotype of K-Ras-/- hepatocytes is restored by expression of K-Ras, but not by H-Ras and N-Ras. Finally, pull-down assays using the Ras-binding domain of Raf1 demonstrate that OSM directly activates K-Ras in fetal hepatocytes. These results indicate that K-Ras specifically mediates cytokine signaling for formation of AJs during liver development (Matsui, 2002).

While these results indicate that among the three Ras proteins, K-Ras specifically mediates OSM signaling to induce the formation of E-cadherin-based adhesion, the molecular basis for the specificity of K-Ras currently is unknown. A structural difference in the C-terminal short stretches may provide a hint. H-Ras and N-Ras have homologous C-terminal stretches, by which both are palmitoylated. This modification enables them to be recruited to a particular subdomain of the plasma membrane, called the caveola. In contrast, K-Ras is not palmitoylated and is anchored to the membrane through the basic domain near the C-terminus. There is no evidence so far that K-Ras is concentrated in a certain subdomain of the plasma membrane. Based on this difference, it is tempting to speculate that K-Ras stimulates a distinct array of effector molecules and thereby elicits cellular responses unique to K-Ras. It is thus possible that OSM induces the localization of E-cadherin through K-Ras by activating unique effector proteins that are not activated by H- or N-Ras (Matsui, 2002).

Ras signaling and neuronal excitability

The Ras signaling pathway is involved in learning and memory. Ras is activated by nucleotide exchange factors, such as the calmodulin-activated guanine-nucleotide releasing factor 1 (Ras-GRF1). To test whether Ras-GRF1 is required for learning and memory, the Ras-GRF1 gene was inactivated in mice. These mutants performed normally in a rota-rod motor coordination task, and in two amygdala-dependent tasks (inhibitory avoidance and contextual conditioning). In contrast the mutants were impaired in three hippocampus-dependent learning tasks: contextual discrimination, the social transmission of food preferences, and the hidden-platform version of the Morris water maze. These studies indicate that Ras-GRF1 plays a role in hippocampal-dependent learning and memory (Giese, 2001).

Ras-GRF1 is a neuron-specific guanine nucleotide exchange factor for Ras proteins. Mice lacking Ras-GRF1 (-/-) are severely impaired in amygdala-dependent long-term synaptic plasticity and show higher basal synaptic activity at both amygdala and hippocampal synapses. The effects were investigated of Ras-GRF1 deletion on hippocampal neuronal excitability. Electrophysiological analysis of both primary cultured neurons and adult hippocampal slices indicated that Ras-GRF1-/- mice display neuronal hyperexcitability. Ras-GRF1-/- hippocampal neurons show increased spontaneous activity and depolarized resting membrane potential, together with a higher firing rate in response to injected current. Changes in the intrinsic excitability of Ras-GRF1-/- neurons can entail these phenomena, suggesting that Ras-GRF1 deficiency might alter the balance between ionic conductances. In addition, mice lacking Ras-GRF1 displayed a higher seizure susceptibility following acute administration of convulsant drugs. Taken together, these results demonstrate a role for Ras-GRF1 in neuronal excitability (Tonini, 2001).

The NMDA subtype of glutamate receptors (NMDAR) at excitatory neuronal synapses plays a key role in synaptic plasticity. The extracellular signal-regulated kinase (ERK1,2 or ERK) pathway is an essential component of NMDAR signal transduction controlling the neuroplasticity underlying memory processes, neuronal development, and refinement of synaptic connections. NR2B, but not NR2A or NR1 subunits of the NMDAR, interacts in vivo and in vitro with RasGRF1, a Ca2+/calmodulin-dependent Ras-guanine-nucleotide-releasing factor. Specific disruption of this interaction in living neurons abrogates NMDAR-dependent ERK activation. Thus, RasGRF1 serves as NMDAR-dependent regulator of the ERK kinase pathway. The specific association of RasGRF1 with the NR2B subunit and study of ERK activation in neurons with varied content of NR2B suggests that NR2B-containing channels are the dominant activators of the NMDA-dependent ERK pathway (Krapivinsky, 2003).

G proteins of the Ras family function as molecular switches in many signalling cascades; however, little is known about where they become activated in living cells. FRET (fluorescent resonance energy transfer)-based sensors have been used to report on the spatio-temporal images of growth-factor-induced activation of Ras and Rap1. Epidermal growth factor activates Ras at the peripheral plasma membrane and Rap1 at the intracellular perinuclear region of COS-1 cells. In PC12 cells, nerve growth factor-induced activation of Ras is initiated at the plasma membrane and transmitted to the whole cell body. After three hours, high Ras activity is observed at the extending neurites. By using the FRAP (fluorescence recovery after photobleaching) technique, it was found that Ras at the neurites turns over rapidly; therefore, the sustained Ras activity at neurites is due to high GTP/GDP exchange rate and/or low GTPase activity, but not to the retention of the active Ras. These observations may resolve long-standing questions as to how Ras and Rap1 induce different cellular responses and how the signals for differentiation and survival are distinguished by neuronal cells (Mochizuki, 2001).

Targets of Ras signaling: Ras cell survival and programmed cell death

Regulation of cell survival is crucial to the normal physiology of multicellular organisms. Growth factors can promote cell survival by activating the phosphatidylinositide-3'-OH kinase and its downstream target, the serine-threonine kinase Akt. PI3'K may also directly interact with and be activated by the small G protein Ras. However, the mechanism by which Akt functions to promote survival is not understood. Growth factor activation of the PI3'K/Akt signaling pathway culminates in the phosphorylation of the BCL-2 family member BAD, thereby suppressing apoptosis and promoting cell survival. Akt phosphorylates BAD in vitro and in vivo, and blocks the BAD-induced death of primary neurons in a site-specific manner. These findings define a mechanism by which growth factors directly inactivate a critical component of the cell-intrinsic death machinery (Datta, 1997).

The phosphorylation of BAD may lead to the prevention of cell death via a mechanism that involves selective association of the phosphorylated forms of BAD with 14-3-3 protein isoforms (see Drosophila Leonardo). This BAD/14-3-3 interaction can occur when BAD becomes phosphorylated at SER-112 and/or Ser-136; the induced association of BAD with 14-3-3 appears to prevent BAD association with BCL-XL or BCL-2. Preliminary evidence suggests that consitutively active Akt can induce the association of BAD and 14-3-3zeta, and that kinase-inactive Akt does not induce this association event. Akt-induced Bad/14-3-3zeta association depends on the presence of BAD Ser-136. It has been proposed that in the absence of BAD phosphorylation, BAD may bind to BCL-XL or BCL-2 and suppress survival by inducing BAX homodimer formation. The prodeath function of BAD could also be a direct consequence of BAD's heterodimerization with BCL-XL or BCL-2, and may not involve BAX heterodimerization. It is possible that BAD brings Akt to the 14-3-3 complex, where Akt may phosphorylate additional signaling molecules to promote cell survival (Datta, 1997).

Caspases are intracellular proteases that function as initiators and effectors of apoptosis. The kinase Akt and p21-Ras, an Akt activator, induce phosphorylation of pro-caspase-9 (pro-Casp9) in cells. Cytochrome c-induced proteolytic processing of pro-Casp9 is defective in cytosolic extracts from cells expressing either active Ras or Akt. Akt phosphorylates recombinant Casp9 in vitro on serine-196 and inhibits its protease activity. Mutant pro-Casp9(Ser196Ala) is resistant to Akt-mediated phosphorylation and inhibition in vitro and in cells, resulting in Akt-resistant induction of apoptosis. Thus, caspases can be directly regulated by protein phosphorylation (Cardone, 1998).

Growth factor deprivation is a physiological mechanism to regulate cell death. An interleukin-2 (IL-2)-dependent murine T-cell line was used to identify proteins that interact with Bad upon IL-2 stimulation or deprivation. Using the yeast two-hybrid system, glutathione S-transferase (GST) fusion proteins and co-immunoprecipitation techniques, it was found that Bad interacts with protein phosphatase 1alpha (PP1alpha). Serine phosphorylation of Bad is induced by IL-2 and its dephosphorylation correlates with the appearance of apoptosis. IL-2 deprivation induces Bad dephosphorylation, suggesting the involvement of a serine phosphatase. A serine/threonine phosphatase activity, sensitive to the phosphatase inhibitor okadaic acid, was detected in Bad immunoprecipitates from IL-2-stimulated cells, increasing after IL-2 deprivation. This enzymatic activity also dephosphorylates in vivo ³²P-labeled Bad. Treatment of cells with okadaic acid blocks Bad dephosphorylation and prevents cell death. Finally, Ras activation controls the catalytic activity of PP1alpha. These results strongly suggest that Bad is an in vitro and in vivo substrate for PP1alpha phosphatase and that IL-2 deprivation-induced apoptosis may operate by regulating Bad phosphorylation through PP1alpha phosphatase, whose enzymatic activity is regulated by Ras (Ayllon, 2000).

Mdm2 acts as a major regulator of the tumor suppressor p53 by targeting its destruction. mdm2 gene is shown here to be regulated by the Ras-driven Raf/MEK/MAP kinase pathway, in a p53-independent manner. Mdm2 induced by activated Raf degrades p53 in the absence of the Mdm2 inhibitor p19ARF. This regulatory pathway accounts for the observation that cells transformed by oncogenic Ras are more resistant to p53-dependent apoptosis following exposure to DNA damage. Activation of the Ras-induced Raf/MEK/MAP kinase may therefore play a key role in suppressing p53 during tumor development and treatment. In primary cells, Raf also activates the Mdm2 inhibitor p19ARF. Levels of p53 are therefore determined by opposing effects of Raf-induced p19ARF and Mdm2 (Ries, 2000).

Thus Mdm2 expression is modulated by the Ras/Raf/MEK/MAP kinase pathway through activation of Ets and AP-1 sites in the P2 promoter, upstream from the p53 responsive element and independent of its activity. Furthermore, Mdm2 induced by the Ras/Raf/MEK/MAP kinase pathway is functionally active and leads to degradation of p53. This signaling pathway is intact in tumor cells expressing activated Ras because Mdm2 protein levels decrease dramatically after inhibiting MEK activity in these cells. Importantly, the effects of induced Mdm2 on p53 are regulated by p19ARF. Ras therefore acts on p53 through two competing pathways. Activation of the Ras/Raf/MEK/MAP kinase cascade results in elevated levels of Mdm2 protein. However, in normal cells, this pathway also induces the expression of p19ARF, which inhibits Mdm2 activity. Thus, in normal cells, levels of p53 are determined by a balance between opposing effects of the Ras/Raf/MEK/MAP kinase pathway. In mouse embryonic fibroblasts (MEFs), these opposing effects are equivalent, and Raf is ineffective at inducing p53, despite its effects in p19ARF. In different cell types, or even in MEFs growing under slightly different conditions, the balance of these opposing pathways is likely to be different. For example, in IMR90 human diploid fibroblasts, activated MEK leads to accumulation of p53, presumably because p14ARF exceeds Mdm2 induction (Ries, 2000 and references therein).

To investigate the role of Ras in the adult central nervous system, constitutively activated V12-Ha-Ras was expressed selectively in neurons of transgenic mice via a synapsin promoter. Ras-transgene protein expression increases postnatally, reaching a four- to five-fold elevation at day 40 and persisting at this level, thereafter. Neuronal Ras is constitutively active and a corresponding activating phosphorylation of mitogen-activated kinase is observed, but there are no changes in the activity of phosphoinositide 3-kinase, the phosphorylation of its target kinase Akt/PKB, or expression of the anti-apoptotic proteins Bcl-2 or Bcl-X(L). Neuronal Ras activation does not alter the total number of neurons, but induces cell soma hypertrophy, which resulted in this case in a 14.5% increase of total brain volume. Choline acetyltransferase and tyrosine hydroxylase activities were increased, as well as neuropeptide Y expression. Degeneration of motorneurons is completely prevented after facial nerve lesion in Ras-transgenic mice. Furthermore, neurotoxin-induced degeneration of dopaminergic substantia nigra neurons and their striatal projections is greatly attenuated. Thus, the Ras signaling pathway mimics neurotrophic effects and triggers neuroprotective mechanisms in adult mice. Neuronal Ras activation might become a tool to stabilize donor neurons for neural transplantation and to protect neuronal populations in neurodegenerative diseases (Heumann, 2000).

The connector enhancer of KSR (CNK) is a multidomain scaffold protein discovered in Drosophila, where it is necessary for Ras activation of the Raf kinase. Recent studies have shown that CNK1 also interacts with RalA and Rho and participates in some aspects of signaling by these GTPases. A novel aspect of CNK1 function has been demonstrated, i.e. reexpression of CNK1 suppresses tumor cell growth and promotes apoptosis. As shown previously for apoptosis induced by Ki-Ras(G12V), CNK1-induced apoptosis is suppressed by a dominant inhibitor of the mammalian sterile 20 kinases 1 and (MST1/MST2). Immunoprecipitates of MST1 endogenous to LoVo colon cancer cells contain endogenous CNK1; however, no association of these two polypeptides can be detected in a yeast two-hybrid assay. CNK1 does, however, bind directly to the RASSF1A and RASSF1C polypeptides, constitutive binding partners of the MST1/2 kinases. Deletion of the MST1 carboxyl-terminal segment that mediates its binding to RASSF1A/C eliminates the association of MST1 with CNK1. Coexpression of CNK1 with the tumor suppressive isoform, RASSF1A, greatly augments CNK1-induced apoptosis, whereas the nonsuppressive RASSF1C isoform is without effect on CNK1-induced apoptosis. Overexpression of CNK1-(1-282), a fragment that binds RASSF1A but is not proapoptotic, blocks the apoptosis induced by CNK1 and by Ki-Ras(G12V). Thus, in addition to its positive role in the proliferative outputs of active Ras, the CNK1 scaffold protein, through its binding of a RASSF1A.MST complex, also participates in the proapoptotic signaling initiated by active Ras (Rabizadeh, 2004).

Evolutionary homologs: Table of contents

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