Ras oncogene at 85D


EVOLUTIONARY HOMOLOGS

Table of contents

Ras activation by growth factor receptors

Treatment of PC12 cells with nerve growth factor (NGF) induces a rapid increase in tyrosine phosphorylation of multiple cellular proteins. Expression of a dominant inhibitory Ras mutant specifically blocks NGF- and TPA-induced tyrosine phosphorylation 42 and 44 kd MAPK proteins. MAPK activation, as measured by in vitro phosphorylation of myelin basic protein, is also regulated by Ras. Ras is not required for NGF-induced activation of Trk or tyrosine phosphorylation of PLC-gamma 1. Thus, NGF-induced tyrosine phosphorylation occurs both prior to and following Ras action, and Ras plays a critical role in the NGF- and TPA-induced tyrosine phosphorylation of MAPKs (Thomas, 1992).

Nerve growth factor (NGF) treatment causes a profound down-regulation of epidermal growth factor receptors during the differentiation of PC12 cells. This process is characterized by a progressive decrease in epidermal growth factor (EGF) receptor level. Treatment of the cells with NGF for 5 days produces a 95% reduction in the amount of EGF receptors. This down-regulation does not occur in PC12nnr5 cells, which lack the p140(trk) NGF receptor. However, in PC12nnr5 cells stably transfected with p140(trk), the NGF-induced heterologous down-regulation of EGF receptors is reconstituted in part. NGF-induced heterologous down-regulation, but not EGF-induced homologous down-regulation of EGF receptors, is blocked in Ras- and Src-dominant-negative PC12 cells. Treatment with either pituitary adenylate cyclase-activating peptide (PACAP) or staurosporine stimulates neurite outgrowth in PC12 cell variants, but neither induces down-regulation of EGF receptors. NGF treatment of PC12 cells in suspension induces down-regulation of EGF receptors in the absence of neurite outgrowth. These results strongly suggest a p140(trk)-, Ras- and Src-dependent mechanism for NGF-induced down-regulation of EGF receptors and separate this process from NGF-induced neurite outgrowth in PC12 cells (Lazarovici, 1997).

p21c-ras plays a critical role in mediating tyrosine kinase-stimulated cell growth and differentiation. In PC12 cells, expression of a dominant inhibitory mutant of ras, c-Ha-ras(Asn-17), antagonizes growth factor- and phorbol ester-induced activation of the erk-encoded family of MAP kinases, the 85-92 kd RSKs, and the kinase(s) responsible for hyperphosphorylation of the proto-oncogene product Raf-1. Expression of the activated ras oncogene is sufficient to stimulate these events. These data indicate that ras mediates nerve growth factor receptor and protein kinase C modulation of MAP kinases, RSKs, and Raf-1 (Wood, 1992).

p21ras is believed to be involved in the neuronal differentiation of cells responsive to nerve growth factor (NGF). NGF stimulates the activation of p21ras in embryonic sensory neurons and in PC12 cells. In the initial 5 min of exposure to NGF, the activation is concentration-dependent. In the sensory neurons and PC12 cells, the apparent maximal activation is reached at 50 and 10 ng/ml, respectively, with half-maximal activation at approximately 5 and 2-3 ng/ml, respectively. Kinetic analysis at low concentrations of NGF show that p21ras activation slowly increases with time in both types of cells, while high concentrations result in rapid activation within 5 min. NGF regulates the activation state of p21ras in these cells and provides evidence suggesting that activation of p21ras is involved in NGF signal transduction. Treatment of PC12 cells with brain-derived neurotrophic factor or neurotrophin-3 (NT-3) fails to activate p21ras, suggesting that binding alone to p75LNGFR is insufficient for ras activation. Treatment with the kinase inhibitor, K252a, which inhibits the NGF tyrosine kinase receptor p140trk, abolishes ras activation, suggesting that p140trk is the major mediator of p21ras activation by NGF (Ng, 1993).

A mechanism by which the nerve growth factor (NGF) signal is transduced to the nucleus to induce gene expression has been characterized. An NGF-inducible, Ras-dependent protein kinase has been identified that catalyzes the phosphorylation of the cyclic AMP response element-binding protein (CREB) at Ser-133. Phosphorylation of Ser-133 stimulates the ability of CREB to activate transcription in NGF-treated cells. These findings suggest that CREB has a more widespread function than previously believed and functions in the nucleus as a general mediator of growth factor responses (Ginty, 1994).

Neurotrophins activate the Trk tyrosine kinase receptors, which subsequently initiate signaling pathways that have yet to be fully resolved, resulting in neuronal survival and differentiation. The ability of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) to activate GTP binding to p21ras was investigated using cultured embryonic chick neurons. In both sympathetic and sensory neurons, the addition of NGF markedly increases the formation of Ras-GTP. The magnitude of the effect depends upon the developmental stage, peaking at embryonic day 11 in sympathetic neurons and at embryonic day 9 in sensory neurons, times when large numbers of neurons depend on NGF for survival. Surprisingly, following the addition of BDNF, no formation of Ras-GTP can be observed in neurons cultured with BDNF. When sensory neurons are cultured with NGF alone, both NGF and BDNF stimulate GTP binding to Ras. In rat cerebellar granule cells, while the acute exposure of these cells to BDNF results in the formation Ras-GTP, no response is observed following previous exposure of the cells to BDNF, as is observed with sensory neurons. However, this desensitization was not observed in a transformed cell line expressing TrkB. In neurons, the mechanism underlying the loss of the BDNF response appears to involve a dramatic loss of binding to cell-surface receptors, as determined by cross-linking with radiolabeled BDNF. Receptor degradation can not account for the desensitization since cell lysates from neurons pretreated with BDNF reveal that the levels of TrkB are comparable to those in untreated cells. These results indicate that in neurons, the pathways activated by NGF and BDNF are differentially regulated and that prolonged exposure to BDNF results in the inability of TrkB to bind its ligand (Carter, 1995).

In rat embryonic sympathetic neurons from the superior cervical ganglia (SCG,) NGF-mediated survival depends on the activation of the trkA receptor tyrosine kinase and on the activity of p21ras, the membrane-anchored small G-protein found in the intracellular plasma. In contrast, chick sympathetic neurons derived from the more caudally located lumbosacral chain ganglia (LSCG) do not respond to activated p21ras (G12V-Ha-ras mutant). In these neurons endogenous p21ras and its downstream effector MAP kinase are activated but are not essential for NGF-dependent survival. Permanently activated p21ras protein does promote neuron survival in chick sympathetic neurons of the SCG. Consistently, their NGF-mediated survival is sensitive to Fab fragments blocking endogenous p21ras activity. These results suggest that sympathetic neurons derived from sympathoenteric (SCG) and sympathoadrenal (LSCG) lineages differ in their requirement for p21ras in the NGF-mediated survival pathways (Markus, 1997).

Fibroblast growth factor receptor (FGFR) activation leads to receptor autophosphorylation and increased tyrosine phosphorylation of several intra cellular proteins. Autophosphorylated tyrosine 766 in FGFR1 serves as a binding site for one of the SH2 domains of phospholipase Cy and couples FGFR1 to phosphatidylinositol hydrolysis in several cell types. There are six additional autophosphorylation sites (Y-463, Y-583, Y-585, Y-653, Y-654 and Y-730) on FGFR1. Autophosphorylation on tyrosines 653 and 654 is important for activation of tyrosine kinase activity of FGFR1 and is therefore essential for FGFR1-mediated biological responses. In contrast, autophosphorylation of the remaining four tyrosines is dispensable for FGFR1-mediated mitogen-activated protein kinase activation and mitogenic signaling in L-6 cells as well as neuronal differentiation of PC12 cells. Interestingly, both the wild-type and a mutant FGFR1 (FGFR1-4F) are able to phosphorylate Shc and an unidentified Grb2-associated phosphoprotein of 90 kDa (pp90). Binding of the Grb2/Sos complex to phosphorylated Shc and pp90 may therefore be the key link between FGFR1 and the Ras signaling pathway, mito-genesis, and neuronal differentiation (Mohammadi, 1996).

The PDGF receptor-beta mediates both mitogenic and chemotactic responses to PDGF-BB. Although the role of Ras in tyrosine kinase-mediated mitogenesis has been characterized extensively, its role in PDGF-stimulated chemotaxis has not been defined. Using cells expressing a dominant-negative ras, Ras inhibition is found to suppress migration toward PDGF-BB. Overexpression of either Ras-GTPase activating protein (Ras-GAP) or a Ras guanine releasing factor (GRF) also inhibits PDGF-stimulated chemotaxis. In addition, cells producing excess constitutively active Ras fail to migrate toward PDGF-BB, consistent with the observation that either excess ligand or excess signaling intermediate can suppress the chemotactic response. These results suggest that Ras can function in normal cells to support chemotaxis toward PDGF-BB and that either too little or too much Ras activity can abrogate the chemotactic response. In contrast to Ras overexpression, cells producing excess constitutively active Raf, a downstream effector of Ras, migrate toward PDGF-BB. Cells expressing dominant-negative Ras are able to migrate toward soluble fibronectin demonstrating that these cells retain the ability to migrate. These results suggest that Ras is an intermediate in PDGF-stimulated chemotaxis but may not be required for fibronectin-stimulated cell motility (Kundra, 1995).

Insulin and insulin-like growth factors (IGF-I and IGF-II) support the survival and differentiation of many types of neurons, including those from fetal chick forebrain. The mechanisms by which these peptides exert their neurotrophic actions are poorly understood. The aims of this study were to determine if insulin and IGF-I activate p21ras in fetal chick forebrain neurons and if activation of p21ras mediates the neurotrophic actions of these peptides. Activation of neuronal p21ras was examined by measuring the amount of GTP bound to p21ras before and after growth factor treatment. Insulin and IGF-I increase the ratio of GTP/GTP + GDP by 31% and 36%, respectively. p21Ras activation by insulin and IGF-I is maximal within 5 min. In the presence of insulin the response is sustained out to 180 min, whereas the response to IGF-I decreases significantly by 180 min. Both peptides stimulate p21ras at low concentrations indicating that insulin and IGF-I activate ras by interacting with their homologous receptor. Pretreatment of neurons with lovastatin, an inhibitor of ras isoprenylation, completely blocks the activation of p21ras by insulin and IGF-I. Lovastatin also blocks the ability of these growth factors to support the survival and differentiation of fetal chick neurons in culture. It is concluded that insulin and IGF-I activate p21ras in fetal chick forebrain neurons by increasing the amount of GTP bound to p21ras. The activation of neuronal p21ras is necessary for insulin and IGF-I to promote survival and differentiation in these neurons (Robinson, 1994).

The cytokines leukemia inhibitory factor (LIF) and ciliary neurotrophic factor (CNTF) have been implicated in determination of neuronal phenotype as well as promotion of neuronal survival. However, the intracellular mechanisms by which their signals are transduced remain poorly understood. In NBFL cells LIF increases activated Ras in a rapid, transient, and concentration-dependent manner. CNTF and a related cytokine, oncostatin M, produce similar increases. CNTF and LIF also increase activated Ras in neuron-enriched dissociated cultures of sympathetic ganglia. Moreover, these cytokines rapidly and transiently induce specific tyrosine-phosphorylated proteins, p165 and p195. In NBFL cells the protein kinase inhibitors K252a and staurosporine block LIF-induced increases in tyrosine phosphorylation, and block activated Ras. These data support a possible role for Ras in the cell differentiation effects of LIF and CNTF (Schwarzschild, 1994).

The Ras signal transduction pathway is activated by a number of hematopoietic cytokines and is implicated in the prevention of apoptotic death in hematopoietic cells. Recent studies have provided evidence that the downstream pathways of Ras are highly divergent; several independent pathways appear to mediate distinct biological functions of Ras. The downstream pathway(s) of Ras responsible for the maintenance of hematopoietic cell survival was examined by using various mutants of signaling molecules. Activation of the Raf/MAPK pathway in interleukin (IL) 3-dependent cells by expression of an oncogenic Raf or a Ras mutant (G12V/T35S) prevents apoptosis following IL-3 deprivation. In contrast, another Ras mutant (G12V/V45E), which is apparently incapable of activating MAPK, efficiently blocks apoptosis as well. It is therefore likely that the activation of the Raf/MAPK pathway is not an absolute requirement for the prevention of apoptosis, and there appears to be a Raf/MAPK-independent pathway requiring Ras that contributes to hematopoietic cell survival. Since Ras(G12V/V45E) is able to cause the phosphorylation of p70/S6 kinase, the S6 kinase pathway was inhibited by rapamycin and by wortmannin. The anti-apoptotic function of Ras(G12V/V45E), but not of Ras(G12V), is critically influenced by both inhibitors. These results indicate that the Raf/MAPK pathway and a rapamycin/wortmannin-sensitive pathway involving phosphorylation of p70S6 are both able mediate Ras function to prevent apoptotic death in hematopoietic cells (Kinoshita, 1997).

During vertebrate embryogenesis, the neuroectoderm differentiates into neural tissues and also into non-neural tissues such as the choroid plexus in the brain and the retinal pigment epithelium in the eye. The molecular mechanisms that pattern neural and non-neural tissues within the neuroectoderm remain unknown. FGF9 is normally expressed in the distal region of the optic vesicle that is destined to become the neural retina, suggesting a role in neural patterning in the optic neuroepithelium. Ectopic expression of FGF9 in the proximal region of the optic vesicle extends neural differentiation into the presumptive retinal pigment epithelium, resulting in a duplicate neural retina in transgenic mice. Ectopic expression of constitutively active Ras is also sufficient to convert the retinal pigment epithelium to neural retina, suggesting that Ras-mediated signaling may be involved in neural differentiation in the immature optic vesicle. The original and the duplicate neural retinae differentiate and laminate with mirror-image polarity in the absence of an RPE, suggesting that the program of neuronal differentiation in the retina is autonomously regulated. In mouse embryos lacking FGF9, the retinal pigment epithelium extends into the presumptive neural retina, indicating a role of FGF9 in defining the boundary of the neural retina (Zhao, 2001).

Ras and adenylyl cyclase

Posttranslational modification of Ras protein has been shown to be critical for interaction with its effector molecules, including S. cerevisiae adenylyl cyclase (See Drosophila Rutabaga). Lipid modification, specifically farnesylation, which accompanies the membrane attachment of Ras, stimulates the Ras-dependent activation of yeast adenylyl cyclase. The leucine rich repeat in the middle repetitive domain of adenyl cyclase interacts with Ras. In this study, a reconstituted system was used with purified adenylyl cyclase and Ras proteins carrying various degrees of the modification to show that the posttranslational modification, especially the farnesylation step, is responsible for a 5- to 10-fold increase in Ras-dependent activation of adenylyl cyclase activity even though it has no significant effect on binding of Ras and adenyl cyclase to one another. The stimulatory effect of farnesylation is found to depend on the association of adenylyl cyclase with 70-kDa adenylyl cyclase-associated protein (CAP) (which is known to be required for proper in vivo response of adenylyl cyclase to Ras protein), by comparing the levels of Ras-dependent activation of purified adenylyl cyclase with and without bound CAP. The region of CAP required for this effect is mapped to CAP's N-terminal segment of 168 amino acid residues, which coincides with the region required for the in vivo effect. The stimulatory effect is successfully reconstituted by in vitro association of CAP with the purified adenylyl cyclase. These results indicate that the association of adenylyl cyclase with CAP is responsible for the stimulatory effect of posttranslational modification of Ras on its activity. This may be the mechanism underlying its requirement for the proper in vivo cyclic AMP response (Shima, 1997).

PI3K is an effector of Ras

The observation that activated c-Ha-Ras p21 interacts with diverse protein ligands suggests the existence of mechanisms that regulate multiple interactions with Ras. This work studies the influence of the Ras effector c-Raf-1 on the action of guanine nucleotide exchange factors (GEFs) on Ha-Ras in vitro. Purified GEFs (the catalytic domain of yeast Sdc25p and the full-length and catalytic domain of mouse CDC25Mm) and the Ras binding domains (RBDs) of Raf-1 [Raf (1-149) and Raf (51-131)] were used. Not only the intrinsic GTP/GTP exchange on Ha-Ras but also the GEF-stimulated exchange is inhibited in a concentration-dependent manner by the RBDs of Raf. Conversely, the scintillation proximity assay, which monitors the effect of GEF on the Ras.Raf complex, shows that the binding of Raf and GEF to Ha-Ras.GTP is mutually exclusive. The various GEFs used yielded comparable results. It is noteworthy that under more physiological conditions mimicking the cellular GDP/GTP ratio, Raf enhances the GEF-stimulated GDP/GTP exchange on Ha-Ras, in agreement with the sequestration of Ras.GTP by Raf. Consistent with these results, the GEF-stimulated exchange of Ha-Ras.GTP is also inhibited by another effector of Ras, the RBD (amino acid residues 133-314) of phosphatidylinositol 3-kinase p110alpha (see Drosophila Pi3K2E). The data show that Raf-1 and phosphatidylinositol 3-kinase can influence the upstream activation of Ha-Ras. The interference between Ras effectors and GEF could be a regulatory mechanism to promote the activity of Ha-Ras in the cell (Giglione, 1998).

Ha-, N-, and Ki-Ras are ubiquitously expressed in mammalian cells and can all interact with the same set of effector proteins. However, in vivo there are marked quantitative differences in the ability of Ki- and Ha-Ras to activate Raf-1 and phosphoinositide 3-kinase. Thus, Ki-Ras both recruits Raf-1 to the plasma membrane more efficiently than Ha-Ras and is a more potent activator of membrane-recruited Raf-1 than Ha-Ras. In contrast, Ha-Ras is a more potent activator of phosphoinositide 3-kinase than Ki-Ras. Interestingly, the ability of Ha-Ras to recruit Raf-1 to the plasma membrane is significantly increased when the Ha-Ras hypervariable region is shortened so that the spacing of the Ha-Ras GTPase domains from the inner surface of the plasma membrane mimics the spacing of Ki-Ras. Importantly, these data show for the first time that the activation of different Ras isoforms can have distinct biochemical consequences for the cell. The mutation of specific Ras isoforms in different human tumors can, therefore, also be rationalized (Yan, 1998).

Ras activation of phosphoinositide 3-kinase gamma (PI3Kgamma) is important for survival of transformed cells. PI3Kgamma is strongly and directly activated by H-Ras G12V in vivo or by GTPS-loaded H-Ras in vitro. A crystal structure of a PI3Kgamma/Ras-GMPPNP complex has been determined. A critical loop in the Ras binding domain positions Ras so that it uses its switch I and switch II regions to bind PI3Kgamma. Mutagenesis shows that interactions with both regions are essential for binding PI3Kgamma. Ras also forms a direct contact with the PI3Kgamma catalytic domain. These unique Ras/PI3Kgamma interactions are likely to be shared by PI3Kalpha. The complex with Ras shows a change in the PI3Kgamma conformation that may represent an allosteric component of Ras activation (Pacold, 2000).

Type I phosphoinositide 3-kinases are responsible for the hormone-sensitive synthesis of the lipid messenger phosphatidylinositol(3,4,5)-trisphosphate. Type IA and IB subfamily members contain a Ras binding domain and are stimulated by activated Ras proteins both in vivo and in vitro. The mechanism of Ras activation of type I PI3Ks is unknown, in part because no robust in vitro assay of this event has been established and characterized. Other Ras effectors, such as Raf and phosphoinositide-phospholipase Cepsilon, have been shown to be translocated into the plasma membrane, leading to their activation. Posttranslationally lipid-modified, activated N-, H-, K-, and R-Ras proteins can potently and substantially activate PI3Kgamma when using a stripped neutrophil membrane fraction as a source of phospholipid substrate. GTPgammaS-loaded Ras can significantly (6- to 8-fold) activate PI3Kgamma when using artificial phospholipid vesicles containing their substrate, and this effect is a result of both a decrease in apparent Km for phosphatidylinositol(4,5)-bisphosphate and an increase in the apparent Vmax. However, neither in vivo nor in the two in vitro assays of Ras activation of PI3Kgamma could any evidence be detected of a Ras-dependent translocation of PI3Kgamma to its source of phospholipid substrate. These data suggest that Ras activates PI3Kgamma at the level of the membrane, by allosteric modulation and/or reorientation of the PI3Kgamma, implying that Ras can activate PI3Kgamma without its membrane translocation. This view is supported by structural work that has suggested binding of Ras to PI3Kgamma results in a change in the structure of the catalytic pocket (Suire, 2002).

Ras proteins signal through direct interaction with a number of effector enzymes, including type I phosphoinositide (PI) 3-kinases. Although the ability of Ras to control PI 3-kinase has been well established in manipulated cell culture models, evidence for a role of the interaction of endogenous Ras with PI 3-kinase in normal and malignant cell growth in vivo has been lacking. This study generated mice with mutations in the Pi3kca gene, encoding the catalytic p110α isoform, that block p110α interaction with Ras. Cells from these mice show proliferative defects and selective disruption of signaling from growth factors to PI 3-kinase. The mice display defective development of the lymphatic vasculature, resulting in perinatal appearance of chylous ascites. Most importantly, they are highly resistant to endogenous Ras oncogene-induced tumorigenesis. The interaction of Ras with p110α is thus required in vivo for certain normal growth factor signaling and for Ras-driven tumor formation (Gupta, 2007).

The creation of mice lacking the ability of their p110α PI 3-kinase catalytic subunit to interact with activated Ras provides an opportunity to definitively address the significance of this interaction in growth factor signaling both in vivo and in vitro. In cultured mouse embryo fibroblasts, loss of p110α binding to Ras strongly reduces PI 3-kinase activation by EGF and FGF-2, but not by PDGF. This differential requirement for Ras may reflect the fact that the activated receptor for PDGF directly binds the p85 regulatory subunit of PI 3-kinase at the plasma membrane, whereas the others do not. EGF receptor is thought to direct PI 3-kinase activation more indirectly, either via Gab1 and Grb2 or via ErbB3. In the case of FGF-2, its receptor phosphorylates the docking protein FRS2, which in turn binds Grb2 and Gab1. Where PI 3-kinase is recruited to receptor complexes indirectly, it is possible that smaller numbers of p110 molecules are activated, perhaps making a costimulatory role for Ras binding more essential. However, other considerations may also be involved, as insulin signaling to PI 3-kinase, which involves IRS family adaptors, does not appear to be majorly dependent on Ras interaction with p110α. Another possible explanation might be the extent to which particular growth factor receptors use different isoforms of p110. Deletion of p110α in mouse embryo fibroblasts results in more complete inhibition of EGF than PDGF signaling to Akt. It has been suggested that this might reflect an ability of PDGF, but not EGF, receptor to regulate p110β, which is also expressed in these cells (Gupta, 2007).

Mice have been made where a similar set of mutations has been introduced into a different isoform of PI 3-kinase, p110γ, whose expression is largely restricted to hematopoietic cells. This resulted in loss of accumulation of PIP3 in neutrophils in response to chemoattractants. In addition, flies with a similar mutation introduced into Dp110 show greatly reduced egg-laying ability and are small in size (Orme, 2006). This suggests that blocking Ras interaction with p110 can attenuate PI 3-kinase regulation in other systems as well (Gupta, 2007).

Interaction with Ras has been shown to allosterically activate PI 3-kinase via a change in the structure of the catalytic pocket, in a manner that is synergistic with binding of p85 to pYXXM peptides. By itself, the interaction of Ras with p110 is insufficient to drive membrane translocation of PI 3-kinase, a necessity for its activation, suggesting that the ability of oncogenic mutant Ras by itself to drive PI 3-kinase activation may be dependent on low-level signaling input from receptor tyrosine kinases, especially EGF receptor family members that respond to autocrine ligands produced in response to activation of the Raf/ERK branch of Ras downstream pathways (Gupta, 2007).

The phenotype of mice homozygous for the p110α RBD mutation suggests that in vivo there is also a requirement for the Ras/PI 3-kinase interaction for some growth factors to signal correctly. Most obviously, there is a partial failure and delay of development of the lymphatic system, resulting in the accumulation of chylous ascites in newborn pups. This developmental phenotype is similar to that of VEGF-C+/− mice and also to some extent angiopoietin 2 knockout mice. The sprouting of the first lymphatic vessels from embryonic veins appears to be highly dependent on VEGF-C signaling, with homozygous deletion of VEGF-C resulting in embryonic edema from E12.5, complete lack of lymphatic vasculature, and death in utero. The similarity of the phenotype of the VEGF-C heterozygotes and p110α RBD mutant homozygotes could suggest that failure of Ras to engage p110α directly might result in vivo in a roughly 50% reduction in the ability of VEGF-C to signal to a critical downstream effector system, such as PI 3-kinase/Akt. It has been demonstrated that VEGF-C promotes survival and proliferation of lymphatic endothelial cells in vitro and induces Akt and ERK activation. VEGF-C signals through two receptor tyrosine kinases, VEGFR2 and VEGFR3, of which VEGFR3 is critical for its role in control of lymphatic development. VEGFR3 makes a good candidate for a receptor that might require Ras to signal to PI 3-kinase as, like EGFR and FGFRs, it lacks good p85-binding motifs and presumably must engage the pathway indirectly. However, it is also possible that signaling through other lymphangiogenic growth factors and their receptors, such as angiopoeitin 2 and Tie2, could also be defective in the p110α RBD mutant mice (Gupta, 2007).

Ras and Protein kinase C

Expression of transforming Ha-Ras L61 in NIH3T3 cells causes profound morphological alterations that include a disassembly of actin stress fibers. The Ras-induced dissolution of actin stress fibers is blocked by the specific PKC inhibitor GF109203X at concentrations that inhibit the activity of the atypical aPKC isotypes lambda and zeta, whereas lower concentrations of the inhibitor that block conventional and novel PKC isotypes are ineffective. Coexpression of transforming Ha-Ras L61 with kinase-defective, dominant-negative (DN) mutants of aPKC-lambda and aPKC-zeta, as well as antisense constructs encoding RNA-directed against isotype-specific 5' sequences of the corresponding mRNA, abrogates the Ha-Ras-induced reorganization of the actin cytoskeleton. Expression of a kinase-defective, DN mutant of cPKC-alpha is unable to counteract Ras with regard to the dissolution of actin stress fibers. Transfection of cells with constructs encoding constitutively active (CA) mutants of atypical aPKC-lambda and aPKC-zeta lead to a disassembly of stress fibers, independent of oncogenic Ha-Ras. Coexpression of (DN) Rac-1 N17 and addition of the phosphatidylinositol 3'-kinase (PI3K) inhibitors wortmannin and LY294002 are in agreement with a tentative model suggesting that in the signaling pathway from Ha-Ras to the cytoskeleton aPKC-lambda acts upstream of PI3K and Rac-1, whereas aPKC-zeta functions downstream of PI3K and Rac-1. This model is supported by studies demonstrating that cotransfection with plasmids encoding L61Ras and either aPKC-lambda or aPKC-zeta results in a stimulation of the kinase activity of both enzymes. Furthermore, the Ras-mediated activation of PKC-zeta is abrogated by coexpression of DN Rac-1 N17 (Uberall, 1999).

A connection between Ras and Notch pathways

E47 (see Drosophila Daughterless) is a widely expressed transcription factor that activates B-cell-specific immunoglobulin gene transcription and is required for early B-cell development. In an effort to identify processes that regulate E47, and potentially B-cell development, it was found that activated Notch1 (see Drosophila Notch) and Notch2 effectively inhibit E47 activity. Only the intact E47 protein is inhibited by Notch. Fusion proteins containing isolated DNA binding and activation domains are unaffected. Although overexpression of the coactivator p300 partially reverses E47 inhibition, results of several assays indicate that p300/CBP is not a general target of Notch. Notch inhibition of E47 does not correlate with its ability to activate CBF1/RBP-Jkappa, the mammalian homolog of Suppressor of Hairless, a protein that associates physically with Notch and defines the only known Notch signaling pathway in Drosophila (Ordentlich, 1998).

E47 is inhibited by Deltex, a second Notch-interacting protein. Evidence is provided that Notch and Deltex may act on E47 by inhibiting signaling through Ras. The EGR-1 promoter (see Huckebein) is known to be stimulated by Ras through the action of mitogen-activated protein kinases (MAPKs) on a ternary complex involving ETS proteins (e.g., ELK1) and Serum response factor. The activity of a CAT reporter under the control of the EGR-1 promoter is inhibited by Deltex, both in the presence and in the absence of Ras stimulation by platelet-derived growth factor. To reduce the complexity of the effects, a series of GAL4 promoter fusions were used and their abilities to activate a minimal promoter containing GAL4 binding sites was assessed. GAL4-Jun includes a portion of the c-Jun protein whose activity is dependent on signaling from Ras to SAPK/JNK. A promoter fragment lacking the CBF1 interaction domain inhibits GAL4-Jun activity but has no effect on GAL4-CREB. Similarly, Deltex inhibits GAL4-Jun activity and has no effect on GAL4-CREB. Although it is likely that N2-IC and Deltex have somewhat different effects on cells, these results clearly show that both Notch and Deltex inhibit signaling by Ras, as measured by the ability to stimulate SAPK/JNK activity. It is proposed that this is the mechanism by which Notch and Deltex inhibit E47 (Ordentlich, 1998).

The coordination of signals from different pathways is important for cell fate specification during animal development. A novel mode of crosstalk between the epidermal growth factor receptor/Ras/mitogen-activated protein kinase cascade and the LIN-12/Notch pathway during Caenorhabditis elegans vulval development has been defined. Six vulval precursor cells (VPCs) are initially equivalent but adopt different fates as a result of an inductive signal mediated by the Ras pathway and a lateral signal mediated by the LIN-12/Notch pathway1. One consequence of activating Ras is a reduction of LIN-12 protein in P6.p, the VPC believed to be the source of the lateral signal. A 'downregulation targeting signal' (DTS) has been identified in the LIN-12 intracellular domain, which encompasses a di-leucine-containing endocytic sorting motif. The DTS seems to be required for internalization of LIN-12, and on Ras activation it might mediate altered endocytic routing of LIN-12, leading to downregulation. If LIN-12 is stabilized in P6.p, lateral signalling is compromised, indicating that LIN-12 downregulation is important in the appropriate specification of cell fates in vivo (Shaye, 2002).

The DTS contains two adjacent leucine residues and several serines that are conserved in all known nematode LIN-12/Notch proteins. 'Di-leucine motifs' are well-characterized sorting signals that usually take the form (-)(2-4)XLL, where (-) is often an acidic residue or phosphoserine, although basic residues have also been seen at this position, and X is usually a polar or bulky residue. Functional motifs that contain M, V or I instead of L have also been found. Di-leucine motifs regulate different aspects of protein trafficking, including the constitutive or ligand-stimulated internalization of transmembrane receptors, and the routing of proteins within the endocytic and/or secretory pathways. Different residues within the same di-leucine motif might modulate different aspects of motif activity (namely internalization versus routing), and the activity of di-leucine motifs that contain serines might be regulated by phosphorylation. In several cases the di-leucine motif leads to routing of the protein to lysosomes for degradation.Mutating the two leucine residues of the DTS to two alanines disrupts downregulation of sTM::LIN-12(intra)::GFP. Because downregulation of GFP-tagged LIN-12 fragments requires both membrane association and a di-leucine motif, it is inferred that the mechanism of downregulation involves increased internalization and/or altered endocytic routing of LIN-12 when Ras is activated (Shaye, 2002).

The gene sur-2 encodes a component of the Mediator complex, which activates transcription in response to Ras/MAPK signalling in mammalian cells. SUR-2 also seems to be activated by the Ras pathway in C. elegans, and hermaphrodites lacking sur-2 activity display a failure in lateral signalling. In sur-2(-) hermaphrodites, LIN-12(+)::GFP is not downregulated. This result suggests that the failure of lateral signalling in sur-2(-) hermaphrodites might result at least in part from the failure to downregulate endogenous LIN-12(+). Furthermore, in sur-2(-) hermaphrodites, as in the wild type, LIN-12(+)::GFP accumulates in puncta, suggesting that SUR-2/Mediator-promoted transcription is not necessary for the initial internalization, but instead might affect the rate of internalization and/or routing of endocytosed LIN-12(+)::GFP (Shaye, 2002).

A model is presented for cross-talk between the Ras and LIN-12 pathways in P6.p. LIN-12, like other transmembrane proteins, seems to be constitutively internalized and might be routed to recycling endosomes or to lysosomes. It is proposed that Ras activation leads to transcription of at least one gene whose product regulates the rate of internalization and/or subcellular routing of LIN-12, leading to its degradation. Internalization (and perhaps, in addition, endocytic routing) of LIN-12 is mediated by the DTS: when the DTS is removed, LIN-12 accumulates in the apical membrane of P6.p. Persistence of LIN-12, achieved by deleting the DTS or by removing sur-2 activity, is correlated with inhibition of lateral signalling, demonstrating that downregulation is functionally important (Shaye, 2002).

The novel mode of crosstalk between the Ras and LIN-12/Notch pathways may be conserved. Vertebrate Notch proteins contain a highly conserved potential di-leucine-like motif (1822KKFRFEEPVVL1832 in human Notch1) that, like the LIN-12 DTS, lies between the transmembrane domain and the first ankyrin motif, and furthermore the large intracellular domain of notch proteins contains additional potential endocytic motifs. Whether these motifs function as such, and whether their function is regulated by Ras or other signals, can only be answered through experiments in other systems. Observations of Wingless endocytosis has led to the speculation that Ras might modulate the endocytic routing of Wingless bound to its receptor Frizzled2 via a di-leucine motif on Frizzled2, dependent on the transcription of an unknown factor in response to epidermal growth factor receptor/Ras/MAPK signalling. If this proves to be so, the future identification of the putative factors that recognize the targeting signals of LIN-12/Notch and Frizzled2 will reveal whether Ras works through a common or distinct mechanism to modulate the activity of these different receptors (Shaye, 2002).

A connection between Ras and Rho pathways

Rho family GTPases act as molecular switches to control a variety of cellular responses, including cytoskeletal rearrangements, changes in gene expression, and cell transformation. In the active, GTP-bound state, Rho interacts with an ever-growing number of effector molecules, which promote distinct biochemical pathways. This study describes the isolation of hCNK1, the human homologue of Drosophila connector enhancer of ksr, as an effector for Rho. hCNK1 contains several protein-protein interaction domains, and Rho interacts with one of these, the PH domain, in a GTP-dependent manner. A mutant hCNK1, which is unable to bind to Rho, or depletion of endogenous hCNK1 by using RNA interference inhibits Rho-induced gene expression via serum response factor but has no apparent effect on Rho-induced stress fiber formation, suggesting that it acts as a specific effector for transcriptional, but not cytoskeletal, activation pathways. Finally, hCNK1 associates with Rhophilin and RalGDS, Rho and Ras effector molecules, respectively, suggesting that it acts as a scaffold protein to mediate cross talk between the two pathways (Jaffe, 2004).

Ras and epigenetic silencing

The conversion of a normal cell to a cancer cell occurs in several steps and typically involves the activation of oncogenes and the inactivation of tumour suppressor and pro-apoptotic genes1. In many instances, inactivation of genes critical for cancer development occurs by epigenetic silencing, often involving hypermethylation of CpG-rich promoter regions. It remains to be determined whether silencing occurs by random acquisition of epigenetic marks that confer a selective growth advantage or through a specific pathway initiated by an oncogene. This study performed a genome-wide RNA interference (RNAi) screen in K-ras-transformed NIH 3T3 cells and identify 28 genes required for Ras-mediated epigenetic silencing of the pro-apoptotic Fas gene. At least nine of these RESEs (Ras epigenetic silencing effectors), including the DNA methyltransferase DNMT1, are directly associated with specific regions of the Fas promoter in K-ras-transformed NIH 3T3 cells but not in untransformed NIH 3T3 cells. RNAi-mediated knockdown of any of the 28 RESEs results in failure to recruit DNMT1 to the Fas promoter, loss of Fas promoter hypermethylation, and derepression of Fas expression. Analysis of five other epigenetically repressed genes indicates that Ras directs the silencing of multiple unrelated genes through a largely common pathway. Last, it was shown that nine RESEs are required for anchorage-independent growth and tumorigenicity of K-ras-transformed NIH 3T3 cells; these nine genes have not previously been implicated in transformation by Ras. These results show that Ras-mediated epigenetic silencing occurs through a specific, complex, pathway involving components that are required for maintenance of a fully transformed phenotype (Gazin, 2007).

Evolutionary homologs: Table of contents

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

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