Ras oncogene at 85D


EVOLUTIONARY HOMOLOGS

Table of contents

Ras and differentiation pathways

RasD, a Dictyostelium homolog of mammalian Ras, is maximally expressed during the multicellular stage of development. Normal Dictyostelium aggregates are phototactic and thermotactic, moving towards sources of light and heat with great sensitivity. Disruption of the gene for rasD causes a near-total loss of phototaxis and thermotaxis in mutant aggregates, without obvious effects on undirected movement. Previous experiments had suggested important roles for RasD in development and cell-type determination. Surprisingly, rasD- cells show no obvious changes in these processes. These cells represent a novel class of phototaxis mutant, and indicate a role for a Ras pathway in the connections between stimuli and coordinated cell movement. Expression of a second Ras protein, RasD, containing an activating mutation (G12T) has a profound effect on development. Although cells aggregated normally, the mounds that form are multitipped and unable to develop further. In addition, pattern formation is markedly disrupted, with prestalk gene expression greatly enhanced and prespore gene expression greatly reduced (Wilkins, 2000).

In C. elegans, the formation of the hermaphrodite vulva is induced by an RTK/Ras signaling pathway. The vulva is generated from six multipotent ventral ectodermal blast cells, P3.p-P8.p. Each of these six P(3-8).p cells can potentially adopt either the 1° vulval cell fate, the 2° vulval cell fate, or the 3° nonvulval cell fate. During wild-type development, a signal from the gonadal anchor cell induces the nearest P(3-8).p cell, P6.p, to adopt the 1° fate and the adjacent P5.p and P7.p cells to adopt the 2° fate. The cells furthest from the anchor cell, P3.p, P4.p, and P8.p, adopt the uninduced 3° fate. Vulval induction acts through a signaling pathway, which includes an EGF-like ligand; a receptor tyrosine kinase, Ras and MAP kinase, to regulate the activities of the ETS transcription factor LIN-1 and the winged-helix transcription factor LIN-31 (Lu, 1998 and references).

Vulval induction is negatively regulated by the synthetic multivulva (synMuv) genes. Loss-of-function mutations in these genes result in a multivulva (Muv) phenotype as a consequence of the expression of vulval cell fates by the P3.p, P4.p, and P8.p cells. The Muv phenotype of these mutants requires mutations in two genes. Specifically, these synMuv mutations fall into two classes, referred to as A and B. Animals carrying a class A and a class B mutation have a Muv phenotype, while animals carrying one or more mutations of the same class have a wild-type vulval phenotype. These mutations appear to define two functionally redundant pathways that negatively regulate the expression of vulval cell fates (Lu, 1998).

Four class A genes (lin-8, lin-15A, lin-38, and lin-56) and ten class B genes (lin-9, lin-15B, lin-35, lin-36, lin-37, lin-51, lin-52, lin-53, lin-54, and lin-55) have been identified. lin-15 encodes both A and B activities in two nonoverlapping transcripts. lin-15A, lin-15B, lin-9, and lin-36 encode novel proteins. Two genes, an Rb related protein and its binding partner, have been characterized in one of these pathways. lin-35 encodes a protein similar to the tumor suppressor Rb and the closely related proteins p107 and p130. lin-53 encodes a protein similar to RbAp48, a mammalian protein that binds Rb. In mammals, Rb and related proteins act as regulators of E2F transcription factors, and RbAp48 may act with such proteins as a transcriptional corepressor. It is proposed that LIN-35 and LIN-53 antagonize the Ras signaling pathway in C. elegans by repressing transcription in the vulval precursor cells of genes required for the expression of vulval cell fates (Lu, 1998).

It is proposed that the class B synMuv genes inhibit vulval induction by a conserved mechanism: LIN-35 Rb forms a complex with a sequence-specific transcription factor, presumably an E2F-like protein, and recruits a corepressor complex containing HDA-1, LIN-53 p48, and other proteins to turn off the transcription of vulval specification genes via E2F-binding sites. In the wild type, in the P3.p, P4.p, and P8.p cells, synMuv gene activity antagonizes the basal activity of the RTK/Ras pathway by repressing transcription of vulval genes. As a result, those cells adopt the nonvulval 3° fate. However, in P5.p, P6.p, and P7.p the antagonistic effect of synMuv gene activity is inactivated or can be overcome by the activated RTK/Ras pathway, thereby releasing transcriptional repression and permitting the expression of vulval fates. In the P(3-8).p cells of a synMuv mutant, repression cannot occur and all six P(3-8).p cells express vulval fates, resulting in a Muv phenotype. The synMuv genes do not appear to exert their effects by regulating cell cycle progression of the P(3-8).p cells, since all six of these cells have very similar cell cycle profiles. The synMuv genes must act genetically upstream of or in parallel to the Ras pathway. Action in parallel would be consistent with recent findings from studies of mammalian cells: dominant-negative Ras and Ras neutralizing antibodies induce an Rb-dependent block in DNA synthesis and G1 arrest, suggesting that Rb functions to inhibit mitogenesis downstream of or in parallel to Ras (Lu, 1998 and references).

Directed cell rearrangements occur during gastrulation, neurulation, and organ formation. Despite the identification of developmental processes in which invagination is a critical component of pattern formation, little is known regarding the underlying cellular and molecular details. C. elegans vulval epithelial cells undergo morphological changes that generate an invagination through the formation of seven stacked rings. The dynamics of ring formation during multivulva morphogenesis of a let-60/ras gain-of-function mutant has been studied as a model system to explore the cellular mechanisms that drive invagination. Using three-dimensional confocal microscopy, the behavior of individual cells was analyzed in a let-60/ras mutant. Stereotyped cell fusion events occur within the rings that form functional and nonfunctional vulvae in a let-60/ras mutant. Expression of let-60/ras gain-of-function results in abnormal cell migration, ectopic cell fusion, and structural fate transformation. Gain-of-function mutations involve the adoption of primary, secondary and tertiary sublineages by vulval precursor cells that normally adopt only the tertiary sublineage and consequently result in a multivulva (Muv) phenotype, in which additional vulva-like structures are generated independently of the presence of the gonadal somatic anchor cell. Within each developing vulva, the anterior and posterior halves develop autonomously. Contrary to prevailing hypotheses which have proposed three cell fates (primary, secondary and tertiary), it was found that each of the seven rings that make up the vulva is a product of a discrete structural pathway that is derived from arrays of seven distinct cell fates. Autonomous ring formation is the morphogenetic force that drives invagination of the vulva (Shemer, 2000).

Three observations indicate that Ras may have a potential role during the entire organogenesis of the vulva in cell fusion and cell migration. (1) In the background of a ras (gf) mutation, at least some of the cells (the daughters of A in the real vulva) have acquired the ability to fuse to the surrounding hypodermis. The early inductive function of Ras could not contribute to this phenomenon since the lineage of these cells is normal. Rather, this mutation affects the cell fate of A. (2) In the let-60/ras (gf) mutant there are abnormal filopodial extensions of A- and B-derived cells. (3) The structurally differentiated state transformation described may be related to the Ras pathway since Ras is normally expressed in all the primordial cells after the execution of the sublineage and throughout vulva formation. Based on these results it is proposed that Ras and other signal transduction processes are involved in late cellular events (e.g., filopodial extensions and cell fusion) required to form a stack of rings that drive invagination (Shemer, 2000).

In Caenorhabditis elegans, Ras/ERK and Wnt/ß-catenin signaling pathways cooperate to induce P12 and vulval cell fates in a Hox-dependent manner. eor-1 and eor-2, two new positively acting nuclear components of the Ras and Wnt pathways, are described. eor-1 and eor-2 act downstream or in parallel to ERK and function redundantly with the Mediator complex gene sur-2 and the functionally related gene lin-25, such that removal of both eor-1/eor-2 and sur-2/lin-25 mimics the removal of a main Ras pathway component. Furthermore, the eor-1 and eor-2 mutant backgrounds reveal an essential role for the Elk1-related gene lin-1. eor-1 and eor-2 also act downstream or in parallel to pry-1 Axin and therefore act at the convergence of the Ras and Wnt pathways. eor-1 encodes the ortholog of Drosophila Tramtrack and human PLZF, a BTB/zinc-finger transcription factor that is fused to RARalpha in acute promyelocytic leukemia. eor-2 encodes a novel protein. EOR-1/PLZF and EOR-2 appear to function closely together and cooperate with Hox genes to promote the expression of Ras- and Wnt-responsive genes. Further studies of eor-1 and eor-2 may provide insight into the roles of PLZF in normal development and leukemogenesis (Howard, 2002).

Chromatin-modifying complexes are important for transcriptional control, but their roles in the regulation of development are poorly understood. Components of the nucleosome remodelling and histone deacetylase (NURD) complex antagonize vulval development, which is induced by the Ras signal transduction pathway. In three of the six equivalent vulval precursor cells, the Ras pathway is active, leading to the production of vulval fates; in the remaining three, the Ras pathway is inhibited and vulval fates repressed. Inhibition of Ras signaling occurs in part through the action of the synthetic multivulval (synMuv) genes, which comprise two functionally redundant pathways (synMuvA and synMuvB). Five C. elegans members of the NURD chromatin remodelling complex inhibit vulval development through both the synMuvA and synMuvB pathways [hda-1, rba-1 (a Caf-1 homolog), lin-53 (another Caf-1 homolog), chd-3 (an Mi-2 homolog) and chd-4]; another two members, the MTA1-related genes egr-1 and egl-27, act only in the synMuvA pathway. It is proposed that the synMuvA and synMuvB pathways function redundantly to recruit or activate a core NURD complex, which then represses vulval developmental target genes by local histone deacetylation. These results emphasise the importance of chromatin regulation in developmental decisions. Furthermore, inhibition of Ras signaling suggests a possible link between NURD function and cancer (Solari, 2000).

Ras-mediated signaling is necessary for the induction of vulval cell fates during C. elegans development. cgr-1 was identified by screening for suppressors of the ectopic vulval cell fates caused by a gain-of-function mutation of the let-60 ras gene. Analysis of two cgr-1 loss-of-function mutations indicates that cgr-1 positively regulates induction of vulval cell fates. cgr-1 is likely to function at a step in the Ras signaling pathway that is downstream of let-60, which encodes Ras, and upstream of lin-1, which encodes a transcription factor, if these genes function in a linear signaling pathway. These genetic studies are also consistent with the model that cgr-1 functions in a parallel pathway that promotes vulval cell fates. Localized expression studies suggest that cgr-1 functions cell autonomously to affect vulval cell fates. cgr-1 also functions early in development, since cgr-1 is necessary for larval viability. CGR-1 contains a CRAL/TRIO domain likely to bind a small hydrophobic ligand and a GOLD domain that may mediate interactions with proteins. A bioinformatic analysis revealed that there is a conserved family of CRAL/TRIO and GOLD domain-containing proteins that includes members from vertebrates and Drosophila. The analysis of cgr-1 identifies a novel in vivo function for a member of this family and a potential new regulator of Ras-mediated signaling (Goldstein, 2006).

The Mi-2 protein is the central component of the recently isolated NuRD nucleosome remodelling and histone deacetylase complex. Although the NuRD complex has been the subject of extensive biochemical analyses, little is known about its biological function. The two C. elegans Mi-2 homologs, LET-418 and CHD-3, play essential roles during development. The two proteins possess both shared and unique functions during vulval cell fate determination, including antagonizm of the Ras signaling pathway required for vulval cell fate induction and the proper execution of the 2° cell fate of vulval precursor cells, a process under the control of LIN-12 Notch signaling. One of the C. elegans Mi-2 homologs, LET-418, plays a role in antagonizing the RTK/Ras/MAP kinase pathway via the synthetic multivulva (synMuv) pathway, supporting the recently proposed link between chromatin remodelling by NuRD-like complexes and the Ras signaling pathway. LET-418 and CHD-3 appear to have a shared role in the proper execution of the LIN-12 Notch dependent 2° cell fate of the P5.p and P7.p vulval precursor cells (von Zelewsky, 2000).

Mesoderm induction is a critical early step in vertebrate development, involving changes in gene expression and morphogenesis. In Xenopus, normal mesoderm formation depends on signalling through the fibroblast growth factor (FGF) tyrosine kinase receptor. One important signalling pathway from receptor tyrosine kinases involves p21ras. Ras associates with the serine kinase c-Raf-1 in a GTP-dependent manner, and this complex phosphorylates and activates MAPK/ERK kinase (MEK), a protein kinase with dual specificity. MEK then activates p42mapk and (at least in mammals) p44mapk, both members of the mitogen-activated protein (MAP) kinase family. FGF activates MAP kinase during mesoderm induction: the use of dominant-negative constructs suggests that mesoderm induction by FGF requires both Ras and Raf. However, these experiments do not reveal whether Ras and Raf do act through MAP kinase to induce mesoderm or whether another pathway, such as the phosphatidylinositol 3-kinase cascade, is involved. It is shown that expression of active forms of MEK or of MAP kinase induces ventral mesoderm of the kind elicited by FGF. Overexpression of a Xenopus MAP kinase phosphatase blocks mesoderm induction by FGF, and causes characteristic defects in mesoderm formation in intact embryos, whereas inhibition of the P13 kinase and p70 S6 kinase pathways has no effect on mesoderm induction by FGF. FGF induces different types of mesoderm in a dose-dependent manner; strikingly, this is mimicked by expressing different levels of activated MEK. Together, these experiments demonstrate that activation of MAP kinases is necessary and sufficient for mesoderm formation (Umbhauer, 1995).

To clarify the role of K-Ras in vivo, K-ras mutant mice were generated by gene targeting. In contrast to the findings that H-Ras-deficient mice and N-Ras-deficient mice are born and grow normally, the K-Ras-deficient embryos die progressively between embryonic day 12.5 and term. At embryonic day 15.5, their ventricular walls are extremely thin. Earlier, at embryonic day 11.5, they demonstrate increased cell death of motoneurons in the medulla and the cervical spinal cord. These results indicate K-Ras is essential for normal development in mice; therefore, residual Ras composed of H-Ras and N-Ras cannot compensate for the loss of K-Ras function in the mutant mice (Koera, 1997).

The transcription factor serum response factor (SRF), a phylogenetically conserved nuclear protein, mediates the rapid transcriptional response to extracellular stimuli, e.g. growth and differentiation signals. Complexes between DNA and protein, that contain SRF or its homologs, function as nuclear targets of the Ras/MAPK signaling network, thereby directing gene activities associated with processes as diverse as pheromone signaling, cell-cycle progression (transitions G0-G1 and G2-M), neuronal synaptic transmission and muscle cell differentiation. So far, the activity of mammalian SRF has been studied exclusively in cultured cells. To study SRF function in a multicellular organism, a Srf null allele was generated in mice. SRF-deficient embryos (Srf -/-) have a severe gastrulation defect and do not develop to term. They consist of misfolded ectodermal and endodermal cell layers, do not form a primitive streak or any detectable mesodermal cells and fail to express the developmental marker genes Bra (T), Bmp-2/4 and Shh. Activation of the SRF-regulated immediate early genes Egr-1 and c-fos, as well as the alpha-Actin gene, is severely impaired. This study identifies SRF as a new and essential regulator of mammalian mesoderm formation. It is therefore suggested that in mammals Ras/MAPK signaling contributes to mesoderm induction, as is the case in amphibia (Arsenian, 1998).

The S. cerevisiae 14-3-3 homologs BMH1 and BMH2 (See Drosophila Leonardo) are not essential for viability or mating-related MAPK cascade signaling, but they are essential for pseudohyphal-development MAPK cascade signaling and other processes. Activated alleles of RAS2 and CDC42 induce pseudohyphal development and MAPK cascade signaling in Bmh+ strains, but not in ste20 (p65PAK) or bmh1 bmh2 mutant strains. Moreover, Bmh1p and Bmh2p associate with Ste20p in vivo. Three alleles of BMH1 encode proteins defective for MAPK cascade signaling and association with Ste20p, yet these alleles complement other 14-3-3 functions. Therefore, the 14-3-3 proteins are specifically required for RAS/MAPK cascade signaling during pseudohyphal development in S. cerevisiae. Ras2, CDC42, 14-3-3 proteins and p65PAK are required for cell elongation that takes place during pseudohyphal development, but the MAPK cascade is not required for cell elongation (Roberts, 1997).

In ascidian embryos, inductive interactions are necessary for the fate specification of notochord cells. Previous studies have shown that notochord induction occurs at the 32-cell stage and that basic fibroblast growth factor (bFGF) has notochord-inducing activity in ascidian embryos. In vertebrates, it is known that bFGF receptors have tyrosine kinase domain; the signaling pathway is mediated by the small-GTP binding protein, Ras. To study the role of Ras in ascidian embryos, dominant negative Ras (RasN17) was injected into fertilized eggs. RasN17 inhibits the formation of notochord, suggesting that the Ras signaling pathway is involved in signal transduction in the induction of notochord cells. When the presumptive-notochord (A6.2) blastomere is co-isolated with the inducer (A6.1) blastomere and then RasN17 is injected into the A6.2 blastomere, notochord differentiation is suppressed. The presumptive-notochord blastomeres injected with RasN17 were treated with bFGF. Many of them fail to develop notochord-specific features. Next to be examined was the effect of injecting constitutively active Ras (RasV12) into the A6.2 blastomeres. However, microinjection of RasV12 into these cells does not bypass notochord induction. These results suggest that the Ras signaling pathway is essential for the formation of notochord and that another signaling pathway must also be activated simultaneously in notochord formation during ascidian embryogenesis (Nakatani, 1997).

Experiments with mammalian tissue culture cells have implicated the small GTPase Ras in the control of cellular proliferation. Evidence is presented here that this is not the case for a living animal, the nematode Caenorhabditis elegans: proliferation late in embryogenesis and throughout the four larval stages is not noticeably affected in animals lacking Ras in various parts of their cell lineages. Instead, genetic mosaic analysis of the let-60 gene suggests that Ras is only required (at least later in development, since a maternal effect cannot be excluded) for establishment of a few temporally and spatially distinct cell fates. Only one of these, the duct cell fate, appears to be essential for viability (Yochem, 1997).

The let-60 ras gene of Caenorhabditis elegans is one of the key elements in a signal transduction pathway that controls the choice between vulval and epidermal differentiation in response to extracellular signals. To identify components acting downstream of let-60 ras in the vulval signaling pathway, a reduction-of-function mutation in the sur-1 gene has been identified that completely suppresses the multivulva phenotype of a hyperactive let-60 ras mutation. About 10% of animals homozygous for the sur-1 mutation also display a specific and intriguing vulval cell lineage defect. In addition, the sur-1 mutation results in a cold-sensitive egg-laying defective phenotype and a partial larval lethal phenotype. The sur-1 gene encodes a protein similar in overall structure to Drosophila and mammalian MAP kinases (See Drosophila Rolled). The functional homology between Sur-1 MAP kinase and mammalian MAP kinases is also demonstrated by the ability of a rat ERK2 kinase to rescue the sur-1 mutant phenotypes. Genetic double-mutant analyses place sur-1 downstream of let-60 ras but upstream of lin-1 in the vulval signaling pathway. These results provide further evidence for the extreme conservation of the Ras-mediated signaling pathway between worms and humans and for the function of MAP kinases in cell signaling processes that control cell differentiation and animal development (Wu, 1994).

The let-60 ras gene of C. elegans is required for multiple aspects of development. The vulvar differentiation pathway is the most intensively studied of these, but the ras pathway has now been shown to also be essential for male spicule development. Using vulval differentiation, molecular genetic techniques are now being used to study structure/function relationships of particular signaling components and to identify new positively and negatively acting proteins of Ras-mediated signaling pathways. Mutations affecting LET-23, a receptor tyrosine kinase homolog, which cause tissue-specific defects, have been localized to the carboxyl terminus. SH2 domain specificity has been analyzed through Src/SEM-5 chimeric proteins in transgenic nematodes. A mitogen-activated protein kinase that acts downstream of LET-60 Ras in vulval differentiation has been identified. Negative regulatory genes have been cloned and found to encode novel proteins and a clathrin adaptor protein (Kayne, 1995).

The Ras signaling pathway specifies a variety of cell fates in many organisms. However, little is known about the genes that function downstream of the conserved signaling cassette, or what imparts the specificity necessary to cause Ras activation to trigger different responses in different tissues. In C. elegans, activation of the Ras pathway induces cells in the central body region to generate the vulva. Vulval induction takes place in the domain of the Hox gene lin-39, a homolog of Drosophila Sexcombs reduced. lin-39 is absolutely required for Ras signaling to induce vulval development. During vulval induction, the Ras pathway, together with basal lin-39 activity, up-regulates lin-39 expression in vulval precursor cells. If lin-39 function is absent at this time, no vulval cell divisions occur. If lin-39 is replaced with the posterior Hox gene mab-5, then posterior structures are induced instead of a vulva (Maloof, 1998).

Animal lacking lin-1, which encodes an ETS-like transcription factor (see Drosophila Pointed), have a multivulva phenotype: in lin-1 mutants, all the vulval precursor cells generate vulval lineages in an anchor-cell independent fashion. Because lin-39 increases in lin-1 mutants, it seems likely that Lin-39 protein acts downstream of lin-1, and thus is required for the multivulva phenotype of lin-1. Lin39 alone cannot trigger vulval development. Thus, the Ras pathway must have other functions in vulval development in addition to inducing lin-39 expression These findings suggest that in addition to permitting vulval cell divisions to occur, lin-39 is also required to specify the outcome of Ras signaling by selectively activating vulva-specific genes (Maloof, 1998).

Fibroblast growth factor (FGF) has been proposed to be involved in the specification and patterning of the developing vertebrate nervous system. There is conflicting evidence, however, concerning the requirement for FGF signaling in these processes. To provide insight into the signaling mechanisms that are important for neural induction and anterior-posterior neural patterning, the dominant negative Ras mutant, N17Ras, was employed, in addition to a truncated FGF receptor (XFD). Both N17Ras and XFD, when expressed in Xenopus laevis animal cap ectoderm, inhibit the ability of FGF to generate neural pattern. They also block induction of posterior neural tissue by XBF2 and XMeis3. However, neither XFD nor N17Ras inhibits noggin, neurogenin, or XBF2 induction of anterior neural markers. MAP kinase activation has been proposed to be necessary for neural induction, yet N17Ras inhibits the phosphorylation of MAP kinase that usually follows explantation of explants. In whole embryos, Ras-mediated FGF signaling is critical for the formation of posterior neural tissues but is dispensable for neural induction (Ribisi, 2000).

Posterior mesoderm tissue induces midbrain and hindbrain fates from prospective forebrain, an activity that is mimicked in explant culture by bFGF. Treatment of early gastrula age animal cap ectoderm with bFGF protein induces the expression of the spinal cord marker hoxB9. Late gastrula-age (stage 11) animal cap ectoderm treated with bFGF expresses the midbrain and hindbrain marker genes en2 and krox20, in addition to hoxB9, and the forebrain marker otx2 is not induced. The combination of somite tissue with animal caps of gastrula age (stage 10.5) induces the expression of hindbrain-specific genes and low levels of spinal cord-specific genes in the animal cap tissue and this induction is partially sensitive to XFD. Keller explants faithfully recapitulate the A-P distribution of neural markers observed in the whole embryo: blocking FGF signaling using XFD eliminates posterior neural development in Keller explants. The claim that FGF signaling is required for the formation of posterior neural tissue is supported by the results of explant assays. The induction of posterior neural markers requires FGF and Ras signaling. Animal caps do not express posterior neural markers in response to either XBF2 or XMeis3 when either FGF or Ras signaling is blocked. In addition, when MAPK activation is directly inhibited by MAP kinase phosphatase, the ability of XMeis3 to induce the expression of posterior neural markers is greatly curtailed (Ribisi, 2000 and references therein).

Mammalian ras genes are thought to be critical in the regulation of cellular proliferation and differentiation; they are mutated in approximately 30% of all human tumors. However, N-ras and H-ras are nonessential for mouse development. To characterize the normal role of K-ras in growth and development, it has been mutated by gene targeting in the mouse. On an inbred genetic background, embryos homozygous for this mutation die between 12 and 14 days of gestation, with fetal liver defects and evidence of anemia. Thus, K-ras is the only member of the ras gene family essential for mouse embryogenesis. The effect of multiple mutations within the ras gene family has also been investigated. Most animals lacking N-ras function and heterozygous for the K-ras mutation exhibit abnormal hematopoietic development and die between days 10 and 12 of embryogenesis. Thus, partial functional overlap appears to occur within the ras gene family, but K-ras provides a unique and essential function (Johnson, 1997).

The products (p21) of the three mammalian H-, N- and K-ras genes play important roles in intracellular signal transduction, linking membrane receptor kinases to the nuclear pathway through raf and mitogen activated protein kinase. They are involved in the regulation of proliferation and differentiation; activating mutations of these genes are commonly associated with human cancers. Two p21 proteins are encoded by the K-ras gene (p21K-rasA and p21K-rasB) due to alternative splicing of the last exon. While the four p21ras proteins are highly homologous, their sequences diverge significantly at the C-termini, to which distinct biochemical and perhaps even functional differences may be ascribed. However, H-, N- and K-rasB appear to be ubiquitously expressed, with little evidence of tissue-specific or developmental regulation. In contrast, the expression of K-rasA is strikingly different. K-rasA is induced during differentiation of pluripotent embryonal stem cells in vitro. Its expression during early embryogenesis is limited temporally and spatially in a tissue-specific distribution that is largely maintained as an adult. This suggests a distinct biological role for p21K-rasA (Pells, 1997).

The ability of basic helix-loop-helix muscle regulatory factors (MRFs) such as MyoD (Drosophila homolog: Nautilus) to convert nonmuscle cells to a myogenic lineage is regulated by numerous growth factor and oncoprotein signaling pathways. Previous studies have shown that H-Ras 12V inhibits differentiation to a skeletal muscle lineage by disrupting MRF function via a mechanism that is independent of the dimerization, DNA binding, and inherent transcriptional activation properties of the proteins. To investigate the intracellular signaling pathway(s) that mediates the inhibition of MRF-induced myogenesis by oncogenic Ras, two transformation-defective H-Ras 12V effector domain variants were tested for their ability to alter terminal differentiation. H-Ras 12V,35S retains the ability to activate the Raf/MEK/mitogen-activated protein (MAP) kinase cascade, whereas H-Ras 12V,40C is unable to interact directly with Raf-1 yet still influences other signaling intermediates, including Rac and Rho. Expression of each H-Ras 12V variant in C3H10T1/2 cells abrogates MyoD-induced activation of the complete myogenic program, suggesting that MAP kinase-dependent and -independent Ras signaling pathways individually block myogenesis in this model system. However, additional studies with constitutively activated Rac1 and RhoA proteins reveal no negative effects on MyoD-induced myogenesis. Similarly, treatment of Ras-inhibited myoblasts with the MEK1 inhibitor PD98059 reveal that elevated MAP kinase activity is not a significant contributor to the H-Ras 12V effect. These data suggest that an additional Ras pathway, distinct from the well-characterized MAP kinase and Rac/Rho pathways known to be important for the transforming function of activated Ras, is primarily responsible for the inhibition of myogenesis by H-Ras 12V (Ramocki, 1997).

Cell fate commitment in a variety of lineages requires signals conveyed via p21(ras). To examine the role of p21(ras) in the development of B lymphocytes, transgenic mice were generated expressing a dominant-negative form of Ras in B lymphocyte progenitors, using a novel transcriptional element consisting of an enhancer and the lck proximal promoter. Expression of dominant-negative Ras arrests B cell development at a very early stage, prior to formation of the pre-B cell receptor. An activated form of Raf expressed in the same experimental system can both drive the maturation of normal pro-B cells and rescue development of progenitors expressing dominant-negative Ras. Hence p21(ras) normally regulates early development of B lymphocytes by a mechanism that involves activation of the serine/threonine kinase Raf (Iritani, 1997).

To investigate the functional relationship between the transforming ability of Ras and its role as an integral component of the differentiative insulin signaling pathway, a leu61-activated ras gene was introduced into a Ras-transformable, C3H10T1/2-derived preadipocytic cell line. The results demonstrate that rasleu61 expression in this line blocks differentiation and that this block appears at lower levels than required for full neoplastic transformation. In addition, to examine whether the inability of Rasleu61 to induce differentiation by replacing the insulin signal could be attributed to its transforming effect in this system, the effect of Rasleu61 was examined at levels below the baseline, by expressing rasleu61 in a series of preadipocytes that were rendered deficient in endogenous c-Ras activity. Even very low Rasleu61 levels, insufficient to restore the growth rate of these cells to normal, block rather than enhance differentiation, indicating that rasleu61 expression alone is not sufficient to promote adipocytic differentiation in this system, even in the absence of neoplastic transformation. Consistent with its established role as a downstream effector of Ras, v-Raf expression mirrors the v-Ras effects upon adipocytic differentiation and transformation (Raptis, 1997).

To evaluate the role of mitogen-activated protein (MAP) kinase and other signaling pathways in neuronal cell differentiation by basic fibroblast-derived growth factor (bFGF), a conditionally immortalized cell line was used from rat hippocampal neurons (H19-7). Activation of MAP kinase kinase (MEK) is insufficient to induce neuronal differentiation of H19-7 cells. To test the requirement for MEK and MAP kinase (ERK1 and ERK2), H19-7 cells were treated with the MEK inhibitor PD098059. Although the MEK inhibitor blocks the induction of differentiation by constitutively activated Raf, the H19-7 cells still undergoes differentiation by bFGF. These results suggest that an alternative pathway is utilized by bFGF for differentiation of the hippocampal neuronal cells. Expression in the H19-7 cells of a dominant-negative Ras (N17-Ras) or Raf (C4-Raf) blocks differentiation by bFGF; this suggests that Ras and probably Raf are required. Expression of dominant-negative Src (pcSrc295Arg) or microinjection of an anti-Src antibody blocks differentiation by bFGF in H19-7 cells, indicating that bFGF also signals through an Src kinase-mediated pathway. Although neither constitutively activated MEK (MEK-2E) nor v-Src is sufficient individually to differentiate the H19-7 cells, coexpression of constitutively activated MEK and v-Src induces neurite outgrowth. These results suggest that (1) activation of MAP kinase (ERK1 and ERK2) is neither necessary nor sufficient for differentiation by bFGF; (2) activation of Src kinases is necessary but not sufficient for differentiation by bFGF; and (3) differentiation of H19-7 neuronal cells by bFGF requires at least two signaling pathways activated by Ras and Src (Kuo, 1997).

Src controls the epidermal growth factor (EGF)-induced dispersion of NBT-II carcinoma epithelial cells. While only Src and Yes are expressed and activated by EGF, microinjected kinase-inactive mutants of Src (SrcK-) and Fyn (FynK-) are able to exert a dominant-negative effect on the scattering response. Both SH2 and SH3 domains of FynK- are required for inhibition of cell scattering. Expression of dominant-negative N17Ras also abrogates EGF-induced dispersion, showing that Ras is another regulator of cell dispersion. Expression of SrcK- does not alter three elements of the Ras pathway: EGF-evoked Shc tyrosine phosphorylation, Shc-Grb2 complex formation and MAPK activation. The expression of Jun-Fos and Slug rescue the block induced by N17Ras but not by SrcK-, showing that Src kinases and Ras operate in separate pathways. Actinomycin D inhibition of RNA synthesis represses the ability of the activated mutant L61Ras to induce epithelial cell scattering, but does not repress the same ability in F527Src. Since tyrosine phosphorylation of cytoskeleton-associated proteins pp125FAK and cortactin are abolished in EGF-stimulated SrcK- cells, it is concluded that, in contrast to Ras, Src kinases may control epithelial cell dispersion in the absence of gene expression, and by directly regulating the organization of the cortical cytoskeleton (Boyer, 1997).

The mes/metencephalic boundary (isthmus) is an organizing center for the optic tectum and cerebellum. Fgf8 is accepted as a crucial organizing signal. Fgf8b can induce cerebellum in the mesencephalon, while Fgf8a transforms the presumptive diencephalon into mesencephalon. Since lower doses of Fgf8b exert similar effects to those of Fgf8a, the type difference can be attributed to the difference in the strength of the signal. It is of great interest to uncover mechanisms of signal transduction pathways downstream of the Fgf8 signal in tectal and cerebellar development, and this report concentrates on the Ras-ERK pathway. In normal embryos, extracellular-signal-regulated kinase (ERK) is activated at the site where Fgf8 mRNA is expressed. Fgf8b activates ERK while Fgf8a or a lower dose of Fgf8b does not activate ERK in the mes/metencephalon. Disruption of the Ras-ERK signaling pathway by a dominant negative form of Ras (RasS17N) changes the fate of the metencephalic alar plate from cerebellum to tectum. RasS17N cancels the effects of Fgf8b, while co-transfection of Fgf8a and RasS17N exerts additive effects. Disruption of Fgf8b, not Fgf8a, by siRNA results in posterior extension of the Otx2 expression domain. These results indicate that the presumptive metencephalon receives a strong Fgf8 signal that activates the Ras-ERK pathway and differentiates into the cerebellum (Satol, 2004).

Mammalian Ras, which is encoded by three independent genes, has been thought to be a versatile component of intracellular signalling. However, when, where and how Ras signalling plays essential roles in development and whether the three Ras genes have overlapping functions in particular cells remain unclear. This study shows that the three Ras proteins dose-dependently regulate lymphatic vessel growth in mice. Lymphatic vessel hypoplasia is a common phenotype in Ras compound knockout mice, and overexpressed normal Ras in an endothelial cell lineage selectively causes lymphatic vessel hyperplasia in vivo. Overexpression of normal Ras in lymphatic endothelial cells leads to sustained MAPK activation, cellular viability and enhanced endothelial network formation under serum-depleted culture conditions in vitro, and knockdown of endogenous Ras in lymphatic endothelial cells impairs cell proliferation, MAPK activation, cell migration and endothelial network formation. Ras overexpression and knockdown result in up- and downregulation of vascular endothelial growth factor receptor (VEGFR) 3 expression, respectively, in lymphatic endothelial cells in vitro. The close link between Ras and VEGFR3 in vitro is consistent with the result that Ras knockout and transgenic alleles are genetic modifiers in lymphatic vessel hypoplasia caused by Vegfr3 haploinsufficiency. These findings demonstrate a cooperative function of the three Ras proteins in normal development, and also provide a novel aspect of VEGFR3 signalling modulated by Ras in lymphangiogenesis (Ichise, 2010).

Evolutionary homologs: Table of contents

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

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