Ras-mediated Signal Transduction Research Articles

Rationale for Ras and raf-kinase as a target for cancer therapeutics.

Improvements in our understanding of the intrinsic aberrancies in cancer cells have enabled the design and development of novel therapeutics that specifically target these changes. Among the many complex cellular pathways and mechanisms which have been unveiled by new molecular techniques, RAS-mediated signal transduction is one met with tremendous research interests. Activation of RAS initiates several signaling cascades, of which the RAS-RAF-MEK-ERK pathway is among the better delineated, and is the main focus of this review. Other cellular consequences of RAS activation including interactions with the RHO-family proteins, the PI3-kinase pathway, and other mitogen activated protein kinase cascades, will be discussed. The intricate balance and coordination of multiple RAS-mediated signals lead to ultimate effects on cell growth, differentiation, cycling and survival. Pharmacological strategies such as analog development, synthesis of small molecule inhibitors, antisense technology, and vaccine therapy have been utilized to intervene with key RAS-signaling proteins, in an attempt to provide rational therapeutic solutions in malignant diseases.

Assay of Raf-1 activity

The Raf-l serine/threonine kinase is a key effector of Ras-mediated signal transduction. In response to a diverse array of extracellular signals, the evolutionarily conserved Ras GTPase is converted from its inactive GDP-bound form to its active GTP-bound form. Activated Ras then interacts directly with Raf-1 and recruits Raf- 1 from the cytosol to the plasma membrane. At the membrane, Raf- 1 becomes activated and phosphorylates MEK1 [mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase] at serine residues 217 and 221. These phosphorylation events result in the activation of MEK 1, which in turn phosphorylates and activates MAPK (ERK1 and ERK2). The cascade culminates in the translocation of activated MAPK to the nucleus, where it phosphorylates the targets needed for the subsequent transcription of genes involved in cell growth and differentiation.

KSR: a MAPK scaffold of the Ras pathway?

Kinase Suppressor of Ras (KSR) is an intriguing component of the Ras pathway that was first identified by genetic studies performed in Drosophila melanogaster and Caenorhabditis elegans. In both organisms, inactivating mutations in KSR suppress the phenotypic effects induced by activated Ras. These findings together with the fact that KSR contains many structural features characteristic of a protein kinase led to early speculation that KSR is a kinase functioning upstream of the Ras pathway component Raf-1 or in a parallel Ras-dependent pathway. However, in the six years since its discovery, KSR has been found to lack several key properties of known protein kinases, which has cast doubt on whether KSR is indeed a functional enzyme. A major breakthrough in our understanding of the role of KSR in signal transduction has come from recent findings that KSR interacts with several components of the MAP kinase cascade, including Raf-1, MEK1/2 and ERK1/2. The model now emerging is that KSR acts as a scaffolding protein that coordinates the assembly of a membrane-localized, multiprotein MAP kinase complex, a vital step in Ras-mediated signal transduction. Thus, while Kinase Suppressor of Ras may be its name, phosphorylation may not be its game.

The Ras-pathway inhibitor, S- trans- trans-farnesylthiosalicylic acid, suppresses experimental allergic encephalomyelitis

Aim: To evaluate the effects of the synthetic Ras-pathway inhibitor, S- trans- trans-farnesylthiosalicylic acid (FTS) on acute and chronic experimental autoimmune encephalomyelitis (EAE and CR-EAE). Background: Treatment of EAE and MS is based on immunosuppression aiming at downregulation of the proliferating myelin-reactive lymphocytes. One of the pathways of lymphocyte activation involves the GTP-binding protein Ras. FTS destabilizes the attachment of Ras to the cell membrane, resulting in an inhibition of the Ras-mediated signal transduction pathways. Materials and methods: EAE was induced in SJL/J mice by immunization with spinal cord homogenate (MSCH) in adjuvant and two i.v. boosts of pertussis antigen and CR-EAE with passive transfer of proteolipid protein (PLP)-activated lymphocytes. Animals were treated daily starting either from the day of EAE-induction (or cell transfer) or at a later stage, with i.p. injections of FTS (5 mg/kg/day). The clinical severity of the disease was evaluated daily and scored using a 0–6 scale. Results: In six separate experiments, 27 of the 38 (71.7%) vehicle-treated animals developed clinical signs of EAE compared to 17/38 (44.7%) of the FTS-treated mice ( p=0.02, t-test). The maximal average score in the control group was 2.94±2.2, whereas in the FTS group it was significantly lower (1.63±2.2, p=0.01). Mortality was 26.3% and 10.5% in the two groups, respectively ( p=0.03). When treatment was initiated at a later stage, just before the onset of the clinical signs, the protective effect was even more pronounced. A significant suppression of clinical signs was also observed in the CR-EAE model ( p=0.02). Lymphocyte proliferation assays demonstrated a more than twofold decrease in the reactivity to myelin antigens (MBP and PLP) and downregulation of the activated lymphocytes (expressing the CD62L, and IA-k-MHC Class I markers and the Vb17 T-cell receptor) in the FTS-treated group; in vitro FTS suppressed the Ras activity of lymphocytes and inhibited the proliferative ability of the lymphocytes in a dose-dependent manner. Conclusions: FTS suppresses EAE by downregulation of myelin-reactive activated T-lymphocytes. Since FTS did not induce generalized immunosuppressive effects, it may offer significant advantages over the broad immunosuppressive modalities and may be a candidate treatment for autoimmune diseases, such as MS.

Combinatorial effect of T-cell receptor ligation and CD45 isoform expression on the signaling contribution of the small GTPases Ras and Rap1.

By using ligands with various affinities for the T-cell receptor (TCR) and by altering the contribution of the CD45 tyrosine phosphatase, the effects of the potency of TCR-induced signals on the function of small GTPases Ras and Rap1 were studied. T cells expressing low-molecular-weight CD45 isoforms (e.g., CD45RO) exhibited the strongest activation of the Ras-dependent Elk-1 transcription factor and the highest sensitivity to the inhibitory action of dominant negative mutant Ras compared to T cells expressing high-molecular-weight CD45 isoforms (ABC). Moreover, stimulation of CD45RO(+), but not CD45ABC(+), T cells with a high-affinity TCR ligand induced suboptimal Elk-1 activation compared with the stimulation induced by an intermediate-affinity TCR-ligand interaction. This observation suggested that the Ras-dependent signaling pathway is safeguarded in CD45RO(+) expressors by a negative regulatory mechanism(s) which prohibits maximal activation of the Ras-dependent signaling events following high-avidity TCR-ligand engagement. Interestingly, the biochemical activity of another small GTPase, the Ras-like protein Rap1, which has been implicated in the functional suppression of Ras signaling, was inversely correlated with the extent of Elk-1 activation induced by different-affinity TCR ligands. Consistently, overexpression of putative Rap dominant negative mutant RapN17 or the physiologic inhibitor of Rap1, the Rap GTPase-activating protein RapGAP, augmented the Elk-1 response in CD45RO(+) T cells. This is in contrast to the suppressive effect of RapN17 and RapGAP on CD45ABC(+) T cells, underscoring the possibility that Rap1 can act as either a repressor or a potentiator of Ras effector signals, depending on CD45 isoform expression. These observations suggest that cells expressing distinct isoforms of CD45 employ different signal transduction schemes to optimize Ras-mediated signal transduction in activated T lymphocytes.

Sem-4 Promotes Vulval Cell-Fate Determination in Caenorhabditis elegans through Regulation of lin-39 Hox

Vulval cell-fate determination in Caenorhabditis elegans requires the action of numerous gene products, including components of the Ras/Raf/MAPK signaling cascade and the hox gene lin-39. sem-4 encodes a zinc finger protein with previously characterized roles in fate specification of sex myoblasts, coelomocytes, and multiple neuronal lineages in C. elegans (M. Basson and R. Horvitz, 1996, Genes Dev. 10, 1953–1965). By characterizing three new alleles of sem-4 that we identified in a screen for vulval-defective mutants, we determined that loss of sem-4 activity results in abnormal specification of the secondary vulval cell lineages. We analyzed sem-4 interactions with other genes involved in vulval differentiation and determined that sem-4 does not function directly in the Ras-mediated signal transduction pathway but acts in close association with and upstream of lin-39 to promote vulval cell fate. We demonstrate that sem-4 regulates lin-39 expression and propose that sem-4 is a regulator of lin-39 in the vulval cell-fate determination pathway that may act to link lin-39 to incoming signals.

Relationship between Ras pathways and cell cycle control.

The ordered execution of the two main events of cellular reproduction, duplication of the genome and cell division, characterize progression through the cell cycle. Cultured cells can be switched between cycling and non-cycling states by alteration of extracellular conditions and the notion that a critical cellular control mechanism presides on this decision, whose temporal location is known as the restriction point, has become the focus for the study of how extracellular mitogenic signalling impinges upon the cell cycle to influence proliferation. This review attempts to cover the disparate pathways of Ras-mediated mitogenic signal transduction that impact upon restriction point control.

Ras signaling is required for serum-induced hyphal differentiation in Candida albicans.

Serum induces Candida albicans to make a rapid morphological change from the yeast cell form to hyphae. Contrary to the previous reports, we found that serum albumin does not play a critical role in this morphological change. Instead, a filtrate (molecular mass, <1 kDa) devoid of serum albumin induces hyphae. To study genes controlling this response, we have isolated the RAS1 gene from C. albicans by complementation. The Candida Ras1 protein, like Ras1 and Ras2 of Saccharomyces cerevisiae, has a long C-terminal extension. Although RAS1 appears to be the only RAS gene present in the C. albicans genome, strains homozygous for a deletion of RAS1 (ras1-2/ras1-3) are viable. The Candida ras1-2/ras1-3 mutant fails to form germ tubes and hyphae in response to serum or to a serum filtrate but does form pseudohyphae. Moreover, strains expressing the dominant active RAS1(V13) allele manifest enhanced hyphal growth, whereas those expressing a dominant negative RAS1(A16) allele show reduced hyphal growth. These data show that low-molecular-weight molecules in serum induce hyphal differentiation in C. albicans through a Ras-mediated signal transduction pathway.

Pseudomonas aeruginosa Exoenzyme S Disrupts Ras-mediated Signal Transduction by Inhibiting Guanine Nucleotide Exchange Factor-catalyzed Nucleotide Exchange

Pseudomonas aeruginosa exoenzyme S double ADP-ribosylates Ras at Arg(41) and Arg(128). Since Arg(41) is adjacent to the switch 1 region of Ras, ADP-ribosylation could interfere with Ras-mediated signal transduction via several mechanisms, including interaction with Raf, or guanine nucleotide exchange factor-stimulated or intrinsic nucleotide exchange. Initial experiments showed that ADP-ribosylated Ras (ADP-r-Ras) and unmodified Ras (Ras) interacted with Raf with equal efficiencies, indicating that ADP-ribosylation did not interfere with Ras-Raf interactions. While ADP-r-Ras and Ras possessed equivalent intrinsic nucleotide exchange rates, guanine nucleotide exchange factor (Cdc25) stimulated the nucleotide exchange of ADP-r-Ras at a 3-fold slower rate than Ras. ADP-r-Ras did not affect the nucleotide exchange of Ras, indicating that the ADP-ribosylation of Ras was not a dominant negative phenotype. Ras-R41K and ADP-r-Ras R41K possessed similar exchange rates as Ras, indicating that ADP-ribosylation at Arg(128) did not inhibit Cdc25-stimulated nucleotide exchange. Consistent with the slower nucleotide exchange rate of ADP-r-Ras as compared with Ras, ADP-r-Ras bound its guanine nucleotide exchange factor (Cdc25) less efficiently than Ras in direct binding experiments. Together, these data indicate that ADP-ribosylation of Ras at Arg(41) disrupts Ras-Cdc25 interactions, which inhibits the rate-limiting step in Ras signal transduction, the activation of Ras by its guanine nucleotide exchange factor.

The amino-terminal domain of Pseudomonas aeruginosa ExoS disrupts actin filaments via small-molecular-weight GTP-binding proteins.

Pseudomonas aeruginosa delivers exoenzyme S (ExoS) into the intracellular compartment of eukaryotic cells via a type III secretion pathway. Intracellular delivery of ExoS is cytotoxic for eukaryotic cells and has been shown to ADP-ribosylate Ras in vivo and uncouple a Ras-mediated signal transduction pathway. Functional mapping has localized the FAS-dependent ADP-ribosyltransferase domain to the carboxyl-terminus of ExoS. A transient transfection system was used to examine cellular responses to the amino-terminal 234 amino acids of ExoS (DeltaC234). Intracellular expression of DeltaC234 elicited the rounding of Chinese hamster ovary (CHO) cells and the disruption of actin filaments in a dose-dependent manner. Expression of DeltaC234 did not inhibit the expression of two independent reporter proteins, GFP and luciferase, or induce trypan blue uptake, which indicated that expression of DeltaC234 was not cytotoxic to CHO cells. Carboxyl-terminal deletion proteins of DeltaC234 were less efficient in the elicitation of CHO cell rounding than DeltaC234. Cytoskeleton rearrangement elicited by DeltaC234 was blocked and reversed by the addition of cytotoxic necrotizing factor 1 (CNF-1). CNF-1 catalyses the deamidation of Gln-63 of members of the Rho subfamily of small-molecular-weight GTP-binding proteins, resulting in protein activation. This implies a role for small-molecular-weight GTP-binding proteins in the disruption of actin by DeltaC234. Together, these data identify ExoS as a cytotoxin that possesses two functional domains. Intracellular expression of the amino-terminal domain of ExoS elicits the disruption of actin, while expression of the carboxyl-terminal domain of ExoS possesses FAS-dependent ADP-ribosyltransferase activity and is cytotoxic to eukaryotic cells.

Control of growth and differentiation by Drosophila RasGAP, a homolog of p120 Ras-GTPase-activating protein.

Mammalian Ras GTPase-activating protein (GAP), p120 Ras-GAP, has been implicated as both a downregulator and effector of Ras proteins, but its precise role in Ras-mediated signal transduction pathways is unclear. To begin a genetic analysis of the role of p120 Ras-GAP we identified a homolog from the fruit fly Drosophila melanogaster through its ability to complement the sterility of a Schizosaccharomyces pombe (fission yeast) gap1 mutant strain. Like its mammalian homolog, Drosophila RasGAP stimulated the intrinsic GTPase activity of normal mammalian H-Ras but not that of the oncogenic Val12 mutant. RasGAP was tyrosine phosphorylated in embryos and its Src homology 2 (SH2) domains could bind in vitro to a small number of tyrosine-phosphorylated proteins expressed at various developmental stages. Ectopic expression of RasGAP in the wing imaginal disc reduced the size of the adult wing by up to 45% and suppressed ectopic wing vein formation caused by expression of activated forms of Breathless and Heartless, two Drosophila receptor tyrosine kinases of the fibroblast growth factor receptor family. The in vivo effects of RasGAP overexpression required intact SH2 domains, indicating that intracellular localization of RasGAP through SH2-phosphotyrosine interactions is important for its activity. These results show that RasGAP can function as an inhibitor of signaling pathways mediated by Ras and receptor tyrosine kinases in vivo. Genetic interactions, however, suggested a Ras-independent role for RasGAP in the regulation of growth. The system described here should enable genetic screens to be performed to identify regulators and effectors of p120 Ras-GAP.

A potential role of Ras-mediated signal transduction for the enhancement of depolarization-induced Ca 2+ responses in hippocampal neurons by basic fibroblast growth factor

Chronic treatment with basic fibroblast growth factor (bFGF) increases the expression of functional L-type voltage-dependent Ca 2+ channels (VDCCs) in fetal rat hippocampal neurons. We investigated the intracellular signaling mechanisms involved in this effect, using high K + depolarization-induced elevation of intracellular Ca 2+ concentrations as a measure. Genistein, a protein tyrosine kinase inhibitor, significantly attenuated the effect of bFGF. The effect of bFGF was also diminished by concurrent application of a Ras inactivator, N-acetyl- S-farnesyl- l-cysteine. In contrast, a phospholipase C inhibitor U73122, a phosphatidylinositol-3 kinase inhibitor wortmannin, Li + which inhibits inositol phospholipid turnover, or a protein kinase inhibitor calphostin C did not inhibit the effect of bFGF. Phorbol 12-myristate 13-acetate, a protein kinase C activator, did not mimic the effect of bFGF. On the other hand, an adenylyl cyclase activator forskolin and a cyclic AMP analog 8-Br-cyclic AMP markedly attenuated the effect of bFGF, which indicates the presence of a cyclic AMP-mediated negative regulatory mechanism, possibly the interference of Ras–Raf interaction. These results suggest that Ras-mediated signal transduction is required for the enhancement by bFGF of VDCC responses in hippocampal neurons.

SUR-8, a Conserved Ras-Binding Protein with Leucine-Rich Repeats, Positively Regulates Ras-Mediated Signaling in C. elegans

We describe the identification and characterization of a novel gene, sur-8, that positively regulates Ras-mediated signal transduction during C. elegans vulval development. Reduction of sur-8 function suppresses an activated ras mutation and dramatically enhances phenotypes of mpk-1 MAP kinase and ksr-1 mutations, while increase of sur-8 dosage enhances an activated ras mutation. sur-8 appears to act downstream of or in parallel to ras but upstream of raf. sur-8 encodes a conserved protein that is composed predominantly of leucine-rich repeats. The SUR-8 protein interacts directly with Ras but not with the Ras(P34G) mutant protein, suggesting that SUR-8 may mediate its effects through Ras binding. A structural and functional SUR-8 homolog in humans specifically binds K-Ras and N-Ras but not H-Ras in vitro.

Pseudomonas aeruginosa Exoenzyme S ADP-ribosylates Ras at Multiple Sites

Pseudomonas aeruginosa exoenzyme S (ExoS) ADP-ribosylated Ras to a stoichiometry of approximately 2 molecules of ADP-ribose incorporated per molecule of Ras, which suggested that ExoS could ADP-ribosylate Ras at more than one arginine residue. SDS-polyacrylamide gel electrophoresis analysis showed that ADP-ribosylated Ras possessed a slower mobility than non-ADP-ribosylated Ras. Analysis of the ADP-ribosylation of in vitro transcribed/translated Ras by ExoS identified two electrophoretically shifted forms of Ras, which was consistent with the ADP-ribosylation of Ras at two distinct arginine residues. Analysis of ADP-ribosylated in vitro transcribed/translated Ras mutants possessing individual Arg-to-Ala substitutions showed that Arg-41 was the preferred site of ADP-ribosylation and that the second ADP-ribosylation event occurred at a slower rate than the ADP-ribosylation at Arg-41, but did not occur at a specific arginine residue. Analysis of bacterially expressed wild-type RasDeltaCAAX and RasDeltaCAAXR41K supported the conclusion that Arg-41 was the preferred site of ADP-ribosylation. Arg-41 is located adjacent to the switch 1 region of Ras, which is involved in effector interactions. Introduction of ExoS into eukaryotic cells inhibited Ras-mediated eukaryotic signal transduction since infection of PC-12 cells with an ExoS-producing strain of P. aeruginosa inhibited nerve growth factor-stimulated neurite formation. This is the first demonstration that ExoS disrupts a Ras-mediated signal transduction pathway.

KSR stimulates Raf-1 activity in a kinase-independent manner.

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

A Ras-mediated signal transduction pathway is involved in the control of sex myoblast migration in Caenorhabditis elegans.

Sex myoblast migration in the Caenorhabditis elegans hermaphrodite represents a simple, genetically amenable model system for studying how cell migration is regulated during development. Two separable components of sex myoblast guidance have been described: a gonad-independent mechanism sufficient for the initial anterior migration to the mid-body region, and a gonad-dependent mechanism required for precise final positioning (J. H. Thomas, M. J. Stern and H. R. Horvitz (1990) Cell 62, 1041-1052). Here, we demonstrate a role for a Ras-mediated signal transduction pathway in controlling sex myoblast migration. Loss-of-function mutations in let-60 ras, ksr-1, lin-45 raf, let-537/mek-2 or sur-1/mpk-1 cause defects in sex myoblast final positions that resemble those seen in gonad-ablated animals, while constitutively active let-60 ras(G13E) trans-genes allow fairly precise positioning to occur in the absence of the gonad. A mosaic analysis demonstrated that let-60 ras is required within the sex myoblasts to control proper positioning. Our results suggest that gonadal signals normally stimulate let-60 ras activity in the sex myoblasts, thereby making them competent to sense or respond to positional cues that determine the precise endpoint of migration. let-60 ras may have additional roles in sex myoblast guidance as well. Finally, we have also investigated genetic interactions between let-60 ras and other genes important for sex myoblast migration, including egl-15, which encodes a fibroblast growth factor receptor tyrosine kinase (D. L. DeVore, H. R. Horvitz and M. J. Stern (1995) Cell 83, 611-623). Since mutations reducing Ras pathway activity cause a different phenotype than those reducing egl-15 activity and since constitutive Ras activity only partially suppresses the migration defects of egl-15 mutants, we argue that let-60 ras and egl-15 do not act together in a single linear pathway.

New insights into the Ras onco-protein and its interactions with the Raf-1-1 kinase

The Ras proteins are members of a large superfamily of nucleotide-binding proteins that cycle between active GTP- and inactive GDP-bound states. They are positively regulated by guanine nucleotide exchange factors (GEFs) that promote formation of the active GTP-bound state and negatively regulated by GTPase activating proteins (GAPs) that stimulate formation of the inactive GDP-complexed protein. Structural mutations that activate Ras oncogenic potential either impair GAP-stimulated GTPase activity or promote enhanced intrinsic nucleotide exchange. The net result of either biochemical effect is to favor elevated levels of Ras-GTP in vivo.We are investigating selected Ras variants containing substitutions at conserved sites thought to be important for guanine nucleotide binding, GTP hydrolysis, GTP/GDP interconversion and protein recognition, to better understand the role of these conserved amino acids in Ras-mediated signal transduction. Recent studies conducted at the U. of North Carolina will be presented, where we have characterized a novel activating mutation in Ras using multi-dimensional NMR spectroscopy and established a region involved in direct interaction with guanine nucleotide exchange factors.

Peptides containing a consensus Ras binding sequence from Raf-1 and theGTPase activating protein NF1 inhibit Ras function.

A key event in Ras-mediated signal transduction and transformation involves Ras interaction with its downstream effector targets. Although substantial evidence has established that the Raf-1 serine/threonine kinase is a critical effector of Ras function, there is increasing evidence that Ras function is mediated through interaction with multiple effectors to trigger Raf-independent signaling pathways. In addition to the two Ras GTPase activating proteins (GAPs; p120- and NF1-GAP), other candidate effectors include activators of the Ras-related Ral proteins (RalGDS and RGL) and phosphatidylinositol 3-kinase. Interaction between Ras and its effectors requires an intact Ras effector domain and involves preferential recognition of active Ras-GTP. Surprisingly, these functionally diverse effectors lack significant sequence homology and no consensus Ras binding sequence has been described. We have now identified a consensus Ras binding sequence shared among a subset of Ras effectors. We have also shown that peptides containing this sequence from Raf-1 (RKTFLKLA) and NF1-GAP (RRFFLDIA) block NF1-GAP stimulation of Ras GTPase activity and Ras-mediated activation of mitogen-activated protein kinases. In summary, the identification of a consensus Ras-GTP binding sequence establishes a structural basis for the ability of diverse effector proteins to interact with Ras-GTP. Furthermore, our demonstration that peptides that contain Ras-GTP binding sequences can block Ras function provides a step toward the development of anti-Ras agents.

Isolation and mapping of RAB2L, a human cDNA that encodes a protein homologous to RalGDS.

We have isolated a novel human cDNA, designated RAB2L, that encodes a protein homologous to Ral guanine nucleotide dissociation stimulator (RalGDS). The predicted sequence of 122 amino acids revealed 39.5% identity with the C-terminal region of human RalGDS. As the C-terminal region of RalGDS is considered to interact with RAS, the product of this novel gene may also play some role in the ras-mediated signal transduction pathway. Northern analysis revealed that the gene RAB2L is expressed in all human tissues examined. We assigned this gene locus to chromosome band 6p21.3 by fluorescence in situ hybridization.

The C. elegans ksr-1 gene encodes a novel raf-related kinase involved in Ras-mediated signal transduction

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