RTK/Ras/MAPK Signaling

Receptor Tyrosine Kinase (RTK)/Ras GTPase/MAP kinase (MAPK) signaling pathways are used repeatedly during metazoan development to control many different biological processes. In the nematode Caenorhabditis elegans, two different RTKs (LET-23/EGFR and EGL-15/FGFR) are known to stimulate LET-60/Ras and a MAPK cascade consisting of the kinases LIN-45/Raf, MEK-2/MEK and MPK-1/ERK. This Ras/MAPK cascade is required for multiple developmental events, including induction of vulval, uterine, spicule, P12 and excretory duct cell fates, control of sex myoblast migration and axon guidance, and promotion of germline meiosis. Studies in C. elegans have provided much insight into the basic framework of this RTK/Ras/MAPK signaling pathway, its regulation, how it elicits cell-type specific responses, and how it interacts with other signaling pathways such as the Wnt and Notch pathways.


Introduction
Receptor Tyrosine Kinase (RTK)/Ras GTPase/MAP kinase (MAPK) signaling pathways are used repeatedly during metazoan development to control many different biological processes (Schlessinger, 2000).Mutations affecting RTK/Ras/MAPK signaling cause many human syndromes and diseases, including cancer (Malumbres and Barbacid, 2002).Studies in C. elegans have provided much insight into the basic framework of this canonical type of Ras pathway, its regulation, how it elicits cell-type specific responses, and how it interacts with other signaling pathways (for recent reviews see Moghal and Sternberg, 2003;Sundaram, 2004;Tan and Kim, 1999;Wang and Sternberg, 2001).
C. elegans Ras is called LET-60 (Han and Sternberg, 1990).LET-60 Ras acts downstream of at least two different RTKs, LET-23 (related to the Epidermal Growth Factor Receptor or EGFR; Aroian et al., 1990) and EGL-15 (related to the Fibroblast Growth Factor Receptor or FGFR;DeVore et al., 1995).The only known role of LET-60 Ras is to stimulate a MAPK cascade consisting of the kinases LIN-45 (Raf;Han et al., 1993), MEK-2 (MEK;Church et al., 1995;Kornfeld et al., 1995;Wu et al., 1995) and MPK-1 (ERK/MAPK; Lackner et al., 1994;Wu and Han, 1994).LET-60 Ras signaling is required for multiple developmental events, the best studied of which is vulval induction (see Vulval development).Genetic screens based on various let-60 mutant phenotypes have identified many generally-acting "core" components of the Ras pathway as well as numerous regulators or targets of the pathway (Figure 1; Table 1).Indeed, many important Ras pathway genes were first identified in the worm.(Moghal and Sternberg, 2003;Schlessinger, 2000).Upon growth factor binding, an RTK such as LET-23 or EGL-15 dimerizes and autophosphorylates its C-terminal region.The resulting phospho-tyrosine residues serve as docking sites for adaptor proteins such as SEM-5 (Grb2) or SOC-1 (similar to Gab1).These adaptors recruit the Guanine Nucleotide Exchange Factor SOS-1 to activate the small GTPase LET-60 Ras.LET-60-GTP then binds to LIN-45 Raf and promotes its stable association with the plasma membrane and/or endomembranes, where other events then activate LIN-45 kinase activity (Chong et al., 2003).The scaffold protein KSR may assist in LIN-45 activation, but also promotes further signal transmission by bringing together different components of the MAPK cascade (Morrison and Davis, 2003).LIN-45 phosphorylates and activates MEK-2, MEK-2 phosphorylates and activates MPK-1, and MPK-1 then phosphorylates and either activates or inactivates various target proteins.In many cases MPK-1 may move into the nucleus to phosphorylate transcription factors such as the Ets domain protein LIN-1, thus leading to changes in gene expression.
This model draws on a large body of data from multiple systems.C. elegans genetics has been most useful for identifying the genes involved in particular signaling events and determining their order of action and cellular focus.In some cases, physical interactions and/or phosphorylation events have been demonstrated for the worm proteins (e.g., Jacobs et al., 1999;Sieburth et al., 1998;Wu et al., 1995), but in many cases such interactions are inferred based on biochemical studies of related proteins in vertebrate cells.Because the core Ras pathway (and much of its regulation) appears highly conserved between C. elegans and vertebrates (Table 1), current models draw on the combined data from these different systems.

Phenotypes of Ras pathway mutants
Ras signaling is not required for cellular viability in C. elegans (Yochem et al., 1997), but it is required for organismal viability and for many different developmental processes.Because of the widespread roles of Ras signaling during development, mutations affecting the Ras pathway can cause many different pleiotropic defects (Figure 2).Lethality: Ras signaling promotes the excretory duct cell fate, and mosaic analysis suggested that loss of this one specific cell can account for the zygotic lethality of let-60 null mutants (Yochem et al., 1997).The excretory duct cell is required for osmoregulation (Nelson and Riddle, 1984).let-60 loss-of-function mutants, which lack the excretory duct cell, die as "rod-like" larvae with a fluid-filled appearance (Figure 2).let-60 gain-of-function mutants sometimes have two excretory duct cells (Yochem et al., 1997).Ras signaling may have other (maternally-rescued) essential roles in addition to excretory duct formation, since mutations in let-23/EGFR, egl-15/FGFR and ptp-2/SHP-2 cause distinct "scrawny" and lethal defects that are rescued by constitutive forms of LET-60 Ras (DeVore et al., 1995;Gutch et al., 1998;Koga and Ohshima, 1995).
Hyperactivation of egl-15/FGFR or let-60/Ras can also lead to lethality with a "Clear", fluid-filled appearance (Kokel et al., 1998;Schutzman et al., 2001).Mosaic analysis suggests that the Clear phenotype is caused by hypodermal defects (Huang and Stern, 2004).Uterine defects: Ras signaling promotes the uterine uv1 fate, which is important for establishing a proper vulval-uterine connection (Chang et al., 1999).In the absence of uv1, hermaphrodites cannot lay eggs.
Male spicule defects: Ras signaling promotes male spicule fates (Chamberlin and Sternberg, 1994; Male development).Reduced Ras signaling causes spicule defects and prevents males from mating.
Sex myoblast migration defects: Ras signaling helps to specify the proper endpoint of sex myoblast migration (Sundaram et al., 1996).In let-60 loss-of-function mutants, sex myoblasts adopt a broadened range of final positions.This defect can affect egg-laying.
Axon guidance defects: Ras signaling controls the paths of certain ventral cord neurons relative to the ventral midline (Bulow et al., 2004).Whereas in wild-type these neurons extend axons along one side of the midline, in let-60 loss-of-function mutants the neurons wander across the midline.
Sterility due to pachytene exit (Pex) defects: Ras signaling is required for progression through the pachytene stage of meiosis (Church et al., 1995; Somatic sex determination).let-60 loss-of-function mutants are sterile because germ cells arrest in pachytene (Figure 2).
Olfaction defects: Ras signaling is required for sensitivity to volatile attractants (Hirotsu et al., 2000).Whereas wild-type C. elegans will chemotax toward volatile attractants such as isoamylalcohol and diacetyl, let-60 loss-of-function mutants fail to chemotax towards such attractants.
Resistance to Microbacterium nematophilum-induced swelling: ksr-1, lin-45, mek-2 and mpk-1 are required for the swelling response to infection by M. nematophilum (Nicholas and Hodgkin, 2004).Interestingly, let-60 does not appear to be important for this response, suggesting that a bacterial toxin may directly activate the Raf/MEK/ERK cascade.

Screens used to identify Ras pathway components
The vast majority of studies on RTK/Ras/MAPK signaling have focused on the role of the Ras pathway in promoting vulval development (see Vulval development), and many known components of the pathway have been identified through forward genetic screens for mutants with Vul or Muv mutant phenotypes (Ferguson and Horvitz, 1985).Some components of the pathway have been identified based on other mutant phenotypes such as sex myoblast migration defects (Stern and Horvitz, 1991) or germline meiosis defects (Church et al., 1995;Ohmachi et al., 2002).Some have been identified through reverse genetic approaches (Dutt et al., 2004;Gutch et al., 1998;Kamikura and Cooper, 2003;Yoo et al., 2004).Finally, some core Ras pathway components as well as a large set of other genes that influence Ras signaling have been identified through genetic suppressor or enhancer screens (Sternberg and Han, 1998).Many modifier genes have essentially wild-type null phenotypes (Table 1), indicating a significant and surprising amount of redundancy amongst Ras pathway regulators.

Growth factors and RTKs that signal through Ras/MAPK
The C. elegans genome contains twenty-eight predicted RTKs (Popovici et al., 1999), only a few of which have been characterized mutationally (see Genomic overview of protein kinases).Of these characterized RTKs, only the EGF receptor LET-23 and the FGF receptor EGL-15 are known to signal positively through Ras/MAPK (see below).The ephrin receptor VAB-1 negatively regulates MAPK activation during oocyte maturation (Miller et al., 2003).Notably, the insulin-like RTK DAF-2 does not appear to signal through Ras/MAPK (G.Ruvkun, personal communication), but instead signals through a PI3-kinase/Akt pathway (see Signaling in the immune response).

Regulators of LIN-3/EGF and EGL-17/FGF
Reverse genetic approaches have identified several factors important for ligand processing and secretion.The ligand LIN-3 exists in both trans-membrane (locally-acting) and diffusible forms (Hill and Sternberg, 1992;Thomas et al., 1990); generation of the diffusible form in vulval cells appears to require cleavage by the Rhomboid ortholog ROM-1 (Dutt et al., 2004).The ligand EGL-17 is secreted via a mechanism that requires the lipoprotein receptor-related proteins LRP-1 and LRP-2 and the Disabled-related adaptor DAB-1 (Kamikura and Cooper, 2003).

Regulators of Ras activity
As is the case for other small GTPases (see Small GTPases), the activity of LET-60 Ras is controlled by Guanine Nucleotide Exchange Factors (GEFs), which activate Ras by stimulating conversion of Ras-GDP to Ras-GTP, and by GTPase activating proteins (GAPs), which inactivate Ras by stimulating conversion of Ras-GTP to Ras-GDP (Figure 3).The GEF SOS-1 appears necessary for most Ras-mediated developmental events (Chang et al., 2000).The GAPs GAP-1 and GAP-2 negatively regulate Ras signaling during vulval development and excretory duct development, respectively (Hajnal et al., 1997;Hayashizaki et al., 1998).Another negative regulator of Ras is SUR-5, a protein of unknown function that resembles acetyl coenzyme A synthetases (Gu et al., 1998).and Barbacid, 2002).Ras-GDP is inactive, whereas Ras-GTP is active and can bind to effectors such as Raf.Guanine nucleotide exchange factors (GEFs) such as SOS-1 positively regulate Ras by promoting GDP dissociation.GTPase activating proteins (GAPs) negatively regulate Ras by stimulating Ras' intrinsic GTP hydrolyzing activity.Gain-of-function (gf) mutations lock Ras in the active, GTP-bound state; the let-60(gf) allele n1046 (G13E) has been widely used for genetic analyses (Beitel et al., 1990;Han and Sternberg, 1990).Dominant-negative (dn) mutations lock Ras in the inactive, GDP-bound state, causing it to bind stably to and titrate out GEFs; a variety of let-60(dn) mutations have been described (Han and Sternberg, 1991).

Regulators of the Raf/MEK/ERK kinase cascade
Genetic modifier screens have identified a large set of genes that promote or inhibit signaling through the MAPK cascade, but that are not individually required for normal development in most tissues.In general, the roles of these genes can only be detected as suppressor or enhancer effects in appropriate double mutant combinations.Raf/MEK/ERK signaling is apparently subject to many different levels of regulation that individually have modest effects on signaling strength.
Negatively acting gene products in this category include the G-protein coupled receptor SRA-13 and its Gα target GPA-5, which may modulate Ras signaling in response to environmental conditions such as food availability (Battu et al., 2003).Also in this category are the kinase PAR-1 (which is thought to modulate KSR localization; Kao et al., 2003;Muller et al., 2001;Yoder et al., 2004), the MPK-1-binding protein LST-1 (Yoo et al., 2004), and the MAP kinase phosphatase LIP-1 (Berset et al., 2001).
It is important to note that some regulatory proteins may influence Raf/MEK/ERK signaling indirectly, by affecting neighboring cells.For example, the Gαq protein EGL-30 and the voltage-gated calcium channel EGL-19 promote vulval induction, but function in neurons and muscle, respectively (Moghal et al., 2003).Similarly, SRA-13 and GPA-5 influence vulval induction but it is unknown whether these genes function in vulval cells or in neurons (Battu et al., 2003).Also, the zinc transporter CDF-1 can influence vulval induction when expressed in either the vulva or the intestine (Bruinsma et al., 2002).How tissues such as the intestine, neurons and muscle can influence Raf/MEK/ERK activity in vulval cells is still unclear.

Targets of MPK-1 ERK, and other factors influencing downstream responses
No single downstream target of MPK-1 can account for all of the effects of RTK/Ras/MAPK signaling.Rather, different tissues seem to require different subsets of potential targets, and the availability of certain targets may control tissue-specific responses.
Four widely important positive factors are the Mediator subunit SUR-2, the BTB/Zinc finger protein EOR-1, and the novel nuclear proteins LIN-25 and EOR-2 (Howard and Sundaram, 2002;Singh and Han, 1995;Tuck and Greenwald, 1995;Figure 1A).None of these proteins are known to be direct targets of MPK-1, but their functions are important for downstream cellular responses.SUR-2 is a conserved component of the Mediator complex, which links certain sequence-specific DNA binding proteins (such as Ets proteins) to the general RNA Polymerase II transcriptional machinery (Boyer et al., 1999;Stevens et al., 2002).SUR-2 and LIN-25 appear to function together, and they have strong effects on vulval development and weaker effects on excretory duct and P12 development (Nilsson et al., 1998;Nilsson et al., 2000;Singh and Han, 1995;Tuck and Greenwald, 1995).EOR-1 is related to known transcriptional activators and repressors (Barna et al., 2002;Collins et al., 2001).EOR-1 and EOR-2 appear to function together, and they have moderate effects on excretory duct and P12 development and weaker effects on vulval development (Howard and Sundaram, 2002).EOR-1 and EOR-2 appear to act redundantly with SUR-2 and LIN-25 (Howard and Sundaram, 2002).
Several transcription factors are required for cell-type specific responses to RTK/Ras/MAPK signaling, and are candidate MPK-1 targets.These include the forkhead transcription factor LIN-31 (Miller et al., 1993;Tan et al., 1998), which both promotes and inhibits vulval development, the Hox protein LIN-39 (Clark et al., 1993;Eisenmann et al., 1998;Maloof and Kenyon, 1998), the zinc-finger protein SEM-4 (Grant et al., 2000) and the GATA factors EGL-18 and ELT-6 (Koh et al., 2004;Koh et al., 2002), which promote vulval development, and the Hox protein EGL-5 (Chisholm, 1991;Jiang and Sternberg, 1998), which promotes P12 development.Ectopic expression of LIN-31 or LIN-39 can cause other tissues to adopt vulval-like characteristics in response to RTK/Ras/MAPK signaling, suggesting that the presence of these distinct transcription factors may be a key determinant of tissue-specific responses to Ras signaling (Maloof and Kenyon, 1998;Tan et al., 1998).
Not all targets of MPK-1 need be transcription factors.For example, none of the widely important transcriptional regulators seem to be involved in controlling germline meiosis or sex myoblast migration (Church et al., 1995;Sundaram et al., 1996;R. Howard and M. Sundaram, unpublished observations;Figure 1B and C).It is likely that MPK-1 targets in these tissues include factors more directly involved in meiosis and motility.

Interactions between the RTK/Ras/MAPK pathway and other signaling pathways
The RTK/Ras/MAPK pathway often interacts with other signaling pathways to control cell fates.For example, the Ras pathway cooperates with a Wnt pathway (see Wnt signaling) to specify P12 fates and vulval fates, possibly by convergent upregulation of common targets such as Hox genes (Eisenmann et al., 1998;Gleason et al., 2002).During vulval development (see Vulval development), the Ras pathway also acts sequentially with a Notch pathway (see LIN-12/Notch signaling in C. elegans) to induce the proper pattern of vulval fates (Simske and Kim, 1995;Sundaram, 2004).Ras signaling affects Notch signaling in at least two ways.First, Ras stimulates LIN-12/Notch endocytosis to downregulate Notch signaling in the same cell (Shaye and Greenwald, 2002).Second, Ras stimulates the transcription of Notch ligand genes to upregulate Notch signaling in adjacent cells (Chen and Greenwald, 2004).Notch signaling also antagonizes Ras signaling by stimulating the transcription of various negative regulators such as lip-1 and lst-1- 4 (Berset et al., 2001;Yoo et al., 2004).RTK/Ras/MAPK signaling in both P12 and the vulva is antagonized by , EFL-1 (E2F) and other "Synthetic Multivulva" (SynMuv) gene products (Ceol and Horvitz, 2001;Fay and Han, 2000;Jiang and Sternberg, 1998;Lu and Horvitz, 1998).There are three classes of SynMuv genes, A, B and C, that function redundantly (Ceol and Horvitz, 2004;Ferguson and Horvitz, 1989).Many of these genes encode nuclear proteins with apparent roles in RTK/Ras/MAPK signaling chromatin remodeling and transcriptional regulation (Fay and Han, 2000).Some SynMuv genes seem to function cell autonomously and could antagonize Ras signaling at the level of downstream transcriptional output (Thomas and Horvitz, 1999).However, other SynMuv genes (including lin-35/Rb) appear to function cell non-autonomously in the syncytial hypodermis hyp7, suggesting that hyp7-derived signals can influence LET-23 RTK activity (Herman and Hedgecock, 1990;Myers and Greenwald, 2005).

Conclusions and future prospects
Although the basic framework of the RTK/Ras/MAPK pathway is well characterized, the pathway is subject to complex regulation that we are only just beginning to understand.A most surprising finding in C. elegans has been the large number of regulatory proteins that have only modest individual effects on signaling.Furthermore, some regulatory proteins (such as LIN-2/LIN-7/LIN-10) have very cell-type specific effects.Downstream targets of MPK-1 also appear to be cell-type specific, and likely control how a particular cell responds to the same basic signaling pathway.Although vulval development has been and will continue to be a powerful model for studying RTK/Ras/MAPK signaling, ongoing studies of other Ras-mediated developmental processes will surely reveal new and different types of regulatory mechanisms and targets.Maloof, J.N., and Kenyon, C. (1998).The Hox gene lin-39 is required during C. elegans vulval induction to select the outcome of Ras signaling.Development 125,[181][182][183][184][185][186][187][188][189][190]

Figure 1 .
Figure 1.Variations of the RTK/Ras/MAPK signaling pathway controlling different developmental processes in C. elegans.The RTKs LET-23 (EGFR) and EGL-15 (FGFR) are activated by different ligands and control different sets of developmental processes.A) LIN-3/LET-23-dependent processes include vulval, excretory duct and P12 development; B) EGL-17/EGL-15-dependent processes include sex myoblast migration; C) Neither LET-23 nor EGL-15 control pachytene progression during germline meiosis.Both RTKs signal through the adaptor SEM-5 (Grb2) to activate the same core Ras/MAPK pathway.EGL-15 and SEM-5 may also activate additional pathways during sex myoblast migration.The scaffold proteins KSR-1 and KSR-2 assist in LIN-45 (Raf) and/or MEK-2 activation; different processes have different requirements for these two KSR proteins.Shown in A) are five nuclear proteins (LIN-1, SUR-2, LIN-25, EOR-1, EOR-2) that are jointly important for vulval, excretory duct and P12 cell fates.Downstream targets of MPK-1 during sex myoblast migration and germline meiosis are not yet known.See text andTable 1 for details and references.

Figure 2 .
Figure 2. let-60 ras mutant phenotypes.Wild-type phenotypes are shown on the left, Ras pathway mutant phenotypes on the right.See text for details.
Vulval defects: Ras signaling cooperates with Notch and Wnt signaling to promote hermaphrodite vulval development (see Vulval development).let-60 loss-of-function mutants lack a vulva (Vulvaless or Vul phenotype; Figure 2) whereas let-60 gain-of-function mutants have extra vulval tissue (Multivulva or Muv phenotype).

Figure 3 .
Figure 3. Regulation of Ras proteins.Ras GTPase regulation has been extensively studied in other systems(Malumbres and Barbacid, 2002).Ras-GDP is inactive, whereas Ras-GTP is active and can bind to effectors such as Raf.Guanine nucleotide exchange factors (GEFs) such as SOS-1 positively regulate Ras by promoting GDP dissociation.GTPase activating proteins (GAPs) negatively regulate Ras by stimulating Ras' intrinsic GTP hydrolyzing activity.Gain-of-function (gf) mutations lock Ras in the active, GTP-bound state; the let-60(gf) allele n1046 (G13E) has been widely used for genetic analyses(Beitel et al., 1990; Han and Sternberg, 1990).Dominant-negative (dn) mutations lock Ras in the inactive, GDP-bound state, causing it to bind stably to and titrate out GEFs; a variety of let-60(dn) mutations have been described(Han and Sternberg, 1991).

Table 1 . Core components, regulators and targets of the C. elegans RTK/Ras/MAPK signaling pathway.
Core components are shown in black, positive regulators or targets are shown in green, and negative regulators or targets are shown in red.