A lin-45 raf Enhancer Screen Identifies eor-1, eor-2 and Unusual Alleles of Ras Pathway Genes in Caenorhabditis elegans

Corresponding author: Meera V. Sundaram, University of Pennsylvania School of Medicine, 709A Stellar-Chance Labs, 422 Curie Blvd., Philadelphia, PA 19104-6100., sundaram{at}mail.med.upenn.edu (E-mail)

Communicating editor: P. ANDERSON

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In Caenorhabditis elegans, the Ras/Raf/MEK/ERK signal transduction pathway controls multiple processes including excretory system development, P12 fate specification, and vulval cell fate specification. To identify positive regulators of Ras signaling, we conducted a genetic screen for mutations that enhance the excretory system and egg-laying defects of hypomorphic lin-45 raf mutants. This screen identified unusual alleles of several known Ras pathway genes, including a mutation removing the second SH3 domain of the sem-5/Grb2 adaptor, a temperature-sensitive mutation in the helical hairpin of let-341/Sos, a gain-of-function mutation affecting a potential phosphorylation site of the lin-1 Ets domain transcription factor, a dominant-negative allele of ksr-1, and hypomorphic alleles of sur-6/PP2A-B, sur-2/Mediator, and lin-25. In addition, this screen identified multiple alleles of two newly identified genes, eor-1 and eor-2, that play a relatively weak role in vulval fate specification but positively regulate Ras signaling during excretory system development and P12 fate specification. The spectrum of identified mutations argues strongly for the specificity of the enhancer screen and for a close involvement of eor-1 and eor-2 in Ras signaling.

RECEPTOR tyrosine kinase (RTK)-Ras-extracellular signal regulated kinase (ERK) signaling pathways control many different aspects of animal development. The basic features of such signaling pathways have now been elucidated through a combination of biochemical studies in mammalian cells and genetic studies in model organisms such as Drosophila and Caenorhabditis elegans (WASSARMAN et al. 1995 ; CAMPBELL et al. 1998 ; STERNBERG and HAN 1998 ; VOJTEK and DER 1998 ). Growth factor binding stimulates dimerization and subsequent autophosphorylation of RTKs, creating docking sites for adaptor proteins such as Grb2 (SCHLESSINGER 2000 ). Grb2 binds to RTK phosphotyrosine sites via its SH2 domain and to the guanine nucleotide exchange factor (GEF) Sos via its SH3 domains. This interaction localizes Sos to the plasma membrane and allows it to catalyze the exchange of GDP for GTP on Ras (DOWNWARD 1996 ). Ras-GTP then binds to the kinase Raf, and other poorly understood events at the plasma membrane stimulate Raf kinase activity (MORRISON and CUTLER 1997 ). Once activated, Raf phosphorylates and activates MEK, which then phosphorylates and activates ERK. ERK can then translocate into the nucleus where it phosphorylates multiple substrates, including Ets domain transcription factors (YORDY and MUISE-HELMERICKS 2000 ), which then cooperate with or antagonize other factors to elicit cell-type-specific responses (TAN and KIM 1999 ; SIMON 2000 ). Since misregulated Ras pathway signaling contributes to many human pathologies, including cancer, it is of great interest to understand the different ways in which this pathway is normally regulated and might be therapeutically controlled. To identify positive regulators of Ras signaling, we conducted a genetic screen for enhancers of lin-45 raf mutant defects in C. elegans.

In C. elegans, Ras signaling is conveniently not required for mitotic cell division during larval development (YOCHEM et al. 1997 ), but it is required for multiple developmental events, including excretory duct cell fate specification (and hence viability; YOCHEM et al. 1997 ), germline meiotic progression (and hence fertility; CHURCH et al. 1995 ), the P12 ectodermal blast cell fate (JIANG and STERNBERG 1998 ), proper sex myoblast migration (SUNDARAM et al. 1996 ), and certain gonadal and vulval cell fates (and hence egg-laying ability; HAN et al. 1990 ; CHANG et al. 1999 ; WANG and STERNBERG 2000 ). The Ras pathway has been best studied for its role in vulval fate specification, where it determines in part which of six initially equipotent vulval precursor cells (VPCs) will adopt vulval fates (STERNBERG and HAN 1998 ). In wild-type animals, three VPCs adopt vulval fates. Activating mutations in Ras pathway genes cause a Multivulva (Muv) phenotype in which more than three VPCs adopt vulval fates, while loss-of-function mutations in Ras pathway genes cause a Vulvaless (Vul) phenotype in which fewer than three VPCs adopt vulval fates. Screens for Muv and Vul mutants (or for suppressors of such mutants) have elucidated a core Ras pathway consisting of lin-3 (epidermal growth factor-like growth factor), let-23 (RTK), sem-5 (Grb2), let-341 (Sos), let-60 (Ras), lin-45 (Raf), mek-2 (MEK), mpk-1 (ERK), and lin-1 (Ets domain; Figure 1). Such screens have also identified genes that regulate signal transmission through the Ras pathway, including ksr-1 (KORNFELD et al. 1995 ; SUNDARAM and HAN 1995 ), and several likely transcriptional regulators that appear to act in parallel to lin-1, including sur-2 (SINGH and HAN 1995 ) and lin-25 (TUCK and GREENWALD 1995 ; Fig 1).

View larger version (9K): In this window In a new window Download PPT slide   Figure 1. The core Ras pathway in C. elegans (STERNBERG and HAN 1998 ) and positive regulators identified in the lin-45 raf enhancer screen. C. elegans genes are shown in italics. Molecular identities or mammalian homologs are indicated in parentheses. Some tissue-specific targets of the pathway, such as lin-31 (MILLER et al. 1993 ) and lin-39 (MALOOF and KENYON 1998 ), are not shown. eor-1 and eor-2 are placed downstream or in parallel to mpk-1 on the basis of epistasis analysis and molecular identities (R. M. HOWARD and M. V. SUNDARAM, unpublished results).

Since most screens for Ras pathway regulators have focused on vulval phenotypes, genes that primarily regulate Ras signaling in other tissues likely have been missed. By screening for enhancers of the egg-laying defective (Egl) and lethal defects of hypomorphic lin-45 raf mutants, we identified multiple alleles of eor-1 and eor-2, two genes that positively regulate Ras signaling during excretory system development and P12 fate specification and play a relatively minor role during vulval development. These genes were identified independently in other genetic screens (M. HERMAN and M. HENGARTNER, personal communication) and hence named eor (egl-1 suppressor, DiO-uptake defective, Raf enhancer). In addition, we identified interesting alleles of several known Ras pathway genes, including sem-5, let-341, and lin-1. The genetic behavior and molecular lesions of these alleles provide insight into the normal regulation of Ras pathway components.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

General methods and alleles:
General methods for the handling, culturing, and ethyl methanesulfonate (EMS) mutagenesis of nematodes were as previously described (BRENNER 1974 ). Experiments were performed at 20° unless otherwise noted. C. elegans var Bristol strain N2 is the wild-type parent for all strains used in this work. Specific genes and alleles are listed below (see RIDDLE et al. 1997 and references therein unless otherwise noted).

• LGI: dpy-5(e61), sur-6(cs24) (SIEBURTH et al. 1999 ), unc-13(e51), unc-54(e190), unc-101(m1).

• LGIV: dpy-13(e184), dpy-20(e1282), let-60(n1046gf), let-277(m262), let-279(m261), let-280(m259), let-281 (m247), let-282(m258), let-284(m267), lin-45(ku51) (Y. HAN and M. HAN, personal communication), lin-45(ku112) (SUNDARAM and HAN 1995 ), mDf8 (ROGALSKI and RIDDLE 1988 ), unc-5(e53), unc-17(e113).

• LGV: dpy-11(e224), eT1(III;V) (ROSENBLUTH and BAILLIE 1981 ), let-341(s1031), lin-25(e1446), unc-46 (e177), unc-76(e936).

• LGX: dpy-6(e14), gap-1(ga133) (HAJNAL et al. 1997 ), ksr-1(n2526), let-4(mn105), lin-15(n765), lon-2 (e678), sem-5(n1779), syc-2 (gm132) (FORRESTER et al. 1998 ), unc-3(e151), mnDf7 (MENEELY and HERMAN 1979 ).

Isolation and preliminary characterization of enhancer mutations:
MH575 [lin-45(ku51) dpy-20] or MH620 [lin-45(ku112) dpy-20] hermaphrodites were mutagenized with 50 mM EMS and allowed to self-fertilize; their F₁ progeny were then picked to individual plates and allowed to self-fertilize. From plates with multiple F₂ Egl animals, individual Egl animals were picked to screen for the presence of rod-like arrested larvae inside the body cavity (bag of rods phenotype) and to establish candidate lin-45 dpy-20; m/m homozygous lines (where m is the new mutation). In cases where the penetrance of the Egl and lethal phenotypes was very high, wild-type siblings were also picked to establish heterozygous lines. Mutations isolated in the ku51 background include cs1, cs7, cs8, cs13, and cs14 (cs14 was subsequently lost). Mutations isolated in the ku112 background include cs15, cs24, cs26, cs28, cs30, cs31, cs40, cs41, cs42, cs43, cs44, cs47, cs48, cs50, cs51, and cs52.

Candidate strains were outcrossed to N2 males, and the broods of lin-45 dpy-20/++; m/+ animals were scored to determine whether m was an enhancer of lin-45 raf. If comparable proportions of Dpy and non-Dpy animals were Egl, then m was inferred to cause an Egl phenotype on its own, and non-Dpy Egl animals were picked to establish putative m/m lines for further outcrossing and analysis. Alleles in this category include sur-2(cs8, cs43, cs48), lin-25(cs13), sem-5(cs15), and five others that resemble sur-2 or lin-25 alleles but have not been further characterized. If Dpy animals were more frequently Egl than non-Dpy animals, then m was inferred to be an enhancer mutation (or else linked to dpy-20), and candidate lin-45 dpy-20/++; m/m animals were identified and used to establish putative m/m lines for further outcrossing and analysis. Each mutation was then crossed back into the lin-45 mutant background to verify its enhancer properties.

Mapping and complementation tests:
Genetic mapping and complementation tests were performed using standard methods. With the exception of lin-1(cs50), all mutations described here are recessive enhancers.

sem-5(cs15) X:

cs15 was initially mapped to the X chromosome on the basis of the observation that male cross-progeny of cs15 mothers were incapable of mating. Of 42 cs15 homozygotes from cs15/lon-2 dpy-6 mothers, 10 segregated lon-2 and one segregated dpy-6. cs15 failed to complement sem-5(n1779) for the Egl phenotype.

let-341(cs41) V:

Two-factor mapping experiments were performed in the lin-45(ku112) background at 20°. Of 10 cs41 homozygotes from cs41/dpy-11 mothers, 3 segregated dpy-11. At 25°, cs41 failed to complement let-341(s1031) for the Egl and lethal phenotypes.

lin-1(cs50) IV:

In the initial outcross of cs50 lin-45(ku112) dpy-20, >25% (35/95) of the Dpy animals but almost no non-Dpy animals (1/161) were Egl, suggesting that cs50 is on chromosome IV. Of 73 cs50 lin-45(ku112) homozygotes from cs50 lin-45(ku112)/unc-17 dpy-13 mothers, none segregated unc-17 and/or dpy-13. cs50 was determined to be an allele of lin-1 by DNA sequencing.

ksr-1(cs1) X:

cs1 was mapped to the X chromosome on the basis of the observation that male cross-progeny of cs1 mothers transmitted cs1 to 100% of their progeny. cs1 failed to complement ksr-1(n2526) for the ability to suppress the Muv phenotype of let-60(n1046gf).

sur-6(cs24) I:

Two-factor mapping experiments and complementation tests were performed in the lin-45(ku112) background. Of 37 cs24 homozygotes from cs24/unc-13 mothers, 1 segregated unc-13. cs24 failed to complement sur-6(ku123) for the enhancer phenotype.

sur-2(cs26, cs31) I:

Two-factor mapping experiments and complementation tests were performed in the lin-45(ku112) background. Of 41 cs31 homozygotes from cs31/unc-54 mothers, 3 segregated unc-54. cs26 failed to complement cs31 and sur-2(ku9) for the enhancer phenotype.

lin-25(cs52) V:

Three-factor mapping experiments and complementation tests were performed in the lin-45(ku112) background. Of 48 cs52 homozygotes from cs52/dpy-11 unc-76 mothers, 4 segregated dpy-11 and 2 segregated unc-76. cs52 failed to complement lin-25(e1446) for the enhancer phenotype.

eor-1(cs28, cs40, cs44) IV:

In initial outcrosses with eor-1 lin-45(ku112) dpy-20 strains, >75% of Dpy animals but almost no non-Dpy animals were Egl, placing eor-1 on chromosome IV. In three-factor mapping experiments, 33/46 Dpy-13 not-Unc-5 recombinants from cs28/dpy-13 unc-5 mothers segregated cs28, placing eor-1 between these two markers. mDf8, cs40, and cs44 failed to complement cs28 for the lethal and/or enhancer phenotypes. eor-1 maps in the vicinity of sur-8; however, both complementation testing and molecular analysis have verified that eor-1 is a distinct locus (R. M. HOWARD and M. V. SUNDARAM, unpublished results). eor-1 (cs28) also complemented let-277, let-279, let-280, let-281, let-282, and let-284.

eor-2(cs7, cs30, cs42, cs47, cs51) X:

eor-2 was initially mapped to the X chromosome on the basis of the observation that male progeny of eor-2 mothers are uncoordinated (Unc) and incapable of mating. Of 58 cs30 homozygotes from cs30/unc-3 mothers, 1 segregated unc-3. In three-factor mapping experiments, 4/4 Eor-2 not-Unc-3 recombinants from cs30 unc-3/lin-15 mothers segregated lin-15, placing eor-2 to the left of unc-3. mnDf7, cs7, cs42, cs47, and cs51 all failed to complement eor-2(cs30) for the Unc and/or lethal phenotypes. eor-2(cs30) complemented let-4 and syc-2.

Phenotypic observations:
General methods for Nomarski differential interference contrast (DIC) microscopy of live animals were as previously described (SULSTON and HORVITZ 1977 ).

Lethal and Egl phenotypes:

Two or more hermaphrodites of the indicated genotype were picked singly to plates and allowed to lay eggs for 8–24 hr. Rod-like arrested larvae were counted and removed after 1–2 days. Surviving adults were scored as Egl if they appeared bloated with late-stage eggs or hatching larvae. One to three percent of eor-1 and eor-2 mutants die as rod-like young adults, suggesting a late defect in excretory system development or function; these are not included in the larval lethal or Egl categories.

Vul phenotype:

The numbers of vulval and nonvulval descendants of P(3-8).p were counted in L4 stage larvae under DIC optics. Animals with <22 vulval descendants and >6 nonvulval descendants were considered Vulvaless. In some sur-6(cs24) mutants, but not in other mutants described here, some P(4-8).p cells failed to divide and may have adopted an abnormal fused fate (SIEBURTH et al. 1999 ).

2 P11.p phenotype:

Under DIC optics, P11.p and P12.pa nuclei are distinguishable on the basis of nuclear size, morphology, and position (JIANG and STERNBERG 1998 ). The number of P11.p-like nuclei anterior to the anus was counted in L3 or L4 stage larvae.

Sequencing of mutant alleles:
The ksr-1(cs1), sem-5(cs15), and lin-1(cs50) lesions were identified by direct sequencing of genomic PCR products, and the lin-45(ku112) and let-341(cs41) lesions were identified by direct sequencing of RT-PCR products. In each case the entire gene coding region was sequenced (except for lin-1, in which only the 3' half was sequenced) and only a single lesion was identified. lin-1 was also sequenced from the MH620 strain to verify that the cs50 lesion was not present in this parental lin-45(ku112) strain. All lesions were verified by sequencing at least two independently derived PCR products.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

lin-45 hypomorphs are sensitized to further reductions in Ras signaling:
The lin-45(ku51) and lin-45(ku112) alleles weakly reduce lin-45 raf activity but do not cause overt phenotypes (Table 1A; SUNDARAM and HAN 1995 ). ku51 is a missense mutation changing leucine 252 to valine (M. HAN, personal communciation). Interestingly, we identified the ku112 lesion as a missense change affecting the C-terminal conserved 14-3-3 binding site (Fig 2A), suggesting that binding of the chaperone protein 14-3-3 to this site normally promotes LIN-45 activity (see DISCUSSION).

View larger version (14K): In this window In a new window Download PPT slide   Figure 2. Molecular lesions associated with the lin-45 (ku112), sem-5(cs15), let-341(cs41ts), and lin-1(cs50) alleles. Schematic drawings represent each protein, with the general positions of each amino acid substitution marked by the allele name. Below each drawing the specific nucleotide and amino acid substitutions are shown above and below the wild-type sequences, respectively. (A) lin-45(ku112) (S754F) affects the C-terminal 14-3-3 binding site. CR1, CR2, and CR3 are conserved regions found in all Raf family members (MORRISON and CUTLER 1997 ). (B) sem-5(cs15) (W192STOP) truncates the protein within the second SH3 domain. (C) let-341(cs41ts) (E980K) affects the CDC25 Ras GEF homology domain. DH, Dbl homology domain; PH, pleckstrin homology domain. (D) lin-1(cs50) (P316L) affects a minimal consensus mitogen-activated protein kinase phosphorylation site. FQFP, ERK docking site (JACOBS et al. 1999 ).

Both lin-45 raf hypomorphic mutant backgrounds are very sensitive to further reductions in Ras pathway signaling. For example, a putative null allele of ksr-1, n2526, causes few defects on its own, but causes partially penetrant larval lethal and Egl defects in the lin-45(ku51) background and more highly penetrant larval lethal, Egl, and Vul defects in the lin-45(ku112) background (Table 1B; SIEBURTH et al. 1999 ). Furthermore, even a very weak allele of mpk-1 causes nearly complete lethality in the lin-45(ku112) background (SUNDARAM and HAN 1995 ). These observations suggested that we might identify new positive regulators of Ras signaling by screening for enhancers of lin-45 hypomorphic defects.

Genetic screens for enhancers of lin-45 hypomorphic alleles:
We screened for recessive mutations that cause a "bag of rods" phenotype in the lin-45(ku51) or lin-45(ku112) mutant backgrounds (MATERIALS AND METHODS). Homozygous lin-45 hermaphrodites were mutagenized with EMS, and F₁ progeny, which were potentially heterozygous for an enhancer mutation, were placed on individual petri plates. F₂ progeny were screened for retention of eggs and the presence of rod-like arrested larvae inside the body cavity. Note that since this screen required that F₂ animals survive to adulthood and give dead F₃ progeny, it would not identify mutations that cause significant F₂ lethality or sterility; we predicted that most mutations in core Ras pathway genes would fall into this category and thus be avoided. From a total of 1316 ku51 and 7254 ku112 F₁ animals, we identified 26 bag of rods candidates. Outcrossing revealed that the new mutations were of two types: those that cause strong Egl defects on their own and cause more severe or additional defects in combination with the lin-45 raf allele (11 alleles) and those that cause weak or no defects on their own, but cause strong Egl and lethal phenotypes in combination with the lin-45 raf allele (15 alleles, 1 of which was subsequently lost; see MATERIALS AND METHODS).

Mutations of the first type include alleles of the previously described genes sur-2 (SINGH and HAN 1995 ) and lin-25 (TUCK and GREENWALD 1995 ; MATERIALS AND METHODS); their enhancer properties reveal that these genes positively regulate Ras signaling in tissues other than the vulva, as recently reported by NILSSON et al. 2000 . These mutations have not been further characterized. Additional mutations of the first type are sem-5(cs15) and let-341(cs41ts), which are described below. Mutations of the second type affect five previously described genes (lin-1, ksr-1, sur-6, sur-2, lin-25) and two new genes (eor-1, eor-2).

Phenotypes analyzed in the mutant strains:
Each new mutation of interest was scored for phenotypic effects in single mutants and in double mutants with lin-45(ku112) (Table 2). We scored four phenotypes that are commonly associated with reduced Ras signaling efficiency.

1. Rod-like larval lethality: Ras pathway loss-of-function mutants die during early larval development with a distinctive fluid-filled, rod-like appearance (Fig 3A and Fig B). Mosaic analysis of let-60 ras suggested that this lethality is caused by a failure of the excretory duct cell to differentiate properly (YOCHEM et al. 1997 ), although mosaic analysis of let-23 suggested that rod-like lethality may also result from distinct excretory system defects (KOGA and OHSHIMA 1995 ).

   
   

   View larger version (90K): In this window In a new window Download PPT slide   Figure 3. let-60 ras-like lethal, Egl, Vul, and 2 P11.p phenotypes seen in enhancer mutants. (A) Wild-type larva. (B) eor-1(cs28) lin-45(ku112) rod-like larva. (C) Wild-type adult hermaphrodite; arrowhead indicates vulva. (D) eor-1(cs28) lin-45 (ku112) adult bloated with late-stage embryos and hatching larvae; arrowhead indicates normal vulva. (E) Wild-type L4 larva showing normal vulval invagination. (F) sur-6(cs24); lin-45(ku112) L4 larva in which P5.pp and P6.pp (arrows) failed to adopt vulval cell fates. (G) Wild-type larva with one P11.p cell (arrow) and one P12.pa cell (arrowhead). (H) eor-1(cs28) lin-45(ku112) larva with 2 P11.p-like cells (arrows).

   
   

   View this table: In this window In a new window   Table 2. Enhancers of lin-45 raf

2. Egg-laying (Egl) defects: Ras pathway loss-of-function mutants are often Egl (Fig 3C and Fig D). A lack of vulval cells (see below), abnormalities in sex myoblast migration (SUNDARAM et al. 1996 ), gonadal differentiation (CHANG et al. 1999 ), or vulval cell differentiation (WANG and STERNBERG 2000 ) may contribute to this phenotype.

3. Vulvaless (Vul) defects: The Ras pathway is required for the vulval precursor cells P5.p, P6.p, and P7.p to adopt vulval fates (STERNBERG and HAN 1998 ). In Ras pathway loss-of-function mutants, one or more of these cells often adopts a nonvulval fate, resulting in an incomplete or absent vulva (Fig 3E and Fig F).

4. 2 P11.p defects: The Ras pathway is required for the ventral ectodermal blast cell P12 to adopt a fate different from its neighbor P11 (JIANG and STERNBERG 1998 ). In Ras pathway loss-of-function mutants, P12 often adopts a P11 fate, resulting in two P11.p-like descendants anterior to the anus (Fig 3G and Fig H).

Mutations in core Ras pathway genes:
Our enhancer screen identifed only three mutations in core Ras pathway components, and all are unusual and informative alleles.

sem-5(cs15) X:

cs15 is an allele of sem-5, which encodes a Grb2-like SH3-SH2-SH3 domain adaptor protein that functions to connect LET-23 receptor tyrosine kinase signaling to LET-60 RAS activation (CLARK et al. 1992 ). cs15 appears to partially reduce sem-5 function, since it causes rod-like lethal, Egl, Vul, and 2 P11.p defects similar to (but less penetrant than) those seen in strong sem-5 mutants (Table 2A). However, cs15 also appears to increase signaling activity, since unlike strong sem-5 alleles, cs15 causes a synthetic Muv phenotype in a gap-1 [GTPase-activating protein (HAJNAL et al. 1997 )] mutant background (Table 3A). cs15 also dominantly enhances the activated let-60 ras Muv phenotype (Table 3B).

To understand the molecular basis for this unusual genetic behavior, we sequenced the cs15 allele and identified its lesion. cs15 is a nonsense mutation predicted to truncate the SEM-5 protein within the second SH3 domain (Fig 2B). This finding suggests that the C-terminal SH3 domain of SEM-5 has an inhibitory or negative signaling function.

let-341(cs41ts) V:

cs41 is an allele of let-341, which encodes a Sos-related guanine nucleotide exchange factor that acts upstream of let-60 ras (JOHNSEN and BAILLIE 1991 ; CHANG et al. 2000 ). Previously described let-341 mutants are 100% embryonic or larval lethal. cs41 is temperature sensitive such that homozygotes appear essentially wild type (but enhance lin-45(ku112) defects) at 20°, but have strong rod-like lethal, Egl, Vul, and 2 P11.p defects at 25° (Table 2B). These pleiotropic defects of let-341(cs41) animals, and the ability of an activated let-60 ras allele to suppress these defects (Table 2B), support a role for Sos in many different Ras-dependent processes in C. elegans.

We sequenced the cs41 allele and found that it contains a missense mutation within the CDC25-like Ras GEF domain (Fig 2C). The affected residue is not well conserved among different Sos family members, but it is located near the tip of the helical hairpin that catalyzes guanine nucleotide exchange on Ras (BORIACK-SJODIN et al. 1998 ).

lin-1(cs50) IV:

cs50 is an allele of lin-1, which encodes an Ets domain transcription factor that negatively regulates Ras signaling (BEITEL et al. 1995 ). lin-1 null mutants are Muv. In contrast, cs50 has no discernible phenotype on its own but strongly enhances the lin-45(ku112) rod-like lethal, Vul, and 2 P11.p phenotypes (Table 2C). Since cs50 has opposite phenotypic effects from those expected for a loss-of-function mutation and is a semidominant enhancer of lin-45(ku112), cs50 appears to be a weak gain-of-function allele of lin-1. We identified the cs50 lesion as a missense mutation that changes proline 316 of a minimal ERK phosphorylation site (S/T-P) to a leucine (Fig 2D). This suggests that serine 315 may be a target for phosphorylation and negative regulation by MPK-1.

Mutations in positive regulatory genes:
Our enhancer screen also identified alleles of four known positive regulatory genes and two new genes.

ksr-1(cs1) X:

cs1 is an allele of ksr-1, which encodes a kinase-like protein that promotes signaling at a step between Ras and Raf (KORNFELD et al. 1995 ; SUNDARAM and HAN 1995 ; SIEBURTH et al. 1999 ). cs1 causes weak Egl and larval lethal phenotypes on its own, strong Egl, Vul, and rod-like lethal phenotypes in the lin-45(ku51) background (where it was recovered), and complete lethality in the lin-45(ku112) background (Table 1C). Notably, ksr-1(cs1) is a stronger enhancer than the putative null allele ksr-1(n2526) (Table 1), indicating that this allele has a dominant-negative character. The cs1 lesion changes arginine 531 to histidine (MATERIALS AND METHODS) and is identical to the previously described ksr-1(ku68) lesion (SUNDARAM and HAN 1995 ).

sur-6(cs24) I:

cs24 is a partial loss-of-function allele of sur-6, which encodes a B regulatory subunit of protein phosphatase 2A that promotes signaling at a step between Ras and Raf (SIEBURTH et al. 1999 ). A partial genetic characterization of cs24 has been described elsewhere (SIEBURTH et al. 1999 ). cs24 causes very weak Egl, Vul, and Unc phenotypes on its own, but causes strong rod-like lethal, Egl, and Vul phenotypes and weak Unc and 2 P11.p phenotypes in the lin-45(ku112) background (Table 2D; Fig 3F).

sur-2(cs26, cs31) I:

cs26 and cs31 are alleles of sur-2, which encodes a possible component of the transcriptional Mediator/Srb complex and functions downstream of mpk-1 during vulval induction (SINGH and HAN 1995 ; LACKNER and KIM 1998 ; BOYER et al. 1999 ). Most sur-2 alleles cause a strong Vul phenotype, but few other defects. cs26 and cs31 cause only weak Egl and Vul phenotypes (Table 2E) and thus appear to be hypomorphic. cs26 and cs31 cause strong Egl, Vul, and rod-like lethal phenotypes in the lin-45(ku112) background (Table 2E).

lin-25(cs52) V:

cs52 is an allele of lin-25, which encodes a novel protein thought to function with SUR-2 (TUCK and GREENWALD 1995 ; NILSSON et al. 1998 ). Most lin-25 alleles cause a strong Vul phenotype, but few other defects. cs52 causes only weak Egl and Vul phenotypes (Table 2F) and thus appears to be hypomorphic. cs52 causes strong Egl, Vul, and rod-like lethal phenotypes and a weak 2 P11.p phenotype in the lin-45(ku112) background (Table 2F).

eor-1(cs28, cs40, cs44) IV:

These three alleles define a new locus (MATERIALS AND METHODS), which we named eor-1. Each allele causes similar weak Unc, Egl, rod-like lethal, and 2 P11.p phenotypes on its own, and weak Unc and Vul but strong Egl, lethal, and 2 P11.p phenotypes in the lin-45(ku112) background (Table 2G; Fig 3B, Fig D, and Fig H). The basis for the Egl phenotype is unknown, as vulval development appears normal in most animals. eor-1(cs28)/mDf8 animals resemble eor-1 homozygotes (Table 2G); therefore, the eor-1 mutations appear to be loss-of-function alleles.

eor-2(cs7, cs30, cs42, cs47, cs51) X:

These five alleles behave identically to the eor-1 mutations described above, but were mapped to a distinct locus (MATERIALS AND METHODS), which we named eor-2. Each allele causes similar weak Unc, Egl, rod-like lethal, and 2 P11.p phenotypes on its own, and weak Unc and Vul but strong Egl, lethal, and 2 P11.p phenotypes in the lin-45(ku112) background (Table 2H). The basis for the Egl phenotype is unknown as vulval development appears normal in most animals. eor-2(cs30)/mnDf7 animals resemble eor-2 homozygotes (Table 2H); therefore, the eor-2 mutations appear to be loss-of-function alleles.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Over the last decade, experiments in multiple systems have elucidated a highly conserved signal transduction pathway involving receptor tyrosine kinases, the Ras GTPase, and the Raf/MEK/ERK kinase cassette. However, many questions remain about how the Raf/MEK/ERK cassette is initially activated, how the strength and duration of signaling are controlled, what key targets ERK phosphorylates, and what other Ras-dependent or Ras-independent factors cooperate with ERK to affect downstream gene expression and specific cellular behaviors. Enhancers of lin-45 raf, in principle, could define genes involved in any of these regulatory processes. Our screen identified mutations in three core components of the Ras pathway (sem-5, let-341, and lin-1), two positive regulators that act at a step between Ras and Raf (ksr-1 and sur-6), two positive regulators that act downstream or in parallel to mpk-1 ERK (sur-2 and lin-25), and two previously uncharacterized positive regulators (eor-1 and eor-2; Fig 1). Although eor-1 and eor-2 also have roles in Ras-independent developmental events (M. HERMAN and M. HENGARTNER, personal communication), the spectrum of mutations we identified argues strongly for the specificity of our enhancer screen and therefore for a close involvement of eor-1 and eor-2 in Ras signaling.

Unusual alleles of lin-45, sem-5, let-341, and lin-1:
Since our primary goal was to identify new regulators of Ras signaling, a positive aspect of our enhancer screen was that it selectively identified mutations in positive regulatory genes, while for the most part avoided mutations in core components of the Ras pathway. This is probably due to the fact that most alleles of core pathway genes would cause strong F₂ lethality and/or sterility in the lin-45 hypomorphic mutant backgrounds. However, we did identify single alleles of three core components, sem-5 Grb2, let-341 Sos, and lin-1 Ets, and these alleles pinpoint domains or residues likely to play important roles in regulating these components. The lin-45 raf allele that we used for most of our screens also contains an interesting lesion.

lin-45(ku112) is a missense mutation in the C-terminal 14-3-3 binding site of LIN-45 RAF. 14-3-3 is a chaperone protein that binds preferentially to the consensus sequence RSXSXP (MUSLIN et al. 1996 ). This sequence is found near the C terminus of all Raf family members, and binding of 14-3-3 to this site positively regulates mammalian B-Raf activity (MACNICOL et al. 2000 ). The hypomorphic nature of lin-45(ku112) (which instead has the sequence RFXSXP) suggests that 14-3-3 binding to this site likely positively regulates LIN-45 Raf activity in C. elegans as well.

sem-5(cs15) truncates the second SH3 domain of the SEM-5 adaptor. While the first SH3 domain of SEM-5/Grb2 is primarily responsible for interactions with Sos (SASTRY et al. 1995 ), the role of the second SH3 domain is less clear. Our finding that cs15 can increase Ras signaling suggests that the second SH3 domain has an inhibitory or negative signaling function. Such a negative function for Grb2 family members has been proposed recently on the basis of physical interactions between Grb2 proteins and negative regulators such as the adaptor protein Disabled (LE and SIMON 1998 ; XU et al. 1998 ; ZHOU and HSIEH 2001 ) or the tyrosine kinase ARK-1 (HOPPER et al. 2000 ). However, genetic evidence for a Grb2 negative function has until now been limited to the case of one Ras-independent RTK-mediated process (HOPPER et al. 2000 ). The sem-5(cs15) allele will be a valuable tool for investigating further the negative function of SEM-5/Grb2, which our data suggest may be of widespread importance.

let-341(cs41ts) contains a missense mutation in the helical hairpin of the Ras GEF domain of Sos and causes a spectrum of defects consistent with reduced Ras signaling. Therefore, cs41 is likely to specifically affect the ability of LET-341 Sos to catalyze guanine nucleotide exchange on LET-60 RAS and may not affect Dbl-domain-mediated exchange activity toward Rho family GTPases (e.g., NIMNUAL et al. 1998 ). This temperature-sensitive allele will be very useful in dissecting the contributions of LET-341 Sos to different Ras-mediated processes.

lin-1(cs50) is an apparent gain-of-function allele and contains a missense mutation that changes the proline of a consensus ERK phosphorylation site to a leucine. All previously described lin-1 gain-of-function mutations appear to disrupt the C-terminal ERK docking site of LIN-1, suggesting that these mutations are able to escape negative regulation by MPK-1/ERK (JACOBS et al. 1998 , JACOBS et al. 1999 ). However, LIN-1 has 18 potential ERK phosphorylation sites (BEITEL et al. 1995 ), and it is not yet known which of these sites is important for LIN-1 regulation. On the basis of the cs50 lesion and its weak gain-of-function effects, we hypothesize that Ser315 is one of multiple MPK-1/ERK phosphorylation sites required to downregulate LIN-1.

Genes that act between Ras and Raf:
Our enhancer screen identified single non-null alleles of ksr-1 and sur-6, two genes that positively regulate Ras signaling at a step between Ras and Raf (Fig 1). These mutations cause only very mild let-60 ras-like defects, but strongly enhance the rod-like lethal and Vul defects of lin-45(ku112) mutants. sur-6(cs24), unlike ksr-1 mutations, also enhances the lin-45(ku112) 2 P11.p defect.

KSR is a conserved Raf-related protein (KORNFELD et al. 1995 ; SUNDARAM and HAN 1995 ; THERRIEN et al. 1995 ) that binds to MEK (DENOUEL-GALY et al. 1997 ; YU et al. 1997 ) and in mammalian cells is found in a large protein complex containing Raf, MEK, ERK, and a number of other proteins (STEWART et al. 1999 ). KSR has therefore been proposed to be a scaffold protein that assembles Raf/MEK/ERK signaling complexes and/or recruits other regulators into such complexes (MORRISON 2001 ). C. elegans has two partially redundant ksr genes, ksr-1 and ksr-2 (OHMACHI et al. 2002 ). The ksr-1(cs1) allele identified in our lin-45(ku51) enhancer screen is a missense allele encoding R531H. The corresponding variant of murine KSR (R615H) is severely compromised for MEK binding, but still interacts with many other proteins in the KSR complex (STEWART et al. 1999 ), perhaps explaining the dominant-negative behavior of this allele. A propensity for ksr-1 missense alleles to be dominant negative (SUNDARAM and HAN 1995 ) and for ksr-2 mutants to be sterile (OHMACHI et al. 2002 ) may explain our failure to recover any ksr alleles in the lin-45(ku112) background.

SUR-6 is a PR55 family B regulatory subunit for protein phosphatase 2A (PP2A) and may direct the PP2A catalytic core to a particular Ras pathway substrate such as LIN-45 RAF or KSR-1 (SIEBURTH et al. 1999 ). The sur-6(cs24) allele identified in our screen is clearly non-null, since RNA-mediated interference indicates that sur-6 is an essential gene (SIEBURTH et al. 1999 ; FRASER et al. 2000 ; PIANO et al. 2000 ). However, even though cs24 is nearly wild type with respect to viability, it behaves as a strong loss-of-function allele with respect to its ras-like phenotypes (SIEBURTH et al. 1999 ). cs24 is a missense mutation affecting one of several highly conserved WD repeats (NEER et al. 1994 ; SIEBURTH et al. 1999 ), and we propose that it could specifically compromise an interaction between SUR-6 and a Ras pathway component or regulator.

Genes that are required primarily for vulval development:
Our screen identified multiple alleles of sur-2 and lin-25, two genes that act downstream of mpk-1 to promote vulval fates (Fig 1). sur-2 encodes a novel but conserved protein that interacts with components of the human Mediator/Srb complex (SINGH and HAN 1995 ; BOYER et al. 1999 ). lin-25 also encodes a novel protein (TUCK and GREENWALD 1995 ) and is thought to function closely with sur-2 (NILSSON et al. 1998 , NILSSON et al. 2000 ). Since the Mediator/Srb complex associates with RNA polymerase II and recruits it to certain promoters (RACHEZ and FREEDMAN 2001 ), these genes are thought to positively regulate Ras target gene transcription. We isolated several strong sur-2 and lin-25 alleles that cause a Vul phenotype like that of previously described mutants, and several hypomorphic alleles that cause few phenotypes on their own but enhance lin-45(ku112) lethal, Egl, and Vul defects. Our results reinforce the findings of NILSSON et al. 2000 , who showed that sur-2 and lin-25 function in several Ras-mediated processes, although they are required primarily for vulval development.

Genes that are required primarily for Ras-mediated processes other than vulval development:
Our screen identified multiple loss-of-function alleles of two genes, eor-1 and eor-2, that have a relatively weak role during vulval development but appear to positively regulate Ras-mediated signaling in multiple other tissues. eor-1 and eor-2 mutations cause a similar spectrum of weakly penetrant rod-like lethal, Egl, and 2 P11.p defects, and these defects are dramatically enhanced in the lin-45 (ku112) mutant background. It is interesting to note that this spectrum of defects is somewhat reciprocal to those caused by sur-2 or lin-25 mutations, suggesting that different Ras-mediated developmental events have different requirements for eor-1 and eor-2 activity vs. sur-2 and lin-25 activity.

Our recent studies have shown that eor-1 and eor-2 function downstream or in parallel to mpk-1 and encode nuclear proteins that likely act at the level of transcriptional regulation (R. M. HOWARD and M. V. SUNDARAM, unpublished results; Fig 1). Like sur-2 and lin-25, eor-1 and eor-2 could be downstream targets of the Ras pathway or could cooperate with the Ras pathway to promote certain cellular outcomes. Further studies of eor-1 and eor-2 should provide insight into the important question of how Ras signaling controls different downstream transcriptional responses.

	FOOTNOTES

¹ These authors contributed equally to this work.

	ACKNOWLEDGMENTS

We thank Yuming Han for isolating the lin-45(ku51) allele and Min Han for sharing unpublished data and for support during the design of this screen. We also thank Michael Hengartner and Michael Herman for sharing unpublished data, the Caenorhabditis Genetics Center for worm strains, and Elizabeth Bucher and members of our laboratory for helpful comments and advice. This work was supported by a Penn-Hughes award and National Institutes of Health grant 1R01GM58540-01A1 to M.V.S. C.E.R. is a post-doctoral fellow of the Jane Coffin Childs Memorial Fund for Medical Research. R.M.H. is a predoctoral trainee supported by the National Institutes of Health Training Program in Developmental Biology grant 5-T32-HD-07516.

Manuscript received December 17, 2001; Accepted for publication February 11, 2002.

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