ARK-1 Inhibits EGFR Signaling in C. elegans

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1 Molecular Cell, Vol. 6, 65 75, July, 2000, Copyright 2000 by Cell Press ARK-1 Inhibits EGFR Signaling in C. elegans Neil A. Hopper,* Junho Lee, and Paul W. Sternberg Excessive or inappropriate signaling by RTKs has * MRC Laboratory of Molecular Biology been implicated in the progression of several cancers Hills Road and can cause a variety of developmental disorders Cambridge CB2 2QH under experimental conditions. In addition, varying the United Kingdom amount and duration of signaling has been shown to Howard Hughes Medical Institute elicit distinct cellular responses (reviewed by Marshall, Division of Biology 1995; see also Katz et al., 1995; Greenwood and Struhl, California Institute of Technology 1997). This suggests that RTK signaling must be very Pasadena, California tightly regulated with mechanisms to prevent ligandindependent activation and to attenuate signaling after activation has occurred. The nematode C. elegans provides Summary an excellent genetic system in which to study negative regulation of EGFR signaling with inappropriate A screen for synthetic enhancers of sli-1 identified signaling being easily assayable at a developmental ark-1 (for Ack-related tyrosine kinase), a novel inhibitor level. of let-23 EGFR signaling in C. elegans.anark-1 mutaanchor During normal development of C. elegans, the gonadal tion synergizes with mutations in other negative regulators cell induces 3 out of 6 vulval precursor cells of let-23, resulting in increased RAS signaling. (VPCs) to divide and differentiate into vulval tissue (Sul- Genetic analysis suggests that ARK-1 acts upstream ston and White, 1980; Kimble, 1981). The inducing signal of RAS and is dependent upon SEM-5. ARK-1 inhibits is encoded by the lin-3 gene, a member of the epidermal LET-23-mediated ovulation, a RAS-independent funcsponse growth factor (EGF) family of growth factors. The retion. ARK-1 physically interacts with SEM-5 in the to this signal is mediated through let-23, which yeast two-hybrid assay. We find that sem-5 also has encodes an EGFR homolog; sem-5; let-60ras; lin-45raf; a negative function in let-23-mediated ovulation and mek-2; and the mpk-1/sur-1 MAP kinase (reviewed by suggest that this negative function is mediated by the Sternberg and Han, 1998). Thus, vulval induction utilizes recruitment of inhibitors such as ARK-1. the evolutionarily conserved RTK/Ras/MAP kinase sig- naling pathway. Introduction In the VPCs, LET-23 EGFR is localized to the basal surface where it is accessible to inductive signal (reviewed by Kim, 1997). Several negative regulators of Polypeptide growth factors commonly act through activation of receptor tyrosine kinases (RTKs) to elicit a wide EGFR-mediated signaling in C. elegans have been iden- range of biochemical responses that can ultimately lead tified as suppressors of the vulvaless phenotype that to mitosis and/or differentiation. To date, a biochemical results when LET-23 is not properly localized. These approach complemented by genetic studies in the fly include sli-1, gap-1, and unc-101, which encode homo- and the worm has led to a fairly sophisticated idea of how logs of the mammalian proteins Cbl, rasgap, and the these responses are mediated. Thus, ligand-induced real., 1994; Jongeward et al., 1995; Yoon et al., 1995; clathrin-associated protein, AP47, respectively (Lee et ceptor dimerization and subsequent autophosphorylation of specific tyrosine residues create high-affinity Hajnal et al., 1997). Though mutations in all of these binding sites for proteins such as Grb2, the regulatory negative regulators are silent by themselves with re- subunit of phosphoinositol-3 kinase, phospholipase spect to vulval induction, it can be argued that these C-, and others (reviewed by Kazlauskas, 1994). Grb2 mutations result in a higher efficacy of LET-23-mediated and its homologs, SEM-5 in Caenorhabditis elegans and signaling. Indeed, animals doubly mutant for sli-1 and Drk in Drosophila, are composed of a single SH2 domain unc-101 or gap-1 display a hyperinduced vulval phenoflanked by two SH3 domains (Clark et al., 1992a; Lotype, with more than three cells adopting vulval fates wenstein et al., 1992; Olivier et al., 1993; Simon et al., (Sternberg et al., 1994; G. Jongeward, C. Chang, and 1993). The SH2 domain mediates binding to the receptor P. W. S., unpublished results). whereas the flanking SH3 domains bind proline-rich modevelopment of the posterior ectoderm, development of In addition to vulval induction, let-23 is required for the tifs in the carboxyl terminus of Sos, an exchange factor for Ras (Buday and Downward, 1993; Chardin et al., the male spicules, proper connection of the uterus to 1993; Egan et al., 1993; Olivier et al., 1993; Simon et al., the vulva, and for viability of young larvae (Aroian and 1993; Raabe et al., 1995). Thus, Grb2/SEM-5/Drk serves Sternberg, 1991; Chamberlin and Sternberg, 1994; Jiang as an adaptor protein linking RTK signaling to Ras actitions are mediated through RAS activation. A sixth func- and Sternberg, 1998; Chang et al., 1999). All these funcvation via the recruitment of Sos. Ras activation in turn stimulates a kinase cascade composed of raf, MEK, and tion for let-23, during ovulation, utilizes distinct tyrosine MAP kinase (reviewed by Marshall, 1994). residues in the cytoplasmic tail of LET-23 and is mediated through a RAS-independent pathway (Han et al., 1990; Jongeward et al., 1995; Hajnal et al., 1997; Lesa To whom correspondence should be addressed ( nah@ mrc-lmb.cam.ac.uk). and Sternberg, 1997). Instead, this fertility function is Present address: Department of Biology, Yonsei University, Shinchon-dong, mediated through inositol trisphosphate signaling (Clan- Seodaemun-ku, Seoul, Korea. dinin et al., 1998).

2 Molecular Cell 66 Table 1. Genetic Interactions between ark-1 and sli-1, and gap-1 and unc-101 Strain 20 C 25 C Proportion Average Proportion Average Hyperinduced Induction Hyperinduced Induction (1) N2 0% 3.0 ( 20) 0% 3.0 (20) (2) ark-1 0% 3.0 (20) 20% 3.1 (25) (3) sli-1 0% 3.0 (20) 3% 3.02 (30) (4) gap-1 0% 3.0 (20) 0% 3.0 (30) (5) unc-101 0% 3.0 (20) (6) ark-1; sli-1 40% 3.35 (40) 84% 4.3 (25) (7) ark-1; gap-1 90% 4.0 (20) 95% 4.7 (22) (8) unc-101; ark-1 29% 3.26 (21) (9) ark-1(sy247)/sdf22 7% 3.05 (44) (10) ark-1( )/sdf22; sli-1 0% 3.0 (21) (11) ark-1(sy247)/sdf22; sli-1 60% 3.6 (55) 95% 4.3 (20) (12) gap-1; ark-1[rna i ] 61% 3.75 (18) Viability to Hatching (20 C) Viability to Hatching (25 C) N2 119/121 (98%) 166/182 (91%) ark-1 214/220 (97%) 47/49 (96%) sli-1 262/284 (92%) 102/119 (86%) gap-1 147/151 (97%) 166/174 (95%) ark-1; sli-1 218/254 (86%) 166/258 (64%) ark-1; gap-1 204/256 (80%) 163/276 (59%) let-60(gf) 219/238 (92%) 155/168 (92%) let-60(gf) ark-1 164/209 (78%) 23/39 (59%) let-60(rf); sli-1 a 170/199 (85%) 93/136 (68%) b let-60(rf) ark-1; sli-1 a 153/158 (97%) 99/156 (63%) b (Top) Vulva induction was scored using Nomarski optics during the early-mid L4 stage. Animals were maintained at the indicated temperature several generations before scoring. Proportion hyperinduced indicates the number of animals having greater than three VPCs induced as a percentage of the total. Average induction indicates the total number of induced VPCs divided by the number of animals assayed (given in parentheses). (Bottom) Eggs were scored as inviable if they failed to hatch after 2 days. Alleles used were unc-101(sy108), ark-1(sy247), let-60(n1046gf), let-60(n2021rf), sli-1(sy143), and gap-1(n1691). a let-60(2021rf) was marked with unc-24(e138). b Lethality observed was distinct from the earlier embryonic lethality observed in ark-1; sli-1, ark-1; gap-1, and let-60(gf) ark-1 animals (see Figures 1C and 1D and text for details). sdf22 is a deficiency that deletes the ark-1 locus. In this report, we describe the isolation, molecular themselves (with respect to vulval induction), and it had cloning, and genetic characterization of ark-1, a negative been observed that animals of the doubly mutant geno- regulator of EGFR-mediated signaling. ark-1 en- types unc-101; sli-1 and sli-1 gap-1 display excessive codes an Ack-related nonreceptor tyrosine kinase. An vulval differentiation, we performed a genetic screen ark-1 mutation synergizes with mutations in other nega- looking for mutations that would result in a hyperinduced tive regulators, resulting in partially penetrant embryonic vulva phenotype in a sli-1 background. From an initial lethality and hyperinduced vulva phenotypes. Both of screen of 10,000 EMS-mutagenized genomes of a slithese phenotypes result from increased RAS activity. 1(sy143) background, a single uncharacterized locus ark-1 suppresses all let-23 functions assayed, including was identified. This novel locus, designated ark-1 (for sterility. Thus, ark-1 activity is not limited to the ras Ack-related kinase, see below), is defined by a single pathway. Genetic analysis suggests that ark-1 is depen- mutation, sy247. dent upon sem-5 activity. Consistent with this, sem-5 also suppresses let-23 sterility. This demonstrates that ark-1 Genetically Interacts with sli-1, gap-1, sem-5 mediates negative as well as positive signaling. and unc-101 in the Vulva The further demonstration that ARK-1 and SEM-5 physi- Animals of the genotype ark-1(sy247); sli-1(sy143) have cally interact in the yeast two-hybrid assay suggests a a hyperinduced vulva phenotype with 40% of animals model where the dependence of ark-1 upon sem-5 may having greater than three VPCs induced at 20 C (average be direct. number of VPCs induced 3.35; Table 1, top, line 6). This hyperinduced phenotype is temperature sensitive Results with greater than 80% of ark-1(sy247); sli-1(sy143) animals having excess vulval tissue at 25 C (average number Isolation of ark-1 of VPCs induced 4.3; Table 1, top, line 6). ark-1 We sought to isolate mutations that increase the efficacy also synergizes with mutations in unc-101 and gap-1, of LET-23-mediated signaling but that might have been resulting in a hyperinduced vulva phenotype (Table 1, missed in the screens looking for suppressors of the top). The strongest interaction is seen between ark-1 vulvaless phenotypes associated with a mislocalization and gap-1, with 90% of animals being hyperinduced at of LET-23. Since sli-1, gap-1, and unc-101 are silent by 20 C (Table 1, top, line 7).

3 ARK-1 Inhibits EGFR Signaling in C. elegans 67 Figure 1. The Early Embryonic Lethality Displayed by ark-1; sli-1 Animals Is Likely to Result from Increased RAS Activity Nomarski photomicrographs of the embryonic lethality at 25 C. (A) Inviable eggs from ark-1(sy247); sli-1 (sy143) parents. (B) Inviable eggs from let-60(n1046gf) ark-1 (sy247) parents. (C) Inviable eggs from unc-24(e138) let-60 (n2021rf); sli-1(sy143) parents. (D) Inviable eggs from unc-24(e138) let-60 (n2021rf) ark-1(sy247); sli-1(sy143) parents. let-60(n2021) suppresses the early arrest phenotype of ark-1(sy247); sli-1(sy143) animals. The ark-1(sy247) mutation is silent by itself at 20 C, but at 25 C, occasional animals with hyperinduced vulva are observed (Table 1, top, line 2). Vulva induction in an ark-1(sy247) background remains dependent upon ligand. Gonad-ablated ark-1(sy247); sli-1(sy143) animals (thus lacking the source of the inductive signal) have no vulval induction (data not shown). Therefore, the excess vulval induction observed in ark-1(sy247); sli-1(sy143) animals is due to either increased production of LIN-3, C. elegans EGF, or increased sensitivity to it. Hyperinduced Vulvae of ark-1; sli-1 Animals Are Qualitatively Different from Those Observed in ark-1; gap-1 Animals In wild-type animals, it is the daughters of P5 7.p that are induced to form the vulva, with the daughters of P3.p, P4.p, and P8.p remaining uninduced (Sulston and Horvitz, 1977). In ark-1; sli-1 animals, the extra vulva induction observed at 20 C is usually in P4.p. In fact, P4.p is induced four times more often than P8.p (P4.p is induced in 26% of animals, P8.p in 6%, n 40). In contrast to this, in ark-1; gap-1 animals, the extra vulva induction observed is usually in P8.p, with P8.p being five times more likely to be induced than P4.p (P4.p is induced in 15% of animals, P8.p in 80%, n 40). Our interpretation is that sli-1 and gap-1 activity is not uniform across the Pn.p cells. We suggest that gap-1 activity is higher in P8.p than in P4.p and that sli-1 activity is higher in P4.p than in P8.p. It is known that the Pn.p cells are not equivalent in their sensitivity to inductive signal (Clandinin et al., 1997). It may be that this sensitivity is determined in part by the level of expression of negative regulators such as sli-1 and gap-1. ark-1 Animals Display a Synthetic Embryonic Lethality While animals singly mutant for sli-1, ark-1,orgap-1 are as viable as wild-type animals, doubly mutant animals of genotypes ark-1; sli-1 and ark-1; gap-1 are subviable, with lethality occuring early during embryogenesis at around 5 hr after the first cell division (Table 1, bottom; Figure 1A). Again, this phenotype becomes more severe at 25 C. We reasoned that this lethality might result from increased RAS activity, perhaps leading to the misspecification of an unidentified embryonic cell fate. Consistent with this, we found that ark-1 synergizes with a weak gain-of-function allele of let-60ras to cause an identical embryonic lethality (Table 1, bottom; Figure 1B). Moreover, we found that the synthetic early embry- onic lethality of ark-1; sli-1 animals is suppressed by a reduction-of-function mutation in let-60ras (Table 1, bottom). This result is more apparent at 25 C where the lethality observed in ark-1; sli-1 animals is enhanced. However, there is a distinct lethality associated with let-60(lf), which results from the failure to specify the excretory duct cell (Yochem et al., 1997). At 25 C, the effect of this is exacerbated, and animals arrest very late in embryogenesis, just prior to hatching (Figure 1C). In let-60(rf) ark-1; sli-1 animals, the early embryonic le- thality is almost completely suppressed, and the dead embryos observed are arrested late (Figure 1D). We infer that the early embryonic lethality seen in ark-1; sli-1 and ark-1; gap-1 animals is a consequence of enhanced RAS signaling. Molecular Cloning of ark-1 ark-1 was mapped to a small genetic region to the left of unc-31 corresponding to 400 kb on the physical

4 Molecular Cell 68 Figure 2. Molecular Characterization of ark-1 (A) Identification of the ark-1 locus. The rescuing cosmid C01C7 contains 26 kb of genomic insert of which 12 kb is shared with the overlapping cosmid K09D4, which fails to rescue. A 14 kb SalI subclone containing an 11.4 kb genomic fragment from the other end of insertion also fails to rescue. A minigene composed of 5.6 kb of genomic sequence fused to 2.6 kb of cdna is sufficient for robust rescue. (B) Domain structure of ARK-1. (C) Alignment of the domains of ARK-1 sharing homology with Ack, B0302.1, and DPR2. DPR2 has not been identified genetically but exists as a partial cdna clone lacking sequences at both the 5 and 3 ends that was initially isolated as a hybridizing cdna to a human -PDGFR low-stringency probe (Ito et al., 1994). B corresponds to the predicted open reading frame determined by the genefinder program. Areas of identity between all four proteins are highlighted in black. Identity between two or three proteins are highlighted with gray tones.

5 ARK-1 Inhibits EGFR Signaling in C. elegans 69 map (see Experimental Procedures). Transformation ex- negative construct retained strong rescue of the hyperinduced periments identified cosmid C01C7 as being able to vulva phenotype (not shown). This suggests rescue the 85% penetrant hyperinduced vulva pheno- that the predicted tyrosine kinase activity of ark-1 is not type of ark-1(sy247); sli-1(sy143) mutant animals at 25 C essential for function. However, since the sy247 muta- (Figure 2A). Other cosmids conferred no rescue. Se- tion truncates ARK-1 after the kinase domain, it is possible quencing by the C. elegans genome sequencing consortium that the tyrosine negative construct displays intraquencing revealed that C01C7 contains just one open reading genic complementation with ark-1(sy247). frame. A full-length cdna corresponding to this open reading frame was placed under the control of a heat shock promoter. This construct was introduced into ark- sy247 Reduces ark-1 Activity 1(sy247); sli-1(sy143) mutant animals by microinjection. The deficiency sdf22 is a 2 Mb deletion of chromosome Transformed animals undergoing a heat shock protocol IV, which includes the region where ark-1 lies (Clark et were fully rescued for the hyperinduced phenotype. An al., 1988). The deficiency sdf22 fails to complement arkark-1 minigene composed of genomic DNA, including 1(sy247) and as a heterozygote does not have a vulval 2.3 kb upstream of the predicted SL1 leader trans-splice phenotype (Table 1, top, lines 10 and 11). Placing the acceptor, fused within the sixth exon to cdna se- ark-1(sy247) mutation in trans to a deficiency, which is quences also tested successfully for rescue (Figure 2A). halving the genetic dose of ark-1(sy247), increases the penetrance and severity of the hyperinduced vulva phenotype observed at 20 C (Table 1, top, compare lines 2 ark-1 Is Predicted to Encode a Tyrosine Kinase Related to Ack and 6 to lines 9 and 11). We do not observe an increase The predicted open reading frame and rescuing cdna in severity of the embryonic lethality by halving the arkencodes a nonreceptor tyrosine kinase of 1043 amino 1(sy247) dose (data not shown), nor do we observe an acids in length. The N-terminal tyrosine kinase domain effect of halving the genetic dose at 25 C with regard is immediately followed by an SH3 domain (Figure 2B). to the vulval phenotype (Table 1, top, compare line 6 C terminal to the SH3 domain is a CRIB (Cdc42/rac to line 11). We conclude that ark-1(sy247) is a severe interactive binding) domain (Figure 2B). The carboxyl reduction-of-function mutation. half of the ARK-1 protein has a number of proline-rich Microinjection of dsrna into a worm specifically de- motifs forming potential SH3 binding sites and potential creases the activity of the cognate gene, both in the MAP kinase phosphorylation sites. Database searching injected worm and its progeny (Fire et al., 1998). We revealed that ark-1 is most similar to a Drosophila partial performed RNA-mediated interference experiments cdna designated DPR2 (46% identity over amino acids [RNA i ] using dsrna derived from ark-1 cdna as inde of ARK-1; Figure 2C). Both ark-1 and DPR2 share pendent verification that the phenotypes observed in a domain structure that is very similar to that of the Ack ark-1(sy247) animals are due to a reduction of ark-1 subfamily of cytoplasmic tyrosine kinases. Though the function. Since the penetrance of the hyperinduced phe- N terminal halves of ark-1 and Ack are 34% identical notype is greatest in ark-1; gap-1 animals, we injected (residues of Ack), they may be distinct, as the gap-1 animals and looked for animals with hyperinduced C. elegans genome sequencing consortium has identifrom vulvae in the offspring. We found that injection of dsrna fied an open reading frame (designated B0302.1) with ark-1 into gap-1 animals resulted in a 60% pene- greater predicted similarity to Ack (39% identical be- trant hyperinduced vulval phenotype (Table 1, top, line tween residues ; Figure 2C). It is perhaps signifivulval 12). This compares to the 90% penetrant hyperinduced cant that the CRIB domain of B shares much phenotype of ark-1(sy247); gap-1 animals (Table higher similarity with Ack than does ARK-1 (Figure 2C). 1, top, line 7). The finding that gap-1; ark-1[rna i ] animals To confirm that the cloned cdna corresponds to are phenotypically similar to ark-1(sy247); gap-1 animals ark-1, genomic DNA corresponding to C01C7.1 was isolated confirms that ark-1(sy247) reduces ark-1 activity. from ark-1(sy247) animals and sequenced. Only one mutation was found. This is a C-to-T transition at position 1638 relative to the SL1 fusion point and introbut ark-1 Suppresses lin-3, let-23, and sem-5, duces a stop codon at position Q 528. Thus, ark-1(sy247) Not let-60 is expected to produce a mutant transcript with a very Vulval induction is mediated by the evolutionarily con- long 3 untranslated region. If translated, this would encomponents served RTK/RAS/MAP kinase pathway. The upstream code a truncated protein missing its C-terminal half but to RAS in C. elegans are defined by lin-3, would still contain the tyrosine kinase, SH3 and CRIB let-23, sem-5, and let-60, which encode homologs of domains (Figure 2B). Egf, Egfr, Grb2, and Ras, respectively. We tested the To ascertain whether the tyrosine kinase domain is ability of ark-1 to suppress mutations in each of these essential for ark-1 activity, sequences encoding the ATP genes. Since vulval induction remains gonad dependent binding site were mutated in the ark-1 minigene (see in an ark-1 mutant background, it follows that ark-1 will Experimental Procedures). The resulting tyrosine kinase not suppress null mutations in lin-3. However, we find Identity between ARK-1 and DPR2 but not Ack or B is shown with white text on a gray background. Identity between Ack and B but not ARK-1 or DPR2 is shown with black text on a gray background. Numbering for DPR2 begins at 1 for the first codon of the cloned cdna (which is not ATG).

6 Molecular Cell 70 Table 2. Suppression of the Vulvaless Genes by ark-1 suppresses sem-5(n1619), it fails to suppress the null Average Number of VPCs Induced mutation sem-5(ay73), which introduces an early stop codon. Genotype ark-1( ) ark-1(sy247) The let-341 gene was initially identified in a screen for / 3.0 (Many) 3.0 (Many) essential genes located on chromosome V and causes lin-3(n378) 0.8 (29) 2.85 (20) a similar rodlike larval lethality as seen in let-23, sem-5, let-23(sy1) 0.8 (20) 2.2 (21) and let-60 (Johnsen and Baillie, 1991). let-341 has also let-23(sy10) (31) 0.15 (21) been isolated as a suppressor of the lin-15 multivulva let-23(sy97) 0.0 (21) a 0.19 (21) phenotype (Clark et al., 1992b). let-341 encodes the C. sem-5(n2019) 0.5 (20) 1.28 (20) sem-5(n2195) 3.0 (20) 3.0 (20) elegans homolog of Sos (Chang et al., 2000). A weak sem-5(n1619) 0.35 (40) 2.89 (23) mutation in let-341 is not suppressed by ark-1. Similarly, sem-5(ay73) 0.0 (18) 0.0 (22) we find that ark-1 does not suppress let-60, which enlet-341(n2023) 2.15 (20) 2.36 (21) codes RAS. Specifically, neither of two strong reductionlet-60(n2021) 2.4 (22) 2.85 (20) of-function mutations is suppressed by ark-1, and there let-60(n1876) 0.0 (62) b 0.0 (21) is only a slight enhancement of signaling by the preslet-60(n2035) (62) b 0.0 (20) ence of the ark-1 mutation of the weak reduction-of- Vulva induction scored at 20 C. The molecular lesions in the sem-5 function let-60(n2021) allele (Table 2). Thus, we find that alleles are as follows: n2019 is a spice donor mutation, n2195 results ark-1 suppresses non-null vulvaless mutations in lin-3, in a G201R substitution within the C-terminal SH3 domain, n1619 results in a P49L substitution in the N-terminal SH3 domain, and let-23, and sem-5 but fails to suppress non-null vul- ay73 is a putative null allele resulting in a Q 10 Stop (Clark et al., 1992a; valess mutations in let-341 and let-60. This, in addition M. Stern, personal communication). to the strongest suppression by ark-1 observed being a Data from Aroian and Sternberg (1991). of sem-5, suggests that ark-1 may act at the sem-5 step b Data from C. Yoon, C. Chang, and P. W. S. (unpublished). to inhibit signaling. Genetic Evidence that ark-1 Is Dependent that ark-1 does suppress a reduction-of-function muta- upon sem-5 for Its Activity tion in lin-3 (Table 2). Likewise, we find that ark-1 supobserved Though ark-1 fails to suppress mutations in ras, we presses non-null mutations in the receptor, let-23 (Table that ark-1 enhances the hyperinduced vulva 2). let-23(sy1) generates a mutant receptor lacking the phenotype of the gain-of-function allele let-60(n1046) last six carboxy-terminal amino acids, which results in (Table 3). let-60(n1046gf) results in a G13E substitution the failure of the receptor to become localized to the that is expected to decrease the GAP-stimulated basal membrane of the vulval precursor cells (Aroian GTPase activity of RAS (Beitel et al., 1990). Gonad ablaet al., 1994; Kaech et al., 1998). This allele is strongly tion experiments have shown that let-60(n1046gf) is sen- suppressed by unc-101, sli-1, and gap-1, resulting in a sitive to upstream signaling (Sundaram and Han, 1995; hyperinduced vulva phenotype (Lee et al., 1994; Jongeamount N. A. H. and P. W. S., unpublished results). Indeed, the ward et al., 1995; Hajnal et al., 1997). We find that ark-1 of vulval induction observed in a let-60(n1046gf) also suppresses let-23(sy1), but no animals with greater background is diminished by mutations in sem-5 (Table than 3.0 VPCs induced were observed (Table 2 and data 3). The fact that let-60(n1046gf) is suppressed by semnot shown). ark-1 also weakly suppresses let-23(sy10) 5(n2195G201R) indicates that the C-terminal SH3 do- and let-23(sy97), two severe reduction-of-function mu- main of SEM-5 does contribute to signaling in the vulva. tations of the receptor. Given our interpretation that ark-1 acts at sem-5 to in- sem-5 encodes the C. elegans homolog of Grb2, an hibit signaling, we sought to determine whether any of adaptor protein composed almost entirely of an SH2 the effects of ark-1 were dependent upon the presence domain flanked by two SH3 domains. Biochemical analwould of sem-5. To this end, we determined whether ark-1 ysis of Grb2 has shown that each of the SH3 domains still synergize with the gain-of-function let- can bind Sos, a guanine nucleotide exchange factor for 60(n1046) allele in the background of mutations in Ras (Chardin et al., 1993; Egan et al., 1993). However, sem-5. We observed that synergy occurs when either genetic analysis suggests that the N-terminal SH3 donot the N- or the C-terminal SH3 domains are disrupted, but main is essential and the C-terminal SH3 domain is resynergy in the background of a sem-5 null (Table 3). Thus, the dundant (Clark et al., 1992a; Raabe et al., 1995; Cheng between ark-1 and let-60(n1046gf) is dependent et al., 1998). Thus, sem-5(n1619), which results in a P49L upon sem-5 activity. substitution in the N-terminal SH3 domain, behaves as a severe reduction-of-function mutation, whereas sem- ark-1 and sem-5 Negatively Regulate the Fertility 5(n2195), which results in a G201R substitution in the Function of let-23 C-terminal SH3 domain, is silent with respect to viability The finding that synergy between ark-1 and letand vulval induction (Clark et al., 1992a). Animals F1 60(n1046gf) is dependent upon sem-5 activity may re- homozygous for sem-5(n1619p49l) have low viability, flect that ark-1 acts upon (or upstream of) sem-5 to and any surviving animals are vulvaless and fail to produce inhibit signaling or that activation of ark-1 itself requires any viable offspring. ark-1 almost completely sup- sem-5 signaling. These two possibilities might be distin- presses sem-5(n1619p49l) (Table 2). This indicates that guished if ark-1 can suppress the fertility defect of cer- in an ark-1 mutant background, the N-terminal SH3 do- tain lin-3 or let-23 alleles. The activity of lin-3 and letmain becomes redundant and the C-terminal SH3 domain 23, but not sem-5 or let-60, is required for ovulation becomes able to signal effectively. Whereas ark-1 (Han et al., 1990; Clark et al., 1992a; Clandinin et al.,

7 ARK-1 Inhibits EGFR Signaling in C. elegans 71 Table 3. Synergy between ark-1(rf) and let-60(gf) in the Vulva Requires sem-5 Activity Proportion of Animals Strain Average Induction with 3 Cells Induced let-60(gf) /20 let-60(gf) ark /20 let-60(gf); sem-5(ay73) 3.2 4/30 let-60(gf) ark-1; sem-5(ay73) 3.1 4/34 let-60(gf); sem-5(n1619p49l) 3.1 2/21 let-60(gf) ark-1; sem-5(n1619p49l) /20 let-60(gf); sem-5(n2195g201r) 3.3 6/20 let-60(gf) ark-1; sem-5(n2195g201r) /22 Vulva Induction scored at 20 C. sem-5(ay73) is a putative null allele. Alleles used were let-60(n1046gf) and ark-1(sy247). 1998; McCarter et al., 1999; N. A. H., unpublished obser- and ovulation. This inhibition by ark-1 appears to be vations). Thus, if ark-1 suppresses let-23 sterility, then dependent upon sem-5 activity. This suggests the gethe function of ark-1 cannot solely be to inhibit sem-5. netic model presented in Figure 3A. The carboxyl half To test whether ark-1 negatively regulates this fertility of the ARK-1 protein contains several PxxP motifs that function, we tested whether ark-1 could suppress the could potentially form binding sites for proteins consterile let-23 allele, sy10. As previously reported, let- taining SH3 domains. A physical model to explain the 60(n1046gf) and sli-1 failed to suppress the fertility de- dependence of ark-1 upon sem-5 activity is that ARK-1 fect of let-23(sy10); however, we found that ark-1 was is recruited by SEM-5 into the LET-23 signaling complex. able to partially suppress the sterility (Table 4). The sup- All the PxxP motifs of ARK-1 are expected to be deleted pression by ark-1 is quite weak with let-23; ark-1 animals in any translated product of sy247. We therefore tested having a low brood size (not shown). However, this may whether ARK-1 and SEM-5 could interact using the yeast be in part due to let-23; ark-1 animals being subviable two-hybrid assay (Fields and Song, 1989). In this assay, (viability 31%, n 183). When the lethality of let-23 we found that the carboxyl terminus of ARK-1 interacts is fully suppressed by the presence of a mutation in with SEM-5 (Figure 3B). This interaction appears to be sli-1, we find that 37% of animals are suppressed for specific as no interaction was observed with the SH3 the sterility (see Table 4). Thus, the activity of ark-1 is domain of ARK-1 (not shown). In addition, the interaction not limited to the ras pathway. The suppression of let-23 with SEM-5 could be mapped to a particular PxxP motif sterility by ark-1 allows us to test whether ark-1 activity is at the extreme carboxyl terminus of ARK-1, with other dependent upon sem-5 signaling. If ark-1 is dependent proline-rich PxxP regions of ARK-1 showing no interacupon sem-5 activity for its inhibition of ovulation, then tion (Figure 3B). This interacting domain of ARK-1 shares a null mutation in sem-5 would also be expected to considerable homology with a region of Drosophila Sos suppress the fertility defect of let-23. Because of the that has been shown by other workers to bind very lethality of sem-5 and let-23, this experiment is perstrongly to the Drosophila ortholog of SEM-5 (Figure 3C; formed in a let-60gf background. We find that a sem-5 Raabe et al., 1995). Despite this, we have been unable null allele does indeed suppress the sterility of let-23 to coimmunoprecipitate SEM-5 with the C terminus of (Table 4). Similar to the suppression seen by ark-1, such ARK-1 in vitro (data not shown). Thus, although the intersuppressed let-23; let-60gf; sem-5 animals have a low action between ARK-1 and SEM-5 observed in the yeast brood size (not shown). This result demonstrates that two-hybrid assay supports a direct model for the depensem-5 has a negative effect, in addition to its well-estabdence of ark-1 upon sem-5, it remains to be proven, lished positive role, in let-23-mediated signaling. and an indirect model cannot be ruled out. A Model for ark-1 Activity The genetic data presented above demonstrates that Discussion ark-1 inhibits let-23-mediated viability, vulva induction, In this study, we have identified and cloned the ark-1 Table 4. The Sterility of let-23(sy10) Is Suppressed by ark-1 gene, which encodes an Ack-related protein that inhibits and sem-5 LET-23-mediated signaling. ark-1(sy247) was isolated Genotype Proportion Fertile in a screen for mutations that result in a hyperinduced vulva in a sli-1 background. sli-1, which encodes a Cbl let-23(sy10) 0% (30) homolog, negatively regulates let-23-mediated let-60ras let-23(sy10); lin-15 0% (23) signaling (Jongeward et al., 1995; Yoon et al., 1995). let-23(sy10); sli-1 0% (59) let-23(sy10); let-60(gf) 0% (42) ark-1; sli-1 animals display partially penetrant embryonic let-23(sy10); ark-1 20% (41) lethality and hyperinduced vulval phenotypes. These let-23(sy10); ark-1; sli-1 37% (30) phenotypes are also observed in ark-1; gap-1 and letlet-23(sy10); let-60(gf); sem-5 55% (31) 60gf ark-1 animals and are suppressed by mutations Fertile is defined as having greater than two offspring. Alleles used reducing let-60ras activity. Thus, both the embryonic were lin-15(e1763), sli-1(sy143), let-60(n1046gf), ark-1(sy247), and lethality and hyperinduced vulva phenotypes are likely sem-5(ay73). unc-4(e120) was used as a marker and mnc1 as a to result from increased RAS activity. Genetic analysis balancer for let-23(sy10). unc-31(e169) was present in some of the demonstrates that the sy247 mutation results in a reducanimals scored for let-23(sy10); ark-1 and let-23(sy10); sli-1 fertility. tion of ark-1 activity. This is confirmed by reverse genetic

8 Molecular Cell 72 Figure 3. A Genetic Model of ark-1 Activity in let-23-mediated Signaling (A) ark-1 inhibits all let-23 functions. The data is consistent with a model whereby ark-1 activity requires sem-5 signaling. lfe-1/itr-1 encodes the IP 3 receptor. Gain-of-function mutations in lfe-1/itr-1 have been isolated as suppressors of the lin-3/let-23 ovulation de- fect (Clandinin et al., 1998). (B) The carboxyl terminus of ARK-1 interacts with SEM-5 in the yeast two-hybrid assay. ARK-1 ( ) fails to interact with SEM-5 despite containing five distinct PxxP motifs (see EMBL accession number AJ271057). ARK-1 constructs containing amino acids do interact. Three clones per each cotransformed yeast line are tested on Trp Leu His restrictive media. The GAL4 DNA binding domain (amino acids 1 147) is fused in-frame to SEM-5 where indicated. The GAL4 activation domain (amino acids ) is fused in-frame to the test sequences. The dash indicates no fusion. Growth indicates interaction between the GAL4 fusions. Interactions were confirmed by testing complementary fusions, where possible. (C) Alignment of the C terminus of ARK-1 with a region of Drosophila SOS that binds strongly to Drk, the Drosophila ortholog of SEM-5 (see Raabe et al., 1995). Amino acids of ARK-1 identified by the yeast two-hybrid assay as being required for interaction with SEM-5. non-null mutations in let-341sos and let-60ras. Together with the observation that ark-1 most strongly suppresses sem-5, this suggests that ark-1 is likely to act at the sem-5 step. ark-1 also synergizes with a weak gain-of-function let-60ras allele. This synergy is not observed in the absence of sem-5, consistent with the hypothesis that ark-1 acts at or upstream of sem-5. The fertility function of let-23 is mediated by a RASindependent pathway and is instead believed to be mediated by the production of IP 3 (Clandinin et al., 1998). The physiological basis of the requirement for let-23 is for the normal control of spermathecal dilation during ovulation. In sterile lin-3 and let-23 mutants, the spermatheca fails to dilate to receive the mature oocytes, and consequently, mature oocytes become trapped in the gonad arm and endomitotically replicate their DNA (Clandinin et al., 1998; McCarter et al., 1999). This characteristic sterile phenotype is not observed in adult animals mutant in genes encoding sem-5 or members of the RAS/MAP kinase pathway (Beitel et al., 1990; Han et al., 1990; Clark et al., 1992a; N. A. H., unpublished observations), though mutations in the RAS/MAP kinase pathway can cause a distinct sterility due to the failure of meiotic germ cells to exit pachytene (Church et al., 1995). Thus, the RAS/MAP kinase pathway is either not activated in the spermatheca, or such activation has no consequence with respect to spermathecal dilation. The finding that ark-1 suppresses let-23 sterility demonstrates that the activity of ark-1 is not limited to the RAS pathway. Since our analysis of ark-1 function in vulval induction suggests that ark-1 acts at the sem-5 step and sem-5 does not have a positive role in the let-23- mediated fertility function, this creates a possible quandary. There are two possible explanations. First, ark-1 could have two separable activities, one negatively regulating let-23-mediated RAS signaling and the other negatively regulating the RAS-independent let-23 fertil- ity function. Alternatively, ark-1 has one activity, to negatively regulate let-23, and that activity is dependent upon sem-5 (Figure 3A). If ark-1 is dependent upon sem-5 for its activity, then sem-5(lf) would also behave as a negative regulator of fertility. This is what we observe. sem-5 suppresses the fertility defect of let-23 in a quali- tatively similar manner to ark-1 (Table 4). In mutants where the receptor is only weakly activated, one would expect that relatively small amounts of SEM-5 would be recruited into the signaling complex, and negative regulators dependent upon SEM-5 recruit- ment would under these circumstances only have a weak effect. The suppression of let-23(sy97) by ark-1 might be an example of such an effect. let-23(sy97) encodes a truncated receptor protein that lacks the canonical SEM-5 binding sites (Songyang et al., 1993; Aroian et al., 1994). In this mutant, RAS activation is severely compromised but not eliminated (Aroian and Sternberg, 1991), consistent with the suggestion that LET-23(sy97) is a very poor substrate for SEM-5 binding. The relatively small suppression of let-23(sy97) by ark-1 is therefore consistent with the model that ark-1 activity is depen- dent upon sem-5 recruitment. ark-1 encodes a cytoplasmic tyrosine kinase related experiments where the activity of ark-1 is reduced by dsrna-mediated interference: gap-1; ark-1 [RNA i ] animals display a hyperinduced vulva phenotype qualitatively similar to that observed in ark-1(sy247); gap-1 animals. Thus, the wild-type function of ark-1 is to inhibit let-23-mediated RAS signaling. to Ack. The function of Ack remains obscure but is be- The ark-1 mutation is able to suppress non-null muta- lieved to be an effector of Cdc42 (Manser et al., 1993; tions in lin-3, let-23, and sem-5 but is unable to suppress Yang and Cerione, 1997). The closest homolog to ark-1

9 ARK-1 Inhibits EGFR Signaling in C. elegans 73 Construction of Transgenic Strains Transgenic strains were produced as described by Mello et al. (1991), using pmh86[dpy-20( )] as a transformation marker. Rescue of ark-1(sy247) was determined by scoring the vulval anatomy of transgenic animals of the genotype dpy-20(e1282) ark-1(sy247); sli- 1(sy143); syex{pmh86[dpy-20( )]; cosmid} in the F3 generation maintained at 25 C. Site-directed mutagenesis was used to construct an ark-1 minigene deficient in predicted tyrosine kinase activ- ity. The following missense mutations were introduced to disrupt ATP binding: G120V, Q121D, and G122A. The presence of the intro- duced mutations was determined by the presence of an additional SalI restriction site. For transgenes that were being expressed from the heat shock promoter (16 41), the vector ppd49.83 was used (Mello and Fire, 1995). Young transgenic adult hermaphrodites were picked to a fresh plate and allowed to lay eggs for 1 hr before being removed. The plates were maintained at 25 C and heat shocked for 20 min at 33 C at 29, 33, and 37 hr after laying. is DPR2 from Drosophila. DPR2 is thought to be distinct from Ack, as the CRIB domain of Ack is specific for Cdc42 whereas the CRIB domain of DPR2 binds both Cdc42 and Rac (Burbelo et al., 1995). ark-1 may also be distinct from Ack, as there exists another C. elegans predicted protein that is more similar to Ack than is ARK-1. However, the presence of a CRIB domain may suggest that ARK-1 functions as an effector of Cdc42 and/or Rac. Using the yeast two-hybrid assay, we have shown that SEM-5 can interact with the carboxyl terminus of ARK-1. Though there is no direct evidence that this interaction is important for ARK-1 activity, it suggests a model whereby the dependence of ark-1 upon sem-5 is direct and ARK-1 itself maybe recruited into the signaling complex by SEM-5. An alternative model of sequestration of SEM-5 by ARK-1 would not explain the suppression of let-23 sterility by ark-1 or sem-5. In Drosophila, Sprouty, which acts as an inhibitor of Ras signaling, has also been suggested to be recruited by Drk (Casci et al., 1999). This implies that Grb2/SEM-5/Drk has an inhibitory role in EGFR signaling and that it is likely to be a general feature. The suppression of let-23 sterility by sem-5 demonstrates that Grb2/SEM-5/Drk does indeed have an inhibitory role in EGFR signaling. This fits findings from a mammalian system. A fusion protein composed of the SH2 domain of Grb2 fused to the Ras exchange factor Sos is able to restore Grb2 function to Grb2-deficient cells and so raises the ques- tion as to the function of the two SH3 domains of Grb- 2/SEM-5/Drk (Cheng et al., 1998). It is suggested that using an adaptor allows for regulation. Here, we demonstrate that sem-5 can act as a negative regulator of EGFR-mediated signaling and suggest that this effect may be brought about by the recruitment or activation of negative regulators such as ARK-1. Experimental Procedures Genetics and Strains Strains were handled according to standard protocols and maintained at 20 C, unless otherwise stated (Brenner, 1974). Ethyl-meth- ane-sulfonate (50 mm) was used as a mutagen (Brenner, 1974). The following alleles were used: LGI, unc-38(x20), unc-101(sy108); LGII, let-23(sy1, sy10, sy17, sy97), unc-4(e120); LGIV, lin-3(n378), let- 60(n1046, n1876, n2021, n2035), dpy-20 (e1282), ark-1(sy247), unc- 31(e169), unc-24(e138); LGV, let-341(n2023), him-5(e1490); and LGX, sli-1(sy143), gap-1(n1691), lin-15(e1763), sem-5(ay73, n1619, n2019, n2195). sem-5(ay73) is a putative null allele resulting from a Q 10 STOP nonsense mutation (M. Stern, personal communication). mnc1[dpy-10 unc-52] is a rearrangement on LGII that was used as a balancer for let-23. szt1 is a reciprocal I;X translocation and is used as a balancer for sem-5. For example, F1 sem-5(ay73) homozygotes were identified as unc-38 progeny from unc-38(x20)/szt1; sem- 5(ay73)/szT1 parents. sdf22 is a deficiency on IV that deletes the ark-1 locus. sli-1(sy143) were mutagenized and mutants selected from F2 ani- mals displaying additional bumps on their ventral surface visible under the dissecting microscope. Candidate mutants were backcrossed five times and mapped using the hyperinduced (Hin) phenotype. ark- 1(sy247) was found to be very tightly linked to unc-31(e169). Threefactor mapping was performed between dpy-20(e1282) and unc- 31(e169) in the presence of sli-1(sy143). 12 from 13 Dpy non-unc animals and 1 from 9 Unc non-dpy animals segregated Hin progeny. This places ark-1 at position 6.18, 0.1 map units to the left of unc- 31 on chromosome IV. The genetic deficiency sdf22 deletes 2 map units of genome from positions 5.87 to 7.82 on chromosome IV and is thus expected to delete the ark-1 locus (Clark et al., 1988). Using the Hin phenotype observed in a sli-1(sy143) background, sdf22 fails to complement ark-1(sy247). Vulval Induction Assay The extent of vulval induction was assayed by examining vulval anatomy in the early to mid-l4 stage of development under Nomarski optics using a Plan 100 objective (Sulston and Horvitz, 1977; Sternberg and Horvitz, 1986). Any animal having greater than three VPCs induced is scored as hyperinduced. Viability Assays Young adult hermaphrodites maintained at the test temperature were transferred to a fresh plate and left to lay eggs for 4 hr. Eggs were then transferred to a fresh plate and counted. The eggs were then incubated at the test temperature for 2 days and the number of viable worms counted. For photography, eggs that were unhatched after 24 hr were assumed to be inviable. Isolation of ark-1 cdna and Determination of Molecular Lesion of sy247 Standard molecular biological techniques were used (Sambrook et al., 1989). A 5.6 kb genomic SalI fragment from the cosmid C01C7 was used to screen a C. elegans mixed stage cdna library (a kind gift from R. Barstead). A full-length cdna was constructed from three overlapping clones. Several cdnas corresponding to ark-1 were cloned and sequenced, and no alternatively spliced forms were detected. Genefinder predicts a slightly different cdna, but such a cdna was not detected in RT-PCR experiments. Single-worm PCR was performed on L1 larvae of the genotype ark-1(sy247) and the product sequenced. The lesion described in the text was confirmed by sequencing independent PCR products, and no other error was reproducibly observed. dsrna-mediated Interference dsrna corresponding to ark-1 was generated by in vitro transcription using 1.6 kb of ark-1 cdna corresponding to the 3 end of the gene inserted into pbluescript (Stratagene). Transcripts were prepared using T3 and T7 RNA polymerase and products pooled prior to injection (Fire et al., 1998). Progeny of injected worm were assayed at 20 C. Two-Hybrid Protocol The yeast two-hybrid protocol was followed essentially as described by Fields and Song (1989). Reagents and the yeast strain, CG1945, were acquired from Clontech, CA (Matchmaker kit). Fragments of sem-5, ark-1, and Drosophila SOS were amplified from cdna clones by PCR and ligated in-frame into pgbt-9 and pgad-424 using restriction sites engineered into the PCR primers. Acknowledgments We thank R. Barstead for providing his cdna library; A. Fire for providing heat shock vectors; M. Stern for providing the sem-5(ay73) allele; members of the Sternberg laboratory for their discussions;

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