Caenorhabditis Elegans Fibroblast Growth Factor Receptor Signaling Can Occur Independently of the Multi-Substrate Adaptor FRS2

Caenorhabditis elegans Fibroblast Growth Factor Receptor Signaling Can Occur Independently of the Multi-Substrate Adaptor FRS2

Te-Wen Lo,*,1 Daniel C. Bennett,*,2 S. Jay Goodman† and Michael J. Stern*,3 *Department of Genetics and †Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8005 Manuscript received December 21, 2009 Accepted for publication March 14, 2010

ABSTRACT The components of receptor tyrosine kinase signaling complexes help to define the specificity of the effects of their activation. The Caenorhabditis elegans fibroblast growth factor receptor (FGFR), EGL-15, regulates a number of processes, including sex myoblast (SM) migration guidance and fluid homeostasis, both of which require a Grb2/Sos/Ras cassette of signaling components. Here we show that SEM-5/Grb2 can bind directly to EGL-15 to mediate SM chemoattraction. A yeast two-hybrid screen identified SEM-5 as able to interact with the carboxy-terminal domain (CTD) of EGL-15, a domain that is specifically required for SM chemoattraction. This interaction requires the SEM-5 SH2-binding motifs present in the CTD (Y 1009 and Y 1087), and these sites are required for the CTD role of EGL-15 in SM chemoattraction. SEM-5, but not the SEM-5 binding sites located in the CTD, is required for the fluid homeostasis function of EGL-15, indicating that SEM-5 can link to EGL-15 through an alternative mechanism. The multi-substrate adaptor protein FRS2 serves to link vertebrate FGFRs to Grb2. In C. elegans, an FRS2-like gene, rog-1, functions upstream of a Ras/ MAPK pathway for oocyte maturation but is not required for EGL-15 function. Thus, unlike the vertebrate FGFRs, which require the multi-substrate adaptor FRS2 to recruit Grb2, EGL-15 can recruit SEM-5/Grb2 directly.

IBROBLAST growth factors (FGFs) play important A large portion of mammalian FGFR signaling is F roles in many developmental and physiological mediated by the multi-substrate adaptor protein FRS2/ processes, including cell migration, angiogenesis, pro- snt-1 (Kouhara et al. 1997; Hadari et al. 1998, 2001; liferation, differentiation, and survival (Ornitz and Lax et al. 2002; Gotoh et al. 2005). FRS2 constitutively Itoh 2001; Polanska et al. 2009). Mammals have a associates with the juxtamembrane region of the FGFR battery of both FGF ligands and high-afﬁnity receptors via its amino-terminal PTB domain (Xu et al. 1998; Ong to carry out this diverse set of important functions. et al. 2000). Upon FGFR activation, FRS2 becomes These ligands and their receptors are generated from a heavily phosphorylated, allowing it to recruit Grb2 set of 18 genes encoding FGFs and 4 genes encoding and Shp2 via their SH2 domains (Kouhara et al. 1997; their receptors (Eswarakumar et al. 2005). Upon li- Eswarakumar et al. 2005). Since these components gand binding, ﬁbroblast growth factor receptors (FGFRs) cannot associate with the receptor in the absence of dimerize, activating their intrinsic tyrosine kinase activ- FRS2 (Hadari et al. 2001), FRS2 serves as an essential ity, which causes both autophosphorylation on intracel- link between the activated receptor and many down- lular tyrosine residues and phosphorylation of additional stream signal transduction pathways. substrates (Eswarakumar et al. 2005). These phosphor- The understanding of FGF-stimulated signal trans- ylation events lead to the assembly of a signaling complex duction pathways has been aided by the study of FGF around the activated receptor, ultimately promoting signaling in model organisms. Powerful genetic screens various downstream signaling pathways (Eswarakumar and the reduced complexity of the set of FGFs and their et al. 2005). receptors in both Drosophila melanogaster and Caenorhab- ditis elegans have helped promote an understanding of the conserved aspects of FGF signaling pathways Supporting information is available online at http://www.genetics.org/ uang tern olanska cgi/content/full/genetics.109.113373/DC1. (H and S 2005; P et al. 2009). In C. 1Present address: Department of Molecular and Cell Biology, University elegans, FGF signaling is mediated by two FGF ligands, of California, 131 Koshland Hall, Berkeley, CA 94720-3204. EGL-17 and LET-756, and a single FGF receptor, EGL-15 2Present address: Department of Therapeutic Radiology, Yale University, (DeVore et al. 1995; Burdine et al. 1997; Roubin et al. 15 York St., HRT 210, New Haven, CT 06510-3221. 1999). The EGL-15 FGFR is structurally very similar to 3Corresponding author: College of Graduate Studies, MH230, University of Central Florida, P.O. Box 160112, 4000 Central Florida Blvd., Orlando, mammalian FGF receptors, with the highest level of FL 32816-0112. E-mail: [email protected] sequence conservation found within the intracellular

Genetics 185: 537–547 ( June 2010) 538 T-W. Lo et al. tyrosine kinase domain and the three extracellular the mispositioning of the SMs. Four egl-15 alleles specif- immunoglobulin (IG) domains. ically affect SM migration; when homozygous, these Similar to mammalian FGFRs, alternative splicing also mutations cause dramatic mispositioning of the SMs, generates functionally distinct EGL-15 isoforms. A ma- but do not cause a Soc phenotype (Goodman et al. jor structural difference between EGL-15 and other 2003). Three of these are nonsense mutations in exon FGFRs lies in an additional domain located between 5A and eliminate the 5A EGL-15 isoform. The phenotype the first IG domain and the acid box of EGL-15. This of these mutants highlights the specific requirement EGL-15-specific insert is encoded by a pair of mutually of the 5A isoform for SM migration guidance. The exclusive fifth exons, generating two EGL-15 isoforms, fourth mutation in this class, egl-15(n1457),isanon- 5A and 5B, with different functions (Goodman et al. sense mutation that truncates the carboxy-terminal 2003). Alternative splicing also affects the sequence at domain, specifically implicating the CTD in SM migra- the very end of the carboxy-terminal domain (CTD) of tion guidance. EGL-15 (Goodman et al. 2003), giving rise to four Immediately following their birth at the end of the distinct C-terminal isoform types, referred to as types first larval stage (L1), the two bilaterally symmetric I–IV (see Figure 1A). SMs undergo anteriorly directed migrations to final EGL-15, like its mammalian orthologs, is also involved positions that flank the precise center of the gonad in a large variety of functions, including cell migration (Sulston and Horvitz 1977). In the middle of the guidance affecting the sex myoblast (SM) (Stern and third larval stage, the SMs divide to generate 16 cells that Horvitz 1991; DeVore et al. 1995; Goodman et al. differentiate into the egg-laying muscles. Multiple 2003) and CAN cells (Fleming et al. 2005), muscle arm mechanisms help guide the migrations of the SMs extension (Dixon et al. 2006), a number of processes (Chen and Stern 1998; Branda and Stern 2000), controlling terminal axon morphology (Bulow et al. including a chemoattraction mediated by EGL-15 that 2004), muscle protein degradation (Szewczyk and guides the SMs to their precise final positions (Burdine Jacobson 2003), and fluid homeostasis (Huang and et al. 1998). The EGL-17 FGF serves as the chemo- Stern 2004). A conserved FGFR signaling pathway in attractive cue, emanating from central gonadal cells C. elegans was established by identifying the genes (Branda and Stern 2000). In the absence of this necessary for the role of EGL-15 in fluid homeostasis, chemoattraction, SMs are posteriorly displaced (Stern and much of this same pathway is utilized in other and Horvitz 1991). While mispositioned SMs still functions of EGL-15 (DeVore et al. 1995; Borland et al. generate sex muscles, these muscles end up too far 2001; Huang and Stern 2004, 2005). The identification posterior to attach properly, causing the animal to be of these genes was facilitated by a temperature-sensitive defective in egg laying (Egl). mutation affecting a receptor tyrosine phosphatase, The signal transduction pathway downstream of EGL-15 CLR-1 (CLeaR), which functions to negatively regulate that mediates SM chemoattraction is not well estab- EGL-15 signaling (Kokel et al. 1998). Mutations in clr-1 lished. Several lines of evidence implicate SEM-5/Grb2, abolish this regulatory constraint on EGL-15, resulting LET-341/Sos, and LET-60/Ras in SM chemoattraction. in fluid accumulation in the pseudocoelomic cavity due The roles of components in the Ras-MAPK cascade in to hyperactive EGL-15 signaling. The buildup of this this event are less clear (Sundaram et al. 1996; Chen clear fluid, resulting in the Clr phenotype, is easily et al. 1997; Chen and Stern 1998). A crucial gap in our scored and can be used to identify suppressors (soc, understanding lies in the link between activated EGL-15 suppressor of Clr) that reduce EGL-15 signaling effi- and the downstream signaling components. Here we ciency. These suppressors define an EGL-15 signaling show that SEM-5, the C. elegans GRB2 ortholog, appears pathway necessary for fluid homeostasis, which involves to bind directly to SH2 binding sites within the carboxy the activation of the Ras/MAPK cascade via the SEM-5/ terminal tail of EGL-15. These interactions are required Grb2 adaptor protein, the let-341/sos-1 Sos-like guanine for SM chemoattraction, but not for the essential nucleotide exchange factor, and a PTP-2-SOC-1/Shp2- function of EGL-15. Gab1 cassette (Borland et al. 2001). Mutations in egl-15 have been identified on the basis of their effects on either fluid homeostasis or the guidance MATERIALS AND METHODS of the migrating SMs (DeVore et al. 1995; Goodman et al. Genetic manipulations: All strains were derived from C. 2003). Most of these alleles fall into an allelic series and elegans var. Bristol, strain N2, using standard genetic protocols affect the general aspects of EGL-15 signaling (Goodman (Brenner 1974) and standard genetic manipulations (Her- et al. 2003). The strongest of these alleles confers an early man 1988). Nematode strains were grown and maintained on larval arrest phenotype, whereas weaker alleles confer NGM agar plates and raised at 20° unless otherwise indicated. either a scrawny body morphology (Scr) or just the ability Transgenic assays were conducted as previously described for the genomic and cassette assays (Lo et al. 2008) and for the to suppress the Clr phenotype (Soc). While the weakest of deletion and truncation assays (Goodman et al. 2003), using these alleles does not affect egg laying, more highly egl-15(n1456) as the null allele. The tm1031 deletion in rog-1 compromised mutants show an egg-laying defect due to was isolated by the knockout consortium at the National EGL-15 Roles in SM Migration 539

Bioresource Project for C. elegans (Tokyo). This allele deletes a NH#1286, egl-15(Y884F); and NH#1287, egl-15(Y1009F, Y884F). critical portion of the PTB domain (supporting information, NH#1354, the EGL-15(Intra IV) yeast two-hybrid bait, was Figure S1), thereby destroying the sole structure on which the created by two-round PCR amplification, using NH#934 for the putative interaction with EGL-15 is based. tm1031 mutants common upstream portion of EGL-15(Intra IV) and the EGL- were maintained as balanced heterozygotes using the semi- 15 type IV cDNA for the unique downstream portion of EGL- dominant sup-9(n1550sd) allele, which lies 3 map units from 15(Intra IV). The resulting PCR product was cloned into the rog-1 on chromosome II (Levin and Horvitz 1993). sup- PstI site of the yeast two-hybrid ‘‘bait’’ vector pBTM116. The 9(n1550sd) causes heterozygotes to display a characteristic following constructs are derivatives of NH#1354: NH#1355, suite of phenotypes, including Lon (Long), Unc (Uncoordi- egl-15(Intra IV, Y1009F); NH#1360, egl-15(Intra IV, Y1009F, nated), Rbr (Rubberband), and Egl (Egg-laying defective); Y1087F). NH#1321, the backbone vector for our cassette assays, n1550 homozygotes are inviable. Mutants were identified as was generated from the NH#112 genomic rescuing construct non-Rbr animals, and recombinants were identified by non- as follows. The C-terminal domain of EGL-15, downstream of Rbr non-Unc animals that had broods of wild-type size. PCR the unique HpaI site at amino acid L932, was replaced with the identification of tm1031 mutants was by duplex PCR, which unc-54 39 UTR from pPD136.61. Various fragments were can differentiate wild-type, mutant, and heterozygote animals cloned into this HpaI site to generate transgenes that corre- by the presence or absence of both alleles. RNA interference spond to specific portions of the alternatively spliced CTD (RNAi) was carried out according to standard protocols (Fire isoforms. Mutations in these constructs were generated using et al. 1998). standard PCR methods. Full-length ced-2 and ptp-2 were cloned Determination and representation of sex myoblast posi- into the XhoI site in pACT2 to generate CED-2 and PTP-2 yeast tion: SM final positions were determined with respect to the two-hybrid constructs. underlying hypodermal Pn.p cells (Pn.p, the posterior daugh- Yeast two-hybrid screen selection procedure: The con- ters of cells P1–P11) as previously described (Thomas et al. structs in the yeast two-hybrid vectors, pBTM116 and pACT or 1990) and are depicted using box-and-whisker plots (Moore pACTII, were used to transform the Saccharomyces cerevisiae L40 and McCabe 1993) aligned to a schematic representation of (MATa, trp1, leu2, his3, LYSTlexA-HIS3, URA3TlexA-LacZ) and the Pn.p cell positions (see Figure 2, Figure 3, and Figure S4). AMR70 (MATa, trp1, leu2, his3, URA3TlexA-LacZ) yeast strains, In brief (Goodman et al. 2003), each set of SMs is ordered respectively. L40 yeast was sequentially transformed with the according to anteroposterior position and divided into quar- EGL-15(Intra I) plasmid, NH#934, and with a C. elegans cDNA tiles. The ‘‘box’’ includes the positions of SMs within the two library using the lithium acetate method and treated as central quartiles. An additional vertical line within the box described (Rose et al. 1990). Transformations were plated to indicates the median SM position at the boundary between the synthetic medium lacking histidine, leucine, and tryptophan. second and third quartiles. Overlap of the line representing The plates were incubated at 30° for 3 days. Colonies that grew the median position with a right or left border of the box is were patched on selective plates to test for regrowth. Colonies depicted as a thickening of that line. A quartile length (1Q) is that regrew were assayed for b-galactosidase activity by a filter determined on the basis of the range of positions covered by assay (Dalton and Treisman 1992), and colonies that turned the 2Q box length. Bars (‘‘whiskers’’) of up to 1.5Q length blue in 24 hr at 30° were retained. Library plasmid DNA was extend from the edges of the box to additional data points. As recovered via yeast miniprep and retested via a mating assay and whisker length does not extend beyond the range of data filter-lift b-galactosidase assays. Library plasmids that passed points, these bars may be shorter than a 1.5Q length or even both retests were sequenced for identification. absent. Data points beyond the edge of the bars (‘‘outliers’’) Yeast two-hybrid interaction assays: Dilution series for yeast are indicated by individual hatch marks. This representation of matings were set up in the following manner. Individual SMs therefore depicts the overall range of SMs as well as their cultures for dilution interaction assays were grown in the general distribution and median position. To score SMs of appropriate selective liquid medium cultures overnight at 30°. tm1031 sterile mutant homozygotes, a large population of Samples were diluted to OD600 ¼ 0.2, 5 ml was dotted on plates tm1031/n1550 heterozygotes was grown to adulthood and then 1 cm apart undiluted and at dilutions of 1:100, 1:1000, and cloned to individual NGM agar plates. The mixed progeny of 1:10,000, and the plate was incubated for 3 days at 30°. these heterozygotes were grown to mid-L3 stage at 20° and Quantitative b-galactosidase assays: Mated yeast cells were individual non-Rbr animals were numbered and mounted, and grown overnight in TRP LEU media at 30°.AnOD600 was their SMs were scored using Nomarski optics. To eliminate taken, and the pellet of 1.0 ml of the overnight culture was nonmutant recombinant progeny, these individually scored resuspended in 1.0 ml of Z buffer (8.52 g anhydrous Na2HPO4, animals were then recovered to PCR tubes and individually 4.8 g anhydrous NaH2PO4, 0.12 g anhydrous MgSO4, 0.74 g genotyped for the tm1031 deletion via duplex PCR, and KCl dissolved in 1.0 liter water, and pH was adjusted to 7.0 with recombinants were excluded from the data set. HCl), pelleted, and resuspended in 150 ml Z buffer supple- Plasmid construction: All plasmids constructed using PCR mented with 2-mercaptoethanol (27 ml of 2-mercaptoethanol/ were confirmed by sequencing. Details of plasmid construc- 10 ml Z buffer); 50 ml of chloroform and 20 ml of 0.1% SDS tion are available upon request. NH#112 is the wild-type egl-15 were added, and the sample was vortexed for 15 sec. A total genomic rescuing fragment (DeVore et al. 1995). NH#838 egl- of 700 ml of prewarmed (30°) ONPG (ortho-nitrophenyl- 15(Q948STOP) was generated by introducing the nonsense muta- B(beta)-galactoside; 1 mg/ml in Z buffer with 2-mercapto- tion in the EGL-15 CTD that corresponds to the egl-15(n1457) ethanol; Sigma) was added, and the reactions were incubated mutation, thereby truncating the CTD from all EGL-15 iso- at 30° until the solutions turned a yellow color. The reactions forms expressed from this construct. The genomic constructs were checked at 1 and 5 min and then every 5 min until 15 min. used for the experiments the results of which are summarized Reactions were stopped by adding 0.5 ml 1 m Na2CO3 and in Figure 2 are derivatives of NH#112. NH#934, the EGL- microcentrifuged for 10 min to pellet debris. OD420 was taken 15(Intra I) yeast two-hybrid bait, was created by PCR amplifi- for each of the reactions. Miller units were calculated using the cation of the intracellular domain of EGL-15 from the EGL-15 following equation: (OD420 3 1000)/OD600 3 time (in min) 3 type I cDNA (NH#324). The resulting PCR product was volume (in ml). All assays were repeated at least three times. cloned into the HpaI sites of the yeast two-hybrid ‘‘bait’’ vec- Genetic assays for putative interactors: To determine if the tor pBTM116. The following constructs are all derivatives of putative EGL-15 interactors played a role in EGL-15 signaling, NH#934: NH#726, egl-15(K672A); NH#1285, egl-15(Y1009F); we examined the available mutants for the Egl phenotype. L4 540 T-W. Lo et al.

TABLE 1 EGL-15 interactors

Genea Function/structure Strength of interaction (MU)b Mutant phenotype SH2-containing interactors F13B12.6 (76) SH2 domain protein 3300 6 1118 Wild type sem-5 (2) Grb2 homolog 2100 6 1050 Vul, Let, Mig vav-1 (3) Vav proto-oncogene 1900 6 398 Let abl-1 (13) Abl homolog 1200 6 724 Wild type aap-1 (1) AGE-1 adapter protein 38 6 11 Wild type csk-1 (3) C-terminal Src kinase 46 6 52 Let, Ste Non-SH2-containing interactors tag-153 (1) NOT2/NOT3/NOT5 domain protein 1300 6 360 Wild type tdc-1 (1) Tyrosine decarboxylase 1000 6 547 Wild type T05E11.3 (3)c Endoplasmic reticulum protein 33 6 13 Wild type c Y48G8AL.9 (1) C2H2-type zinc-finger protein 32 6 12 Wild type a The number of screen isolates of each gene is shown in parentheses. b Strength of interaction is measured by a quantitative b-gal assay and reported in Miller units (MU) as the mean 6 SD. c For T05E11.3 and Y48G8AL.9, the RNAi phenotype is listed because mutant alleles were not available. animals were picked and observed 48 hr later for the presence clones of this gene represented the majority of isolates of accumulated eggs in the uterus. For interactors in which a from this screen (76/104). genetic mutant was not available, we utilized standard RNAi A number of phenotypic tests were conducted to techniques (Fire et al. 1998). To determine if the interactors played a role in the essential function pathway, we screened the determine whether any of the genes corresponding to mutants for the ability to suppress the Clr phenotype. We used the identified interactors shared functions with EGL-15. RNAi (against the interactors) in a clr-1(e1745ts) background. L4 These tests (see materials and methods) measured animals were placed on RNAi feeding clones, and their progeny effects on either fluid homeostasis or SM chemoattrac- were shifted to 25° (the restrictive temperature) and examined tion when the interactor genes were compromised using for the presence of Soc animals. egl-15 and L4440 RNAi were used as a positive and negative control, respectively. RNA interference or existing mutant alleles. The only assays that revealed egl-15-like phenotypes were those conducted with sem-5 (data not shown); these assays RESULTS confirmed the common functions shared between SEM- A yeast two-hybrid screen reveals an important 5 and EGL-15 (DeVore et al. 1995). interaction between the C. elegans Grb2 homolog, An alternative way to authenticate the yeast two-hybrid SEM-5, and EGL-15: To identify potential signaling interactors was to show the specificity of the observed components that might bind directly to EGL-15,we interaction. Because SH2 domains have a well-defined performed a yeast two-hybrid screen using the intracel- interaction mechanism, it was possible to test the SH2 lular domain of EGL-15. Since the identification of domain-containing interactors further for the mecha- interactors might be dependent on tyrosine autophos- nism by which they bound EGL-15. Phosphorylated phorylation, the entire intracellular domain, including tyrosines, like those present in the EGL-15 intracellular the juxtamembrane domain, the full kinase domain, domain, often serve as high-affinity binding sites for SH2 and the CTD, was inserted in a two-hybrid bait vector. domain-containing proteins (Pawson 2004). We tested This EGL-15 ‘‘bait’’ construct (NH#934) contains the whether EGL-15-interactor binding activity was depen- type I CTD [EGL-15(Intra I)], since it is the most dent on the kinase activity of EGL-15(Intra I). Mutating prevalent C-terminal isoform (Goodman et al. 2003), the ATP-binding site (K672A) of the kinase domain and preliminary analysis indicated that this CTD isoform within the EGL-15(Intra I) bait construct abolishes could mediate SM chemoattraction (Branda 2001). EGL-15 autophosphorylation in yeast (Figure S2). This This bait is tyrosine-phosphorylated in yeast (Figure mutation is also known to abolish the phenotypic effects S2). We screened 2.0 3 108 transformants and identified of otherwise hyperactivated egl-15 constructs (Kokel 104 clones that specifically interact with EGL-15(Intra et al. 1998). When tested with this EGL-15(K672A Intra I) I). These clones define 10 putative interactors (Table 1). bait (NH#726), all 10 interactors failed to show yeast two- Six of the putative interactors have an SH2 domain, hybrid binding activity (Figure 1B and data not shown), including the C. elegans homologs of Grb2 (SEM-5), Vav indicating that all 10 require an active kinase domain to (VAV-1), Abl (ABL-1), and Csk (CSK-1) and the p85 sub- interact with EGL-15(Intra I). unit of PI3K (AAP-1). The sixth SH2 domain-containing To test the specificity of these interactions further, we interactor is a protein of unknown function (F13B12.6); investigated whether the six SH2-containing proteins EGL-15 Roles in SM Migration 541

of the 10 interactors. Deletion of the C-terminal domain had a variable effect on the strength of the interactions between EGL-15 and all of the interactors (Table S1). However, 2 of the 10 interactors, SEM-5 and CSK-1, showed the most dramatic reductions, consistent with their interactions requiring the C-terminal domain of EGL-15(Intra I). Of these 2 interactors, only SEM-5 has been observed to exhibit egl-15-like phenotypes, with established roles in both the essential and the SM migration functions of EGL-15 (Clark et al. 1992; Stern et al. 1993; Chen et al. 1997). Within the C-terminal domain of EGL-15, there are two consensus binding sites (YXNX) for the SH2 domain of SEM-5 (see Figure 1A; Songyang et al. 1993). Y 1009 (YCND) lies in the amino-terminal portion of the CTD within a fragment that is common to all EGL-15 isoforms, including the type I isoform that was used for the yeast two-hybrid screen. Y 1087 (YYNT) lies within a fragment that is unique to the type IV isoform. An additional consensus SEM-5 binding site sequence is found at Y 884 (YANL) within the kinase domain of EGL- 15. We tested the relevance of each of these potential binding sites by mutating these tyrosines to phenylalanines, both individually and in combination, and by testing the constructs in yeast two-hybrid assays (Figure 1B, Table S1). Mutating Y1009 to phenylalanine in the igure T F 1.—The SEM-5 EGL-15 interaction is dependent type I construct abolishes the EGL-15TSEM-5 interac- upon the SEM-5 SH2 binding sites in the CTD of EGL-15. (A) Important residues in the EGL-15 CTD isoforms. The tion. By contrast, the Y884F mutation does not, which is C-terminal portion of the kinase domain and the CTD are consistent with the CTD being necessary for this in- shown. The gray box is common to all C-terminal isoforms. teraction. The type IV isoform contains both Y1009 and Sequences of the CTD isoforms can be found in Figure S5. Y1087 (Figure 1A). Mutating both of these sites is (B) SEM-5 binds directly to EGL-15 Y1009 and Y1087. A necessary to abolish the yeast two-hybrid interaction full-length SEM-5 prey was mated to eight EGL-15 baits: empty vector control (pBTM116) and derivatives containing variants between EGL-15 and SEM-5. These data indicate that of either the type I or the type IV EGL-15(Intra). Growth is an SEM-5 can interact with EGL-15 via either Y 1009 or Y 1087. indication of an interaction between SEM-5 and the EGL-15 The two SEM-5 binding sites in the EGL-15 CTD bait. Dilutions of the culture mixture are indicated above. redundantly mediate SM chemoattraction: Two types of hFGFR1, a bait containing the corresponding portion of transgenic rescue assays were used to assess the mech- the intracellular domain of the human FGFR1. Similar results were obtained using the human SEM-5 ortholog, Grb2. anism by which the CTD mediates SM chemoattraction. In the first of these, a genomic rescuing construct was used to identify CTD sites that are necessary for SM identified in our screen [of 61 predicted from the C. chemoattraction. The wild-type EGL-15 genomic con- elegans genome sequence (http://www.wormbase.org)] struct (NH#112) rescues both the larval arrest and the indicated binding specificity in these assays. Two addi- SM migration defects of egl-15(null) animals when in- tional SH2-containing constructs, which encode CED- troduced in transgenic assays (Figure 2A). By contrast, 2/CrkII and PTP-2/Shp2, were built and tested for their a construct that contains the mutation found in egl- ability to interact with EGL-15(Intra I). Both CED-2 and 15(n1457) that truncates the CTD rescues only the larval PTP-2 fail to interact with EGL-15(Intra I), despite being arrest defect and fails to rescue the SM migration defect, expressed and able to interact with another EGL-15 resulting in severely posteriorly displaced SMs (Figure isoform (Figure S3; see below). Taken together, these 2B). The results of a series of CTD deletion and trun- data indicate that the mode ofthese interactions is specific cation constructs tested using this assay indicated the and suggest that they may be mechanistically relevant. presence of a site in the alternatively spliced region that The migration-specific phenotype of egl-15(n1457) can efficiently mediate SM chemoattraction (Figure S4). suggests that the CTD might be an important site for These results are consistent with a possible role of Y 1087 protein–protein interactions required for SM chemo- in SM chemoattraction. attraction. Thus, we tested whether deletion of the The second transgenic rescue assay tested whether C-terminal domain of the EGL-15(Intra I) bait [EGL- portions of the CTD are sufficient for mediating SM 15(DCTD); NH#1278] would abolish the binding of any chemoattraction. This assay has the benefit of eliminat- 542 T-W. Lo et al.

ing the complexities due to redundancy and alternative splicing. We created a CTD-sufficiency ‘‘cassette’’ in which we deleted all egl-15 sequences downstream of the kinase domain within the genomic rescuing fragment and appended the commonly used 39 UTR sequences from the unc-54 gene (Figure 3). This deleted the EGL-15 CTD and all C-terminal alternative splicing from the egl-15 genomic rescuing fragment. Fragments could be tested for sufficiency by introducing them into a unique HpaI site located just after the kinase domain. The ‘‘empty cassette’’ (without any fragments inserted), when expressed in an egl-15(null) strain, can rescue the essential function, but not the SM chemoattraction function of EGL-15 (Figure 3A). Thus, short sequences can be inserted into this ‘‘cassette’’to serve as an artificial CTD to test whether they are sufficient to restore proper SM chemoattraction. To distinguish this assay from the normal genomic rescuing assay, we refer to this as the ‘‘cassette assay.’’ All constructs tested in this assay rescue the essential function of EGL-15. Individual DNA fragments spanning the Y 1009 SEM-5 binding site (Figure 3B) and the Y1087 SEM-5 binding site (Figure 3D) are each capable of efficient SM chemoattraction rescue. SMs in these rescued lines are centered, unlike the empty vector control where the SMs are posteriorly displaced. To test the relevance of the SEM-5 binding sites within these fragments, we introduced tyrosine-to-phenylalanine mutations into the respective fragments. The mutated constructs fail to rescue SM migration (Figure 3, C and E), resulting in SMs that are posteriorly displaced to a similar extent to those observed in the empty cassette control lines. Thus, Y 1009 and Y 1087 are specifically required for these fragments to rescue, suggesting that SEM-5 binding at these sites can provide the links to EGL-15 that mediate SM chemoattraction. Removal of both C-terminal domain SEM-5 binding sites abolishes SM chemoattraction: To test the roles of the Y 1009CND and Y 1087YNT binding sites in SM chemoattraction, we introduced tyrosine-to-phenylalanine mutations into the genomic rescuing construct, singly and in combination, and determined the effects on SM migration guidance. Mutation of Y 1009 and Y 1087 together abolishes SM chemoattraction rescue (Figure 2H). By contrast, individual mutations do not show the same dramatic effect on SM positions (Figure 2, D and E). Figure 2.—Genomic assay results of the EGL-15 CTD role Consistent with the inability of Y 884 to bind SEM-5 in the in SM chemoattraction. EGL-15 Y1009 and Y1087 are func- yeast two-hybrid assay, the presence of Y 884 does not tionally redundant. Sex myoblast distributions for three lines permit SM chemoattaction (Figure 2H), and mutation for each construct are shown. The indicated transgenes were 884 all tested in an egl-15(null) background. (A) Wild-type EGL-15 of Y to phenylalanine does not significantly affect SM (NH#112). (B) A truncated version of EGL-15 that correlates positions, either alone or in combination with other to the egl-15(n1457) mutant (NH#838). (C) The single Y884F mutation fails to disrupt sex myoblast migration (NH#1379). (D and E) The single Y1009F (NH#818) or Y1087F (NH#1382) mutations do not account for the posterior SM positions of The Y1009F mutation in conjunction with either the Y1087F DCTD. (F and G) Double mutations with Y884 (Y884F/ mutation (NH#1370) or with both Y1087F and Y884F Y1009F, NH#841; Y884F/Y1087F, NH#1380) are not more (NH#1356) (DSEM-5) abolishes SM chemoattraction. n, num- affected than the respective single mutations. (H and I) ber of sex myoblasts scored. EGL-15 Roles in SM Migration 543

tyrosine-to-phenylalanine mutations (Figure 2, C, F, G, and I). Thus, our yeast two-hybrid data show that SEM-5 can bind directly to EGL-15 at both Y 1009 and Y 1087. Both Y 1009 and Y 1087 are consensus Y-X-N-X SEM-5 binding sites. Analyses of these sites in the cassette assay demonstrate that both are sufficient for SM chemoattraction, while the genomic assays demonstrate that the sites act redundantly to mediate SM chemoattraction. An additional factor is implicated in the EGL-15- SEM-5 fluid homeostasis pathway: SEM-5 is a required EGL-15 signaling component not only for SM chemoattraction, but also for fluid homeostasis. While mutations in sem-5 result in a Soc phenotype, the egl-15(n1457) truncation of the EGL-15 CTD, which is predicted to eliminate both SEM-5 binding sites, does not confer a Soc phenotype (Goodman et al. 2003). The phenotype of this mutant thus indicates that an additional site must be able to recruit SEM-5 to EGL-15, either directly or indirectly. Y 884 is another predicted direct binding site for the SEM-5 SH2 domain, even though it cannot bind SEM-5 in the yeast two-hybrid system. To test formally whether it might function to recruit SEM-5 to mediate fluid homeostasis, the EGL-15(DSEM-5) transgene, which mutates all three potential SEM-5 SH2 binding sites, was expressed in a clr-1(e1745ts); egl-15(null) background. clr- 1(e1745ts); egl-15(null); ayEx[EGL-15(DSEM-5)] animals are Clr at the nonpermissive temperature for clr-1(e1745ts), formally demonstrating that EGL-15(DSEM-5) efficiently mediates fluid homeostasis. Thus, EGL-15 transduces its signal to SEM-5 independently of all of its canonical SEM-5 SH2 binding sites, implicating another mechanism that links EGL-15 to SEM-5 to mediate the essential/ fluid homeostasis function. One possible alternate mechanism could utilize an additional adaptor protein. The yeast two-hybrid EGL- 15 interactors might provide this additional link to SEM- 5. These were tested using either RNAi or existing viable mutations in a background that truncates the SEM-5 binding sites in the EGL-15 CTD. None of the nine other putative interactors confers a Soc phenotype when tested in a clr-1(e1745ts); egl-15(n1457) background, suggesting that none provides the link to SEM-5 (data igure F 3.—The cassette assay (‘‘sufficiency’’) results. (Top) not shown). Schematic of the cassette constructs in comparison with the wild-type genomic rescuing construct. (Bottom) Cassette con- Another candidate that could serve as the adaptor structs with the indicated CTD fragments were all tested in an that links EGL-15 to SEM-5 is rog-1, the C. elegans FRS2 egl-15(null) background. (A) Empty cassette (NH#1321, nega- homolog. FRS2 is a key component of the mammalian tive control). (B) E994-Q1026 (the gray fragment in Figure 1A) is FGFR signaling pathway that acts as the main adaptor sufficient for mediating sex myoblast migration (NH#1322). 1009 994 1026 protein linking FGF receptors to downstream signaling (C) Y is required within the gray (E -Q ) fragment to adari mediate sex myoblast migration (NH#1325). (D and E) Sex molecules (H et al. 2001). FRS2 is constitutively myoblast distribution for the wild-type (D) unique portion complexed with FGF receptors. Upon FGF receptor of the type IV isoform (NH#1348) or the same unique portion activation, tyrosines present within FRS2 are phosphor- with the Y1087F mutation (E; NH#1349). ylated, enabling the recruitment of additional signaling components. Phosphorylation of FRS2 on Y 196,Y306,Y349, and Y 392 allows Grb2 to bind (Kouhara et al. 1997), thus linking activated FGF receptors to Grb2. In C. elegans, rog-1 encodes a 599-amino-acid protein containing an 544 T-W. Lo et al.

N-terminal PTB domain that is 34% identical and 60% evidence linking them either to SM chemoattraction or similar to the PTB domain of murine FRS2a; the carboxy- fluid homeostasis. For example, Abl has a well-studied terminal 350 amino acids are less similar (Figure S1) role in actin remodeling (Koleske et al. 1998; Plattner (Matsubara et al. 2007). et al. 1999; Grevengoed et al. 2001; Sini et al. 2004), We tested whether rog-1 in C. elegans plays a similar role while Csk plays a role in integrin-mediated cell adhesion to FRS2 in mammals and serves as an adaptor protein and migration in human colon cancer cells (Rengifo- linking SEM-5 to EGL-15.Mutationofrog-1 alone does not Cam et al. 2004). Vav, like other Dbl homology-containing confer any egl-15-like phenotypes (Table S2)(Matsubara Rho-GEFs, has a cytoskeletal role and also has known et al. 2007; Bennett 2009). Both rog-1(RNAi) and the roles in F-actin polymerization (Fischer et al. 1998) and putative null allele, rog-1(tm1031), are sterile (Table S2) Rac-mediated lamellipodia formation (Crespo et al. and have been shown to have defects in a Ras/MAP kinase 1996, 1997). The AGE-1 adaptor protein, AAP-1, serves pathway that mediates germline progression (Matsubara to link C. elegans PI3-kinase (Paradis et al. 1999) to the et al. 2007; Bennett 2009). These tests did not result in DAF-2 insulin receptor and potentially could recruit either a Soc or SM migration phenotype (Table S2). AGE-1 to EGL-15. PI3 kinase has been shown to act However, for the fluid homeostasis function, this might downstream of vertebrate FGFRs; however, no role for be because of redundancy with the direct binding of PI3 kinase has been established in EGL-15 functions SEM-5 via the EGL-15 CTD. To circumvent this poten- in C. elegans. As tantalizing as these possibilities seem, a tial redundancy, we tested whether compromising rog-1 in variety of genetic tests failed to corroborate the associ- a clr-1(e1745ts); egl-15(n1457) background could suppress ation indicated by the yeast two-hybrid results. It is the Clr phenotype (Soc). clr-1(e1745ts); rog-1(RNAi); possible that some of these potential interactors are egl-15(n1457) animals are not Soc, indicating that ROG-1 involved in some of the other functions of EGL-15. does not play a role in a redundant mechanism. These By contrast, not only does SEM-5 have well-established animals were sterile, similar to the rog-1(tm1031) single roles in the functions mediated by EGL-15, but a large mutant, confirming the effectiveness of the rog-1(RNAi). amount of evidence points to the specificity of the yeast These data support the model that ROG-1 is not the two-hybrid interaction. First, not all SH2-containing adaptor that links SEM-5 to EGL-15 for the fluid homeo- proteins interact with the yeast two-hybrid ‘‘bait’’ EGL- stasis pathway. 15(Intra I), which was used in this screen. C. elegans CED-2 and PTP-2 each have SH2 domains that fail to interact with EGL-15(Intra I), although they interact strongly with EGL-15(Intra-IV) (Figure S3). This dem- DISCUSSION onstrates some degree of specificity of the screen itself. The C. elegans Grb2 ortholog, SEM-5, was originally Second, the interaction between SEM-5 and EGL-15 identified in screens for EGF receptor signaling pro- requires the EGL-15 CTD, the domain required for SM cesses and the fluid homeostasis function mediated by chemoattraction. Third, the binding to EGL-15(Intra I) the FGF receptor, EGL-15 (Clark et al. 1992). SEM-5 is specific to Y 1009, and the binding to EGL-15(Intra-IV) binding sites on the C. elegans LET-23 EGF receptor itself specifically requires a pair of tyrosine residues, Y 1009 and mediate some of the functions of this receptor tyrosine Y 1087; the conservative mutation of these tyrosines to kinase, similar to the mechanism by which the EGF phenylalanines completely abolishes the interaction. receptor and GRB2 function in mammalian systems Fourth, the interaction is dependent on EGL-15 kinase (Lesa and Sternberg 1997). By contrast, Grb2 is linked activity and thus likely requires the tyrosine phosphor- to the activated FGF receptor signaling complex via the ylation known to be essential for SH2 binding. These FRS2 adaptor protein (Hadari et al. 2001). Here we characteristics of the yeast two-hybrid interaction in- show that SEM-5 can bind directly to EGL-15 to mediate dicate its relevance to the physiological functions of SM chemoattraction and that SEM-5 associates with EGL-15 and SEM-5. EGL-15 via a different mechanism for the signal trans- The requirement of the EGL-15 CTD for its interac- duction mechanism that regulates fluid homeostasis. tion with SEM-5 suggested that the interaction would be The direct association between SEM-5 and EGL-15 was necessary for SM chemoattraction, which specifically initially indicated by the results of a yeast two-hybrid requires the EGL-15 CTD. Using two different trans- screen designed to identify C. elegans proteins with the genic rescue assays, we were able to show that both of potential to interact directly with the intracellular these SEM-5 binding tyrosines are important for SM domain of EGL-15. This screen identified 10 potential chemoattraction. Alternative splicing generates a num- direct interactors, including SEM-5 and 5 other inter- ber of EGL-15 isoforms that differ in the C-terminal actors with SH2 domains. Although a number of these portion of their CTDs (Goodman et al. 2003). Y 1009 is potential interactors have known functions that offer located in a region that is common to all CTD isoforms. tantalizing possible connections to migration guidance Although Y 1009 is sufficient to allow SM chemoattraction, or other signaling functions of EGL-15, none, in ad- it is not necessary. By contrast, Y 1087 is specific to the type dition to SEM-5, could be corroborated with genetic IV CTD, but is also sufficient for SM chemoattraction. EGL-15 Roles in SM Migration 545

Figure 4.—Comparison of FGFR and FRS2 signaling complexes in ver- tebrates and C. elegans. (A) The interaction between the vertebrate FGF receptor and Grb2 requires the signaling adaptor protein, FRS2. (B) The C. elegans FGF receptor EGL-15 can bind SEM-5 directly at Y1009 and Y1087, which are required for SM chemoattraction. (C) The C. elegans FRS2 homolog, ROG-1, is involved in a RAS/MAP kinase signaling pathway that is required for germline progression, but does not involve EGL-15.

These two sites function redundantly for SM chemo- mechanism. Although SEM-5 is required for the gonad- attraction; only when both the Y 1009 and the Y 1087 binding dependent repulsion, removing the CTD binding sites sites are mutated is SM chemoattraction abolished. The does not compromise this function. Therefore, the presence of both sites within the type IV isoform suggests mechanism by which SEM-5 and EGL-15 mediate SM that this isoform may play a particularly important role chemorepulsion has yet to be determined. Similarly, in SM chemoattraction. The conservation of this site SEM-5 and EGL-15 also have a shared role in the fluid within Caenorhabditis briggsae EGL-15 provides further homeostasis/essential function. Mutations in SEM-5 can support for its importance. Thus, despite the prevalence suppress the Clr phenotype; however, the egl-15(DSEM-5) of type I transcripts within mixed cDNA isolates, it is mutation cannot, suggesting that SEM-5 must link to possible that the type IV isoform might be more highly EGL-15 by a different mechanism for this function. expressed in the migrating SMs. The average relative While it is possible that SEM-5 could interact directly abundance of the isoforms might be accounted for by a with EGL-15 via its SH3 domains to mediate these skew toward type I expression within the hypodermis, functions, the lack of two-hybrid interactions in the which is a large syncytium that contains many nuclei. absence of the SH2 binding sites in the CTD suggests The intracellular domain of EGL-15 contains an that this is not the case. Rather, these functions may additional YXNX motif at Y 884 that could act as another require an additional adaptor protein to transduce binding site for the SH2 domain of SEM-5. However, Y 884 signals between EGL-15 and SEM-5. We have identified does not appear to play a role in recruiting SEM-5 or in a number of potential candidates that could play this SM chemoattraction. First, SEM-5 does not associate role, including the rog-1-encoded FRS2-like adaptor and with EGL-15 via this site by yeast two-hybrid analysis. the nine remaining yeast two-hybrid interactors. Genetic Second, mutation of the two CTD SEM-5 binding sites tests failed to reveal the involvement of these genes in abolishes SM chemoattraction, indicating that Y 884 is not EGL-15-mediated processes. The multi-substrate adap- sufficient to carry out this recruiting function. The tor protein SOC-1 is known to function in EGL-15- inability of Y 884 to recruit SEM-5 is likely due to the mediated fluid homeostasis (Schutzman et al. 2001). It structural role that it plays within the highly conserved G is possible that it serves to link SEM-5 to EGL-15 a-helix of the EGL-15 kinase domain. The structural independently of the CTD direct binding sites, although importance of this residue is underscored by the fact the mechanism by which SOC-1 itself links to EGL-15 that the corresponding residue in all human FGFRs is remains to be elucidated. phenylalanine instead of tyrosine. By contrast, Y 1009 and Conclusions: Many signal transduction components Y 1087 are C-terminal to the kinase domain and are likely have been conserved throughout the evolution of to be in flexible segments that can be readily phosphor- metazoa. Nevertheless, there is considerable flexibility ylated to trigger SEM-5 recruitment. in how they are used (Figure 4). Data in C. elegans in- Other EGL-15 functions require a different mecha- dicate that the FRS2-like protein, ROG-1, is not involved nism to signal to SEM-5: Similar to the egl-15(n1457) in FGF signal transduction, but rather is involved in a mutation that truncates the EGL-15 CTD, disruption of different RAS/MAP kinase pathway that is critical for the two CTD SEM-5 binding sites, Y 1009 and Y 1087, germline progression. D. melanogaster also has an FRS2- specifically affects SM chemoattraction, leaving intact like protein, but, like C. elegans, no evidence links it to other common functions shared by SEM-5 and EGL-15. FGF signaling. Instead, the Dof protein is critical to One of these shared roles is the SM chemorepulsion FGF signal transduction in Drosophila (Michelson et al. 546 T-W. Lo et al.

1998; Vincent et al. 1998; Imam et al. 1999). C. elegans of the sex myoblasts in C. elegans hermaphrodites. Cell 83: 611– does not have a Dof-like gene in its genome and thus 620. Dixon, S. J., M. Alexander,R.Fernandes,N.Ricker and P. J. Roy, must utilize yet other mechanisms. Here we show that C. 2006 FGF negatively regulates muscle membrane extension in elegans uses direct binding to link SEM-5 to EGL-15 for Caenorhabditis elegans. Development 133: 1263–1275. swarakumar ax chlessinger SM chemoattraction, obviating the need for the FRS2- E , V. P., I. L and J. S , 2005 Cellular signaling by fibroblast growth factor receptors. Cytokine Growth like multi-substrate adaptor protein for this function. Factor Rev. 16: 139–149. For other EGL-15 functions, an additional, yet to be Fire,A.,S.Xu,M.K.Montgomery,S.A.Kostas,S.E.Driver discovered, mechanism links SEM-5 to the EGL-15 sig- et al., 1998 Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 391: naling complex. Identifying that mechanism will further 806–811. show the breadth of evolutionary flexibility in these Fischer, K. D., Y. Y. Kong,H.Nishina,K.T edford,L.E.Marengere signal transduction systems. et al., 1998 Vav is a regulator of cytoskeletal reorganization mediated by the T-cell receptor. Curr. Biol. 8: 554–562. We thank J. Schutzman for initiating the EGL-15 yeast two-hybrid Fleming,T.C.,F.W.Wolf and G. Garriga, 2005 Sensitized ge- experiments, I. Sasson for initiating the ROG-1 experiments, Y. Kohara netic backgrounds reveal a role for C. elegans FGF EGL-17 as for cDNAs, S. Hubbard for discussions about the structure of the EGL- a repellent for migrating CAN neurons. Development 132: 15 kinase domain, and members of our lab for critical reading of this 4857–4867. oodman randa obinson urdine manuscript. Some nematode strains used in this work were provided G , S. J., C. S. B ,M.K.R ,R.D.B and M. J. Stern, 2003 Alternative splicing affecting a novel domain by the Caenorhabditis Genetics Center, which is funded by the in the C. elegans EGL-15 FGF receptor confers functional spec- National Institutes of Health (NIH) National Center for Research ificity. Development 130: 3757–3766. Resources. tm1031 was provided by the National Bioresource Project Gotoh, N., K. Manova,S.Tanaka,M.Murohashi,Y.Hadari et al., for C. elegans (Tokyo). This work was supported by NIH grant GM50504 2005 The docking protein FRS2alpha is an essential compo- (M.J.S.) and by the following NIH training grants: T32 GM07223 nent of multiple fibroblast growth factor responses during early (T-W.L. and D.C.B) and T32 DK07259 (S.J.G.). mouse development. Mol. Cell. Biol. 25: 4105–4116. Grevengoed, E. E., J. J. Loureiro,T.L.Jesse and M. Peifer, 2001 Abelson kinase regulates epithelial morphogenesis in LITERATURE CITED Drosophila. J. Cell Biol. 155: 1185–1198. Hadari, Y. R., H. Kouhara,I.Lax and J. Schlessinger, Bennett, D. C., 2009 Identifying interactors of C. elegans FGFR sig- 1998 Binding of Shp2 tyrosine phosphatase to FRS2 is essential naling. Ph.D. Thesis, Yale University, New Haven, CT. for fibroblast growth factor-induced PC12 cell differentiation. Borland, C. Z., J. L. Schutzman and M. J. Stern, 2001 Fibroblast Mol. Cell. Biol. 18: 3966–3973. growth factor signaling in Caenorhabditis elegans. Bioessays 23: Hadari, Y. R., N. Gotoh,H.Kouhara,I.Lax and J. Schlessinger, 1120–1130. Branda, C. S., 2001 Mechanisms of sex myoblast migration in C. ele- 2001 Critical role for the docking-protein FRS2 alpha in FGF gans hermaphrodites. Ph.D. Thesis, Yale University, New Haven, CT. receptor-mediated signal transduction pathways. Proc. Natl. Branda, C. S., and M. J. Stern, 2000 Mechanisms controlling sex Acad. Sci. USA 98: 8578–8583. Herman, R. K., 1988 Genetics, pp. 17–45 in The Nematode Caenorhab- myoblast migration in Caenorhabditis elegans hermaphrodites. Wood Dev. Biol. 226: 137–151. ditis elegans, edited by W. B. . Cold Spring Harbor Labora- renner tory Press, Cold Spring Harbor, NY. B , S., 1974 The genetics of Caenorhabditis elegans. Genetics uang tern 77: 71–94. H , P., and M. J. S , 2004 FGF signaling functions in the hy- Bulow, H. E., T. Boulin and O. Hobert, 2004 Differential func- podermis to regulate fluid balance in C. elegans. Development 131: 2595–2604. tions of the C. elegans FGF receptor in axon outgrowth and uang tern maintenance of axon position. Neuron 42: 367–374. H , P., and M. J. S , 2005 FGF signaling in flies and worms: Burdine, R. D., E. B. Chen,S.F.Kwok and M. J. Stern, 1997 egl-17 more and more relevant to vertebrate biology. Cytokine Growth encodes an invertebrate fibroblast growth factor family member Factor Rev. 16: 151–158. mam utherland uang ransnow required specifically for sex myoblast migration in Caenorhabdi- I , F., D. S ,W.H and M. A. K , tis elegans. Proc. Natl. Acad. Sci. USA 94: 2433–2437. 1999 stumps, a Drosophila gene required for fibroblast growth Burdine, R. D., C. S. Branda and M. J. Stern, 1998 EGL-17(FGF) factor (FGF)-directed migrations of tracheal and mesodermal expression coordinates the attraction of the migrating sex myo- cells. Genetics 152: 307–318. okel orland e ong orvitz blasts with vulval induction in C. elegans. Development 125: K , M., C. Z. B ,L.DL ,H.R.H and M. J. tern 1083–1093. S , 1998 clr-1 encodes a receptor tyrosine phosphatase that Chen, E. B., and M. J. Stern, 1998 Understanding cell migration negatively regulates an FGF receptor signaling pathway in Caeno- guidance: lessons from sex myoblast migration in C. elegans. rhabditis elegans. Genes Dev. 12: 1425–1437. oleske ifford cott ee ronson Trends Genet. 14: 322–327. K , A. J., A. M. G ,M.L.S ,M.N ,R.T.B Chen, E. B., C. S. Branda and M. J. Stern, 1997 Genetic enhancers et al., 1998 Essential roles for the Abl and Arg tyrosine kinases in of sem-5 define components of the gonad-independent guidance neurulation. Neuron 21: 1259–1272. mechanism controlling sex myoblast migration in Caenorhabdi- Kouhara, H., Y. R. Hadari,T.Spivak-Kroizman,J.Schilling,D. tis elegans hermaphrodites. Dev. Biol. 182: 88–100. Bar-Sagi et al., 1997 A lipid-anchored Grb2-binding protein Clark, S. G., M. J. Stern and H. R. Horvitz, 1992 C. elegans cell- that links FGF-receptor activation to the Ras/MAPK signaling signalling gene sem-5 encodes a protein with SH2 and SH3 do- pathway. Cell 89: 693–702. mains. Nature 356: 340–344. Lax, I., A. Wong,B.Lamothe,A.Lee,A.Frost et al., 2002 The Crespo, P., X. R. Bustelo,D.S.Aaronson,O.A.Coso,M.Lopez- docking protein FRS2alpha controls a MAP kinase-mediated neg- Barahona et al., 1996 Rac-1 dependent stimulation of the ative feedback mechanism for signaling by FGF receptors. Mol. JNK/SAPK signaling pathway by Vav. Oncogene 13: 455–460. Cell 10: 709–719. Crespo, P., K. E. Schuebel,A.A.Ostrom,J.S.Gutkind and X. R. Lesa, G. M., and P. W. Sternberg, 1997 Positive and negative tissue- Bustelo, 1997 Phosphotyrosine-dependent activation of Rac-1 specific signaling by a nematode epidermal growth factor recep- GDP/GTP exchange by the vav proto-oncogene product. Nature tor. Mol. Biol. Cell 8: 779–793. 385: 169–172. Levin,J.Z.,andH.R.Horvitz, 1993 Three new classes of mutations Dalton, S., and R. Treisman, 1992 Characterization of SAP-1, a in the Caenorhabditis elegans muscle gene sup-9. Genetics 135: 53–70. protein recruited by serum response factor to the c-fos serum re- Lo, T. W., C. S. Branda,P.Huang,I.E.Sasson,S.J.Goodman et al., sponse element. Cell 68: 597–612. 2008 Different isoforms of the C. elegans FGF receptor are re- DeVore, D. L., H. R. Horvitz and M. J. Stern, 1995 An FGF recep- quired for attraction and repulsion of the migrating sex myo- tor signaling pathway is required for the normal cell migrations blasts. Dev. Biol. 318: 268–275. EGL-15 Roles in SM Migration 547

Matsubara,Y.,I.Kawasaki,S.Urushiyama,T.Yasuda,M.Shirakata Schutzman, J. L., C. Z. Borland,J.C.Newman,M.K.Robinson,M. et al., 2007 The adaptor-like protein ROG-1 is required for ac- Kokel et al., 2001 The Caenorhabditis elegans EGL-15 signaling tivation of the Ras-MAP kinase pathway and meiotic cell cycle pathway implicates a DOS-like multisubstrate adaptor protein progression in Caenorhabditis elegans. Genes Cells 12: 407– in fibroblast growth factor signal transduction. Mol. Cell. Biol. 420. 21: 8104–8116. Michelson, A. M., S. Gisselbrecht,E.Buff and J. B. Skeath, Sini, P., A. Cannas,A.J.Koleske,P.P.Di Fiore and G. Scita, 1998 Heartbroken is a specific downstream mediator of FGF re- 2004 Abl-dependent tyrosine phosphorylation of Sos-1 mediates ceptor signalling in Drosophila. Development 125: 4379–4389. growth-factor-induced Rac activation. Nat. Cell Biol. 6: 268–274. Moore, D. S., and G. P. Mccabe, 1993 Introduction to the Practice of Songyang, Z., S. E. Shoelson,M.Chaudhuri,G.Gish,T.Pawson Statistics. Freeman and Company, New York. et al., 1993 SH2 domains recognize specific phosphopeptide se- Ong, S. H., G. R. Guy,Y.R.Hadari,S.Laks,N.Gotoh et al., quences. Cell 72: 767–778. 2000 FRS2 proteins recruit intracellular signaling pathways by Stern, M. J., and H. R. Horvitz, 1991 A normally attractive cell in- binding to diverse targets on fibroblast growth factor and nerve teraction is repulsive in two C. elegans mesodermal cell migra- growth factor receptors. Mol. Cell. Biol. 20: 979–989. tion mutants. Development 113: 797–803. Ornitz, D. M., and N. Itoh, 2001 Fibroblast growth factors. Ge- Stern, M. J., L. E. Marengere,R.J.Daly,E.J.Lowenstein,M.Kokel nome Biol. 2: REVIEWS3005. et al., 1993 The human GRB2 and Drosophila Drk genes can Paradis, S., M. Ailion,A.Toker,J.H.Thomas and G. Ruvkun, functionally replace the Caenorhabditis elegans cell signaling gene 1999 A PDK1 homolog is necessary and sufficient to transduce sem-5. Mol. Biol. Cell 4: 1175–1188. AGE-1 PI3 kinase signals that regulate diapause in Caenorhabdi- Sulston,J.E.,andH.R.Horvitz, 1977 Post-embryonic cell lineages tis elegans. Genes Dev. 13: 1438–1452. of the nematode, Caenorhabditis elegans. Dev. Biol. 56: 110–156. Pawson, T., 2004 Specificity in signal transduction: from phospho- Sundaram, M., J. Yochem and M. Han, 1996 A Ras-mediated signal tyrosine-SH2 domain interactions to complex cellular systems. transduction pathway is involved in the control of sex myoblast mi- Cell 116: 191–203. gration in Caenorhabditis elegans. Development 122: 2823–2833. Plattner, R., L. Kadlec,K.A.DeMali,A.Kazlauskas and A. M. Szewczyk, N. J., and L. A. Jacobson, 2003 Activated EGL-15 FGF Pendergast, 1999 c-Abl is activated by growth factors and receptor promotes protein degradation in muscles of Caeno- Src family kinases and has a role in the cellular response to PDGF. rhabditis elegans. EMBO J. 22: 5058–5067. Genes Dev. 13: 2400–2411. Thomas, J. H., M. J. Stern and H. R. Horvitz, 1990 Cell interac- Polanska, U. M., D. G. Fernig and T. Kinnunen , 2009 Extracellular tions coordinate the development of the C. elegans egg-laying interactome of the FGF receptor-ligand system: complexities and system. Cell 62: 1041–1052. the relative simplicity of the worm. Dev. Dyn. 238: 277–293. Vincent, S., R. Wilson,C.Coelho,M.Affolter and M. Leptin, Rengifo-Cam, W., A. Konishi,N.Morishita,H.Matsuoka,T.Yamori 1998 The Drosophila protein dof is specifically required for et al., 2004 Csk defines the ability of integrin-mediated cell adhe- FGF signaling. Mol. Cell 2: 515–525. sion and migration in human colon cancer cells: implication for a Xu, H., K. W. Lee and M. Goldfarb, 1998 Novel recognition motif potential role in cancer metastasis. Oncogene 23: 289–297. on fibroblast growth factor receptor mediates direct association Rose, D. M., F. Winston and P. Hieter, 1990 Laboratory Course Man- and activation of SNT adapter proteins. J. Biol. Chem. 273: ual for Methods in Yeast Genetics. Cold Spring Harbor Laboratory, 17987–17990. Cold Spring Harbor, NY. Roubin, R., K. Naert,C.Popovici,G.Vatcher,F.Coulier et al., 1999 let-756, a C. elegans fgf essential for worm development. Oncogene 18: 6741–6747. Communicating editor: D. I. Greenstein

Supporting Information http://www.genetics.org/cgi/content/full/genetics.109.113373/DC1

Caenorhabditis elegans Fibroblast Growth Factor Receptor Signaling Can Occur Independently of the Multi-Substrate Adaptor FRS2

Te-Wen Lo, Daniel C. Bennett, S. Jay Goodman and Michael J. Stern

2 SI T-W. Lo et al.

FIGURE S1The structure of the rog-1 gene. (A) ROG-1 is encoded by two similar cDNAs of either 1800 or 1809 bp, differing by a minor splicing difference at the exon 5 splice acceptor, marked by an asterisk. The rog-1 gene contains nine exons covering approximately 6 kb of genomic sequence (top). The 3’ ends of our rog-1 clones differ from the cDNA reported by MATSUBARA et al. 2007, which terminates after six exons (bottom). (B) The tm1031 deletion is predicted to truncate ROG-1 in the middle of the PTB domain (shown in black), after 83 amino acids. Four novel amino acids are inserted before a premature stop codon. T-W. Lo et al. 3 SI

FIGURE S2EGL-15 DNA binding domain baits are expressed in yeast. -LexA western blot (left) shows that both the EGL- 15(IntraI) bait used in the two-hybrid screen (WT, NH#934) and the kinase-compromised version (K672A, NH#726) are expressed in yeast (open arrowhead). The LexA (empty vector, pBTM116) control is also expressed (solid arrowhead). - phosphotyrosine blot (right) shows that only the WT EGL-15(Intra I) bait is phosphorylated in yeast. 4 SI T-W. Lo et al.

FIGURE S3PTP-2 and CED-2 interact with EGL-15(Intra IV) but not EGL-15(Intra I). Yeast with full-length PTP-2 and CED-2 prey were mated to eight EGL-15 baits: empty vector control (pBTM116), and derivatives containing variants of either the Type I, II, III or IV EGL-15(Intra). SEM-5 was included as a positive control. Growth is an indication of an interaction between PTP-2, CED-2, SEM-5 and the EGL-15 baits. Dilutions of the culture mixture are indicated above. T-W. Lo et al. 5 SI

6 SI T-W. Lo et al.

FIGURE S4Genomic assay of CTD deletion/truncation constructs. The indicated transgenes were all tested in an egl-15(null) background. The portion of the CTD denoted in gray is common to all CTD isoforms and spans E994-Q1026. (A) Controls. Wild-type EGL-15 restores SM chemoattraction (NH#112), while the negative control, that correlates to the egl-15(n1457) mutation (NH#838), does not. (B) Deletion constructs that retain the unique regions for types 2-4 all restore SM chemoattraction (NH#1166, M934-Y993; NH#1186, M934- C1010; NH#1187, M934- I1020). All deletions start after the kinase domain at M934. The region that was deleted is indicated by a short bent line. The first amino acid after the deletion is indicated with an arrowhead. These results indicate the presence of a site in the alternatively-spliced region that can efficiently mediate SM chemoattraction. (C) Conversely, truncation mutations that exclude the alternatively-spliced region compromise rescue activity. The amino acid that was mutated to a STOP is indicated with an arrowhead (NH#1072, Q1026STOP; NH#1184, N1011STOP; NH#1071, E994STOP). The E994STOP mutation truncates EGL-15 upstream of Y1009 and any of the sites for alternative splicing. Although the N1011STOP mutation leaves Y1009 intact, it removes the N1011 residue, which is critical for the binding of the SH2 domain of SEM-5 (SONGYANG et al. 1993). It is unclear why the Q1026STOP truncation rescues less well than the common fragment alone in the cassette assay (Fig. 3B); this fragment leaves intact the entire common fragment, including the Y1009 SEM-5 binding site. It is possible that the premature translational stop due to the Q1026STOP mutation within the endogenous mRNA may destabilize egl-15 transcripts. NH#1072 egl-15(Q1026STOP), NH#1184 egl-15(N1011STOP), and NH#1071 egl-15(E994STOP) were created similarly to NH#828 (see Materials and Methods). egl-15 deletion constructs (NH#1166 [M934-Y993], NH#1186 [M934-C1010], and NH#1187 [M934-I1020]) were created using standard 2-step PCR methods. T-W. Lo et al. 7 SI

FIGURE S5Amino acid sequences of EGL-15 CTD isoforms. The structures of each of the four EGL-15 CTD isoforms, Type I through Type IV, are shown at the top. Alternative splicing generates the various CTD components. These are color- coded to portray the common elements of these isoforms. The sequences of each of the color-coded fragments are shown below. 8 SI T-W. Lo et al.

TABLE S1

SEM-5 and CSK-1 interactions require the EGL-15 CTD

a Strength of interaction is measured by a quantitative ß-gal assay, and reported in Miller Units (MU) as the mean. b Comparison between the strengths of interaction between EGL-15(Intra I) and either EGL-15(CTD) or EGL- 15(Y1009F) as represented as a ratio, CTD/Intra I or Y1009F/Intra I, respectively. T-W. Lo et al. 9 SI

TABLE S2

ROG-1 does not participate in EGL-15 signaling pathways

1 n 30 for all genotypes except rog-1(tm1031) where n=12. 2 Suppression of the Clr phenotype was scored in a clr-1(e1745ts) background; nd, not determined. 3 n 30 for all genotypes.