SIGNAL transduction via receptor tyrosine kinases levels of receptor activity. For example, dimerization,

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1 Copyright 2004 by the Genetics Society of America Conservation of an Inhibitor of the Epidermal Growth Factor Receptor, Kekkon1, in Dipterans Frederick A. Derheimer, 1 Christina M. MacLaren, Brandon P. Weasner, Diego Alvarado and Joseph B. Duffy 2 Department of Biology, Indiana University, Bloomington, Indiana Manuscript received July 1, 2003 Accepted for publication October 10, 2003 ABSTRACT Regulation of epidermal growth factor receptor (EGFR) signaling requires the concerted action of both positive and negative factors. While the existence of numerous molecules that stimulate EGFR activity has been well documented, direct biological inhibitors appear to be more limited in number and phylogenetic distribution. Kekkon1 (Kek1) represents one such inhibitor. Kek1 was initially identified in Drosophila melanogaster and appears to be absent from vertebrates and the invertebrate Caenorhabditis. To further investigate Kek1 s function and evolution, we identified kek1 orthologs within dipterans. In D. melanogaster, kek1 is a transcriptional target of EGFR signaling during oogenesis, where it acts to attenuate receptor activity through an inhibitory feedback loop. The extracellular and transmembrane portion of Kek1 is sufficient for its inhibitory activity in D. melanogaster. Consistent with conservation of its role in EGFR signaling, interspecies comparisons indicate a high degree of identity throughout these regions. During formation of the dorsal-ventral axis Kek1 is expressed in dorsal follicle cells in a pattern that reflects the profile of receptor activation. D. virilis Kek1 (DvKek1) is also expressed dynamically in the dorsal follicle cells, supporting a conserved role in EGFR signaling. Confirming this, biochemical and transgenic assays indicate that DvKek1 is functionally interchangeable with DmKek1. Strikingly, we find that the cytoplasmic domain contains a region with the highest degree of conservation, which we have implicated in EGFR inhibition and dubbed the Kek tail (KT) box. SIGNAL transduction via receptor tyrosine kinases levels of receptor activity. For example, dimerization, (RTKs) constitutes one of the major modes of cellular coupled with the expansion of family members in verte- communication in metazoans (Schlessinger 2000). brates, allows for the formation of distinct heterodim- One of the first RTKs to be identified was the epidermal eric complexes, thereby diversifying signaling outputs growth factor receptor (EGFR) and it represents, argu- (Alroy and Yarden 1997). Diversity also exists at the ably, the most extensively studied RTK (Schlessinger ligand level, as numerous ligands have been identi- 2000). The EGFR family is highly conserved and repre- fied consistent with the concept of combinatorial signaling sentatives have been identified in invertebrates, such as activities. With one exception, all of these ligands Drosophila melanogaster and Caenorhabditis elegans, as well function in a positive fashion. The exception, Argos as in vertebrates (Wadsworth et al. 1985; Schejter et (Aos), acts in an inhibitory fashion and has not been al. 1986; Aroian et al. 1990). Invertebrates appear to described outside the dipteran lineage (Schweitzer et have a single EGF receptor, while four receptors, EGFR al. 1995; Howes et al. 1998). In addition to Aos, another (or ErbB1), ErbB2, ErbB3, and ErbB4, exist within verte- inhibitor of EGFR, Kekkon1 (Kek1) was identified in brates (Stein and Staros 2000). Modulation of receplione D. melanogaster (Musacchio and Perrimon 1996; Ghig- tor activity is essential to normal development and abergenomic et al. 1999). One possibility is that in lieu of the rant regulation has been linked to oncogenic situations duplications in vertebrates, which expanded (Blume-Jensen and Hunter 2001). The classical nolineages the receptor family, inhibitors have arisen within other tion of EGFR signaling invokes receptor dimerization to provide additional signaling diversity (Perri- and activation in a ligand-dependent fashion (Schles- mon and Duffy 1998). singer 2000). However, numerous mechanisms, both Such diversity in output is crucial to the develop- stimulatory and inhibitory, exist in vivo to govern the mental role of the EGFR family, which functions in numerous cellular events including determination, proliferation, migration, survival, and differentiation. In D. 1 Present address: Department of Cellular and Molecular Biology, University melanogaster, EGFR s role in axis patterning has been of Michigan, 1500 E. Medical Center Dr., Ann Arbor, MI well characterized and provides a useful system in which to investigate the regulation of receptor activity (Riech- Corresponding author: Department of Biology, Indiana University, 1001 E. 3rd St., Jordan Hall, Bloomington, IN mann and Ephrussi 2001). During oogenesis, commu- jduffy@bio.indiana.edu nication between the germline-derived oocyte and its Genetics 166: ( January 2004)

2 214 F. A. Derheimer et al. overlying somatically derived epithelium or follicle cells the evolutionary history of Kek1 and its role as an inhibitor orients axial polarity (Schüpbach 1987; Gonzalez- of EGFR signaling. To address these questions and Reyes et al. 1995; Roth et al. 1995; Wasserman and better define the role of Kek1 in EGFR signaling we undertook Freeman 1998; Riechmann and Ephrussi 2001). Central an evolutionary analysis of Kek1. Here we describe to this process is the establishment of follicle cell the identification and characterization of Kek1 orthologs fates by EGFR in response to a germline signal from the from D. virilis, D. pseudoobscura, and Anopheles gambiae. ligand Gurken (Grk; Neuman-Silberberg and Schüp- We report that the expression and function of DvKek1 bach 1993). Subsequently, the follicle cells are responsible supports conservation of its role as an inhibitor of EGFR for the generation and morphology of the chorion, signaling. Finally, interspecies comparisons reveal a re- which manifests the underlying polarity of the oocyte gion of unexpected conservation in the carboxy-termi- (Spradling 1993). During the latter stages of oogenesis, nal tail, which we have termed the Kek tail (KT) box. EGFR is stimulated in the follicle cells by Grk secretion from the oocyte (Neuman-Silberberg and Schüpbach 1993). This initiates a suite of transcriptional responses MATERIALS AND METHODS and autoregulatory loops responsible for directing dor- Molecular techniques: Standard molecular techniques were sal patterning. Interestingly, two of the transcriptional followed throughout the course of this work (Sambrook et al. targets during this process, kek1 and aos, function as 1989). To obtain Dvkek1 a D. virilis genomic phage library inhibitors of EGFR signaling (Schweitzer et al. 1995; (kindly provided by J. Tamkun) was screened with a digoxy- Wasserman and Freeman 1998; Ghiglione et al. 1999). genin-labeled probe to kek1. Positive phage clones were carried through secondary and tertiary screens and four positive kek1 is expressed in a dorsal anterior gradient within clones (C1-2-2, C2-1-1, C3-4-1, and D1-1-1) representing Dvkek1 the follicle cells, while aos is expressed along the dorsal were selected for subsequent analysis. Using a combination midline (Musacchio and Perrimon 1996; Wasserman of restriction mapping, PCR, and sequencing, a general map and Freeman 1998). In contrast, other transcriptional of the genomic region containing the kek1 gene in D. virilis was targets (e.g., rhomboid) function in a stimulatory manner, generated. The region corresponding to the entire predicted open reading frame (ORF) was then sequenced from phage revealing the existence of tiers of feedback regulation clone C1-2-2 using cycle sequencing according to the manufac- (Ruohola-Baker et al. 1993). Through this complex turer s instructions (Applied Biosystems, Foster City, CA). Sequence network of positive and negative feedback mechanisms, was extended at the 5 end for 20 bp until the EGFR signaling is refined to establish dorsal follicular presence of a CA minisatellite region was detected. Generation fates. of a chimeric Dvkek1-green fluorescence protein (GFP) P{UAS- Dvkek1-gfp} construct was undertaken using Gateway cloning Within this autoregulatory network, kek1 functions technology (Invitrogen, Carlsbad, CA). Briefly, DvKek1 was to attenuate receptor activity. Supporting this, loss- flanked with AttB sites by PCR and recombined in frame into of-function mutations in kek1 suppress egfr mutations ap{uas-gfp} destination vector (W. Wang and J. B. Duffy, in a dose-dependent fashion and display phenotypes unpublished data). To construct P{UAS-kek1 IC -gfp} the extra- indicative of an increase in receptor activity (Ghiglione cellular and transmembrane portion of Kek1 was amplified and cloned into EGFPN1 using a 5 kek1 primer flanked with et al. 1999). In addition, Kek1 misexpression throughout an EcoRI site and a 3 kek1 primer flanked with a KpnI site. development results in phenotypes similar to those of This kek1-gfp fusion was then shuttled into puast using EcoRI loss-of-function mutations in egfr (Ghiglione et al. and XbaI. P{UAS-kek1 IC T -gfp} was cloned using primers encod- 1999). While analysis of Kek1 in oogenesis has provided ing the KT box flanked by a 5 EcoRI and 3 KpnI sites and insight into a specific function, its discovery was based fused in frame to gfp flanked by 5 KpnI and 3 XbaI sites. The N-terminal portion of Kek1 was amplified by PCR and on expression in the embryonic nervous system, where nondirectionally cloned, taking advantage of an EcoRI site it may function in axonal pathfinding (Musacchio and present at the junction with the KT box. Agkek1 was isolated Perrimon 1996; Speicher et al. 1998). Moreover, the by using BLAST to identify kek1-related sequences from raw related gene kekkon2 (kek2) was recovered on the basis of sequence reads deposited into GenBank by the A. gambiae its similarity to kek1 (Musacchio and Perrimon 1996). genome project (Holt et al. 2002). These sequences were subsequently organized into contigs using BLAST and Kek2 exhibits the same arrangement of structural do- Sequencher 4.1 to identify overlapping clones and generate mains and a similar expression profile in the embryonic a continuous ORF. Dpkek1 was identified in a similar manner nervous system (Musacchio and Perrimon 1996). The using the sequences available from the Human Genome Se- completion of the D. melanogaster genome has revealed quencing Center at Baylor College of Medicine. The programs that Kek1 is the founding member of a family of six ProtScale and SignalP were utilized to analyze the presence of signal peptides and transmembrane domains. Hydropathy Drosophila proteins sharing a related extracellular strucplots were performed using Kyte-Doolittle values for hydroture (Figure 1; Musacchio and Perrimon 1996; Adams phobicity. Protein alignments were performed with ClustalW et al. 2000). Each family member contains leucine-rich and manually edited. For in situ hybridizations, the templates repeats (LRRs), an amino (N) and carboxy (C) cysteineto NB1 (for Dmkek1) and the coding region for Dvkek1 were used generate digoxygenin-labeled RNA probe and hybridiza- rich region (N and C cysteine caps) flanking the LRRs, tions were carried out according to Klingler and Gergen and a single C2-type immunoglobulin (Ig) domain. The (1993), with minor modifications and 65 as the hybridization identification of a Kek family of molecules within D. temperature (Musacchio and Perrimon 1996). melanogaster raises a number of questions concerning Drosophila genetics: D. melanogaster transgenics were gener-

3 Conservation of Kek1 215 ated by co-injecting the P{UAS-Dvkek1-gfp} construct with the ecules represent orthologs of kek1 and its overall structransposase-encoding plasmid puchs 2-3 at a 4:1 ratio into ture as a single-pass transmembrane molecule has been w 1118 embryos. Transformant lines were mapped with w - ; Sp/ CyO ; Sb/Tm6, Hu and subsequently balanced. Misexpression well conserved (Figure 2). In its extracellular region analysis was performed by mating transgenic lines to the follicle DmKek1 contains N and C cysteine caps (N and C flanks) cell driver P{GawB}CY2 (CY2-GAL4)(Queenan et al. 1997). flanking a set of LRRs, followed by a single Ig domain Phenotypic analysis: Eggs from adult females raised at 27 (Figures 2 4). These features and their relative orientawere collected, mounted in Lacto-Hoyers, and cleared at 60 tion are all conserved in DvKek1, DpKek1, and AgKek1 for 48 hr. Images were captured under dark-field illumination on a Zeiss Axiophot microscope. For GFP fluorescence im- (Figures 2 4). In agreement with their identification as aging, ovaries from the same females were dissected in PBS, Kek1 orthologs, these molecules display 60% identity fixed in 3.7% formaldehyde for 10 min, washed four times with DmKek1 throughout this region, but 40% identity in PBT, and mounted in 70% glycerol/pbs with SlowFade when compared with other Kek family members. In (Molecular Probes, Eugene, OR). Images were obtained with contrast, the N-terminal insert, a feature distinguishing a Leica TCS SP confocal microscope. Cell culture and co-immunoprecipitations: Drosophila S3 Kek1 from other members of the Kek family in D. melanocells were grown, maintained, and transfected by electroporation gaster, is not well conserved (Musacchio and Perrimon as described in Cherbas and Cherbas (1998). Co-immu- 1996). In DmKek1, the insert lies between the signal noprecipitations were performed as follows. Briefly, S3 cells peptide and the N cysteine cap, but is absent from Agwere transiently cotransfected with GFP-tagged constructs, Kek1 and displays only two short stretches of identity P{UAS-egfr1} orp{uas-egfr2}, and mt-gal4 and induced with 1mmCuSO with DvKek1 and DpKek1 (Figures 3 and 4). 4 (Klueg et al. 2002). GFP-tagged proteins were immunoprecipitated from lysed cells with anti-gfp (CLON- Sequence conservation among Kek1 orthologs: Con- TECH, Palo Alto, CA), coupled to protein A Sepharose beads servation of the extracellular cysteine caps and LRRs: Al- (Amersham-Pharmacia, Piscataway, NJ), and washed thor- though DmKek1 was first characterized as a nonvital oughly. Samples were subjected to SDS-PAGE and transferred locus with no overt phenotypes, subsequent studies have to nitrocellulose membranes (Amersham-Pharmacia). Blots were probed with mouse anti-gfp (CLONTECH) at 1:1000 ascribed two functional roles. In oogenesis Kek1 funcand rabbit anti-egfr (kindly provided by Nick Baker) at tions to attenuate EGFR signaling during axial pat- 1:5000 (Lesokhin et al. 1999). terning, while in neuronal development it appears to function in axonal guidance (Speicher et al. 1998; RESULTS Ghiglione et al. 1999). Kek1 s function during the former process requires the extracellular and transmembrane Identification of Kek1 orthologs within the Diptera: portion of the molecule and is mediated by a In D. melanogaster, kek1 is a member of a multigene direct interaction with the EGFR (Ghiglione et al. family and represents a nonvital function (Figure 1; 1999). This portion of Kek1 contains seven LRRs and Musacchio and Perrimon 1996). The best-character- it has recently been revealed that they are essential to ized contribution of Kek1 to development is its ability Kek1 s interaction with the receptor, with the second to attenuate EGFR signaling (Ghiglione et al. 1999). LRR appearing to play a particularly crucial role (Ghiglione Current evidence suggests that this function is likely to et al. 2003; Alvarado et al. 2004a, accompanying be unique to Kek1 among the six Kek family members article). LRRs have been described as a tandem array identified in D. melanogaster (Alvarado et al. 2004b). of 24 amino acid repeats, which is often flanked by Thus, one possibility is that Kek1 s role in EGFR signal- cysteine-rich caps. LRRs represent the second most prevalent ing is not representative of a conserved or ancestral repeat within the Drosophila proteome, where function. To gain insight into this possibility we under- they are present in a diverse set of secreted, membranebound, took the identification of kek1 orthologs. We searched for and cytoplasmic proteins (Pruess et al. 2003). kek1 in a related drosophilid, D. virilis, with an estimated Structurally, LRRs are believed to mediate protein-protein divergence time from D. melanogaster of million interactions through a characteristic horseshoedivergence years ago (MYA; Beverly and Wilson 1984; Russo et shaped structure (Kobe and Deisenhofer 1994, 1995; al. 1995). To identify Dvkek1, ad. virilis genomic phage Kobe and Kajava 2001). Throughout the N and C flanks library was screened under moderately stringent condi- and LRRs, a high degree of conservation is observed tions with a probe to the Dmkek1 coding region. From among all four Kek1 orthologs, exhibiting 66% amino this screen four kek1-positive phage clones were selected acid identity overall (Figure 3; Table 1). Conservation for further characterization. Analysis of the D. virilis in Kek1 orthologs is apparent in the organization of genomic kek1 sequence predicts an uninterrupted ORF these motifs, as well as of residues known to impart of 910 amino acids (aa) for the DvKek1 protein, as key structural information. For example, the sequence compared to 880 aa for DmKek1. Subsequently, we were Lx 2 LxLx 2 N/C is believed to represent the smallest element able to identify kek1 orthologs in D. pseudoobscura and A. of a full LRR that may be essential to impart gambiae (Holt et al. 2002). Analysis of the kek1 genomic the characteristic horseshoe structure of LRR proteins sequences in these species predicts uninterrupted ORFs (Kobe and Kajava 2001). In all four species the first of 886 aa and 769 aa for DpKek1 and AgKek1, respec- LRR of Kek1 is missing the initial leucine of this element, tively. Interspecific comparisons indicate that these mol- suggesting the first LRR represents only a partial

4 216 F. A. Derheimer et al. Figure 1. Diagrammatic representation of Kek family members in D. melanogaster. Kek family members are single transmembrane pass proteins that share sequence and structural identity in their extracellular domains. This family is defined by the presence in the extracellular domain of seven leucine-rich repeats (LRRs), flanked by N- and C-terminal cysteine-rich caps (N and C flanks), and followed by a single immunoglobulin (Ig) domain. In contrast, their cytoplasmic domains share little identity. Two features distinguish Kek1 from other Kek proteins. The first is the presence of a small insert between the signal peptide and the N flank. This insert is partially conserved in other Kek1 orthologs. The second is a highly conserved region in the carboxy tail of all Kek1 orthologs. Conservation of the transmembrane domain of Kek1: The transmembrane (tm) region spans 21 residues posi- tioned approximately in the middle of the coding sequence and exhibits 62% identity among all four orthologs (Figure 3; Table 1). Structurally, tm domains are typically defined as -helical stretches composed of hydrophobic amino acids. Given such a general definition, the high degree of conservation manifested by the Kek1 tm regions is striking. In contrast, the signal sequence, similarly constrained to a hydrophobic nature, is only 14% identical (Figure 3; Table 1). Thus, the tm region is likely to contribute more specifically to Kek1 function, in addition to its role in anchoring Kek1 within the lipid bilayer. Identification of a novel motif, the intracellular KT box: Previous data suggested that the cytoplasmic domain of Kek1 was not essential for its role in EGFR signaling (Ghiglione et al. 1999). Thus, it was somewhat surpris- ing to discover that the cytoplasmic tail represented the most highly conserved region of Kek1 (Figure 4). Spanning the last 48 residues of the cytoplasmic domain, the Kek tail (KT box) exhibits 92% identity across the four Kek1 orthologs (Figure 4; Table 1). Within repeat. The subsequent six repeats contain this element in full, although the last (seventh) repeat also appears to represent an abbreviated version of a complete LRR. Conservation of the Ig domain: A single C2-type Ig domain of 100 amino acids is present in all Kek1 orthologs identified, as well as in all Kek family members (Figures 1 4). Ig domains represent the second most common domain in D. melanogaster and mediate a wide array of protein-protein interactions (Pruess et al. 2003). These modules are structurally defined by seven to nine antiparallel -sheets, which fold to form a -sandwich. Although a strict consensus sequence does not exist, a number of sequence elements aid in defining an Ig fold. Depending on the Ig domain subtype, the presence of specific disulfide bonds, aromatic residues, and turns are thought to help nucleate and stabilize the fold into an energetically stable conformation. Thus, specific cysteine, proline, and tryptophan residues are often indicative of the Ig signature (Bork et al. 1994; Barclay 1999; Steward et al. 2002). Within the Ig domain Kek1 orthologs share 63% identity, where cysteine, proline, and tryptophan residues are highly conserved (Figure 3; Table 1).

5 Conservation of Kek1 217 Figure 2. Diagrammatic representation of Kek1 orthologs in D. melanogaster (Dm), D. pseudoobscura (Dp), D. virilis (Dv), and A. gambiae (Ag). Kyte-Doolitle hydropathy plots are depicted on the right (positive numbers depict hydrophobicity). All four Kek1 proteins share a similar distribution of hydrophobic regions, centered about the signal peptide (SP) and a transmembrane domain (TM). AgKek1 is the shortest and most divergent protein, displaying a truncated N-terminal insert and cytoplasmic domain. Remarkably, the KT box is present in AgKek1 and highly conserved with respect to the other Kek1 molecules.

6 218 F. A. Derheimer et al. Figure 3. Protein sequence alignment of the extracellular and transmembrane domains of Kek1 in Diptera. Sequence alignment of Kek1 orthologs in D. melanogaster, D. pseudoobscura, D. virilis, and A. gambiae reveals a high degree of conservation at the amino acid level. This conservation is manifested predominantly throughout the LRRs and the Ig domain. In addition, the transmembrane and juxtamembrane regions display remarkable conservation. In contrast, the N terminus is highly divergent, due in part to the presence of a variable signal sequence. Vertical arrows denote putative cleavage sites for the signal sequence. Solid circles represent consensus residues in the LRRs. indicates conserved cysteine residues in the Ig domain. this novel motif, two potential elements exist. The first role for the cytoplasmic domain of Kek1 in subcellular encompasses the last three amino acids (TDV) of Kek1 trafficking. Deletion of the cytoplasmic domain results and represents a putative type I binding site (S/T-X-I/ in aberrant Kek1 localization and less efficient inhibi- V/G) for PDZ domains (Figure 4). PDZ domains most tion of EGFR signaling in oogenesis (Ghiglione et al. commonly function in subcellular trafficking and signal- 2003; Figure 5). Strikingly, we find that addition of a ing (Hung and Sheng 2002). The second proposed portion of the KT box, including the SPDEGY element, element within the KT box is the sequence SPDEGY restores full inhibitory capability, thus providing one previously noted in Kek1 and Kek2 in D. melanogaster possible explanation for its high degree of conservation (Musacchio and Perrimon 1996). The SPDEGY ele- (Figure 5). ment is conserved in all four Kek1 orthologs, as well as Besides the KT box, conservation within the cytoplasmic Kek2 in both D. melanogaster and A. gambiae (Figure 4; domain is limited to small pockets for which data not shown). Functional conservation of the KT box no functional attributes have been identified. However, is supported by recent work, which has uncovered a these pockets encompass most of the tyrosine residues

7 Conservation of Kek1 219 Figure 4. Protein sequence alignment of the cytoplasmic domains of Kek1 in four dipterans. The cytoplasmic domains of Kek1 orthologs are more divergent overall than the extracellular domains. However, of particular interest is the terminal 50 amino acids of all proteins, which exhibit remarkable conservation. In addition, short stretches of identity tend to be centered on tyrosine residues. within the cytoplasmic domain, as 9 of 11 tyrosines are sumably as a result of an expansion in the aos expression conserved and may reflect functional significance (Figure domain (Ghiglione et al. 1999). 4). While species in the subgenus Sophophora, including Expression of Dvkek1 supports a conserved role in D. melanogaster, have only two chorion appendages, a oogenesis: During the latter stages of oogenesis, EGFR wide range of patterns is displayed among the drosophilids. functions to pattern the dorsal epithelium. Its role in For example, D. virilis chorions display four apfunctions this process has been well studied and represents an pendages and initial results suggest that EGFR signaling excellent example of the role of feedback loops in regulating modulates dorsal patterning in this species (Peri et al. signaling outputs and directing pattern formation 1999). We asked if kek1 s expression profile and, thus, (Perrimon and Duffy 1998; Wasserman and Freeman regulation by EGFR signaling was conserved during oo- 1998; Freeman and Gurdon 2002). Briefly, a gradient genesis in D. virilis. During the early stages of dorsalof Grk emanating from the oocyte initiates EGFR activaanterior ventral (DV) axis formation, kek1 is expressed in a dorsal tion within the follicle cells. EGFR activation then initiates gradient in both species (Figure 6, A, B, F, and a transcriptional response that leads to an amplification G). During the ensuing stages, specific elements of the of EGFR activity within the dorsal region through the production of two additional ligands, Spitz and Vein, in the follicle cells. Amplification results in a peak of TABLE 1 EGFR activity along the anterior-dorsal midline, which then directs expression of the inhibitory ligand Aos. Aos Sequence conservation among Kek1 orthologs expression effectively abolishes receptor activity along % identity the midline, thereby splitting the single broad peak (no. identical residues/ of EGFR signaling into two domains, representing the Region no. residues in region) bilateral pattern of dorsal appendages on the chorion Signal peptide 14 (3/21) (Wasserman and Freeman 1998). Throughout this pro- N flank 56 (18/32) cess, expression of kek1 is under the control of EGFR LRRs 68 (106/156) signaling and provides a measure of feedback inhibition C flank 65 (33/51) (Ghiglione et al. 1999). Loss of Kek1 activity results in Ig 63 (64/102) Transmembrane 62 (13/21) an overall increase in EGFR signaling leading to an KT 92 (44/48) increase in the spacing between the appendages, pre-

8 220 F. A. Derheimer et al. Figure 5. A portion of the conserved KT box rescues defects associated with loss of the Kek1 cytoplasmic domain. (A) Graphical representation of full-length Kek1 tagged to GFP (Kek1- GFP), a form of Kek1 lacking the entire cytoplasmic domain (Kek1 IC -GFP), and a similar truncation where the last 15 amino acids of the KT box have been added back (Kek1 IC T -GFP; see also C). The KT box is depicted in gray. (B) Misexpression of these constructs in follicle cells with the CY2- GAL4 driver generates ventralized chorions. V1 represents weak ventralization as indicated by appendages that are closer together or fused at the base. V2 represents moderate ventralization indicated by a single thin appendage, whereas V3 represents severely ventralized chorions with only a small patch of appendage material. Deletion of the cytoplasmic domain results in a phenotype (V1- V2) weaker than full-length Kek1-GFP (V3). Addition of the terminal 15 amino acids of Kek1 displays a phenotype comparable to full-length Kek1 (V3). The number of independent transgenic lines tested is in parentheses. (C) Alignment of the KT box in the four Kek1 orthologs. The bar indicates the portion of the KT box added back in Kek1 IC T -GFP. pattern diverge, although expression remains dorsally results in the production of two isoforms of the EGFR. restricted in both species (Figure 6, C E and H J). These isoforms differ solely at the amino terminus pre- Dmkek1 expression remains primarily in a dorsal anterior ceding the ligand-binding domain and are capable of gradient, until stage 11 when it begins to resolve into binding DmKek1 (Figure 7). Similarly, association of two bilateral stripes (Figure 6E). In contrast, from its DvKek1 with either isoform of DmEGFR can be detected initial dorsal anterior gradient, Dvkek1 develops a broad in S3 cells (Figure 7). To directly test the ability of and distinctive domain of expression during later stages. DvKek1 to inhibit EGFR activity we took advantage of Two small regions of bilateral repression disrupt the the well-documented role of Kek1 in DV patterning broad domain of Dvkek1 expression (Figure 6, H and I). (Ghiglione et al. 1999). During oogenesis, DmKek1 acts This results in the appearance of two triangular patches to inhibit EGFR signaling in the dorsal follicle cells along the outer edges of the expression domain and a (Ghiglione et al. 1999). This inhibitory activity is easily central dorsal patch of expression flanked on either observed through the effects of Kek1 misexpression in side by the regions of repression (Figure 6, H and I). the follicle cells, which results in a loss of dorsal fates and This pattern is then resolved into two bilateral domains ventralization of the chorion (Ghiglione et al. 1999). of expression that appear to extend laterally around Using the same misexpression system, we assayed the the egg chamber (Figure 6J). The dorsally restricted ability of DvKek1 to inhibit DmEGFR signaling in vivo. expression of Dvkek1 supports the conservation of its Misexpression of UAS-Dvkek1 in the follicle cells of D. role as a transcriptional target of EGFR signaling. melanogaster results in ventralization, similar to that ob- Functional conservation of DvKek1 as an inhibitor of served with DmKek1 (Figure 8, C and D). Consistent EGFR: Dorsal expression of kek1 in D. virilis, together with this and its predicted transmembrane structure, with the extensive sequence conservation noted among DvKek1 exhibits a subcellular localization pattern identical all Kek1 orthologs, supports but does not directly demonstrate to DmKek1 (Figure 8, A and B). Likewise, misexall conservation of its feedback role in EGFR sig- pression of DvKek1 in other tissues also results in inhibition naling. To address this we tested the ability of the of EGFR signaling, causing phenotypic effects DvKek1 to both associate with and inhibit the DmEGFR. analogous to those observed with DmKek1 (data not In D. melanogaster the utilization of distinct 5 exons shown).

9 Conservation of Kek1 221 Figure 6. kek1 is expressed in a dynamic pattern during oogenesis in D. melanogaster and D. virilis, as shown by in situ hybridization of speciesspecific kek1 RNA probes in ovaries of D. melanogaster (A E) and D. virilis (F J). In A and F, dorsal is up. In all other images, dorsal is facing outward. Anterior is to the left. (A) In stage 10 egg chambers of D. melanogaster, kek1 RNA is present in the dorsal anterior follicle cells overlying the oocyte nucleus. Expression begins as a large patch that includes lateral follicle cells. (B) Stage 10 egg chamber is rotated slightly to show dorsolateral follicle cells. (C) The area of expression becomes more refined throughout stage 10. (D) Eventually it becomes more focused along the dorsal midline. (E) In stage 11 egg chambers, kek1 RNA is limited to two small patches on either side of the dorsal midline. (F) In D. virilis, kek1 RNA is also expressed in the dorsal anterior follicle cells during stage 10. (G) In early stage 10 it is restricted to a small dorsal triangle. (H) This pattern begins to broaden in the dorsolateral follicle cells during stage 10 and repression starts to appear in two bilateral patches. (I) Repression increases in the two bilateral patches (arrows) leaving a center spot of kek1 expression (arrowhead), as well as two outer dorsolateral patches. ( J) By stage 11, kek1 expression resolves into two bilateral domains that extend laterally around the egg chamber. DISCUSSION Feedback loops constitute an important mechanism for the regulation of EGFR signaling during develop- ment and contribute to its role in pattern formation (Freeman and Gurdon 2002). Many of the underlying molecular components of this pathway are conserved, but in a few instances regulatory components with lim- ited phylogenetic distributions have been identified. The antagonistic ligand Aos represents one such exam- ple and has been suggested to be an example of the independent coevolution of a receptor and a ligand (Stein and Staros 2000). On the surface, invertebrates, with a single EGF receptor and fewer than five ligands, appear to have more restrictions on their ability to gen- erate equally diverse sets of signaling output when compared to vertebrates, which have four receptors and at least twice as many ligands. However, novel regulatory components such as Aos, whose phylogenetic distribution is more limited, might represent alternative ap- proaches to signal diversification. Here we add to comparative studies of EGFR signal- ing through an analysis of Kek1, a transmembrane inhib- itor of the Drosophila EGFR (Ghiglione et al. 1999). Null mutations in kek1 are viable and display only subtle phenotypic effects, consistent with a minor role in EGFR signaling (Musacchio and Perrimon 1996; Ghiglione et al. 1999). Thus, Kek1 s ability to bind and inhibit the receptor might represent a modern co-option event, whereby Kek1 only recently acquired the potential to attenuate receptor activity. To investigate this possibility and to identify conserved Kek1 sequence elements that would serve to inform functional studies, we character- ized kek1 from three dipteran species with an estimated evolutionary divergence time of 250 MYA (Gaunt and Miles 2002). Initially, we chose to search for an ortholog of kek1 in D. virilis and, during the course of our studies, the genome sequences for A. gambiae and D. pseudo- obscura became available (Holt et al. 2002). We were able to identify Kek1 in all three species, supporting an ancient presence for kek1 within the Diptera. Moreover, the functional interchangeability of DvKek1 and DmKek1 in EGFR signaling also suggests that Kek1 s ability to inhibit EGFR was present over MYA and is not a recent acquisition. Consistent with the known requirement of the extracellular and transmembrane domains of Kek1 for EGFR binding and inhibition, interspecies comparison indicated a high degree of conservation throughout this region, with the exception of the N-terminal insert. The conservation of the transmembrane domain relative to the signal peptide (62 vs. 14% identity) was particularly striking and supports the notion of an essential function

10 222 F. A. Derheimer et al. Figure 7. DmKek1 and DvKek1 associate with DmEGFR by co-immunoprecipitation. Drosophila S3 cells were cotransfected with EGFR isoforms 1 or 2 in combination with GFP-tagged forms of D. melanogaster Kek1 (DmKek1-GFP) and Kek2 (DmKek2-GFP), as well as D. virilis Kek1 (DvKek1- GFP). GFP-tagged constructs were immunoprecipitated (IP) with anti-gfp and assayed for the presence of EGFR (top) and GFP (middle) by immunoblot (IB). EGFR loading levels were assayed directly from whole-cell lysates (bottom). DmKek1-GFP and DvKek1-GFP associate with DmEGFR1 and -2, whereas DmKek2-GFP does not. enhancing Kek1 s ability to inhibit EGFR signaling. Whether conservation is solely representative of a contri- bution to Kek1 s role in EGFR signaling or of an alternative function, perhaps contributing to Kek1 s role in neuronal pathfinding, awaits further analysis. DvKek1 is functionally interchangeable with DmKek1 in assays for EGFR binding and inhibition supporting the notion that Kek1 s role in EGFR signaling might contribute to its conservation. However, at least one alternate function for Kek1 has been noted and might also play a role in constraining sequence divergence (Speicher et al. 1998). The expression pattern of Dvkek1 provides additional support for a conserved role for kek1 in EGFR signaling. In D. melanogaster, kek1 expression in the follicle cells is regulated by EGFR signaling and for this region. It will be interesting to determine if this conservation is the result of an EGFR-dependent or -independent function. We have also noted that the transmembrane and juxtamembrane portion of Kek1 displays limited identity with some transmembrane receptor-like kinases from Arabidopsis, also of the LRR superfamily (our unpublished observations). Future functional studies will be required to directly assess the relevance of such conservation and the contribution of this region to the in vivo function of Kek1. Finally, the highest degree of conservation we detected in Kek1 was in the KT box. This striking conservation (92% identity across 48 aa) over 250 MYA argues strongly for an essential role in Kek1 function. Here we demonstrate that this conservation might be due in part to its role in Figure 8. DvKek1 inhibits DmEGFR in vivo. DmKek1 and DvKek1 were tagged with GFP and misexpressed during D. melanogaster oogenesis with the follicle cell driver CY2-GAL4. (A and B) Both GFP-tagged proteins are present at the apical surface of follicle cells during stage 10. (C and D) Misexpression of DmKek1-GFP and DvKek1- GFP results in strongly ventralized chorions, consistent with Kek1-mediated EGFR inhibition.

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