Bipartite Inhibition of Drosophila Epidermal Growth Factor - Genetics

Copyright 2004 by the Genetics Society of America

Bipartite Inhibition of Drosophila Epidermal Growth Factor Receptor by theExtracellular and Transmembrane Domains of Kekkon1

Diego Alvarado,1 Amy H. Rice1 and Joseph B. Duffy2

Department of Biology, Indiana University, Bloomington, Indiana 47405

Manuscript received October 1, 2003Accepted for publication January 14, 2004

ABSTRACTIn Drosophila, signaling by the epidermal growth factor receptor (EGFR) is required for a diverse array

of developmental decisions. Essential to these decisions is the precise regulation of the receptor’s activityby both stimulatory and inhibitory molecules. To better understand the regulation of EGFR activity weinvestigated inhibition of EGFR by the transmembrane protein Kekkon1 (Kek1). Kek1 encodes a moleculecontaining leucine-rich repeats (LRR) and an immunoglobulin (Ig) domain and is the founding memberof the Drosophila Kekkon family. Here we demonstrate with a series of Kek1-Kek2 chimeras that whilethe LRRs suffice for EGFR binding, inhibition in vivo requires the Kek1 juxta/transmembrane region.We demonstrate directly, and using a series of Kek1-EGFR chimeras, that Kek1 is not a phosphorylationsubstrate for the receptor in vivo. In addition, we show that EGFR inhibition is unique to Kek1 amongKek family members and that this function is not ligand or tissue specific. Finally, we have identified aunique class of EGFR alleles that specifically disrupt Kek1 binding and inhibition, but preserve receptoractivation. Interestingly, these alleles map to domain V of the Drosophila EGFR, a region absent fromthe vertebrate receptors. Together, our results support a model in which the LRRs of Kek1 in conjunctionwith its juxta/transmembrane region direct association and inhibition of the Drosophila EGFR throughinteractions with receptor domain V.

CELLULAR communication by the epidermal growth and Yarden 1997; Olayioye et al. 2000). Within theextracellular region the vertebrate ErbBs are composedfactor receptor (EGFR) pathway is widely utilized

throughout development to specify cellular fates and of four domains. Domains I and III (also known as L1and L2) mediate ligand binding, whereas the cysteine-behaviors (Schweitzer and Shilo 1997; Dominguezrich domains II and IV (also named CR1 and CR2) areet al. 1998; Nilson and Schupbach 1999; Van Buskirkinvolved in dimerization and auto-inhibition, respec-and Schupbach 1999). Work in invertebrates and verte-tively (Lax et al. 1988; Garrett et al. 2002; Ogiso et al.brates has implicated this pathway in numerous develop-2002; Ferguson et al. 2003). Interestingly, the extracel-mental processes including axial patterning in Drosoph-lular domain of Drosophila EGFR contains an additionalila, vulval development in Caenorhabditis elegans, andcysteine-rich domain distal to CR2, herein referred to ascardiac development in vertebrates (Aroian et al. 1990;domain V (Price et al. 1989). Distal to the kinase domain,Lee et al. 1995; Nilson and Schupbach 1999). In addi-a noncatalytic carboxy-terminal tail (C-tail) contains tyro-tion, mutation and misregulation of the EGFR familysines that become phosphorylated in response to ligandof receptor tyrosine kinases (RTK) is one of the hall-binding (Yarden and Schlessinger 1987; Ullrichmarks of oncogenic transformation and its associatedand Schlessinger 1990). In contrast to the four verte-alterations in cellular behavior: immortalization, prolif-brate receptors, Drosophila contains only two isoforms,eration, migration, and invasion (Stoscheck and KingEGFR1 and EGFR2, derived from a single locus, torpedo/1986; Harari and Yarden 2000; Blume-Jensen andegfr (Clifford and Schupbach 1994; Lesokhin et al.Hunter 2001; Holbro et al. 2003). With the exception1999). These isoforms differ only at their N termini inof the orphan receptor ErbB2 and ErbB3, which encodesthe signal sequence and a short stretch of flankinga catalytically inactive kinase domain, the vertebrate andamino acids, but are identical through the ligand-bind-invertebrate receptors are type I transmembrane pro-ing, transmembrane, and cytoplasmic domains.teins composed of an extracellular ligand-binding do-

The long-standing model for activation of EGFR sig-main, a transmembrane region, and a cytoplasmic tyro-naling involves receptor homo- or heterodimerizationsine kinase domain (Yarden and Ullrich 1988; Alroyupon ligand binding (Yarden and Schlessinger 1987;Ullrich and Schlessinger 1990). This allows for trans-phosphorylation of a specific subset of tyrosine residues

1These authors contributed equally to this work. in the C-terminal tail to occur. Transphosphorylation2Corresponding author: Department of Biology, Indiana University,

initiates the recruitment of distinct phosphotyrosine1001 E. 3rd St., Bloomington, IN 47405.E-mail: [email protected] binding adaptor/effector molecules and triggers activa-

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188 D. Alvarado, A. H. Rice and J. B. Duffy

tion of a variety of cytoplasmic kinase cascades [e.g., RAS- molecule Kek1 is induced in a graded fashion by recep-tor activity (Musacchio and Perrimon 1996; Ghig-RAF-mitogen-activated protein kinase (MAPK) cascade;

Yarden and Sliwkowski 2001; Schlessinger and Lem- lione et al. 1999). kek1 was initially identified in anenhancer trap screen for genes involved in the develop-mon 2003]. However, a wealth of data obtained in the

past few years supports a more complex scenario that ment of the Drosophila nervous system. Subsequentmolecular and genomic analyses indicated that Kek1distinguishes the EGFR family among RTKs and indi-

cates that long-held notions about the mechanism of is the founding member of a family of six relatedtransmembrane proteins in Drosophila that contain leu-EGFR activation need to be reexamined (Schlessinger

2000, 2002; Burgess et al. 2003). For example, the re- cine-rich repeats (LRRs) and a single C2-type immu-noglobulin (Ig) domain in their extracellular regionscently solved crystal structures of apo- and ligand-bound

soluble forms of EGFR reveal striking differences in the (Musacchio and Perrimon 1996; Adams et al. 2000).Three lines of evidence support a role for Kek1 in atten-mechanism of ligand binding with respect to other RTKs

(Garrett et al. 2002, 2003; Ogiso et al. 2002; Ferguson uating EGFR activity during DV patterning (Ghiglioneet al. 1999). First, loss of Kek1 activity results in wideret al. 2003). Ligand binding may induce a conforma-

tional shift involving rotation about the juxta/trans- spacing between the appendages. This phenotype is dra-matically different from that observed in aos mutants,membrane domain of the receptor, thereby resulting

in the access of substrate to the kinase domain (Moriki which produce a single, wide appendage, suggestingthat Kek1 and Aos utilize distinct mechanisms to inhibitet al. 2001). Perhaps most surprisingly, structural and

functional studies indicate that the EGFR kinase domain EGFR. Second, reduced EGFR activity can be sup-pressed by the simultaneous elimination of Kek1 activity.is likely catalytically active in a monomeric state (Gotoh

et al. 1992; Stamos et al. 2002). This is in stark contrast Last, misexpression of Kek1 in follicle cells results ininhibition of EGFR signaling, observed phenotypicallyto other members of the RTK family (e.g., insulin recep-

tor), which require tyrosine phosphorylation in the acti- as ventralization of the chorion. More recently, loss-of-function studies in the eye, as well as misexpressionvation loop (A-loop) of the kinase domain for full cata-

lytic activity (Hubbard and Till 2000). While kinase studies in both the eye and the wing, indicate that Kek1is likely to function as a general inhibitor of EGFRactivity is essential to signaling, receptor dimerization,

ligand binding/dissociation, subcellular localization, throughout development (Ghiglione et al. 2003; Alva-rado et al. 2004). In addition, deletion analyses andtrafficking, and effector transduction all play important

roles in regulating signaling strength (Olayioye et al. mutagenesis experiments have demonstrated that theLRRs of Kek1 play an essential role in receptor inhibi-2000). The integration of such regulatory mechanisms

is therefore essential to specify the appropriate level of tion (Ghiglione et al. 2003; Alvarado et al. 2004).Despite recent progress in elucidating the mechanismEGFR signaling within a given developmental context.

Although the relevance of positive effectors of EGFR of Kek1 function, a number of questions remain unan-swered. Currently, it is unclear what elements of Kek1signaling has long been appreciated, only in the past

few years has the importance of inhibitory molecules in suffice for inhibition, whether other Kek moleculesfunction redundantly with Kek1, and if Kek1 is a sub-regulating signaling strength and duration come to the

forefront (Freeman 1998). Molecules such as Kekkon1 strate for EGFR-mediated phosphorylation. Here wepresent evidence addressing these questions. Impor-(Kek1), Argos (Aos), D-Cbl, and Sprouty (Spry) inhibit

EGFR signaling, resulting in a refinement of signaling tantly, we show that while the Kek1 LRRs promote bind-ing, the juxta/transmembrane region of Kek1 activelyoutput (Schweitzer et al. 1995; Hime et al. 1997; Was-

serman and Freeman 1998; Casci et al. 1999; Ghigli- contributes to receptor inhibition. We demonstrate thatEGFR inhibition is unique to Kek1 among the Kek fam-one et al. 1999, 2003; Kramer et al. 1999; Pai et al. 2000).

Furthermore, all these inhibitors exert their effects via ily members, that Kek1 is not a substrate for EGFRphosphorylation, and confirm that Kek1 inhibits EGFRdifferent mechanisms, resulting in distinct effects on

receptor signaling. Characterizing the mechanisms by in multiple developmental contexts. Finally, we haveisolated a unique class of EGFR alleles that specificallywhich all EGFR regulators work will be essential to un-

derstanding the balanced interplay between these mole- disrupt its association with Kek1.cules and their contribution to EGFR-mediated develop-mental decisions.

MATERIALS AND METHODSThe specification of dorsal-ventral (DV) polarity inDrosophila provides one well-characterized example of Drosophila genetics: Flies were raised at 27� on standard

media. The following stocks were used: follicle cell driversthe interplay between positive and negative effectors ofP{GawB}CY2 (Queenan et al. 1997) and P{GawB}T155 (Free-EGFR signaling. During the latter stages of oogenesis,man 1996); eye driver P{GAL4-ninaE.GMR} (Freeman 1996);receptor activity is modulated by positive and negativeP{w � mC � Act5C-GAL4}; P{UAS-Egfr.DN.B}29-77-1(Free-

feedback loops to pattern the DV axis (Stevens 1998; man 1996); P{UAS-grk�TC}, grk HK36 (Neuman-Silberberg andVan Buskirk and Schupbach 1999). Throughout dor- Schupbach 1993); egfr 2X51 (Wieschaus et al. 1984); egfr QY1

(Clifford and Schupbach 1994); egfr CO (Clifford andsal fate specification, expression of the transmembrane

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189Bipartite Inhibition of EGFR

Schupbach 1989); Df(2R)egfr 3F18; kek1RA5, and kek1RM2 (Musac- pUAST-Kek2-gfp was made by amplifying the full-length genefrom the NB7 cDNA and subcloning into a basal pUAST-gfpchio and Perrimon 1996). Transgenic lines were generated

by coinjecting each pUAST construct with transposase (pUChs using 5� EcoRI and 3� KpnI sites. Additional details and mapsare available upon request.� �2-3) at a 4:1 ratio into w 1118 embryos.

All pUAST constructs were misexpressed in follicle cells Kek1-Kek2 swaps were generated in vitro by overlappingPCR. Purified fragments were cloned into a Gateway-compati-using CY2-GAL4 or T155-GAL4 and in the developing eye with

GMR-GAL4. For chorion preparations, eggs were collected, ble pUAST-GFP destination vector, according to manufactur-er’s instructions (Invitrogen, Carlsbad, CA).washed, and cleared in lacto-Hoyer’s solution for 48 hr at 60�.

Images were captured under darkfield on a Zeiss Axiophot egfr SOK1–4 were sequenced from genomic DNA isolated from8–10 hemizygous adult flies [SOK/Df(2R)Pu-D17] with QIA-microscope. Ovaries from females expressing green fluores-

cent protein (GFP) constructs used in egglays were dissected GEN DNeasy columns according to manufacturer’s instruc-tions (QIAGEN, Beverly, MA). For egfr SOK5, DNA was isolatedin PBS and fixed for 10 min in 3.7% formaldehyde/PBS,

washed three times in PBT (0.1% Tween-20), and brought to from 30–50 homozygous embryos selected from egfr SOK5/CyO,P{Act-GFP} stock. Likewise, genomic DNA was also isolatedvolume in 70% glycerol/PBS with SlowFade (Molecular

Probes, Eugene, OR). GFP fluorescence images were captured from w;iso2;iso3 adults. egfr exons were amplified individuallyby PCR, using primers specific to noncoding sequence flank-with a Leica TCS SP confocal microscope. Wings were dehy-

drated in 100% ethanol and mounted in polyvinyl lactophe- ing each exon, purified using a gel purification kit (QIAGEN),and sequenced using cycle sequencing according to the manu-nol. Scanning electron microscopy was performed as in Kim-

mel et al. (1990). facturer’s instructions (Applied Biosystems, Foster City, CA).Primer sequences are available upon request. Sequence align-To identify suppressors of Kek1 misexpression in the eye,

P{GAL4-ninaE.GMR}, P{UAS-kek1}/CyO recombinants were ments were performed with MAP (http://searchlauncher.bcm.tmc.edu/multi-align/multi-align.html), using the extracellu-generated by standard methods from stocks containing indi-

vidual P elements. w;iso2;iso3 males were mutagenized with 25 lar and transmembrane portion of the receptor for each ofthe indicated species. The start codon corresponds to the firstmm EMS according to previously described methods (Ash-

burner 1989). Mutagenized males were crossed to P{GAL4- residue with respect to numbering for the alignments.pUAST-egfr SOK constructs were generated using PCR-basedninaE.GMR}, P{UAS-kek1}/CyO females and maintained at 27�.

A total of 110,000 chromosomes from straight-winged F1 males site-directed mutagenesis with mismatch primers encodingchanges R738Q (egfr SOK1–3) and E718K (egfr SOK4). PCR frag-and females were screened for suppression of the P{GAL4-

ninaE.GMR}, P{UAS-kek1}/CyO phenotype. Five straight- ments encoding point mutations and flanked by 5� FseI and3� PshAI restriction sites were cloned into the correspondingwinged strong suppressors were isolated independently and

crossed to w1118; the segregation of the suppressor away from region of the template pUAST-egfr1 (courtesy of Nick Baker).All pUAST-egfr SOK subclones were sequenced to confirm pres-the P{GAL4-ninaE.GMR}, P{UAS-kek1} chromosome in the F2’s

demonstrated that all five suppressors were on the second ence of the point mutations. Primer sequences and constructmaps are available upon request.chromosome. Stocks were generated by crossing F1 w; SOK/�

males to w; P{GAL4-ninaE.GMR}, P{UAS-kek1}/CyO females; Cell culture and immunoprecipitations: Drosophila S3 cellswere grown and maintained as described in Cherbas andw; SOK/P{GAL4-ninaE.GMR}, P{UAS-kek1} males were then

crossed to yw; Sco/Cyo and balanced stocks established. The Cherbas (1998). Cells were grown to a density of �5 � 106

cells/ml and transfected by electroporation. Cells were cotrans-suppressors were then mapped using standard recombinationmapping techniques to genetic position 97–99 on 2R with the fected with 5 �g of metallothionein-GAL4 (mt-GAL4; Klueg et al.

2002), a copper-inducible GAL4 driver, and 5 �g of responderfollowing stocks: al1 dp ov1 b1 pr 1 c1 px1 sp1 and crossveinless-2.The egfr alleles egfr QY1, egfr CO, and Df(2R)egfr 3F18 were used for DNA and induced with 1 mm CuSO4 for 22 hr. Cells were

collected and gently pelleted by centrifugation at 2000 rpmcomplementation tests.Molecular cloning and sequence analysis: keg-based chime- and subsequently lysed in 1 ml of ice-cold Fehon buffer

(Fehon et al. 1990) containing 1 mm PMSF, 1 �m leupeptin,ras were subcloned into pUAST in three steps. The kek1 extra-cellular and transmembrane region was amplified by PCR and 1 �m pepstatin A, and 0.3 �m aprotinin. Lysed cells were

cleared by centrifugation at 14,000 rpm for 5 min at 4�. Super-subcloned into pUAST using 5� EcoRI and 3� BglII sites (pUAST-k1et). The gfp gene was excised from the pEGFP-N1 vector natant was brought up to 5 ml in buffer, and antigen was

immunoprecipitated with 0.5 �l of rabbit anti-GFP (CLON-(CLONTECH, Palo Alto, CA), using 5� KpnI and 3� XbaI re-striction sites, and fused in frame to pUAST-k1et (pUAST-k1et- TECH). Samples were rotated for 2 hr at 4� and subsequently

incubated with 150 �l of a 1:5 slurry of protein A Sepharosegfp). Next, variants of the cytoplasmic domain of egfr wereflanked with 5� BglII and 3� KpnI sites by PCR and incorporated beads (Amersham-Pharmacia, Piscataway, NJ) in Fehon buffer

for 1 hr at 4�. Beads were collected by gentle centrifugationinto pUAST-k1et-gfp. Fragments encode the full-length cyto-plasmic domain (keg), the kinase region (ke�Cg), and the C-tail (3000 rpm for 2 min at 4�) and washed five times in Fehon

buffer. The last two washes were performed in Fehon buffer(ke�Kg). Point mutations encoding kinase-dead chimeras weregenerated by PCR-based site-directed mutagenesis utilizing lacking detergent. Samples were resuspended in 30 �l of a

3:2 mix of 5� sample buffer and TBS, boiled for 5 min, andmismatch primers encoding G901R (ke*gg) and K923M (ke*kg)changes in the kinase region. pUAST-mCD8E �C-gfp was con- loaded in 8% polyacrylamide gels. Transfer to nitrocellulose

membranes (Amersham-Pharmacia) was followed by Ponceaustructed by substituting the kek1 region in pUAST-ke �Cg with aPCR-based fragment from pUAST-mCD8-gfp (courtesy of Liqun staining and subsequently blocked for 1 hr at room tempera-

ture (RT) in 5% nonfat dry milk (NFDM) with TBST (100Luo; Lee and Luo 1999) encoding extracellular and trans-membrane regions of the murine CD8 gene flanked by 5� MfeI mm Tris pH 7.5, 150 mm NaCl, 0.1% Tween-20). Membranes

were incubated with primary antisera at the following concen-and 3� Bgl II restriction sites.pUAST-kek1-gfp was made by first subcloning a PCR-based trations: rabbit anti-EGFR (courtesy of Nick Baker; Lesokhin

et al. 1999) at 1:5000 (2% NFDM in TBST), monoclonal anti-fragment flanked by 5� SpeI and 3� KpnI sites encoding thetransmembrane-intracellular regions of kek1 into pUAST-gfp GFP (CLONTECH) at 1:1000 (5% NFDM in TBST), and

guinea pig anti-Delta (courtesy of Marc Muskavitch; Huppert(pUAST-kek1tm-intra-gfp). Next, the extracellular domain ofkek1 was amplified by PCR from its cDNA (NB1) and cloned et al. 1997) at 1:5000 (5% NFDM). Incubations were done

overnight at 4�, followed by five washes in TBST. Secondaryinto pUAST-kek1tm-intra-gfp using 5� EcoRI and 3� SpeI sites.

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horseradish peroxidase-conjugated goat-anti-rabbit, mouse, pressed them during oogenesis using CY2-GAL4. Misex-and guinea pig antibody incubations (Jackson Immunore- pression of these Keks in follicle cells has no effect onsearch, West Grove, PA) were done at 1:20,000 in 5% NFDM

DV patterning, indicating Kek2, Kek4, Kek5, and Kek6for 1 hr at RT, followed by five washes in TBST. Detectionare not functionally analogous to Kek1 with respect towas performed by chemiluminescence (West Pico; Pierce,

Rockford, IL), according to manufacturer’s instructions, utiliz- EGFR inhibition (Figure 1C). Thus, the ability to associ-ing Kodak Biomax MR-1 autoradiography film. Stripping and ate with and inhibit the EGFR is not a common featurereprobing was performed according to manufacturer’s instruc- shared by Kek family members and is unlikely to providetions.

an explanation for subtlety of the kek1 null phenotype.Phosphorylation assays: Ten ovaries per genotype were dis-Kek1 attenuates receptor signaling in multiple tissues:sected in PBS and homogenized in 500 �l of RIPA buffer with

protease and phosphatase inhibitors (150 mm NaCl, 100 mm Initially, the subtlety of the loss-of-function phenotypeTris pH 7.4, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, for kek1 hindered identification of its role in EGFR sig-1 mm PMSF, 1 �m leupeptin, 1 �m pepstatin A, 0.3 �m aproti- naling. Subsequently, however, a role for kek1 in attenu-nin, 5 mg/ml �-glycerolphosphate, 1 mm sodium orthovana-

ating EGFR signaling during oogenesis was uncovereddate). Ovaries were homogenized and cleared by centrifuga-in DV patterning, as determined by misexpression analy-tion for 10 min. Supernatant was brought up to 1 ml and

immunoprecipitations and Western blots were performed as sis and confirmed through loss-of-function studies (Ghig-described above. Anti-phosphotyrosine (pY99; Santa Cruz Bio- lione et al. 1999). In addition to its role in DV pat-technologies, Santa Cruz, CA) was used at 1:1000 in 4% BSA. terning, the EGFR participates in a multitude of cellularRabbit anti-EGFR and mouse anti- tubulin (Accurate Chemi-

decisions throughout development, where distinct li-cal, Westbury, NY) antibodies were used at 1:5000.gands regulate receptor activity in a tissue-specific fash-ion. It has recently been shown through misexpressionexperiments that Kek1 can inhibit EGFR signaling inRESULTSthe wing and eye (Ghiglione et al. 2003; Alvarado et

Within the Kek family, EGFR inhibition is unique to al. 2004). To determine if endogenous kek1 functionsKek1: In its extracellular region Kek1 is composed of more generally throughout development to attenuatean N-terminal insert, a set of seven LRRs flanked by receptor activity, we generated flies with reduced egfrcysteine-rich caps, and a single Ig domain, which to- activity and asked whether the resulting phenotypesgether with the transmembrane domain suffice to in- could be rescued by simultaneous removal of kek1. Priorhibit EGFR signaling. Within the Drosophila proteome, to DV patterning, EGFR activity in the posterior folliclesignificant sequence similarity and a similar arrange- cells results in the establishment of the anterior-poste-ment of motifs are found in five additional transmem- rior (AP) axis. Hypomorphic combinations of egfr resultbrane proteins that together with Kek1 constitute the in abnormal AP axis specification and mislocalizationKekkon (Kek) family (Musacchio and Perrimon 1996; of the posterior determinant oskar (osk ; Roth et al. 1995;Derheimer et al. 2004). The identification of these struc- Figure 2D). Simultaneous removal of kek1 activity sup-turally related molecules coupled with the subtlety of presses this phenotype, allowing the proper establish-kek1 null phenotype raised the possibility of functional ment of the AP axis (Figure 2F). Likewise, a similarredundancy among Kek family members. To address role for kek1 is observed during wing vein specification,this we tested four of the five additional members, Kek2, which also requires EGFR signaling (Diaz-BenjumeaKek4, Kek5, and Kek6, for effects on EGFR signaling in and Garcia-Bellido 1990; Sturtevant et al. 1993).vitro and in vivo. Initially, we confirmed that both EGFR Hypomorphic combinations of egfr result in vein lossisoforms (1 and 2) associate with Kek1 and each other (Figure 2C) and simultaneous removal of kek1 also sup-by co-immunoprecipitation (co-IP) experiments from presses this phenotype (Figure 2E). Consistent with anDrosophila S3 cells (Figure 1, A and B). Kek1, in addi- increase in EGFR activity in a kek1 background, patchestion to binding the receptor, is also able to associate of ectopic vein are also present in kek1 wings (Figurewith itself (Alvarado et al. 2004). Indicating specificity 2F). Strong dose-dependent inhibitory effects for en-to binding, Kek1 does not associate with the transmem- dogenous kek1 on EGFR signaling have also been ob-brane molecule Delta (Dl; Figure 1B). In contrast to the served in the developing eye (Alvarado et al. 2004).strong interaction detected between Kek1 and EGFR, Since EGFR activation within each tissue is initiated byKek2, Kek4, and Kek5 interact either weakly or not at distinct ligands, our results suggest that Kek1’s inhibi-all with EGFR in co-IPs (Figure 1B). tory activity is neither ligand nor tissue specific, consis-

Consistent with the ability of Kek1 to physically associ- tent with misexpression analyses (Neuman-Silberbergate with the receptor, its misexpression during oogen- and Schupbach 1993; Roth et al. 1995; Simcox 1997;esis with CY2-GAL4 results in inhibition of EGFR sig- Tio and Moses 1997; Ghiglione et al. 1999, 2003;naling and ventralization phenotypes similar to those Guichard et al. 1999; Alvarado et al. 2004). However,observed with loss-of-function alleles of the receptor. it is important to note that these results do not excludeTo determine if other Kek family members displayed the possibility that Kek1 binds all EGFR ligands andinhibitory effects similar to Kek1 in vivo, we generated might function as a ligand sink. Given that Kek1 can

also associate with itself, one possibility is that Kek1 actsUAS lines for Kek2, Kek4, Kek5, and Kek6 and misex-

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Figure 1.—EGFR inhibition is not a common feature of Kek family members. (A) Graphical representation of Kek familymembers and Kek1 variants fused to GFP (circle). Kek family members are single transmembrane proteins consisting of anextracellular domain composed of seven LRRs and a single Ig domain. Their intracellular regions share little similarity. (B) Co-immunoprecipitation experiments from Drosophila S3 cells. GFP-tagged constructs were immunoprecipitated with a polyclonalanti-GFP antibody (IP) and immunoblotted with anti-EGFR (IB; top left), anti-Delta (top right), and a monoclonal anti-GFPantibody as a control (middle). Whole-cell lysates were directly immunoblotted with anti-EGFR and anti-Delta as a loading control(bottom). Kek1GFP associates with both isoforms of EGFR (iso1 and iso2) whereas Kek2GFP, Kek4GFP, and Kek5GFP displayminimal or no affinity for EGFR. (C) Chorion phenotypes and subcellular distribution of GFP-tagged constructs misexpressedin follicle cells with CY2-GAL4. Kek1GFP exhibits strongly ventralized chorions, whereas Kek2GFP, Kek4GFP, Kek5GFP, andKek6GFP do not display ventralized chorions when misexpressed with CY2-GAL4, demonstrating that the extracellular andtransmembrane domains of Kek1 are unique in their ability to inhibit EGFR among Kek family members.

as a homodimer and competes with the receptor for tivity, we were able to test the ligand sink model, usinga chimeric Kek1-EGFR molecule. The extracellular andligand binding (Alvarado et al. 2004). Work with Kek1

and the vertebrate receptor has recently provided sup- transmembrane portions of Kek1 were fused to a por-tion of the cytoplasmic domain of the EGFR includingport against such a model by indicating that Kek1 does

not appear to act simply by binding to vertebrate EGF the kinase domain, but lacking all residues followingthe kinase domain—the C-tail. In Drosophila, as with(Ghiglione et al. 2003). Below we also rule out the

ligand sink model in Drosophila. the vertebrate receptors, the EGFR C-tail contains aseries of tyrosines that recruit adaptor proteins uponKek1 acts in association with EGFR in vivo and not

as a ligand sink: To address the potential for Kek1 to phosphorylation and is essential for signaling activity(Raz et al. 1991; Clifford and Schupbach 1994). Thefunction as a competitive inhibitor of EGFR through

ligand binding in Drosophila, we took advantage of the absence of the C-tail from this chimera prevents it fromundergoing autophosphorylation as a homodimer. Thisknowledge that chimeric proteins have been widely used

in Drosophila to study the function of transmembrane Kek1-EGFR chimera was tagged with GFP and termedKE�CG (Figure 3A).receptors (Dickson et al. 1992; Reichman-Fried et al.

1994; Murphy et al. 1995; Queenan et al. 1997; Schnepp If Kek1 functions as a competitive inhibitor throughhomodimerization and ligand binding, the KE�CG chi-et al. 1998; Boutros et al. 2000; Keleman and Dickson

2001; Keleman et al. 2002). Since the extracellular and mera would still function as an inhibitor, since it couldbind ligand but would fail to activate EGFR signalingtransmembrane portions of Kek1 mediate inhibitory ac-

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Figure 2.—Kek1 effects are not ligand or tissuespecific. Loss-of-function combinations of egfr re-sult in loss of the L4 wing vein and oskar mRNAmislocalization. In each situation, reduction orelimination of kek1 rescues the associated egfr mu-tant phenotype. (A, C, E, and G) Dark-field imagesof wings from (A) wild-type, (C) egfr QY1/egfr CO, and(E) kek1 RA5 egfr QY1/kek1 RM2 egfr CO males. As ob-served in C, loss of the anterior cross-vein (100%,n � 49) and partial loss of the L4 wing vein (69%,n � 49) are evident. In E, restoration of L4 inegfr mutants is observed in response to elimina-tion of kek1 as no gaps were observed in the doublemutants (0%, n � 47). (G) kek1 mutants (kek1RM2/kek1RA5) display ectopic wing vein patches. Thisphenotype is 72.3% penetrant in kek1RM2/kek1RA5

flies (n � 176), whereas in kek1RM2/� (n � 105)and kek1RA5/� (n � 69), this phenotype is notobserved. (B, D, and F) Nomarski images of oskarmRNA localization in egg chambers from (B)wild-type, (D) egfr QY1/egfr CO, and (F) kek1 RA5 egfr QY1/kek1 RM2 egfr CO females. As seen in D, mislocaliza-tion of osk to the middle of the oocyte is evident.In F, osk localizes properly in egfr mutants in re-sponse to elimination of kek1.

due to the absence of the C-tail. Alternatively, if Kek1 transmembrane molecule CD8 was similarly tagged withthe EGFR intracellular domain lacking its C-tail and GFPand EGFR function as a heterodimer in vivo, KE�CG

would bind and cross-phosphorylate the endogenous (mCD8E�CGFP; Figure 3A). Misexpression of either thismolecule or a form of EGFR lacking the extracellularreceptor (Figure 3B). In this scenario, misexpression of

KE�CG would result in EGFR activation, rather than domain (�TOP; Queenan et al. 1997) did not activatesignaling in our assay (Figure 3C and data not shown),inhibition, confirming the presence of a Kek1/EGFR

complex in vivo and providing evidence against the li- indicating that the kinase domain of EGFR alone doesnot trigger signaling. Therefore, the extracellular andgand sink model. Misexpression of KE�CG in follicle

cells resulted in dorsalized chorions, an EGFR gain- transmembrane portions of Kek1 are sufficient to directassociation with EGFR in vivo in a ligand-independentof-function phenotype, strongly suggesting that it is

able to activate signaling via a direct interaction with fashion. A similar conclusion was also made using aKek1-EGFR chimera that includes the C-tail of EGFRthe receptor (Figure 3C). Consistent with this, KE�CG

associates strongly with EGFR1 by co-immunoprecipita- (Ghiglione et al. 2003).Kek1 forms an inhibitory complex with EGFR: Thetion (Figure 3D). Similar stimulatory effects were also

observed in the wing and eye (data not shown). If this physical association of Kek1 with the receptor noted invivo is consistent with a model for inhibition in whichchimera requires endogenous EGFR for its activity, then

reducing access to EGFR should result in suppression receptor dimerization and autophosphorylation areprecluded by association with Kek1 (Ghiglione et al.of its activity. Consistent with this, hemizygosity for egfr,

heterozygosity with receptor alleles lacking the C-tail, 2003). Although this provides a simple explanation forthe lack of receptor signaling, the status of receptoror misexpression of a dominant-negative allele of egfr

(DNegfr) resulted in partial or complete suppression kinase activity when complexed with Kek1 remains animportant and unresolved issue (Burgess et al. 2003).of the KE�CG phenotype, respectively, indicating that

KE�CG requires endogenous EGFR to activate signaling Current data indicate that, unlike most RTKs, the kinasedomain of a monomeric EGFR is catalytically active,(Table 1). In contrast, reduced activity of the EGFR

ligand Gurken (Grk) did not suppress the KE�CG pheno- which is further supported by the observation that theEGFR A-loop does not require tyrosine phosphorylationtype, indicating that association of KE�CG with EGFR is

ligand independent (Table 1). To demonstrate that the for catalytic activity (Gotoh et al. 1992; Stamos et al.2002; Burgess et al. 2003). In contrast, most RTKs re-interaction between KE�CG and EGFR is not directed

by the presence of the kinase domain in the chimera, quire tyrosine phosphorylation in the A-loop of the ki-nase domain for full catalytic activity (Gotoh et al. 1992;a control chimera encoding the murine Ig-containing

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Figure 3.—Kek1 physically associates with EGFR in vivo. (A) Chimeric constructs designed to distinguish between the ligandsink and the heterodimerization models (see B). The extracellular and transmembrane domains of Kek1 and mCD8 were fusedto the cytoplasmic domain of EGFR lacking the C-tail (�C) and tagged with GFP. (B) Functional representation of KE�CGmisexpression in follicle cells. If Kek1 functions as a ligand sink, then misexpression of KE�CG would titrate out EGFR ligandsand result in ventralized chorions. However, if Kek1 functions as a heterodimer with EGFR, misexpression of KE�CG wouldassociate with and activate the endogenous receptor, resulting in dorsalized chorions. (C) Chorion and GFP fluorescence imagesof chimera misexpression in follicle cells with CY2-GAL4. KE�CG displays dorsalized chorions, a hallmark of EGFR overactivation.In contrast, misexpression of mCD8E�CGFP or mCD8GFP (not shown) exhibits no chorion phenotype. (D) KE�CG associatesstrongly with EGFR by co-IP, whereas mCD8E�CGFP binds weakly. This is likely due to a weak affinity of the EGFR kinase domainfor itself, since a similar mCD8 construct lacking the EGFR portion does not associate with EGFR.

Stamos et al. 2002). Thus, we were prompted to investi- strong inhibition of EGFR signaling. In ovaries fromCY2-GAL4, UAS-Kek1-GFP females, no detectable tyro-gate two possible models for Kek1 inhibition of EGFR.

In one model, the receptor kinase domain has access sine phosphorylation of Kek1 is evident, supporting thelatter model (Figure 4). To provide additional supportto and phosphorylates Kek1, which then acts as a “dead

end” substrate by failing to bind the appropriate down- for this, we tested a series of chimeric Kek1/EGFR mole-cules designed to functionally assay the activity of thestream adaptors. Alternatively, Kek1 may not be phos-

phorylated when complexed with EGFR, suggesting that receptor when complexed with Kek1. If Kek1 is phos-phorylated by EGFR and acts as a dead end substrate,the receptor kinase domain is unable to gain access to

Kek1 for trans-phosphorylation. Such a scenario would then exchanging the cytoplasmic domain of Kek1 witha phosphorylation and signaling-competent substratebe consistent with evidence suggesting that rotation of

the receptor transmembrane domain is necessary for (EGFR cytoplasmic domain) would enable recruitmentof the appropriate adaptor proteins, thereby convertingactivation (Moriki et al. 2001). To address these two

models we utilized both a biochemical and a genetic Kek1 to an activator (Figure 5, A and B). In contrast,if Kek1 is not a substrate for phosphorylation, no trans-approach to examine the status of Kek1 as a phosphory-

lation substrate in vivo. phosphorylation of this Kek1/EGFR chimera would oc-cur, thereby maintaining Kek1’s function as an inhibitorFirst, we assayed for tyrosine phosphorylation of Kek1

under conditions in which its misexpression results in (Figure 5B). These chimeras contain the extracellular

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TABLE 1

Effects of egfr and grk on Kek1 and KE�CG misexpression phenotypes in follicle cells

UAS-kek1 UAS-DNegfr Df(2R)egfr 3F18/ egfr 2X51/ egfr QY1/ UAS-grk �TC grk HK36

(V4) (V4) � � � (D3) (V4)

UAS-kek1 (V4) — — — — — V4 —UAS-ke �Cg (D3) V4 V4 D2 D2 D3 — D3

Females misexpressing either Kek1 or KE�CG in follicle cells using CY2-GAL4 were combined with the allelesdepicted in each column. Chorion phenotypes are represented in parentheses, denoting a range from severelyventralized (V4) to severely dorsalized (D3). Misexpression of Kek1 in follicle cells suppresses the dorsalizationeffect of a secreted form of Grk (UAS-grk �TC). The dorsalization effect of KE�CG is completely suppressed whenmisexpressed with kek1 (UAS-kek1) or a dominant negative form of EGFR (DNegfr) and partially suppressed bya deficiency that uncovers the egfr locus [Df(2R)egfr 3F18] and by the loss-of-function allele egfr 2X51, which lacksthe C-tail. In contrast, the KE�CG misexpression phenotype is not suppressed by egfr QY1, a hypomorphic allelethat contains an intact C-tail, indicating that egfr QY1 is a valid substrate for KE�CG. In addition, the ability ofKE�CG and KEG (not shown) to activate signaling is not affected by a background homozygous for grk HK36.

and transmembrane domains of Kek1 fused to kinase- third chimera KE�KG contains an internal deletion en-compassing the entire kinase domain (Qian et al. 1995;deficient forms of the intracellular domain of EGFR

tagged with GFP (KEG; Figure 5A). Three distinct ki- Klingbeil and Gill 1999).As with Kek1, all three chimeras associate with thenase-deficient forms of KEG (KEgG, KEkG, and KE�KG;

Hanks et al. 1988) were engineered to prevent the chi- receptor by co-IPs, but are not phosphorylation sub-strates in vivo (Figure 4 and Figure 5, A and C; data notmeras from phosphorylating the endogenous receptor.shown). Demonstrating that these chimeras are func-Previous reports have demonstrated that a receptor withtional inhibitors, their misexpression in follicle cellsan intact kinase can trigger signaling by trans-phosphor-resulted in strongly ventralized chorions, a phenotypeylation of a kinase-deficient EGFR, indicating that acomparable to that observed with Kek1 misexpressionkinase-deficient receptor is capable of acting as a func-(Figure 5D). Similar inhibitory effects were also ob-tional signaling substrate in vivo (Raz et al. 1991; Clif-served in other tissues (data not shown). Similar resultsford and Schupbach 1994; Guichard et al. 2002). Like-were obtained with a form of KEgG that lacks the GFPwise, the Kek1/EGFR chimeras could act in a mannertag (KEg), indicating that GFP does not interfere withanalogous to the vertebrate kinase-deficient receptorthe function or structure of the chimeras (Figure 5D).ErbB3, which signals by serving as a phosphorylationAs an additional control, we confirmed that the cyto-substrate for other ErbB family members (Sliwkowskiplasmic domain of EGFR is able to act as a functionalet al. 1994; Kim et al. 1998). The first two chimeras, KEgGphosphorylation substrate in a chimeric situation. Weand KEkG, contain point mutations that disrupt ATPtook advantage of the fact that Kek1 is able to homo-binding and phospho-transfer, respectively, whereas thedimerize and created a control chimera, KEG, with afunctional EGFR kinase domain. KEG is heavily phos-phorylated in vivo and its misexpression results in dor-salization of the chorion in the presence or absenceof receptor activity, consistent with the formation ofhomodimers and EGFR-independent activation of sig-naling (Figure 4 and Figure 5, A, C, and D). This demon-strates that the cytoplasmic domain of EGFR in a Kek1chimera is able to act as a functional signaling substratein vivo and that the kinase activity of the inhibitorychimeras was effectively abolished by the kinase domainpoint mutations. Together our results argue that Kek1,as well as the chimeras, is not a phosphorylation sub-strate for EGFR. This supports the latter model in whichthe receptor kinase domain is unable to access Kek1Figure 4.—Kek1 is not phosphorylated in vivo. GFP-tagged

constructs were misexpressed and immunoprecipitated (IP) for phosphorylation and argues against a model infrom ovaries and immunoblotted (IB) with anti-phosphotyro- which Kek1 acts as a pseudo-substrate.sine (top). The blot was stripped and reprobed with anti- The juxta/transmembrane portion of Kek1 is essen-GFP (middle). A whole-lysate sample was probed with anti-

tial for inhibition: One potential explanation for thetubulin as a loading control (bottom). With the exception ofinability of the receptor to phosphorylate Kek1 is thatthe control chimera KEG, none of the molecules tested were

tyrosine phosphorylated. rotation through the receptor’s transmembrane region

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Figure 5.—Kek1/EGFR chimeras support the “inactive receptor” model. (A)Kek1-EGFRGFP (KEG) chimeras are composed of the extracellular and trans-membrane domains of Kek1 fused to EGFR cytoplasmic variants and taggedwith GFP. KEG contains the entire EGFR cytoplasmic domain. Two KEiG chime-ras differ by the incorporation of point mutations that abolish its kinase activity,KEgG (G950R) and KEkG (K971M). In KE�KG, the entire kinase domain isdeleted. (B) These chimeras were generated to differentiate between two modelsfor Kek1 function. In the “dead-end substrate” model, EGFR retains kinasefunction and phosphorylates the C-tail in KEgG, resulting in activation of thepathway and dorsalized chorions. In the “inactive receptor” model, the receptoris incapable of phosphorylating KEgG, resulting in inhibition of signaling andventralized chorions. (C) Misexpression of all kinase-dead chimeras in folliclecells with CY2-GAL4 exhibits ventralized chorions. Misexpression of KEG (wild-type kinase domain) results in dorsalized chorions, demonstrating that the kinaseactivity of KEiG chimeras was effectively abolished. (D) All three chimeras interactwith EGFR by co-IP in S3 cells.

is required to appropriately position the kinase domain degrees of inhibitory activity when assayed in vivo (Fig-ure 6, B and C). The LRR (L), Ig (I), and jt/tm (T)relative to its substrate. In this scenario, the structure

of the Kek1 juxta/transmembrane (jt/tm) region might swap (LIT), which includes the entire extracellular andtransmembrane portion of Kek1 in place of the corre-act to sterically hinder rotation. Consistent with an active

role for the Kek1 jt/tm region, analysis of a series of sponding portion of Kek2, displays inhibition equivalentto full-length Kek1 (refer to Figures 1 and 6C). In con-domain swaps between Kek1 and Kek2 indicates that

while the LRRs suffice for binding, the jt/tm region of trast, the next two swaps, which include only the LRRsand Ig (LI) or the LRRs (L) of Kek1, respectively, butKek1 is necessary for full inhibitory activity in vivo (Fig-

ure 6). Three swaps, each containing progressively less lack the jt/tm portion of Kek1, have minimal inhibitoryactivity in the ovary, eye, and embryo (Figure 6C). Thus,of the extracellular/transmembrane portion of Kek1

fused to Kek2, were constructed and assayed for binding while the LRRs of Kek1 suffice for receptor binding,the jt/tm region of Kek1 is specifically required, in addi-and inhibition of EGFR activity in vivo (Figure 6). All

three swaps bind to the receptor, but display different tion to the LRRs, for full inhibition of the receptor in

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Figure 6.—The juxta/transmembrane region ofKek1 actively contributes to-ward EGFR inhibition. (A)Three constructs were gen-erated where increasinglysmaller portions of Kek1were swapped with the cor-responding regions of Kek2.LITGFP encodes Kek1’sLRRs, Ig, and juxta/tm re-gions and Kek2’s cytoplasmicdomain. LIGFP encodes theLRRs and Ig domain ofKek1 and the juxta/tm andcytoplasmic regions ofKek2. LGFP contains onlythe Kek1 LRRs (includingthe cysteine-rich flanks). (B)Like full-length Kek1, thesechimeras bind EGFR (top),demonstrating that theKek1 LRRs are sufficient forassociating with EGFR. Incontrast, Kek2 binds EGFRweakly. (C) Misexpressionof LITGFP in follicle cellswith CY2-GAL4 results instrongly ventralized chorions,a phenotype similar to thatof full-length Kek1. LIGFP

and LGFP inhibit weakly, demonstrating that the juxta/tm region of Kek1 is necessary for full inhibition. Similar activities werealso observed with GMR-GAL4 and Act5C-GAL4. V1–V3 denotes increasing degrees of ventralization, while R1–R3 denotesincreasingly stronger rough eye phenotypes. Parentheses represent the number of independent transgenic lines tested.

vivo. Supporting this, secreted forms of Kek1 (sKek1, shown). The SOKs do not interact indiscriminately withother molecules, but rather act in a dominant fashionsKek1GFP), display no inhibitory activity when assayed

with CY2-GAL4 (data not shown; Ghiglione et al. 2003). to suppress Kek1-mediated increases or decreases inEGFR signaling.Novel alleles of EGFR disrupt binding and inhibition

by Kek1: One simple interpretation of the current data The first hint that the SOKs were not standard loss-of-function (LOF) mutations was their ability to sup-is that the LRRs direct association with the receptor,

while the jt/tm region of Kek1 facilitates inhibition. In press increases and decreases in EGFR signaling. Fur-ther supporting the notion that the SOKs representlight of this possibility and the knowledge that Kek1

and EGFR interact directly, we reasoned that identifica- unique mutations, the SOKs all map to the same regionand deficiencies for this region fail to suppress the GMR-tion of suppressors of Kek1 misexpression phenotypes

would likely identify mutations in EGFR that disrupt this kek1 eye phenotype. Since the SOKs mapped to the vicin-ity of the receptor, we hypothesized that they representassociation, providing further insight to the mechanism

underlying their association. To identify such suppres- unique alleles of the receptor, defective primarily intheir ability to associate with Kek1. By eliminating thesors, an F1 mutagenesis screen was employed to detect

dominant mutations that suppress the effects of Kek1 receptor’s ability to associate with Kek1, these alleleswould effectively prevent Kek1 or the Kek1/EGFR chi-misexpression in the eye (Freeman 1996; Figure 7A).

From this screen five dominant suppressors of Kek1, or meras from affecting receptor signaling. Confirmingthis, SOK5, the only homozygous lethal SOK mutation,SOKs, that suppress the effects of Kek1 misexpression

both in the eye (GMR-GAL4; UAS-kek1) and in the ovary fails to complement alleles of egfr and contains a singlemissense mutation altering codon 750 (TAC to TGC),(CY2-GAL4; UAS-kek1), were recovered (Figure 7, B–K).

To further address the specificity of the SOKs, we exam- converting tyrosine to a cysteine (Y750C; Figure 9A).Thus, SOK5 represents a novel allele of the receptor thatined their ability to suppress phenotypes associated with

misexpression of DN-EGFR, Aos, Kek family members, acts in a dominant fashion to suppress Kek1-dependenteffects on EGFR signaling. Likewise, molecular analysisand the Kek1/EGFR chimeras. Strikingly, the SOK muta-

tions suppress only the effects of molecules containing indicated that the remaining four SOKs, SOK1, SOK2,SOK3 and SOK4, also contain missense mutations inthe extracellular and transmembrane domain of Kek1,

including both the inhibitory chimera KEgG and the the receptor. SOK4 alters codon 718 (GAG to AAG),converting glutamic acid to a lysine (E718K), whileactivating chimera KE�CG (Figure 8 and data not

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Figure 7.—SOK muta-tions suppress misexpres-sion of kek1 in multiple tis-sues. (A) A screen wasaimed at identifying F1 sup-pressors of a Kek1 misex-pression. (B–K) Adult eyeSEMs and chorion images.(B and G) Wild type. Kek1misexpression in the eyewith GMR-GAL4 (C) and infollicle cells with CY2-GAL4(H) is rescued by SOK3 (Dand I), SOK4 (E and J), andto a lesser extent by SOK5(F and K). GMR-kek1 refersto the [GMR-GAL4], [UAS-kek1] chromosome.

SOK1, SOK2, and SOK3 alter codon 738 (CGA to CAA), while concomitantly disrupting its ability to associatewith Kek1. To test this directly, we assayed the abilityconverting arginine to a glutamine (R738Q; Figure 9A).of the EGFRSOK molecules to associate with full-lengthTogether the five SOKs represent changes to three resi-Kek1, the activating chimera KE�CG, or wild-type EGFRdues spanning only 32 amino acids in the extracellularin co-immunoprecipitations. Supporting our model,portion of the receptor. Moreover, they all lie withinEGFRSOK has reduced affinity for both full-length Kek1domain V, a region absent from all vertebrate orthologsand KE�CG, but is able to associate efficiently with theof the receptor (Figure 9C). Finally, all of the SOKs arewild-type receptor (Figure 9B). Consequently, domainable to promote eye development (in the presence ofV is crucial in mediating the receptor’s interaction withKek1 misexpression) and therefore must retain the abil-and subsequent inhibition by Kek1.ity to dimerize and initiate downstream signaling events.

Consistent with this, the egfr SOK1–4 alleles are viable muta-tions and hence do not affect any vital functions of the

DISCUSSIONreceptor. In contrast, the lethality of egfr SOK5 indicatesthat it disrupts functions of the receptor essential for Throughout development, EGFR activity specifies dis-viability, in addition to its effects on Kek1. tinct cellular responses. Essential to this ability is the

On the basis of our model, we predict that the SOK existence of an integrated network of regulatory mole-cules that direct receptor activity. Kek1, a member of aalleles retain the receptor’s ability to homodimerize,

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Figure 8.—SOK mutants suppress bothactivating and inhibitory Kek1-EGFR chime-ras. (A and D) Misexpression of DN-EGFR re-sults in a severe rough eye phenotype in theabsence (A) and presence (D) of SOK2. (Band E) Misexpression of KE gG results in a phe-notype similar to DN-EGFR (B) but is stronglysuppressed by one copy of SOK2 (E). (C and F)Misexpression of KE�CG activates EGFR, alsoresulting in a rough eye (C); in the presenceof a SOK this phenotype is suppressed (F). (Gand H) Misexpression of KEgG in the ovary re-sults in ventralized chorions (G); in the presenceof a single SOK this phenotype is fully suppressed(H). (I and J) Misexpression of KE�CG in theovary with CY2-GAL4 results in strongly dor-salized chorions (I). A single copy of a SOK par-tially suppresses the dorsalization (J).

family of LRR- and Ig-containing molecules, represents inhibition in vivo. Indeed, full inhibition is restoredonly when the entire extracellular and transmembanea component of this network through its role as a feed-

back inhibitor of receptor activity. Deletion and muta- regions of Kek1 are placed in the context of a Kek2backbone. This result supports an active role for thegenesis studies have now demonstrated that the LRRs

of Kek1, specifically LRR2 and G160, are essential for Kek1 jt/tm domain in inhibition, as a chimera contain-ing the Kek1 LRRs in a Kek2 backbone is membraneits association with, and consequently inhibition of, the

receptor (Ghiglione et al. 2003; Alvarado et al. 2004; tethered, but a weak inhibitor. This indicates that LRR-mediated binding alone is insufficient for receptor inhi-Figure 10). The Kek1 cytoplasmic domain and associ-

ated Kek1 tail (KT) box have also been implicated in bition. Rather, our results suggest that Kek1-mediatedinhibition of EGFR signaling is a bipartite process, inthe inhibitory process, possibly through effects on sub-

cellular trafficking (Ghiglione et al. 2003; Derheimer which the LRRs dictate EGFR binding and the jt/tmregion facilitates inhibition (Figure 10). Phylogeneticet al. 2004; Figure 10).

Bipartite inhibition—the LRRs and jt/tm of Kek1 are analysis has indicated that this region is well conservedin Kek1 orthologs, supporting an important functionalrequired for EGFR inhibition: While it is clear that the

Kek1 LRRs are essential for EGFR binding and inhibi- role for this region (Derheimer et al. 2004). Given thisrequirement for the Kek1 jt/tm region in inhibition, ittion, secreted forms of Kek1 are nonfunctional, indicat-

ing that membrane anchoring is likely to be an essential was interesting to note that the SOK alleles identifythree amino acids present in domain V of the receptor.element to the inhibitory mechanism (Ghiglione et al.

2003; this article). Directly testing this, our Kek1-Kek2 Alteration of these three residues renders the receptorrefractory to inhibition by Kek1 and activation by KE�CG,swaps demonstrate that while the Kek1 LRRs are suffi-

cient for binding in vitro, they provide only minimal respectively. Moreover, two of the changes, R738Q and

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Figure 9.—The SOKs are EGFR alleles that impair association with Kek1’s. (A) Schematic representation of the Drosophilaand human EGFR. Functional domains are labeled as I–V. Domains I and III are the ligand-binding domains while II and IVregulate dimerization. The SOK mutations all map to domain V within a 32-amino-acid stretch. (B) Mutations corresponding toSOK1–3 and SOK4 disrupt binding to Kek1 and KE�CG, but not to a GFP-tagged form of EGFR1 (top). GFP and EGFR loadingcontrols are displayed in the middle and bottom, respectively. (C) Sequence alignment of Drosophila EGFR with orthologs fromA. gambiae, C. elegans, and Homo sapiens. Domain V is conserved in the invertebrate receptors, but is absent in the vertebratereceptor. The SOK residues are highly conserved between Drosophila and Anopheles. In contrast, LET-23 contains one of thethree SOK residues. Residues altered in the SOK mutants are indicated with arrows.

E718K, represent viable alleles of the receptor, capable brate receptor has indicated that the EGFR kinase do-main is in a catalytically open configuration (Stamos etof ligand binding and receptor activation. Together with

our binding data, these results assign a role for domain al. 2002). Such a configuration is unique in that recep-tor tyrosine kinases normally require activation loopV in mediating regulation by Kek1. It is interesting to

note that EGFR domain V represents a third cysteine- phosphorylation to relieve autoinhibitory interactionsthat prevent substrate binding and phosphorylationrich domain in Drosophila, which is absent in the verte-

brate ErbBs. This raises intriguing structural and evolu- (Gotoh et al. 1992; Burgess et al. 2003). In light of adistinct mechanism for activation of EGFR, one pro-tionary questions, as Kek1 has been reported to associate

with all human ErbBs (Ghiglione et al. 2003). It will posal put forth is the rotation twist model, in whichligand binding induces dimerized receptors to pivot inbe important in the future to define those elements in

the receptor that suffice for its inhibition by Kek1 and or near the transmembrane domain, thereby reorient-ing the kinase domains to their substrates. One potentialdetermine if additional distinctions in the interactions

between Kek1 and the different receptor family mem- explanation for the inability of the receptor to phos-phorylate Kek1 is that the structure of the Kek1 jt/tmbers exist.

We also provide both direct (absence of phosphoryla- region might act to hinder such a rotation.Inhibition of EGFR is not a common Kek family func-tion) and indirect evidence (chimeras) that Kek1 is not

a phosphorylation substrate for the receptor. This was tion: Considering that kek1 knockouts exhibit subtle anddose-dependent phenotypes, one important questionsomewhat surprising, as structural work with the verte-

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Kaufman for advice and equipment; K. Klueg and L. Cherbas foradvice in cell culture experiments; F. Rudi Turner for assistance withthe SEMs; Tim Evans, Brandon Weasner, and Christopher Skipwithfor their contributions to this work; and C. Ghiglione and K. CarrawayIII for sharing results prior to publication. We gratefully acknowledgethe help of W. Forrester, A. Prieto, J. Kumar, and K. Cook in criticalreadings of the manuscript. This work was supported by a NationalInstitutes of Health genetics training grant fellowship (GM07757) toD.A. and A.H.R. and by a National Science Foundation grant (IBN-0131707) to J.B.D.

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We thank the Bloomington Drosophila Stock Center, N. Baker, L. Ferguson, K. M., M. B. Berger, J. M. Mendrola, H. S. Cho, D. J.Leahy et al., 2003 EGF activates its receptor by removing interac-Luo, A. Michaelson, N. Perrimon, and T. Schupbach for reagents; T.

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Bipartite Inhibition of Drosophila Epidermal Growth Factor - Genetics