In vivo functional analysis of the Daughter of Sevenless protein in receptor tyrosine kinase signaling

Mechanisms of Development 90 (2000) 205±215 www.elsevier.com/locate/modo

In vivo functional analysis of the Daughter of Sevenless protein in receptor tyrosine kinase signaling Burkhard S. Bausenwein, Marc Schmidt 1, BjoÈrn Mielke, Thomas Raabe* Department of Genetics, University of WuÈrzburg, Biozentrum, Am Hubland, D-97074 WuÈrzburg, Germany Received 24 August 1999; accepted 27 September 1999

Abstract One mechanism used by receptor tyrosine kinases to relay a signal to different downstream effector molecules is to use adaptor proteins that provide docking sites for a variety of proteins. The daughter of sevenless (dos) gene was isolated in a genetic screen for components acting downstream of the Sevenless (Sev) receptor tyrosine kinase. Dos contains a N-terminally located PH domain and several tyrosine residues within consensus binding sites for a number of SH2 domain containing proteins. The structural features of Dos and experiments demonstrating tyrosine phosphorylation of Dos upon Sev activation suggested that Dos belongs to the family of multisite adaptor proteins that include the Insulin Receptor Substrate (IRS) proteins, Gab1, and Gab2. Here, we studied the structural requirements for Dos function in receptor tyrosine kinase mediated signaling processes by expressing mutated dos transgenes in the ¯y. We show that mutant Dos proteins lacking the putative binding sites for the SH2 domains of Shc, PhospholipaseC-g (PLC-g ) and the regulatory subunit of Phosphoinositide 3kinase (PI3-K) can substitute the loss of endogenous Dos function during development. In contrast, tyrosine 801, corresponding to a predicted Corkscrew (Csw) tyrosine phosphatase SH2 domain binding site, is essential for Dos function. Furthermore, we assayed whether the Pleckstrin homology (PH) domain is required for Dos function and localization. Evidence is provided that deletion or mutation of the PH domain interferes with the function but not with localization of the Dos protein. The Dos PH domain can be replaced by the Gab1 PH domain but not by a heterologous membrane anchor, suggesting a speci®c function of the PH domain in regulating signal transduction. q 2000 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Daughter of Sevenless; Multisite adaptor protein; PH domain; Receptor tyrosine kinase signaling

1. Introduction Many developmental processes are regulated by intercellular signaling mechanisms that employ the activation of receptor tyrosine kinases (RTKs). One model system that has been useful in determining the mechanisms of RTKmediated signaling processes is the developing Drosophila eye (Dickson and Hafen, 1993; Zipursky and Rubin, 1994). Each of the 800 single eye units (ommatidia) of the Drosophila eye contains a stereotypic arrangement of eight photoreceptor cells (R1±R8), four lens secreting cone cells and a number of accessory cells which are sequentially recruited during larval and pupal stages in the eye-antennal imaginal disk (Wolff and Ready, 1993). Differentiation of the different cell types in each ommatidium is controlled by at least two RTKs, the Drosophila EGF receptor (DER) and the * Corresponding author. Tel.: 149-931-888-4471; fax: 149-931-8884452. E-mail address: [email protected] (T. Raabe) 1 Present address: Institute of Radiobiology and Cell Research, University of WuÈrzburg, Germany.

Sevenless (Sev) RTK. Whereas DER controls differentiation of all photoreceptor cells (Freeman, 1997; Kumar et al., 1998; Spencer et al., 1998), Sev is only required for speci®cation of the R7 photoreceptor cell. The Sev RTK in the R7 precursor cell is activated by binding to the Boss protein which is localized on the neighboring R8 cell. In the absence of this signal, the R7 precursor cell develops into a nonneuronal cone cell. A major route by which Sev, as well as RTKs in general, transduce signals from the plasma membrane to the nucleus involves the small GTPase Ras and the mitogen-activated protein kinase (MAPK) cassette which comprises the kinases Raf, Dsor1/MEK and Rolled/MAPK (Marshall, 1995; Dominguez and Hafen, 1996). RTKs are coupled to Ras via adaptor proteins. In the case of Sev, the Drk protein interacts via its src homology 2 (SH2) domain with a speci®c phosphotyrosine residue (Y2546) on the intracellular domain of the activated Sev RTK while both Drk SH3 domains bind to the guanine-nucleotide exchange factor Sos (Olivier et al., 1993; Simon et al., 1993; Raabe et al., 1995).

0925-4773/00/$ - see front matter q 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0925-477 3(99)00252-X

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However, this simple linear model of signal transduction has been questioned by the observation that a Sev protein lacking Y2546 is unable to bind to the Drk protein, yet still can transduce the signal in a Ras dependent manner (Raabe et al., 1995, 1996). Further complexity was introduced by the identi®cation of proteins that act upstream or in parallel to Ras to regulate signal transmission from the activated Sev receptor. The PhospholipaseC-g (PLC-g ) protein acts as a negative regulator of R7 cell development (Thackeray et al., 1998), whereas the tyrosine phosphatase Corkscrew (Csw) is a positively acting component (Allard et al., 1996; Perkins et al., 1996). The emerging picture is one of a multi-protein complex being assembled at the Sev receptor leading to the activation of different signaling pathways. This is similar to the situation found in vertebrate RTKs. For example, the PDGF receptor contains multiple tyrosine autophosphorylation sites that serve as docking sites for a whole variety of SH2 domain containing proteins (Pawson, 1995). A second strategy employed by a number of RTKs to recruit signaling molecules is to use multisite adaptor proteins that provide additional docking sites for SH2 domain containing proteins. Vertebrate members of the multisite adaptor protein family include the Insulin Receptor Substrate (IRS) proteins, Gab1, and Gab2 (Holgado-Madruga et al., 1996; Yenush and White, 1997; Gu et al., 1998). In Drosophila, two proteins of this family have been described so far, Chico, a homolog of the IRS proteins (Boehni et al., 1999) and Daughter of Sevenless (Dos) which is involved in signaling from the Sevenless, DER and Torso RTKs (Herbst et al., 1996; Raabe et al., 1996). Characteristic features of multisite adaptor proteins are an N-terminally located Pleckstrin Homology (PH) domain and multiple tyrosine residues within consensus sequences to allow speci®c binding of SH2 domain containing proteins. In the case of Dos, this includes putative binding sites for the Drk and Shc adaptor proteins, PLC-g , the regulatory subunit of Phosphoinositide 3-kinase (PI3-K) and the Csw tyrosine phosphatase. Thus, Dos potentially may serve as an integration point for proteins that trigger Ras dependent and Ras independent signaling events. First evidence for this model was provided by the identi®cation of Csw as a Dos interacting protein (Herbst et al., 1996). This association was shown to be dependent on both SH2 domains of Csw (Allard et al., 1998). However, it is still not known which of the multiple putative SH2 domain binding sites are crucial for the in vivo function of the Dos protein. A second structural feature of the Dos protein is the Nterminally located PH domain. The PH domain is a protein module of about 120 amino acids present in a large variety of proteins that are involved in regulating intracellular signaling pathways (Shaw, 1996; Lemmon et al., 1997). Despite limited amino acid sequence homology, the tertiary structure between different PH domains is remarkable similar. The common architecture comprises seven b -strands forming two antiparallel b -sheets with a C-terminally a helix. Many proteins that contain PH domains require

membrane localization for proper function and several lines of evidence suggest that PH domains themselves contribute to membrane association by binding to phospholipids. PH domains can be grouped into different subclasses with respect to their binding speci®city to different phosphoinositides and recent evidence has indicated that conserved amino acids within the b 1±b 2 loop region are important for binding speci®city (Fruman et al., 1999). In a yeast assay, the Dos and Gab1 PH domains bind with high af®nity to the products of PI3-kinase, PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (Isakoff et al., 1998). Consistent with these results, Dos is localized to the apical membranes of the developing photoreceptor cells (Raabe et al., 1996). However, for Gab1 there is evidence for PH domain-dependent and independent mechanisms of membrane localization (Maroun et al., 1999). Furthermore, mutations in the PH domain of the Sos protein that impair phospholipid binding do not interfere with membrane localization of the Sos protein (Chen et al., 1997). In this study, we have addressed the question whether the PH domain is the only determinant for Dos membrane localization. We show that deletion or mutation of the PH domain does not interfere with membrane localization of Dos in the photoreceptor cells, yet, the PH domain is essential for Dos function. Furthermore, the Dos PH domain was replaced by the vertebrate Gab1 PH domain or a heterologous membrane anchor. Whereas the Gab1 PH domain could substitute for the function of the Dos PH domain, simple membrane localization of the Dos protein was not suf®cient. These results are consistent with a conserved and speci®c function of the PH domains from different multisite adaptor proteins in regulating RTK mediated signaling processes. To determine the relevance of speci®c SH2 domain binding sites on Dos in the context of different RTK dependent developmental processes, endogenous Dos function was removed and replaced by SH2 domain binding de®cient dos transgenes expressed under the endogenous dos enhancer/promotor. Only tyrosine 801, corresponding to a predicted Csw SH2 domain binding site, was found to be critical for Dos function.

2. Results 2.1. Identi®cation of the dos enhancer/promotor element Heat shock induced ubiquitous expression of the dos cDNA in transgenic ¯ies is suf®cient to rescue the lethality of homozygous dos R31, dos P115 or transheterozygous dos R31/ dos P115 animals (Raabe et al., 1996). This lethality rescue assay can be used for a structure/function analysis of the Dos protein by expressing mutated dos transgenes in dos R31 or dos P115 animals. However, ubiquitous expression of the transgenes upon heat shock induction is unlikely to re¯ect the expression pattern of endogenous dos gene. This invokes

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the danger that any phenotype could be caused by ectopic expression of the transgene. To circumvent this problem, we wished to express our transgenes also under the control of the endogenous dos enhancer/promotor element. From the genomic structure of the dos gene as well as the localization of the next transcription unit in close proximity to sequences corresponding to the 5 0 end of our longest dos cDNA (Fig. 1A) we reasoned that all regulatory elements of the dos transcription unit may be included in the short intergenic

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sequence and the large ®rst intron. Therefore, a genomic fragment encompassing sequences from 500 bp upstream of the 5 0 end of the dos cDNA to exon 3 was fused to the remaining dos cDNA (Fig. 1B). The resulting contruct (genE-dos) was introduced into ¯ies by germ-line transformation and several independent transformant lines were tested for their ability to rescue the lethality of homozygous dos R31 or dos P115 animals. A single copy of the genE-dos transgene resulted in rescued ¯ies at the expected Mendelian ratio which were fully viable and showed no phenotypic abnormalities indicating that the genE-dos minigene contains all regulatory sequences. 2.2. Tyrosine 801 is essential for the in vivo function of the Dos protein

Fig. 1. (A) Genomic structure of the dos locus. The horizontal line represent the genomic DNA. Below, the dos cDNA is diagrammed with closed boxes representing translated regions and open boxes indicating nontranslated sequences. The next transcription unit on the 5 0 side of dos is indicated by an arrow. The insertion site of the P element in dos P115 is indicated by a triangle. The dos R31 mutation results in a stop codon at position 463 of the Dos amino acid sequence. Restriction sites for BamHI (B), EcoRI (E), XhoI (X) and RsrII (R) are indicated. (B) Schematic drawing of the genE-dos construct. A genomic BamHI-RsrII fragment was fused within the third exon to the remaining coding region of the dos cDNA. (C) Diagram of the structure of the Dos protein. The PH domain is localized at the Nterminus followed by a number of tyrosine residues that potentially could serve as SH2 domain binding sites. The tyrosine residues exchanged for phenylalanine and tryptophan 104 within the PH domain are indicated. For details see text. (D) Summary of the different constructs used to determine the function of the Dos PH domain. The Dos PH domain was either mutated by a single amino acid exchange (Dos W104A-GFP), completely deleted (Dos DPH-GFP) or replaced by the Gab1 PH (PH Gab1-Dos-GFP) or the Torso extracellular and transmembrane domain (Torso 4021-Dos-GFP). All constructs carry a C-terminal GFP tag and were expressed under the control of the sE/hsp70 element.

To address the question which of the putative SH2 domain binding sites are critical for Dos function we replaced the tyrosine residues corresponding to the predicted binding sites for Shc (Y316), the regulatory subunit of PI3-kinase (Y631), Csw (Y801) or PLCg (Y3581Y537) with phenylalanine (Fig. 1C). Two strategies were employed for transgene expression in ¯ies. Each binding site mutation was introduced into the dos cDNA and placed under the control of the sevenless enhancer and hsp70 promotor sequences (sE/hsp) to allow ubiquitous expression of the transgene upon heat shock induction or cell type speci®c expression in all Sev expressing cells during eye development. As a second approach, the same amino acid substitutions were introduced into the genE-dos construct (Fig. 1B). Expression of the different transgenes was veri®ed by Western Blot analysis (data not shown). First, we tested the ability of the different transgenes to substitute for the lack of endogenous Dos function. Transgenes bearing mutations in the predicted binding sites for Shc, for the PI3-kinase regulatory subunit or in both predicted binding sites for PLCg rescued the lethality of dos R31 and dos P115 animals (data not shown). The results indicate that these single binding site de®cient Dos proteins provide all the functions required for signaling in the absence of a functional endogenous Dos protein. In contrast, expression of the sE/hsp-dos Y801F or genE-dos Y801F transgenes in a homozygous dos R31 or dos P115 background failed to yield viable ¯ies. Since Csw and Dos are required for signaling from the Torso, DER and Sev RTKs (Allard et al., 1996; Perkins et al., 1996; Raabe et al., 1996), the nonfunctional behavior of the Dos Y801F protein suggests that the interaction between Csw and Dos is a crucial step for signaling. Consistent with our results, Y801 has been identi®ed as the binding site for one of the two Csw SH2 domains (M. Simon, pers. commun.). One drawback of the lethality assay is that it is not possible to determine the importance of Y801 in the context of different developmental processes that require different RTKs. To determine the functional properties of the Dos Y801F protein with respect to DER signaling during eye

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development, we set up a genetic test system using the genE-dos constructs in combination with removal of endogenous Dos function in clones of cells (Fig. 2A). Induction of homozygous dos R31 mutant cell clones in the developing eye results in the frequent absence of photoreceptor cells of

different identities (Fig. 2B). This phenotype is consistent with a role of Dos as a mediator of DER signaling in the eye. Flies were generated that carried in addition to the dos R31 mutation a single copy of the wild-type genE-dos transgene. The genE-dos transgene can fully compensate for the lack of endogenous Dos function in dos R31/dos R31 ommatidia. Sections through adult eyes revealed that the ommatidia within the clonal tissue all have the wild-type composition of photoreceptor cells (Fig. 2C). As a further control, we tested the genE-dos Y358F1Y537F transgene which substituted for endogenous Dos function in the lethality assay (see above). Again, the same perfect rescue of the ommatidial structure was observed in dos R31/dos R31 cells expressing the genE-dos Y358F1Y537F transgene (Fig. 2D). In contrast, the genE-dos Y801F construct bearing a mutation in the Csw binding site could not compensate the loss of the endogenous Dos function. Eye sections revealed the absence of a variable number of photoreceptor cells in most ommatidia within the clonal tissue (Fig. 2E). In summary, the lethality assay and the clonal system not only allowed us to identify Y801 as crucial for Dos function but also to determine its speci®c function for photoreceptor cell development which requires activation of the DER RTK. 2.3. Requirement of the PH domain for normal Dos function in the Sevenless signaling pathway

Fig. 2. Tyrosine 801 is essiential for photoreceptor development. (A) Schematic diagram of the experimental strategy to determine the function of wild-type and mutated genE-dos transgenes for eye development. On the left side, a cell heterozygous for the dos R31 mutation on the third chromosome and carrying a P[w 1] marked homologous chromosome is shown. In addition, the second chromosome bears a wild-type or mutated genE-dos transgene (P[genE-dos *]). Mitotic recombination between the FRT sites by heat shock induced expression of the FLP enzyme (located on the X, not shown) results in cells that are homozygous for the P[w 1] marker chromosome or homozygous for the dos R31 mutation. In the latter cells, the only source for Dos is provided by the genE-dos transgene. Homozygous dos R31 cells can be recognized by the loss of the P[w 1] marker, resulting in a patch of cells that lack pigment granules. (B) Histological sections through the retina of an adult ¯y containing tissue homozygous for the dos R31 mutation. Within the clonal tissue, the ommatidial structure is disturbed and only few photoreceptor cells develop. (C±E) Clones of homozygous dos R31 cells that in addition express the wild-type genE-dos (C), the genE-dos Y358F1Y537F (D) or the genE-dos Y801F (E) transgenes. Expression of the genE-dos and the genE-dos Y358F1Y537F transgenes fully restores the wild-type architecture of the ommatidia within the clonal tissue. In contrast, the genE-dos Y801F transgene can not substitute for the loss of endogenous Dos function (E). Scale bar: 10 mm.

The N-terminal located PH domain is the only conserved structural element between the multisite docking proteins Gab1, Gab2, and Dos. The speci®c binding of the Dos and Gab1 PH domains to PtdIns(3,4)P2 and PtdIns(3,4,5)P3 (Isakoff et al., 1998) correlates with a high sequence homology between the PH domains of Dos, Gab1, and Gab2 when compared to other members of the multisite adaptor family (Fig. 3). To analyze whether the PH domain is critical for Dos function and membrane localization, the Dos open reading frame was fused to the GFP coding sequence and the resulting contruct (sE/hsp-dos-GFP) was transformed into ¯ies. Upon heat shock induction, the sE/hsp-dos-GFP transgene was able to rescue the lethality of dos R31 or dos P115 animals in a manner similar as the original sE/hsp-dos construct (data not shown). The sE/hsp-dos-GFP construct was further modi®ed either by completely deleting the PH domain (sE/hsp-dos D PH-GFP) or introducing a tryptophan to alanine mutation at position 104 in the C-terminally located a -helix region of the PH domain (sE/hsp-dos W104AGFP). In the case of the b -adrenergic receptor kinase (b ARK) PH domain, mutation of this highly conserved tryptophan residue affects binding to both phospholipids and the bg subunits of heterotrimeric G-proteins, presumably by disturbing the overall PH domain structure (Touhara et al., 1995). In order to perform a clonal analysis in the eye, the W104A mutation was also introduced into the genE-dos construct (genE-dos W104A). First we assayed the ability of the Dos-GFP, Dos DPH-GFP, and Dos W104A-GFP proteins to compensate the removal of

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Fig. 3. Sequence comparison of the PH domains of human IRS-1, the Drosophila IRS homolog Chico, human Gab1, human Gab2, and Dos. In general, PH domains share only few highly conserved amino acids. However, within the multisite adaptor family, the IRS/Chico subgroup and the Gab/Dos subgroup display a signi®cant higher degree of homology, suggesting common functional roles. Highly conserved amino acids are indicated in black, the amino acids only conserved between IRS-1/Chico and Gab1/Gab2/Dos are shown in light grey and dark grey, respectively. The highly conserved tryptophan residue mutated in the dos W104A construct is indicated by an asterisk.

the wild-type Dos protein in the Sev pathway. Expression of a constitutively activated version of the Sev RTK under the control of the sev enhancer (sE-sev S11) causes a rough eye phenotype due to the transformation of cone cells into additional R7 cells (Fig. 4B) (Basler et al., 1991). The number of supernumerary R7 cells is dependent on the expression level

of the activated Sevenless protein and can be modulated by altering the gene dosis of downstream signaling molecules. In the heterozygous state, the dos R31 and dos P115 mutations largely suppress the rough eye phenotype (Fig. 4C). One copy of the sE/hsp-dos-GFP construct expressed under the control of the sE is suf®cient to restore the multiple R7

Fig. 4. Requirement of the PH domain for Dos function in the Sev signaling pathway. Tangential sections of eyes of the following genotypes: (A) wild-type, (B) sE-sev S11/1, (C) sE-sev S11/1; dos R31/1, (D) sE-sev S11/1; sE=hsp-dos-GFP=1; dos R31/1, (E) sE-sev S11/1; sE=hsp-dos D PH-GFP=1; dos R31/1, (F) sE-sev S11 /1; sE/hsp-dos W104A-GFP/1; dos R31/1, (G) sE-sev S11/1; sE=hsp-PH Gab1-dos-GFP=1; dosR31 =1; (H) sE-sev S11/1; sE=hsp-torso 4021-dos-GFP=1; dos R31=1. In the wild-type (A), each ommatidium contains 8 photoreceptor cells. R1±R6 have large rhabdomeres, the central R7 rhabdomere is small. The rhabdomere of the R8 cell is not visible in this apical section. Expression of a constitutively activated version of the Sev kinase (Sev S11) results in recruitment of multiple R7 photoreceptor cells (B). The multi-R7 cell phenotype is almost completely suppressed in the presence of one copy of the dos R31 allele (C). A single copy of the sE/hsp-dos-GFP (D) or the sE/hsp-PH Gab1-dos-GFP (G) transgenes expressed under the control of the sE are suf®cient to restore the multi-R7 cell phenotype. In contrast, expression of the sE/hsp-dos D PH-GFP (E), sE/hsp-dos W104A-GFP (F) or the sE/hsp-torso 4021dos-GFP transgenes (H) in a sE-sev S11/1; dos R31/1 background did not result in a multi-R7 phenotype, suggesting that the corresponding proteins cannot replace the function of endogenous Dos in Sev signaling. Scale bar: 10 mm.

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phenotype in sE-sev S11/1; sE/hsp-dos-GFP/1; dos R31/1 ¯ies (Fig. 4D). In contrast, expression of the sE/hspdos D PH-GFP (Fig. 4E) or the sE/hsp-dos W104A-GFP (Fig. 4F) construct in a sE-sev S11/1; dos R31/1 background did not result in a multi-R7 cell phenotype. Compared to sEsev S11/1; dos R31/1 ¯ies, the average number of R7 cells per ommatidium is not increased, suggesting that a Dos protein lacking the PH domain or carrying a mutated PH domain is unable to provide normal Dos function in the Sev pathway. 2.4. Localization of the Dos protein to the membrane is not affected by deletion of the PH domain The speci®c binding of the Dos PH domain to PtdIns(3,4)P2 and PtdIns(3,4,5)P3 correlates well with our observation that the Dos protein is localized to the apical membranes of the developing photoreceptor cells in the eye imaginal disk (Raabe et al., 1996; Isakoff et al., 1998). Hence, the failure of the Dos W104A-GFP and the Dos DPHGFP proteins to substitute normal Dos function could be due to mislocalization of the mutated proteins. Therefore, we compared the subcellular localization of the Dos-GFP, Dos W104A-GFP and the Dos DPH-GFP proteins in eye imaginal disks. Upon expression under the control of the sE, the DosGFP protein accumulates at the apical membrane in all Sev expressing cells (Fig. 5A,E). Only a minor fraction of the Dos protein is localized at other membrane sites and in the cytoplasm. Surprisingly, the same localization pattern was observed for the Dos W104A-GFP (Fig. 5B,F) and the Dos DPHGFP proteins (Fig. 5C,G). In summary, these results show that recruitment of the Dos protein to the membrane cannot be solely mediated by the PH domain, however, the PH domain is essential for normal Dos function in Sev mediated signaling. 2.5. The Gab1 PH domain but not a heterologous transmembrane anchor can replace the function of the Dos PH domain The Dos PH domain shows the highest homology to the Gab1 and Gab2 PH domains (Fig. 3). Mutational analysis has demonstrated a requirement for the Gab1 PH domain for localization of Gab1 at sites of cell±cell contact and for proper function of Gab1 downstream of the Met RTK during epithelial morphogenesis (Maroun et al., 1999). To determine whether the sequence similarities between the Gab1 and Dos PH domains re¯ect similar functions, we replaced the Dos PH domain by the Gab1 PH domain and generated transgenic ¯ies (sE/hsp-PH Gab1-dos-GFP). Upon expression under the sE enhancer, the sE/hsp-PH Gab1-dos-GFP transgene restores the multi-R7 phenotype in an sE-sev S11/1; dos R31/1 background to a similar degree as the sE/hspdos-GFP construct (Fig. 4G). Thus, the Gab1 PH domain has similar functional properties as the Dos PH domain. If the only feature of the PH domain is to contribute to membrane localization of Dos, then a constitutively membrane tethered version of Dos should be functional.

Fig. 5. Subcellular localization of the Dos-GFP (A and E), Dos W104A-GFP (B and F), Dos DPH-GFP (C and G) and Torso 4021-Dos-GFP (D and H) proteins in the eye imaginal disk of third instar larvae. Confocal images of the GFP auto¯uorescent staining patterns in anterior±posterior sections (A±D) and in apical to basal cross sections (E±H). (A) Upon expression under the sE, the Dos-GFP protein accumulates at the apical tip of all sevexpressing cells behind the morphogenetic furrow (Tomlinson et al., 1987). An apical to basal cross section of the disk shown in (A) highlights the apical localization of the Dos-GFP protein (E). Apical localization is also observed for the Dos W104A-GFP (B and F), Dos DPH-GFP (C and G), and Torso 4021-Dos-GFP (D and H) proteins.

To test this hypothesis, the PH domain of Dos was replaced by the extracellular and transmembrane domain of the Torso RTK (sE/hsp-torso 4021-dos-GFP). This strategy has been used to localize Raf to the membrane and thereby activating it (Dickson et al., 1992b). The chimeric Torso 4021-Dos-GFP protein is localized to the apical portion in all Sev expressing cells in a similar manner as the Dos-GFP protein (Fig. 5D,H). However, sE induced expression of the sE/hsptorso 4021-dos-GFP transgene failed to restore the multi-R7 cell phenotype in sE-sev S11/1; dos R31/1; sE/hsp-torso 4021dos-GFP/2 ¯ies (Fig. 4H). Together with the results obtained with the sE/hsp-dos D PH-GFP and sE/hspdos W104A-GFP constructs this suggests that Dos function is dependent on the presence of a PH domain which seems to have a more complex function in regulating signaling from the Sev RTK than to act as a simple localization module. 2.6. Dos requires the PH domain for non-Sev RTK signaling To investigate whether there is a general requirement of the Dos PH domain for normal function of the Dos protein,

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we tried to rescue the lethal phenotype caused by the dos R31 and dos P115 mutations through heat shock induced ubiquitous expression of the Dos-GFP, Dos DPH-GFP, PH Gab1-DosGFP or Dos W104A-GFP proteins. In the case of Dos-GFP and PH Gab1-Dos-GFP, fully viable ¯ies were recovered. In contrast, expression of the Dos DPH-GFP or Dos W104A-GFP proteins in homozygous dos R31, dos P115 or transheterozygous dos R31/dos P115 animals was not suf®cient. Most animals died as pharate adults and only few poorly viable ¯ies were recovered. These escapers showed defects in eye morphology and in wing vein formation (Fig. 6A,B). In general, rescued ¯ies bearing the Dos DPH-GFP construct showed stronger defects than ones expressing the Dos W104A-GFP transgene. To exclude the possibility that the eye phenotype was caused by misexpression of the Dos DPH-GFP or the Dos W104A-GFP protein, we induced clones of cells homozygous for the dos R31 mutation in amimals that also carried a genE-dos W104A transgene (see above). Eye sections revealed the absence of a variable number of photoreceptor cells in many ommatidia (Fig. 6C). This phenotype is somewhat weaker but reminiscent to the phenotypes caused by removal of DER, Dos, or other components of the Ras/ MAPK signaling cassette during eye development. 3. Discussion We have performed an in vivo functional characterization of Dos, a putative multisite adaptor protein that functions in signal transduction from a variety of RTKs. To determine not only a general requirement of de®ned structural elements for Dos function but also more speci®cally in the context of different developmental processes that require activation of several RTKs, we used a clonal assay system that allowed us to express mutated transgenes under the control of the dos enhancer/promotor in the absence of

Fig. 6. The PH domain is essentiell for Dos function during eye and wing development. (A) wild-type wing. (B) Ubiquitous expression of the dos W104A or the dos D PH transgene occasionally rescues the lethality of homozygous dos R31 animals. However, these rare escapers show defects in wing vein formation (arrowheads). (C) Marked clones of homozygous dos R31 cells were generated in ¯ies expressing the genE-dos W104A transgene (for the experimental design see Fig. 2A). The Dos W104A protein is not able to fully substitute the lack of endogenous Dos function in the eye (compare Fig. 2B and C). Within the clonal tissue, many ommatidia lack photoreceptor cells (arrows). Scale bar: 10 mm.

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endogenous Dos function. We have provided evidence that a single tyrosine residue that would be predicted to bind a Csw SH2 domain is critical for Dos function. Furthermore, we showed that the PH domain is crucial for the function of the Dos protein in RTK mediated signal transduction, yet it is not the only element required for membrane localization of the Dos protein during eye development. 3.1. The role of the PH domain for Dos function The Dos PH domain belongs to a subgroup within the PH domain family that share several highly conserved amino acids in the b 1±loop±b 2 region critical for high af®nity binding to PtdIns(3,4)P2 and PtdIns(3,4,5)P3. This interaction requires an Arginine at position 14 in the b 2-strand, a residue found to be mutated in the PH domain of the Bruton tyrosine kinase (BTK) in patients suffering from X-linked agammaglobulinemia (XLA) (Salim et al., 1996). The corresponding mutation in the Dos PH domains (R27C) blocks binding to phospholipids in a yeast assay as well as membrane targeting of an isolated PH domain expressed in Cos cells (Isakoff et al., 1998). These experiments supported the idea that the Dos PH domain is critical for membrane localization of the protein. Hence, it was intriguing to ®nd that our PH domain mutant constructs expressed in ¯ies were still localized to the membrane. One important difference between our analysis and the studies demonstrating binding of the Dos PH domain to phospholipids is that the latter were done with isolated PH domains whereas our transgenes contained all Dos sequences outside the PH domain and were expressed in cells that normally require Dos function. Therefore, one explanation for membrane localization of the Dos DPH and Dos W104A proteins could be a direct or indirect link to other membrane bound proteins, mediated by structural elements outside the PH domain. For IRS-1 and IRS-2 it has been shown that both, the PH domain and the structurally related phosphotyrosine-binding domain (PTB), contribute to insulin receptor binding (Yenush and White, 1997). A third insulin receptor interaction domain on IRS-2, the kinase regulatory loop binding (KRLB) domain, requires the presence of three tyrosine phosphorylation sites on the activated insulin receptor (Sawka-Verhelle et al., 1996). In the case of Gab1, the interaction with the Met RTK can be mediated by Grb2. The Grb2 SH2 domain interacts with the Met receptor whereas one of the Grb2 SH3 domains binds to Gab1 (Bardelli et al., 1997; Nguyen et al., 1997). In addition, a proline-rich sequence motif in Gab1, referred to as the Met binding domain (MBD) can directly interact with a phosphotyrosine containing peptide of the Met receptor (Weidner et al., 1996). A similar scenario can also be envisioned for Dos. The localization of our PH mutant Dos constructs at the apical membranes closely resembles the localization of the Sev RTK (Tomlinson et al., 1987). Although Dos becomes tyrosine-phosphorylated upon Sev activation (Herbst et al., 1996) and contains several proline-rich

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sequence motifs, a direct association of Dos with Sev or DER has not so far been demonstrated. Indirect binding of Dos to Sev could be mediated by the Csw tyrosine phosphatase which binds to Dos in an SH2 dependent manner whereas both Csw SH2 domains are dispensible for binding to the Sev receptor (Allard et al., 1998). A direct test of whether Sev and DER are responsible for the PH domain independent recruitment of Dos to the apical membrane in the photoreceptor cells would require complete removal of Sev and DER. Whereas sev is a non-essential gene, the involvement of DER in cell proliferation (Xu and Rubin, 1993), its requirement for differentiation of all photoreceptor cells (Freeman, 1997; Kumar et al., 1998; Spencer et al., 1998), and its role in cell survival (Bergmann et al., 1998; Kurada and White, 1998) did not allow us to perform localization studies for Dos in the absence of both RTKs. If a Dos protein without a PH domain is able to localize to the membrane, what then is the function of the PH domain Recently, it has been shown that the Gab1 protein is recruited to the cell membrane by two distinct mechanisms. One mechanism depends on the PH domain and requires PI3-kinase activity. A second, PH domain independent mechanism involves the binding of Gab1 to the stimulated Met receptor. Most importantly, recruitment of Gab1 to the Met receptor in the absence of the PH domain is not suf®cient to provide normal Gab1 function in epithelial morphogenesis (Maroun et al., 1999). Our results correlate well with these observations. The Dos DPH and Dos W104A proteins are membrane localized but are not able to fully substitute the function of the endogenous Dos protein. Furthermore, the Dos PH domain function can be replaced by the Gab1 PH domain whereas a heterologous membrane anchor is not suf®cient. In a similar manner, the IRS-1 PH domain function can only be substituted by homologous PH domains derived from IRS-2 or Gab1 but not by the b -ARK, PLCg or spectrin PH domains (Burks et al., 1997). Although we cannot entirely rule out the possibility that the Torso 4021 membrane anchor interferes with Dos protein function, our data support the idea of a speci®c function of the Gab1/Dos PH domains in signal transduction. Whether this function is dependent on the selective binding of phospholipids or involves additional interactions with other targets remains to be determined. 3.2. The function of Dos as a multisite adaptor protein The sequence context of a number of tyrosine residues suggested that the signaling potential of Dos depends on binding of SH2 domain containing proteins such as Drk, Shc, PI3-kinase, PLCg , and Csw. The different SH2 domain binding sites may serve to link the activated receptor to diverse signaling pathways. In vertebrates, multiple SH2 domain containing proteins bind to tyrosine phosphorylated Gab1 and Gab2 in response to various extracellular stimuli (Holgado-Madruga et al., 1996; Ingham et al., 1998; Takahashi-Tezuka et al., 1998; Lehr et al., 1999). This suggested

that Dos ful®lls a similar function in signaling from the Sev, DER and Torso RTKs. Consistent with this model, Dos becomes phosphorylated by a constitutively activated form of Sev on sites that allow interaction with Csw in an SH2 domain dependent manner (Herbst et al., 1996). Furthermore, the sequence context of tyrosine 801 (LQYFDL) matches the consensus binding site (V/LxYxxV/L) for the Csw SH2 domains (Huyer and Alexander, 1999). Our mutational analysis proved the in vivo relevance of this tyrosine residue for Dos function and recent experiments demonstrated that this site is indeed required for binding of Csw to Dos (M. Simon, pers. commun.). However, Csw as well as the vertebrate homolog SHP-2 contain tandem N-terminally SH2 domains. In the case of IRS-1 and Gab2, two tyrosine residues were identi®ed as binding sites of the SH2 domains of SHP-2 (Gu et al., 1998; Myers et al., 1998). Analysis of the Dos sequence also revealed a second putative binding motif (VVY854RSV) for the Csw SH2 domain. Consistent with a model of two binding sites for Csw on Dos, mutations in either Csw SH2 domain results in partial loss of Dos binding, whereas simultaneous mutation of both SH2 domains completely disrupts binding to Dos (Allard et al., 1998). Although several models have been discussed (Herbst et al., 1996; Van Vactor et al., 1998), it still remains unclear how binding of Csw to Dos generates a positive signal for signal transduction. One intriguing aspect of our experiments is the ®nding that only one tyrosine residue (Y801) is crucial for the in vivo function of the Dos protein, whereas removal of the predicted binding sites for PI3-kinase, Shc and PLCg did not interfere with normal Dos function during development. At least in the case of PLCg , there is genetic evidence for an involvement in DER and Sev signaling (Thackeray et al., 1998). A mutation in the PLCg gene, small wing (sl), results in supernummary R7 photoreceptor cells indicating that PLCg is a negative regulator of RTKs signaling. Further evidence is provided by the observation that sl mutations strongly enhance the phenotype of mutated forms of the GTPase activating protein Gap1 (Powe et al., 1999) whereas the sl phenotype is suppressed by mutations in the dos gene (J. Riesgo Escovar and T. Raabe, unpublished observations). PLCg comprises a catalytic domain, three PH domains, two SH2 domains and one SH3 domain. Together with the observation that there are two consensus binding sites for the PLCg SH2 domains on Dos (Y358DTP and Y537DIP) but none in the intracellular domain of DER and Sev, this suggested a role for Dos in localizing PLCg to the activated RTK. However, in our in vivo assays, a Dos protein lacking both putative PLCg binding sites provides all functions. There are several possible explanations for this result. First, it has to be shown that PLCg binds to Dos in a SH2 domain-dependent manner. Second, the presence of a SH3 domain on PLCg and proline-rich SH3 domain binding motifs on Dos may provide an additional link. Third, disruption of the catalytic activity of PLCg by the sl mutation might cause a much

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stronger phenotype than disturbing the binding properties of the protein. In general, the importance of single SH2 domain binding sites for signaling is underscored by our functional analysis. Disruption of one branch of a signaling network may be compensated by alternative signaling pathways. This is exampli®ed by our previous studies which showed that a mutant form of the Sev RTK unable to bind to Drk still transduces the signal in a Ras dependent manner (Raabe et al., 1995). The in vivo functional analysis of the Dos protein presented here has to be complemented by biochemical studies demonstrating the interaction of Dos with proteins other than Csw as well as the functional characterization of these proteins for receptor tyrosine kinase mediated signal transduction. In this respect, our clonal test system is of general use for assaying the in vivo function of putative protein±protein interaction domains within the Dos protein for eye development irrespectively of their requirement for other developmental processes.

4. Materials and methods 4.1. Plasmid construction and in vitro mutagenesis The complete dos cDNA (Raabe et al., 1996) was cloned into a pBluescriptw II KS (Stratagene) vector. For in vitro mutagenesis of potential SH2 domain binding sites, the QuickChangee Site-Directed Mutagenesis kit (Stratagene) was used. The following oligonucleotides were used to introduce tyrosine to phenylalanine substitutions. 5 0 -CACCGCATCCGGACCGTTCATACCCATCAGCGAGTG-3 0 for Y316F (Shc), 5 0 -CAACTTGGATCCCAAGTTCTTCGATACGCCGCGTAGTCAT-3 0 for Y358F (PLCg ), 5 0 -CTTTCATCGAGGAAAGCTTTGACATTCCAAGGTCCCACCA-3 0 for Y537F (PLCg ), 5 0 -GGAGGGCAATGTTTTTCGATTCGACTTCATGGAGCAGGCG-3 0 for Y631F (PI3-K), 5 0 -GACTGAGCACAAGCTGCAGTTCTTTGATCTGGATGTGACCAAC-3 0 for Y801F (Csw). For generation of the dos-GFP constructs, the TGA stop codon of the dos coding sequence (pos. 2680±2682) and the 3 0 untranslated region was replaced with a GGA codon, immediately followed by unique BglII and XbaI sites. These sites were used for insertion of the coding sequence of a modi®ed version of the GFP protein carrying the S65A, V68L and S72A substitutions for enhanced auto¯uorescence (Cormack et al., 1996). The W104A substitution in the PH domain was introduced by site directed mutagenesis using the oligonucleotide 5 0 -GCGGATATGCGCGACGCAGTGAACTGCATCTGC-3 0 . To exchange the Dos PH domain for the Gab1 PH domain in the dos-GFP construct, an unique NdeI site was inserted at position 386 at the 3 0 end of the PH domain encoding sequence. The region from posi-

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tion 11 (EcoRI) to 1386 (NdeI) of the dos cDNA was replaced by a PCR fragment encoding amino acids 1±116 of the Gab1 protein (Holgado-Madruga et al., 1996). The Dos DPH-GFP contruct was produced by deleting the PH domain encoding sequences with EcoRI and NdeI. The EcoRI and NdeI sites were polished and religated thereby creating a new translational start site for the remaining dosGFP coding sequence. To replace the Dos PH domain with the Torso extracellular and transmembrane domains, an additional EcoRI site was inserted at positions 395±400 of the dos cDNA. The sE/hsp-torso 4021-dos-GFP construct was prepared by replacing the Sevenless protein encoding sequences of the sE/hsp-torso 4021-sev vector (Dickson et al., 1992a) for an EcoRI-XbaI fragment encoding the 759 C-terminally amino acids of Dos fused to the GFP S65A,V68L,S72A protein. Each modi®cation of the dos cDNA was veri®ed by sequencing using the DyeDeoxye Terminator Cycle Sequencing kit (Perkin Elmer). For assembly of a dos minigene (genE-dos), genomic clones encompassing the whole dos transcription unit were isolated from a l GEM11 library using the dos cDNA as a probe. A 4.3 kb genomic BamHI fragment containing sequences from 500nt upstream of the 5 0 end of the longest dos cDNA sequence to exon 3 was fused within exon 3 to the dos cDNA using a unique RsrII restriction site. All SH2 domain binding site mutations and the W104A mutation described above were introduced into the genE-dos construct. 4.2. Germline transformation All dos cDNA constructs were cloned as Asp718-NotI fragments into a modi®ed pW8 vector (Klemenz et al., 1987) under the control of the hsp70 promotor and a duplicated 1.2 kb sev enhancer element (Basler et al., 1991). As a transformation vector for the genE-dos construct, we used a modi®ed pUC8 vector carrying the yellow gene as a selectable marker (kindly provided by E. Hafen). Transgenic lines were generated by injecting Qiagen-puri®ed plasmid DNA into w 1118 or y,w 1118 embryos. For each construct, several independent transformant lines were established. Transgene expression was veri®ed by Western Blot analysis. 4.3. Genetics To assay whether the wild-type or the mutated sE/hspdos*-GFP transgenes can substitute for the function of the wild-type Dos protein in the Sev pathway, the average number of R7 cells per ommatidium from ¯ies of the genotype sE-Sev S11/1, or sE-Sev S11/1; dos R31 /1, or sE-Sev S11/ 1; sE/hsp-dos*-GFP/1; dos R31/1 was compared. For each sE/hsp-dos*-GFP construct, three heads of two independent transgenic lines were analyzed. To test whether ubiquitous expression of the sE/hsp-dos*-GFP transgenes can rescue the lethality of the dos R31 or dos P115 mutations, animals carrying the appropriate transgene in a homozygous dos R31, dos P115 or transheterozygous dos R31/dos P115 back-

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ground were shifted for 60 min to 378C every 6 h during development. The different genE-dos contructs were tested in the same mutant backgrounds without heat shock induction. 4.4. Clonal analysis To induce somatic clones in the eye, the Flp/FRT recombinase system was used (Xu and Rubin, 1993). The dos R31, FRT80B chromosome has been described previously (Raabe et al., 1996). Males of the genotype y,w,hsFlp1/Y; P[w 1]75C, FRT80B/1 were crossed with y,w; dos R31, FRT80B/TM6B females that carried in addition a single copy of either the P[y 1, genE-dos], P[y 1, genE-dos Y801F], P[y 1, genE-dos Y3581537F], or P[y 1, genE-dos W104A] transgenes on the second chromosome. First instar larvae of these crosses were heat-shocked for 1 h at 378C to induce mitotic recombination between the FRT-bearing homologous chromosomes. Females of the progeny with the genotype y,w,hsFlp1/1; P[y 1, genE-dos*]/1; dos R31, FRT80B/ P[w 1]75C, FRT80B were selected. Cells in the eye homozygous for the dos R31 mutation were identi®ed by the lack of pigmentation, the yellow gene served as a marker for the presence of the transgene. Only clones spanning more than one ommatidium were taken for eye sections. 4.5. Histology and microscopy For histological sections, heads were ®xed with OsO4 as described previously (Basler et al., 1991) and embedded in EPON. 1 mm eye sections were cut with an Ultracut microtom (Reichert and Jung) and stained with Toluidine Blue/ Borax solution for microscopy (Leitz, Aristoplan). Confocal microscopy of eye imaginal disks expressing GFP-tagged dos transgenes was carried out with a Leitz Aristoplan Confocal Laser Scanning Microscope. Acknowledgements We would like to thank B. Cormack, B. Dickson and E. Hafen for providing reagents, M. Simon for sharing unpublished data and S. Kramer and T. Zars for critical reading of the manuscript. We are grateful to K. Rein for technical advice in confocal microscopy. This work was supported by grants from the Deutsche Forschungsgemeinschaft (Ra561/3-2 and SFB465/A-9). References Allard, J.D., Chang, H.C., Herbst, R., McNeill, H., Simon, M.A., 1996. The SH2-containing tyrosine phosphatase corksrew is required during signaling by sevenless, Ras1 and Raf. Development 122, 1137±1146. Allard, J.D., Herbst, R., Carroll, P.M., Simon, M.S., 1998. Mutational analysis of the SRC homology 2 domain protein-tyrosine phosphatase corkscrew. J. Biol. Chem. 273, 13129±13135. Bardelli, A., Longati, P., Gramaglia, D., Stella, M.C., Comoglio, P.M., 1997. Gab1 coupling to the HGF/Met receptor multifunctional docking

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