INTRODUCTION

Top Abstract Introduction Materials and methods Results Discussion References

The Ras family of small GTPases play pivotal roles in the regulation of signal transduction pathways downstream of receptor tyrosine kinases (RTKs), G protein-coupled receptors, and cytokine receptors. Ras proteins cycle between the inactive GDP-bound state and the active GTP-bound conformation that interacts with downstream effector proteins such as the Raf family of protein kinases and phosphoinositide 3'-kinase (42). This GTPase cycle is regulated by guanine nucleotide exchange factors (GEFs) that activate Ras by stimulating release of GDP and binding to GTP and by GTPase-activating proteins (GAPs) that inhibit Ras by increasing the intrinsic rate of GTP hydrolysis (5).

Three types of GAP protein for Ras have been identified in mammalian cells: p120 Ras-GAP (64, 68), neurofibromin (the product of the human tumor suppressor gene NF1) (46, 71), and two closely related proteins, Gap1^m (45) and Gap1^IP4B (16). Although it is clear that all three types can act as GAPs and thus inhibit the activity of Ras proteins, p120 Ras-GAP has been also implicated as an effector of Ras. Notably, p120 Ras-GAP has an amino-terminal region similar in structural organization to "adapter" proteins. This region contains Src homology 2 and 3 (SH2 and SH3) domains involved in interactions with other proteins and pleckstrin homology (PH) and C2 (or CalB) domains that may promote membrane association by binding to phospholipids (31, 56). Studies using mammalian tissue culture cells suggest a role for the SH2 and SH3 domains of p120 Ras-GAP in cell transformation, changes in gene expression, K⁺ channel opening, and cytoskeletal rearrangements, and in Xenopus oocytes there is evidence that the SH3 domain of p120 Ras-GAP is required for meiotic maturation in response to Ras or insulin (63).

The identification of proteins that bind to the amino-terminal adapter-like region of p120 Ras-GAP has provided further evidence that it has roles independent of its GAP activity. The SH2 domains bind to certain plasma membrane-associated RTKs such as the platelet-derived growth factor  receptor (2) and to the cytosolic proteins p190 Rho-GAP (59) and p62dok, a "docking" protein of unknown function (10, 72). Proteins that bind to the SH3 domain of p120 Ras-GAP have proved difficult to identify but G3BP, a cytoplasmic protein that contains RNA binding motifs, may be such an SH3 ligand (55). The biochemical and physiological consequences of the interaction of p120 Ras-GAP with these proteins are poorly understood.

An important advance in understanding the physiological roles of p120 Ras-GAP has come from the analysis of mice carrying a null mutation in the gene for p120 Ras-GAP (Gap) (36). Embryos homozygous for this mutation die in utero with abnormal vasculature and increased apoptosis in the nervous system. Fibroblasts from Gap^/ embryos can proliferate in vitro and show enhanced accumulation of Ras-GTP upon growth factor stimulation (67). This genetic analysis shows that p120 Ras-GAP is not necessary for mitogenesis, at least for most cell types, but is required to downregulate Ras following exposure to growth factors and to protect neuronal cells from apoptosis. Whether p120 Ras-GAP is involved in other Ras-regulated processes such as cell migration or differentiation is unresolved.

To study the role of p120 Ras-GAP in a genetically tractable organism we set out to identify a homolog from the fruit fly Drosophila melanogaster, which has proved to be a powerful model system for the genetic analysis of Ras signaling pathways (19, 41). Using an expression cloning strategy in the fission yeast Schizosaccharomyces pombe we cloned a cDNA encoding Drosophila RasGAP, which is similar in both sequence and biochemical properties to mammalian p120 Ras-GAP. Although loss-of-function mutations in the Drosophila RasGAP gene are not available we have begun to analyze its function in vivo by ectopic expression. We show that overexpression of RasGAP in the wing imaginal disc downregulates signaling through RTKs and inhibits wing growth. The system we have developed will prove useful in understanding the physiological role of RasGAP and the function of each of its modular domains.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and methods Results Discussion References

Methods for S. pombe. Yeast extract supplements (YES) and Edinburgh minimal medium (EMM) were used for growth of S. pombe cultures (49), and synthetic sporulation agar (SSA) was used to promote mating and sporulation (22). The gap1 mutant strain JZ446 (h⁹⁰ gap1::ura4⁺ ade6-M216 leu1 ura4-D18) (40) was used in the screen for the identification of novel GTPase-activating proteins and was transformed by the lithium acetate method (53). Plasmids were rescued from yeast into Escherichia coli as described previously (49).

Isolation of a full-length RasGAP cDNA and plasmid constructions. The RasGAP cDNA isolated in the yeast screen (RasGAP78) was truncated at the 5' end and predicted to initiate translation at Met266. Through screening a gt10 embryonic cDNA library (Stratagene) and by rapid amplification of cDNA ends (RACE) PCR with Drosophila polyA⁺ RNA (Clontech) as template for the Marathon cDNA Amplification Kit (Clontech), a cDNA including 291 bp of 5' untranslated sequence was assembled. A myc tag epitope recognized by the 9E10 monoclonal antibody, followed by two TAA termination codons, was engineered at the C terminus of the open reading frame by using the Exsite Mutagenesis Kit (Stratagene). To generate the GAP catalytic domain construct, RasGAPCat, the C-terminal 443 codons were amplified with a sense primer incorporating an NcoI site which was used to fuse the resulting PCR product in-frame to the first 85 codons of the RasGAP cDNA, just N terminal to the first SH2 domain. The full-length cDNA and the C terminus myc-tagged constructs were subcloned as NotI fragments into the Drosophila expression vector pUAST (6) and the S. pombe expression vector pREPCD1 (24), a derivative of the pREP1 plasmid with a NotI site in the polylinker (47). The C-terminal part of RasGAP (amino acids 429 to 954) was cloned as a ClaI/BamHI fragment into the E. coli expression vector pGEX-KG (33). The SH2-SH3-SH2 region (amino acids 83 to 343) and the N-terminal SH2 domain (amino acids 83 to 182) were amplified by PCR and cloned as XbaI/XhoI fragments into pGEX-KG. Missense mutations were introduced by PCR-mediated mutagenesis (37) into each SH2 domain, changing a highly conserved Arg residue to Leu (amino acid 110 in the N-terminal domain and amino acid 278 in the C-terminal domain) to give the GST-SH2*32* mutant and the RasGAPSH2* mutant. Sequences of the oligonucleotides used for PCR and site-directed mutagenesis are available upon request from the corresponding author. All PCR-derived fragments were sequenced to ensure that no mutations were introduced. Sequences were determined with oligonucleotide primers with the TaqFS Terminator Cycle Sequencing Kit on an ABI 377 DNA sequencer (Applied Biosystems) and were analyzed using the Genetics Computer Group package (32). The UAS-tor⁴⁰²¹-btl and UAS-tor⁴⁰²¹-htl constructs were generated by using KpnI and XbaI to remove the fusion genes from P[sE-tor⁴⁰²¹-FGFR1] and P[sE-tor⁴⁰²¹-FGFR2], respectively (57), and the inserts were subcloned into pUAST.

General biochemical and immunological techniques. Protein samples were separated by sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis (PAGE) and electroblotted to Immobilon membranes (NEN) as previously described (35). Anti-RasGAP antibody incubations were for 1 to 2 h in 5% skimmed milk-TBST (25 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.1% Tween-20) with washes in TBST. Antiphosphotyrosine blots were blocked in 3% bovine serum albumin (BSA)-TBST at room temperature for 1 to 2 h, incubated overnight at 4°C with the primary antibody 4G10 (Upstate Biotechnology) at a concentration of 0.3 µg/ml in 3% BSA-TBST, and washed in TBST. Horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence substrates were used as recommended by the manufacturer (Pierce). Protease inhibitors were the Complete mix (Boehringer Mannheim) together with 1 mM phenylmethylsulfonyl fluoride.

Purification of GST fusion proteins and preparation of antibodies. Glutathione S-transferase (GST) fusion proteins were expressed in E. coli by induction with 0.5 mM isopropyl-1-thio--D-galactopyranoside for 2 to 4 h at 37°C. To purify wild-type and Val12 H-Ras GST fusion proteins (69), cells were resuspended in TMD buffer (50 mM Tris-HCl [pH 7.5], 5 mM MgCl₂, 1 mM dithiothreitol) plus protease inhibitors and lysozyme added to a concentration of 1 mg/ml, and the cell suspension was incubated for 20 min at 4°C. Cells were lysed by adding Triton X-100 and DNase I to concentrations of 0.5% and 20 µg/ml, respectively, and the lysate was incubated for 20 min at 4°C. Following centrifugation at 12,000 × g for 15 min, the supernatants were mixed with glutathione-Sepharose beads and washed extensively with TMD, and the fusion proteins were eluted with 50 mM Tris-HCl (pH 8.0)-5 mM MgCl₂-10 mM reduced glutathione. The purified proteins were dialyzed against TMD and stored in aliquots at 70°C. The GST-SH232 and GST-NSH2 fusion proteins were purified by glutathione-Sepharose affinity chromatography following solubilization with N-lauroylsarcosine (26). The purified GST-NSH2 fusion protein was dialyzed against phosphate-buffered saline and used to raise rat monoclonal antibodies by using standard protocols (35). The GST-RasGAP C-terminal region was purified with glutathione-Sepharose beads following solubilization with N-lauroylsarcosine, and the insoluble RasGAP polypeptide was released from the fusion protein by cleavage with thrombin. Following SDS-PAGE purification the RasGAP polypeptide was used to immunize a rabbit by using standard protocols (35). The rabbit polyclonal antibody was purified by applying the crude antiserum to the GST-fusion protein cross-linked to glutathione-Sepharose resin (3) and eluting the bound immunoglobulin with 100 mM glycine, pH 2.5 (35). The purified antibody was titered to determine its optimum concentration for Western blotting and immunoprecipitation.

Immunoprecipitations and GST pull-downs from Drosophila cell extracts. Soluble extracts from Drosophila embryos, larvae, pupae, and adults in Nonidet P-40 (NP-40) lysis buffer plus protease inhibitors were prepared as previously described (15). For immunoprecipitations, 0.1% SDS-0.5% deoxycholate were added to the extracts and 1 mg of protein was incubated with the purified anti-RasGAP antibody for 2 h followed by incubation with protein G-Sepharose for 1 h. The beads were washed three times in the same buffer before analysis by SDS-PAGE and Western blotting. For pull-downs with GST-SH232 fusion proteins, cell extracts (0.5 mg of protein) were incubated with the fusion proteins (10 µg) bound to glutathione-Sepharose for 1 h at 4°C, washed three times in lysis buffer, and analyzed by SDS-PAGE and Western blotting.

Immunostaining of embryos. Embryos were dechorionated, fixed with 4% paraformaldehyde, devitellinized in methanol, and incubated with antibodies as previously described (73). Incubation was overnight with the anti-RasGAP monoclonal antibody (1/2 diluted tissue culture supernatant) and for 2 h with Cy3-conjugated anti-rat immunoglobulin G (IgG) (Jackson ImmunoResearch). Images were collected with a BioRad MRC-1024 confocal laser scanning microscope and assembled in Adobe Photoshop 4.0.

GAP assay. S. pombe cell extracts were prepared from the gap1 mutant strain JZ446 transformed with a vector plasmid (pREPCD1) or the full-length RasGAP cDNA in pREPCD1 by vortexing with glass beads in 50 mM HEPES [pH 7.4]-100 mM NaCl-5 mM MgCl₂-1 mM sodium phosphate (pH 7.4)-1-mg/ml BSA plus protease inhibitors. Following centrifugation at 15,000 × g for 15 min, the supernatant was concentrated to ~20-mg/ml protein by ultrafiltration (Centricon 30; Amicon). GAP assays were performed by a method described previously (20) with [-³²P]GTP-loaded GST-Ras or GST-RasV12 fusion proteins and separation of GTP and GDP bound to Ras by thin-layer chromatography, except that following the incubation with cell extract the GST-Ras fusion proteins were purified with glutathione-Sepharose. A PhosphorImager (Molecular Dynamics) was used to image and quantify the [-³²P]-labeled guanine nucleotides, and the percent GTP remaining was calculated as 100(GTP/GDP + GTP).

Drosophila stocks and phenotypic analysis. Flies were raised and crossed at 25°C according to standard procedures. Transgenic lines were made by P element-mediated transformation into a y w stock. The GAL4 lines used were MS-1096 (X chromosome) for expression in the dorsal pouch of the wing disc (9) and hsp70-GAL4 (3rd chromosome) for expression upon heat shocking (51). The Ras1^e2F mutant allele was used (60). Wings were dissected, dehydrated in ethanol, and mounted in Euparal (Agar Scientific). Photographs of wings were digitized with a flat-bed scanner, processed in Adobe Photoshop 4.0, and analyzed using NIH Image 1.60 software.

Nucleotide sequence accession number. The RasGAP cDNA nucleotide sequence has been submitted to the EMBL database under accession no. AJ012609.

	RESULTS

Top Abstract Introduction Materials and methods Results Discussion References

Expression cloning in fission yeast of a Drosophila p120 Ras-GAP homolog. An expression cloning screen with S. pombe was devised to identify inhibitors of Ras function from D. melanogaster. In S. pombe, mutation of the gap1 gene (also known as sar1), which encodes a member of the RasGAP family of proteins, results in hypersensitivity to mating pheromones, formation of extended mating projections, and inefficient mating (Fig. 1A, i, and 1B, i) (40, 70). These phenotypes result from hyperactivation of Ras1, the S. pombe homolog of mammalian Ras oncoproteins, because mutations in Ras1 that inhibit its GTPase activity cause identical effects (28, 50). We reasoned that expression of inhibitors of Ras function from higher organisms in a gap1 mutant strain would restore efficient mating by downregulating the activities of Ras1-regulated signaling pathways. Such a screen might identify both proteins that bind directly to Ras1, such as GAPs and effectors, and proteins that interfered with the function of downstream components of the Ras1 signaling pathways.

View larger version (53K): [in this window] [in a new window]   FIG. 1.   Complementation of the S. pombe gap1 mutant phenotype by the Drosophila RasGAP cDNA. The homothallic h90 gap1 mutant strain (JZ446) was transformed with the vector pREPCD1 (i), pST200-1(gap1+) carrying the S. pombe gap1+ gene (ii), pREPRasGAP78 containing the original cDNA isolated from the library screen (iii), pREPRasGAP containing the full-length cDNA (iv), and pREPRasGAPCat containing the GAP catalytic domain (v). A wild-type homothallic h90 strain was used as control (vi). Transformants were grown on sporulation agar (SSA) at 30°C for 3 days. Differential interference contrast microscopy (A) and iodine staining (B), which stains spores, revealed that the three RasGAP cDNA constructs allowed the gap1 mutant strain to undergo mating and sporulation. The arrows in panel A indicate typical zygotic asci.

Structure of Drosophila RasGAP. Drosophila RasGAP and human p120 Ras-GAP share 47% amino acid identity (68% similarity) overall (Fig. 2A). Like its mammalian homologs, the Drosophila protein can be divided into two regions. The amino-terminal part consists of protein-protein (SH2 and SH3) and protein-lipid (PH and C2/CalB) interaction domains. The carboxy-terminal part contains the GAP catalytic domain which is conserved in RasGAPs from yeast to mammals (5).

View larger version (76K): [in this window] [in a new window]   FIG. 2.   RasGAP amino acid sequence and genomic organization of the RasGAP gene. (A) Alignment of the predicted amino acid sequence of RasGAP (top) with human p120 Ras-GAP (bottom). The comparison was generated with the GAP program of the Genetics Computer Group package (32). |, identical amino acids; :, amino acid pairs with Dayhoff matrix scores greater than 0.5; ., nonidentical amino acids having a positive Dayhoff matrix score of <0.5. The SH2 domains are shown by shading and the SH3 domain is boxed. The PH domain is underlined by a solid line and the C2/CalB domain is underlined by a dotted line. The asterisk indicates the conserved tyrosine that is phosphorylated in human p120 Ras-GAP. (B) The exon-intron structure of the RasGAP transcription unit. The white boxes represent noncoding regions, and the black boxes represent coding regions.

GAP activity of RasGAP. The original RasGAP cDNA (RasGAP78) identified in the expression cloning screen was amino-terminally truncated and predicted to initiate at Met266. We examined whether constructs carrying the full-length cDNA or just the GAP catalytic domain (amino acids 553 to 954, designated RasGAPCat) could also rescue the gap1 mutant strain (JZ446). Both constructs could restore efficient mating to the gap1 mutant strain (Fig. 1A, iv and v and B, iv and v) and in fact both rescued better than overexpression of the S. pombe gap1⁺ gene (Fig. 1A, ii and B, ii), restoring mating to nearly wild-type levels (Fig. 1B, vi). These results show that the RasGAP catalytic domain is responsible for downregulation of Ras1 activity in S. pombe. The ability of RasGAP to rescue the Ras "hyperactivation" phenotype caused by loss of gap1 function, but not that caused by a GAP-insensitive ras1 mutant (ras1-Val17), suggested that RasGAP was acting as a GAP on the S. pombe Ras1 protein in vivo, rather than as a "dominant negative" by binding to Ras1-GTP and preventing interaction with Ras1 effector proteins.

View larger version (38K): [in this window] [in a new window]   FIG. 3.   GAP activity of RasGAP. (A) Expression of full-length RasGAP in S. pombe. Cell extracts prepared from a gap1 mutant strain transformed with pREPRasGAP (lane 1) or a vector control (lane 2) were subjected to SDS-PAGE and Western blotted with an anti-RasGAP polyclonal antibody (-RasGAP). Expression of the full-length RasGAP protein migrating close to the predicted molecular weight (108 kDa) can be detected in lane 1. Migration of molecular weight markers (in kilodaltons) is shown. (B) The RasGAP and vector cell extracts were assayed for GAP activity on wild-type H-Ras (hatched bars) or Val12 H-Ras (filled bars); a control assay without extract was done to determine the intrinsic GTPase activity of the Ras proteins. The percent GTP remaining after a 15-min incubation was measured and plotted in a bar graph. Error bars show standard deviations of assays done in triplicate. Results are representative of three independent experiments.

Characterization of the RasGAP protein. A monoclonal antibody raised against the N-terminal SH2 domain of RasGAP detected the 108-kDa RasGAP protein by immunoblotting. The levels of expression of RasGAP in embryos, larvae, and adults were assessed by immunoblotting equal amounts of total protein from each developmental stage. This showed that the protein is expressed throughout development but with significantly higher levels of embryos (Fig. 4A). Immunostaining of embryos with the anti-RasGAP monoclonal antibody detected staining in precellularized embryos (0 to 2 h), before zygotic transcription begins, indicating that the RasGAP protein is supplied maternally (Fig. 4D). The RasGAP protein was cytoplasmic and appeared to be present in all cells at the cellular blastoderm stage (Fig. 4E).

View larger version (82K): [in this window] [in a new window]   FIG. 4.   Characterization of RasGAP and interacting proteins. (A) Developmental expression of RasGAP protein. Equal amounts of total cell lysates from embryos (0 to 18 h) (Em), mixed-stage larvae (L), and adults (Ad) were Western blotted with anti-RasGAP monoclonal antibody. A control lysate (Ct) was made from adult flies expressing a heat-shock-inducible RasGAP transgene after heat shock for 1 h at 37°C. (B) Tyrosine phosphorylation of RasGAP in vivo. RasGAP was immunoprecipitated from embryonic extracts and Western blotted with either an anti-RasGAP polyclonal antibody (-RasGAP) or a monoclonal antiphosphotyrosine antibody (-pTyr). Migration of molecular weight markers (in kilodaltons) is shown. (C) In vitro association of the SH2 domains of RasGAP with tyrosine-phosphorylated proteins from different developmental stages. Extracts were prepared from 0 to 4-h embryos (Em1), 12- to 24-h embryos (Em2), mixed-stage larvae (L), pupae (P), and adult females (Ad). The wild-type GST-SH232 or mutant GST-SH2*32* fusion proteins bound to glutathione-Sepharose were used to precipitate proteins which were subsequently Western blotted with the antiphosphotyrosine antibody. (D) Immunostaining of precellularized embryo with anti-RasGAP monoclonal antibody showing early expression of RasGAP protein during embryonic development. (E) Immunostaining of cellularized blastoderm embryo showing cytoplasmic localization of RasGAP protein.

Inhibition of wing growth by ectopic RasGAP expression. To gain insight into the in vivo role of RasGAP, we generated transgenic Drosophila expressing the full-length RasGAP cDNA, tagged with the myc epitope, under the control of the yeast GAL4 upstream activating sequence (UAS) (6). Several transgenic lines were obtained by P element germ-line transformation. To determine which lines were able to express RasGAP in a GAL4-dependent manner, we crossed the UAS-RasGAP lines with a line expressing GAL4 under the control of a heat-shock-regulated promoter from the Hsp70 gene. Progeny carrying both UAS-RasGAP and hsp70-GAL4 were heat shocked, and the expression levels of the ectopically expressed RasGAP were analyzed by Western blotting of whole-fly lysates with both the anti-RasGAP polyclonal antibody and the anti-myc antibody (24). On the basis of this analysis, we selected three independent UAS-RasGAP lines expressing the transgene at different levels for further experiments. The UAS-RasGAP lines were crossed to a number of stocks expressing GAL4 in various spatial and temporal patterns throughout development, and the progeny from these crosses were analyzed for phenotypes dependent on RasGAP expression.

View larger version (67K): [in this window] [in a new window]   FIG. 5.   Ectopic expression of RasGAP in the wing imaginal disc leads to a decrease in size of the adult wing. GAL4 and RasGAP were expressed in the dorsal pouch of the wing with the MS-1096 GAL4 line. The genotypes are GAL4/+ (A), GAL4/+ UAS-RasGAP/+ (B), GAL4/Y (C), and GAL4/Y UAS-RasGAP/+ (D). (A and B) Wings from females; (C and D) wings from males. Bar, 250 µm.

Downregulation of receptor tyrosine kinase signaling by ectopic RasGAP expression. Inhibition of the Ras1-MAPK pathway also disrupts the formation of wing veins, a process that requires the Drosophila EGF receptor homolog (DER) (18). Ectopic expression of RasGAP, however, did not disrupt the longitudinal wing veins (Fig. 5B and D). (Loss of the anterior cross vein as seen in Fig. 5D is also seen at a variable frequency in males hemizygous for the MS-1096 GAL4 insertion but not carrying UAS-RasGAP.) However, differentiation of wing veins occurs during the pupal stage when the level of expression from the MS-1096 GAL4 line might be lower than during the larval stages (11).

View larger version (145K): [in this window] [in a new window]   FIG. 6.   Ectopic expression of RasGAP inhibits signaling by activated FGF receptor homologs (Breathless and Heartless) in the wing. The genotypes are GAL4/+ UAS-tor4021-btl/+ (A), GAL4/+ UAS-tor4021-btl/+ UAS-RasGAP/+ (B), GAL4/+ UAS-tor4021-htl/+ (C), GAL4/+ UAS-tor4021-htl/+ UAS-RasGAP/+ (D), GAL4/+ UAS-tor4021-htl/+ UAS-RasGAPSH2*/+ (E), and GAL4/+ UAS-tor4021-htl/+ Ras1e2F/+ (F). The GAL4 line was MS-1096. Bar, 250 µm.

	DISCUSSION

Top Abstract Introduction Materials and methods Results Discussion References

The p120 Ras-GAP homolog we have identified, RasGAP, is the third GAP for Ras proteins to be identified in Drosophila and shows that this invertebrate has one of each of the types of RasGAPs found in mammals (30, 62). The most conserved region of the protein encompasses the SH2 and SH3 protein-protein interaction domains in the amino-terminal adapter-like region, which is 57% identical in amino acid sequence between the Drosophila and human proteins. The SH2 domains of mammalian p120 Ras-GAP mediate binding to certain RTKs, p190 Rho-GAP, and the docking protein p62dok (see the introduction). In work described elsewhere we show that the SH2 domains of RasGAP bind in vitro to an autophosphorylation site on Torso (14), a RTK required during early embryonic development (61). The sequences surrounding the phosphotyrosine residue (phosphoYLEP) on Torso match the optimal binding site for the SH2 domains of mammalian p120 Ras-GAP (38, 39). The RasGAP SH2 domains bind in vitro to a small number of tyrosine-phosphorylated proteins from different developmental stages. Whether these are RTKs and the Drosophila homologs of p190 Rho-GAP and p62dok remains to be established.

One notable difference between RasGAP and the mammalian p120 Ras-GAP isoform is that the latter has a proline-rich sequence near the amino terminus which is absent in the Drosophila protein. This region appears to be required for in vitro association of p120 Ras-GAP with Src family tyrosine kinases (Src, Hck, Lck, and Fyn) via their SH3 domain (7) and could be involved in the in vivo association of p120 Ras-GAP with Src family tyrosine kinases (13). Drosophila RasGAP also lacks an ~70-amino-acid hydrophobic region found at the amino terminus of p120 Ras-GAP. The p100 Ras-GAP isoform expressed in placenta does not have the polyproline region or a hydrophobic amino terminus (65).

We have begun to study the function of RasGAP in vivo by ectopic expression of the full-length protein in the wing imaginal disc with the GAL4-UAS system. Overexpression of RasGAP inhibits growth of the wing, resulting in a decrease in size of the adult wing of up to 45% as a consequence of having fewer rather than smaller cells. This suggests that RasGAP overexpression reduces the rate of proliferation or increases the rate of apoptosis in the wing imaginal disc. Is this effect due to inhibition of Ras1 function? This could occur either by reducing the amount of Ras1-GTP, which would be consistent with the biochemical properties of RasGAP in vitro, or by competition between RasGAP and Ras1 effectors for binding to Ras1-GTP.

The ability of RasGAP to suppress ectopic wing vein formation caused by hyperactivation of the Breathless and Heartless FGF receptor homologs strongly suggests that RasGAP can indeed downregulate Ras1 activation by these RTKs. Both Drosophila FGF receptors have exact matches to the optimal binding site (phospho YxxPxD, where x is any amino acid) for the SH2 domains of mammalian p120 Ras-GAP (38, 39). Given our demonstration in this study that the SH2 domains of RasGAP are essential for its activity in vivo it is likely that the activated FGF receptors directly recruit RasGAP to the plasma-membrane where it is well placed to interact with its substrate, Ras1-GTP.

The DER-Ras1-MAPK pathway regulates both cell proliferation and wing vein differentiation in the wing disc (18). It was puzzling, therefore, why RasGAP overexpression did not affect wing vein formation in otherwise wild-type flies. One possible explanation is that the MS-1096 GAL4 line does not drive sufficient expression of RasGAP in the pupal disc when vein differentiation occurs. However, the ectopic veins caused by the activated FGF receptors were efficiently suppressed by coexpression of RasGAP. We favor the explanation that RasGAP is not efficiently recruited to the plasma membrane by the DER RTK, and this is consistent with our failure to detect an effect of RasGAP overexpression on photoreceptor development in the eye, a process controlled by the DER and Sev RTKs, neither of which contains the RasGAP SH2 binding consensus. The insulin receptor (Inr), another RTK, is also implicated in the control of cell proliferation in the wing disc (12, 25), but we did not see any enhancement of the RasGAP overexpression phenotype in flies heterozygous for an inr mutation (24). The insensitivity of the RasGAP-induced small wing phenotype to the gene dosage of Ras1 suggests that RasGAP could be inhibiting proliferative signals that are not dependent on Ras1.

One important and unresolved issue is the specificity of RasGAPs in vivo. Mammalian p120 Ras-GAP can act as a GAP in vitro for the Ras-related protein R-Ras (29), and also, more surprisingly, for the much more distantly related small GTPase Rab5 (43), which is involved in the regulation of endocytosis. A further twist is that the Ras-related protein Rap1/Krev-1 binds to p120 Ras-GAP with high affinity but is not a substrate (27). Drosophila homologs of R-Ras (Ras2), Rap1 (Roughened), and Rab5 (Drab5) have been identified (34, 52, 58). Their roles in imaginal disc growth and differentiation are not clear, although it has been observed that expression of a constitutively active Ras2 mutant in the wing disc gives rise to ectopic veins (6, 17). Therefore, it is possible that some of the effects of RasGAP overexpression are the consequence of interactions with Ras2, Roughened, or Drab5.

Alternatively, the functions of proteins that interact with the amino-terminal adapter region of RasGAP may be affected by RasGAP overexpression and cause growth inhibition. However, overexpressing just the SH2-SH3-SH2 region of RasGAP by using the same UAS-GAL4 system does not affect wing growth (24). Further studies of the effects of expressing RasGAP transgenes with mutations in the modular domains and the isolation of RasGAP mutants should help to clarify the physiological roles of this important signaling protein. The extensive structural and functional similarities between the Drosophila and mammalian RasGAPs indicates that insights gained from genetic approaches in flies will also be relevant to the role of p120 Ras-GAP in mammals.