Mutational Analysis of the SRC Homology 2 Domain Protein-tyrosine Phosphatase Corkscrew
1998; Elsevier BV; Volume: 273; Issue: 21 Linguagem: Inglês
10.1074/jbc.273.21.13129
ISSN1083-351X
AutoresJohn Allard, Ronald Herbst, Pamela M. Carroll, Michael A. Simon,
Tópico(s)Cytokine Signaling Pathways and Interactions
ResumoThe SRC homology 2 (SH2) domain protein-tyrosine phosphatase, Corkscrew (CSW) is required for signaling by receptor tyrosine kinases, including the Sevenless receptor tyrosine kinase (SEV), which directs Drosophila R7 photoreceptor cell development. To investigate the role of the different domains of CSW, we constructed domain-specific csw mutations and assayed their effects on CSW function. Our results indicate that CSW SH2 domain function is essential, but either CSW SH2 domain can fulfill this requirement. We also found that CSW and activated SEV are associatedin vivo in a manner that does not require either CSW SH2 domain function or tyrosine phosphorylation of SEV. In contrast, the interaction between CSW and Daughter of Sevenless, a CSW substrate, is dependent on SH2 domain function. These results suggest that the role of the CSW SH2 domains during SEV signaling is to bind Daughter of Sevenless rather than activated SEV. We also found that although CSW protein-tyrosine phosphatase activity is required for full CSW function, a catalytically inactive CSW is capable of providing partial function. In addition, we found that deletion of either the CSW protein- tyrosine phosphatase insert or the entire CSW carboxyl terminus, which includes a conserved DRK/GRB2 SH2 domain binding sequence, does not abolish CSW function. The SRC homology 2 (SH2) domain protein-tyrosine phosphatase, Corkscrew (CSW) is required for signaling by receptor tyrosine kinases, including the Sevenless receptor tyrosine kinase (SEV), which directs Drosophila R7 photoreceptor cell development. To investigate the role of the different domains of CSW, we constructed domain-specific csw mutations and assayed their effects on CSW function. Our results indicate that CSW SH2 domain function is essential, but either CSW SH2 domain can fulfill this requirement. We also found that CSW and activated SEV are associatedin vivo in a manner that does not require either CSW SH2 domain function or tyrosine phosphorylation of SEV. In contrast, the interaction between CSW and Daughter of Sevenless, a CSW substrate, is dependent on SH2 domain function. These results suggest that the role of the CSW SH2 domains during SEV signaling is to bind Daughter of Sevenless rather than activated SEV. We also found that although CSW protein-tyrosine phosphatase activity is required for full CSW function, a catalytically inactive CSW is capable of providing partial function. In addition, we found that deletion of either the CSW protein- tyrosine phosphatase insert or the entire CSW carboxyl terminus, which includes a conserved DRK/GRB2 SH2 domain binding sequence, does not abolish CSW function. Receptor tyrosine kinases (RTKs) 1The abbreviations used are: RTK, receptor-tyrosine kinase; SH2, SRC homology 2; CSW, corkscrew; PTP, protein-tyrosine phosphatase; PDGF, platelet-derived growth factor; DOS, Daughter of Sevenless.1The abbreviations used are: RTK, receptor-tyrosine kinase; SH2, SRC homology 2; CSW, corkscrew; PTP, protein-tyrosine phosphatase; PDGF, platelet-derived growth factor; DOS, Daughter of Sevenless. regulate cellular growth and differentiation in response to extracellular signals. The binding of a specific ligand to the extracellular domain of an RTK leads to a well characterized series of biochemical events, including RTK dimerization, activation of the cytoplasmic tyrosine kinase domain, and phosphorylation of the RTK on specific tyrosine residues (reviewed in Ref. 1van der Geer P. Hunter T. Lindberg R.A. Annu. Rev. Cell Biol. 1994; 10: 251-337Crossref PubMed Scopus (1232) Google Scholar). The activated and phosphorylated RTK then regulates key cellular target proteins. Frequently, these targets contain either SRC homology 2 (SH2) domains or phosphotyrosine binding domains that bind to specific phosphorylated tyrosine residues of the RTK. The recruitment of such SH2- and phosphotyrosine binding domain-containing proteins regulates their function and leads to the activation of important intracellular signaling pathways. For example, the interaction of the SH2 domain-containing protein GRB2 with activated RTKs leads to the stimulation of the RAS pathways (2McCormick F. Curr. Opin. Genet. Dev. 1994; 4: 71-76Crossref PubMed Scopus (208) Google Scholar, 3Egan S.E. Weinberg R.A. Nature. 1993; 365: 781-783Crossref PubMed Scopus (523) Google Scholar). Among the key signaling proteins, the function of which is required during RTK signaling, is a class of protein-tyrosine phosphatases that includes SHP2 in mammals and Corkscrew (CSW) in Drosophila(reviewed in Ref. 4Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Crossref PubMed Scopus (731) Google Scholar). In addition to their protein-tyrosine phosphatase (PTP) domains, these proteins are characterized by the presence of two SH2 domains at their amino terminus and a poorly conserved carboxyl-terminal “tail” region. In addition, CSW contains a domain of approximately 150 amino acids that interrupts its PTP domain and is not present in SHP2 (5Perkins L.A. Larsen I. Perrimon N. Cell. 1992; 70: 225-236Abstract Full Text PDF PubMed Scopus (328) Google Scholar). The first evidence that SHP2/CSW proteins were important for RTK signaling came from the identification of CSW as a maternally contributed, positively acting component during signaling by the Drosophila TORSO RTK (5Perkins L.A. Larsen I. Perrimon N. Cell. 1992; 70: 225-236Abstract Full Text PDF PubMed Scopus (328) Google Scholar). Subsequent studies have implicated CSW in signaling by several other Drosophila RTKs including SEV and the Drosophila epidermal growth factor receptor (6Allard J.D. Chang H.C. Herbst R. McNeill H. Simon M.A. Development. 1996; 122: 1137-1146Crossref PubMed Google Scholar, 7Perkins L.A. Johnson M.R. Melnick M.B. Perrimon N. Dev. Biol. 1996; 180: 63-81Crossref PubMed Scopus (100) Google Scholar). In vertebrates, SHP2 has been shown to participate in signaling by the epidermal growth factor, PDGF, fibroblast growth factor, and insulin receptors (reviewed in Refs. 4Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Crossref PubMed Scopus (731) Google Scholar and 8Streuli M. Curr. Opin. Cell Biol. 1996; 8: 182-188Crossref PubMed Scopus (164) Google Scholar). Two potential mechanisms for the action of SHP2/CSW during RTK signaling have been proposed. In the case of certain RTKs, such as the PDGF receptor, RTK autophosphorylation creates binding sites for the SH2 domains of SHP2 (9Bennett A.M. Tang T.L. Sugimoto S. Walsh C.T. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7335-7339Crossref PubMed Scopus (341) Google Scholar, 10Li W. Nishimura R. Kashishian A. Batzer A.G. Kim W.J. Cooper J.A. Schlessinger J. Mol. Cell Biol. 1994; 14: 509-517Crossref PubMed Google Scholar). Once bound to receptor, SHP2 becomes phosphorylated at a particular tyrosine residue in its tail region. Phosphorylated SHP2 can then be bound by the SH2 domain of GRB2. The recruitment of GRB2 to the SHP2·RTK complex is proposed to lead to the activation of the RAS signaling pathway. Studies of signaling by the SEV RTK in Drosophila have led to an additional model for CSW action during RTK signaling (6Allard J.D. Chang H.C. Herbst R. McNeill H. Simon M.A. Development. 1996; 122: 1137-1146Crossref PubMed Google Scholar, 11Herbst R. Carroll P.M. Allard J.D. Schilling J. Raabe T. Simon M.A. Cell. 1996; 85: 899-909Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). The activation of SEV induces neuronal differentiation of the R7 photoreceptor precursor cell (for reviews, see Refs. 12Hafen, E., Dickson, B., Raabe, T., Brunner, D., Oellers, N., and van der Straten, A. (1993) Dev. Suppl. 41–46Google Scholar, 13Simon M.A. Dev. Biol. 1994; 166: 431-442Crossref PubMed Scopus (48) Google Scholar, 14Zipursky S.L. Rubin G.M. Annu. Rev. Neurosci. 1994; 17: 373-397Crossref PubMed Scopus (232) Google Scholar). In the R7 cell, CSW acts as a positive component of the signaling pathway initiated by SEV (6Allard J.D. Chang H.C. Herbst R. McNeill H. Simon M.A. Development. 1996; 122: 1137-1146Crossref PubMed Google Scholar). Genetic epistasis experiments have demonstrated that CSW function is required in the developing R7 cell even when constitutively activated forms of RAS and RAF are used to initiate the SEV signaling cascade. These experiments have suggested that although CSW may be involved in RAS activation, CSW must also play a role either downstream of RAS/RAF activation or in a parallel signaling pathway that acts in conjunction with the RAS/MAPK pathway. One approach to understanding the role of CSW/SHP2 during RTK signaling has been to identify substrates for their phosphatase activity. A CSW substrate, the Daughter of Sevenless protein (DOS), was identified as a tyrosine-phosphorylated protein trapped in a complex with a catalytically inactive version of CSW (11Herbst R. Carroll P.M. Allard J.D. Schilling J. Raabe T. Simon M.A. Cell. 1996; 85: 899-909Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). DOS contains an amino-terminal pleckstrin homology domain and is most similar to the family of multiadaptor proteins that includes GAB-1 and IRS-1 (15Raabe T. Riesgo-Escovar J. Liu X. Bausenwein B.S. Deak P. Maroy P. Hafen E. Cell. 1996; 85: 911-920Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). The analysis of mutations that inactivate the dos gene has indicated that DOS is a positive component of the SEV pathway (11Herbst R. Carroll P.M. Allard J.D. Schilling J. Raabe T. Simon M.A. Cell. 1996; 85: 899-909Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 15Raabe T. Riesgo-Escovar J. Liu X. Bausenwein B.S. Deak P. Maroy P. Hafen E. Cell. 1996; 85: 911-920Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Together, these biochemical and genetic studies have led to the proposal that DOS must be dephosphorylated by CSW during SEV signaling. Searches for SHP2 substrates in mammalian cells have also led to the identification of a number of potential substrates. At present, these include the transmembrane protein SHPS1/SIRP1α, IRS-1, the PDGF receptor, and several tyrosine-phosphorylated proteins with sizes ranging from 80 to 120 kDa (reviewed in Refs. 4Neel B.G. Tonks N.K. Curr. Opin. Cell Biol. 1997; 9: 193-204Crossref PubMed Scopus (731) Google Scholar and 8Streuli M. Curr. Opin. Cell Biol. 1996; 8: 182-188Crossref PubMed Scopus (164) Google Scholar). In order to define further the role of CSW/SHP2 during RTK signaling, we have mutated each of the domains of CSW and investigated their effects on SEV signaling and Drosophila development. In this report, we provide evidence that the the PTP domain and at least one functional SH2 domain are essential for CSW function. Analysis of the ability of either mutant or wild type CSW to interact with DOS and SEV indicates that the association in vivo of CSW with SEV is not dependent on either the tyrosine phosphorylation of SEV or the presence of functional SH2 domains in CSW. In contrast, the association of DOS and CSW requires functional CSW SH2 domains. These results suggest that the role of the CSW SH2 domains is not in binding to SEV but is instead in the recognition of substrates, such as DOS. In addition, we found that a deletion of either the unique CSW PTP insert region or the conserved GRB2 binding site-containing carboxyl terminus of CSW does not appreciably reduce CSW function during signaling by SEV, TORSO, and perhaps all RTKs. Drosophila culture and crosses were carried out using standard procedures. The csw 13–87and csw C114 alleles used in this study have been characterized as genetic nulls, whereas the csw 6allele retains partial activity. All three alleles are recessive lethal (5Perkins L.A. Larsen I. Perrimon N. Cell. 1992; 70: 225-236Abstract Full Text PDF PubMed Scopus (328) Google Scholar). Tissue sectioning and scanning electron microscopy was performed as described (6Allard J.D. Chang H.C. Herbst R. McNeill H. Simon M.A. Development. 1996; 122: 1137-1146Crossref PubMed Google Scholar). Mutations in csw were generated using oligonucleotide mutagenesis as described previously (6Allard J.D. Chang H.C. Herbst R. McNeill H. Simon M.A. Development. 1996; 122: 1137-1146Crossref PubMed Google Scholar). The Arg to Lys substitutions in the SH2 domains of CSW were generated using oligonucleotide 5′-AGGAGGAGAGCTTGGCGAGGAAG-3′ forcsw R32K and oligonucleotide 5′-TCTGAGATTCCTTGACGAGAAAC-3′ for csw R137K. Thecsw R32K/R137K construct was made by subcloning fragments from the constructs containing the single point mutations. Deletions of the CSW PTP domain insert (amino acids 290–444) and the carboxyl terminus (amino acids 551–841) were generated using the oligonucleotides 5′-GATGTAGGTCTTAAACATCTCCAGGTCGCCGTCGGTGGGCAG-3′ and 5′-GACTTAGAAATTCATTTGTCAGGCGATCAGGGTCTGTATATA-3′, respectively. Thecsw construct encoding only the SH2 domains (csw SH2SH2) was made by placing a stop codon after residue 231 in the csw cDNA. The csw mutant cDNAs were subcloned into the transformation vector pKB267, which contains a hybrid promoter (SE) consisting of sevenless enhancers and thehsp70 promoter (16Basler K. Siegrist P. Hafen E. EMBO J. 1989; 8: 2381-2386Crossref PubMed Scopus (72) Google Scholar). This vector directs gene expression in a subset of cells in the eye, including R3, R4, and R7, as well as general expression in all cells following heat shock induction. Thecsw constructs were introduced into the germline by P element-mediated transformation using standard procedures. All flies used in this study were w 1118. The constructs were marked by the presence of the [w +] gene in the P-element. To assay for rescue of the zygotic lethality associated with homozygous csw loss-of-function mutations, larvae from a cross between males carrying the mutant P[SE-csw] construct and csw 13–87/FM7 females were heat shocked daily for 1 h at 37 °C until eclosion. The progeny were scored for the presence ofcsw 13–87/Y males, which indicated that the mutant P[SE-csw] construct could provide zygotic CSW function. To assay maternal CSW function, the csw 13–87/Y; P[SE-csw] males obtained from the zygotic rescue experiment were crossed with csw13–87/FM7 females. The resulting progeny were heat shocked daily during their development to induce expression of the mutant P[SE-csw] construct. This resulted in the production of homozygouscsw 13–87 females that carried the mutant P[SE-csw] construct. These zygotically rescued females were then scored for their ability to produce normal progeny when crossed to wild type flies. The production of normal progeny indicated that the mutant CSW was able to provide maternal CSW function. To measure the effect of mutant P[SE-csw] constructs on the ability of P[SE-csw G547E] to inhibit R7 development, ommatidia from flies heterozygous for the mutant P[SE-csw] construct and P[SE-cswG547E] were sectioned and the number of R7 cells was quantitated. Immunoblotting, immunoprecipitation, and glutathione S-transferase fusion protein precipitation experiments were carried out as described previously (11Herbst R. Carroll P.M. Allard J.D. Schilling J. Raabe T. Simon M.A. Cell. 1996; 85: 899-909Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). The csw and dos cDNAs were cloned into pAT-Hygro, which drives constitutive expression from theActin5C promoter (6Allard J.D. Chang H.C. Herbst R. McNeill H. Simon M.A. Development. 1996; 122: 1137-1146Crossref PubMed Google Scholar). The SEVS11 and SEVS11KM constructs were described previously (17Simon M.A. Dodson G.S. Rubin G.M. Cell. 1993; 73: 169-177Abstract Full Text PDF PubMed Scopus (364) Google Scholar). The SL2 cells were selected in either Hygromycin B (0.2 mg/ml) or G418 (1 mg/ml). For heat shock induction of SEVS11 and SEVS11KM, cells were incubated at 37 °C for 30 min and then returned to 23 °C. The antibodies used in this work were anti-CSW-CT (6Allard J.D. Chang H.C. Herbst R. McNeill H. Simon M.A. Development. 1996; 122: 1137-1146Crossref PubMed Google Scholar), anti-DOS-1, which was raised against the carboxyl terminus of DOS (amino acids 856–878), and anti-PY20 (Signal Transduction Laboratories). The pGEX-TK-CSWSH2SH2 domain construct contains amino acids 1–217 of CSW (11Herbst R. Carroll P.M. Allard J.D. Schilling J. Raabe T. Simon M.A. Cell. 1996; 85: 899-909Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). The csw R32K andcsw R137K SH2 domain mutations were transferred as restriction fragments into pGEX-TK-CSWSH2SH2. The fusion proteins were affinity purified on glutathione-agarose beads (Sigma). For precipitation of DOS, 4 μg of glutathioneS-transferase fusion protein was incubated with cell lysates (107 cell equivalents) that were prepared from pervanadate-treated SL2-DOS cells. Our approach to investigating the functional role of each of the domains of CSW was to introduce domain-specific mutations into a construct that contained a csw cDNA expressed under the control of a hybrid sevenless enhancer/heat shock promoter transcriptional control element (16Basler K. Siegrist P. Hafen E. EMBO J. 1989; 8: 2381-2386Crossref PubMed Scopus (72) Google Scholar). This transcription unit (SE) directs constitutive expression in a subset of cells in the Drosphila eye, including the photoreceptor R7, R3, and R4 precursor cells and all four of the cone cell precursors. In addition, the SE cassette also provides pulses of ubiquitous expression when the flies are grown at 37 °C for short periods. The mutated SE-csw constructs were introduced into the genome by P element-mediated germline transformation and assayed for function. In order to control for possible variation in transcription due to P element insertions at different sites in the genome, several different insertions were analyzed for each construct. In each case, equivalent results were obtained. The function of the mutated CSW proteins was tested in three ways. The first assay took advantage of a particular allele of csw that we had previously isolated in a genetic screen for mutations that attenuate signaling by SEV (18Simon M.A. Bowtell D.D. Dodson G.S. Laverty T.R. Rubin G.M. Cell. 1991; 67: 701-716Abstract Full Text PDF PubMed Scopus (649) Google Scholar). Although in vitro analysis of the CSWG547Eprotein indicates that it retains the ability to dephosphorylate model substrates such as p-nitrophenyl phosphate (data not shown), the mutant protein in vivo lacks normal CSW function and inhibits the functioning of wild type CSW (6Allard J.D. Chang H.C. Herbst R. McNeill H. Simon M.A. Development. 1996; 122: 1137-1146Crossref PubMed Google Scholar). The expression of CSWG547E in the developing eye under SEtranscriptional control blocks development of the R3, R4, and R7 photoreceptors and leads to flies with small eyes that are rough and disordered (6Allard J.D. Chang H.C. Herbst R. McNeill H. Simon M.A. Development. 1996; 122: 1137-1146Crossref PubMed Google Scholar) (see Fig. 3). Because expression of wild type CSW underSE control suppresses the effects ofSE-csw G547E, we assayed the signaling ability of mutant CSW proteins by determining whether their expression in thesevenless transcriptional pattern could also suppress theSE-csw G547E phenotype. The second assay took advantage of previous observations that animals that lack zygotic CSW function die during pupation due to severe developmental abnormalities in many of their tissues (5Perkins L.A. Larsen I. Perrimon N. Cell. 1992; 70: 225-236Abstract Full Text PDF PubMed Scopus (328) Google Scholar, 7Perkins L.A. Johnson M.R. Melnick M.B. Perrimon N. Dev. Biol. 1996; 180: 63-81Crossref PubMed Scopus (100) Google Scholar). Heat shock promoter-driven expression was therefore used to determine whether the mutant proteins could provide sufficient CSW function to rescue these defects in animals that otherwise lacked CSW function. The test was conducted by crossing flies carrying the SE-csw transgene to females heterozygous for a loss-of-function csw allele in order to generate males that were hemizygous for the defectivecsw allele but carried the SE-csw transgene. For these assays, we generally used both the csw 13–87and csw C114 alleles. Each of these alleles has been genetically characterized as a loss-of-function allele (5Perkins L.A. Larsen I. Perrimon N. Cell. 1992; 70: 225-236Abstract Full Text PDF PubMed Scopus (328) Google Scholar). The third assay was applicable only to the cases in which expression of the mutated protein was able to rescue the lethality of cswanimals. In these cases, the resulting csw 13–87/Y males carrying the SE-csw transgene were crossed tocsw 13–87/FM7c females in order to generate females that were homozygous for thecsw 13–87 allele but were alive due to rescue by the expression of the mutant CSW. These females were then tested for their fertility. Since maternal contribution of CSW is essential for TORSO signaling and proper embryonic development, the fertility of these females is a sensitive assay for the ability of the mutant CSW to function during TORSO signaling (5Perkins L.A. Larsen I. Perrimon N. Cell. 1992; 70: 225-236Abstract Full Text PDF PubMed Scopus (328) Google Scholar). SH2 domains possess an invariant arginine residue that is contained in a highly conserved motif (FLVRES) (19Koch C.A. Anderson D. Moran M.F. Ellis C. Pawson T. Science. 1991; 252: 668-674Crossref PubMed Scopus (1427) Google Scholar). Mutagenesis and crystallography studies have shown that this arginine residue is crucial for the interaction between an SH2 domain and the phosphotyrosine of its target binding sequence (20Schaffhausen B. Biochim. Biophys. Acta. 1995; 1242: 61-75PubMed Google Scholar). We therefore tested the importance of the CSW SH2 domains by mutating the corresponding arginines of each SH2 domain (Arg32 and Arg137) to lysines and assaying the ability of the resulting proteins to function in the assays described earlier. A construct containing mutations in both SH2 domains (CSWR32K/R137K) was unable to either rescue the zygotic lethality of male flies lacking csw function (csw13–87/Y) or to suppress the eye defects in flies carrying the SE-cswG547Ecassette (Fig.1 and TableI).Table IRescue of R7 photoreceptor development by mutant csw constructsGenotypeOmmatidia containing R7%P[SE-csw]>99P[SE-csw G547E]2.5P[SE-csw SH2SH2]<1P[SE-csw C583S]45P[SE-csw G547E]/P[SE-csw]99P[SE-csw G547E]/P[SE-csw R32K]16P[SE-csw G547E]/P[SE-csw R137K] 99P[SE-csw G547E]/P[SE-csw Δtail]96P[SE-csw G547E]/P[SE-csw SH2SH2] 99P[SE-csw G547E]/P[SE-csw src90]98Ommatidia were sectioned in the posterior portion of the eye and scored for the presence of the R7 photoreceptor cell. At least 250 ommatidia were counted for each genotype. Open table in a new tab Ommatidia were sectioned in the posterior portion of the eye and scored for the presence of the R7 photoreceptor cell. At least 250 ommatidia were counted for each genotype. These results demonstrated the importance of CSW SH2 domain function but did not indicate whether both SH2 domains are required. We addressed this question by testing the function of CSW proteins in which only a single SH2 domain was inactivated. Similar results were obtained with both CSWR32K and CSWR137K. In each case, the mutant protein was capable of providing sufficient CSW function to rescue the lethality of male flies (csw13–87/Y) otherwise lacking CSW function and of providing maternal CSW function in eggs derived from homozygouscsw 13–87 females (Fig. 1). Furthermore, the rescued male flies had smooth eyes with normally constructed ommatidia (data not shown). These results indicated that each of the individual SH2 mutant proteins was capable of providing sufficient CSW function to fulfill the normal roles of CSW and suggest that the two SH2 domains may perform redundant functions. In contrast to their ability to function as the sole maternal or zygotic source of CSW function during normal development, neither of the single SH2 domain mutated proteins was effective at suppressing the rough-eyed phenotype of SE-csw G547E flies (Table I). This may indicate that expression of CSWG547Einterferes with normal CSW function by using both of its SH2 domains to bind to an essential regulator or target of CSW. The CSWR32K and the CSWR137K proteins may be less effective competitors than wild type CSW because of the reduced binding affinity caused by the loss of one of the SH2 domain interactions. One possible role for the SH2 domains of CSW during SEV signaling is to allow CSW to bind to activated SEV. Previous studies of the role of SHP2 in signaling by the PDGF and epidermal growth factor receptors has indicated that the SHP2 SH2 domains bind directly to sites of autophosphorylation on the activated RTKs (21Vogel W. Lammers R. Huang J. Ullrich A. Science. 1993; 259: 1611-1614Crossref PubMed Scopus (488) Google Scholar, 22Feng G.S. Pawson T. Trends Genet. 1994; 10: 54-58Abstract Full Text PDF PubMed Scopus (168) Google Scholar, 23Lechleider R.J. Freeman Jr., R.M. Neel B.G. J. Biol. Chem. 1993; 268: 13434-13438Abstract Full Text PDF PubMed Google Scholar) and that this interaction can lead to the tyrosine phosphorylation of SHP2. We therefore asked whether the SH2 domains of CSW might be required to allow CSW to interact with activated SEV. We first sought to examine whether SEV and CSW can be found in a complex in Drosophilatissue culture cells (Schneider line 2, SL2 cells). Because SL2 cells do not express detectable levels of SEV and express only barely detectable levels of CSW, we used SL2 cells that had been engineered to constitutively express elevated levels of CSW under the control of theActin5C promoter and an activated form of SEV (SEVS11) under the control of a heat shock promoter (SL2-CSWSEVS11 cells). Heat shocking of these cells leads to the production of large amounts of catalytically active and tyrosine-phosphorylated SEV (11Herbst R. Carroll P.M. Allard J.D. Schilling J. Raabe T. Simon M.A. Cell. 1996; 85: 899-909Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar). Extracts of SL2-CSWSEVS11cells were immunoprecipitated with monoclonal antibodies to SEV. Examination of these anti-SEV immunoprecipitates by immunoblotting with anti-CSW antisera revealed the presence of CSW (Fig.2 A). While only a portion of the CSW within the SL2-CSWSEVS11 cells were found in the anti-SEV immunoprecipitates, the results nevertheless indicate that CSW and SEV can associate in Drosophila cells. Among the possible explanations for the low stoichiometry of the CSW·SEV complex are limited stability during the immunoprecipitation and the possible requirement for another cellular protein that was not overexpressed in the SL2-CSWSEVS11 cells. Despite the association of CSW and SEVS11 in SL2-CSWSEVS11cells, CSW was not detectably phosphorylated and thus does not appear to be a target for activated SEV (data not shown). Furthermore, a catalytically inactive CSW (CSWC583S) also associated with SEVS11 and failed to be tyrosine-phosphorylated, indicating that the lack of CSW phosphorylation was not due to autodephosphorylation (data not shown). To further investigate whether CSW and SEV interact in a manner similar to that reported for SHP2 and the PDGF receptor, we asked whether the association of CSW and SEVS11 depends on both the tyrosine phosphorylation of SEV and the SH2 domains of CSW. Two experiments were conducted. In the first, a line of SL2 cells (SL2-CSWSEVS11KM cells) was produced that constitutively expressed wild type CSW and inducibly expressed a catalytically inactive version of the SEVS11 protein. Anti-CSW immunoblots were then used to compare the level of CSW found in anti-SEV immunoprecipitates from SL2-CSWSEVS11 and SL2-CSWSEVS11KM cells. Contrary to our expectations, the amount of CSW complexed with SEV was not significantly different between the extracts of the two cell lines despite the lack of detectable tyrosine phosphorylation of SEVS11KM (Fig.2 A and data not shown). These results indicated that the complex between CSW and SEV is unlikely to involve direct interactions between CSW SH2 domains and SEV phosphorylation sites. In order to more directly assay the participation of the SH2 domains of CSW in the interaction with SEV, we conducted similar co-immunoprecipitation assays with extracts from a SL2 cell line (SL2-CSWR32K/R137KSEVS11) expressing CSW in which the SH2 domains were inactivated by mutation as described earlier. We found that the presence of the SH2 domain mutations did not markedly affect the CSW-SEV interaction. Together, these results indicate that the presence of functional CSW SH2 domains is not required for the association of CSW and SEV. Furthermore, these results raise the possibility that CSW and SEV might interact through another protein rather than directly with each other. The existence of an SH2- and phosphotyrosine-independent complex containing both CSW and SEV suggests that a crucial role of the SH2 domains of CSW might be in some aspect of CSW function besides recognition of phosphorylated SEV. One possible role might be in the recognition of phosphorylated substrates. Because the only known substrate for the PTP activity of CSW is the DOS protein, we investigated whether the SH2 domains of CSW might interact directly with phosphorylated DOS. Because our mutational analysis of the SH2 domain requirements for CSW indicated that either of the SH2 domains can at least partially fulfill the SH2 domain requirements for CSW, our expectation was that each SH2 domain would be capable of binding to phosphorylated DOS. We tested this prediction by assaying the ability of glutathione S-transferase fusion proteins containing either wild type
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