Interaction of the Grb10 Adapter Protein with the Raf1 and MEK1 Kinases*

André NantelDagger §, Khosro Mohammad-Ali, Jennifer SherkDagger , Barry I. Posner, and David Y. ThomasDagger par

From the Dagger  Eukaryotic Genetics Group, Biotechnology Research Institute, National Research Council, 6100 Royalmount, Montreal, Quebec H4P 2R2, Canada, the  Department of Experimental Medicine, McGill University, Montreal, Quebec H3A 2B2, Canada, and the par  Biology Department and Department of Anatomy and Cell Biology, McGill University, Montreal, Quebec H3A 2B2, Canada

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Grb10 and its close homologues Grb7 and Grb14, belong to a family of adapter proteins characterized by a proline-rich region, a central PH domain, and a carboxyl-terminal Src homology 2 (SH2) domain. Their interaction with a variety of activated tyrosine kinase receptors is well documented, but their actual function remains a mystery. The Grb10 SH2 domain was isolated from a two-hybrid screen using the MEK1 kinase as a bait. We show that this unusual SH2 domain interacts, in a phosphotyrosine-independent manner, with both the Raf1 and MEK1 kinases. Mutation of the MEK1 Thr-386 residue, which is phosphorylated by mitogen-activated protein kinase in vitro, reduces binding to Grb10 in a two-hybrid assay. Interaction of Grb10 with Raf1 is constitutive, while interaction between Grb10 and MEK1 needs insulin treatment of the cells and follows mitogen-activated protein kinase activation. Random mutagenesis of the SH2 domain demonstrated that the Arg-beta B5 and Asp-EF2 residues are necessary for binding to the epidermal growth factor and insulin receptors as well as to the two kinases. In addition, we show that a mutation in Ser-beta B7 affects binding only to the receptors, while a mutation in Thr-beta C5 abrogates binding only to MEK1. Finally, transfection of Grb10 genes with specific mutations in their SH2 domains induces apoptosis in HTC-IR and COS-7 cells. These effects can be competed by co-expression of the wild type protein, suggesting that these mutants act by sequestering necessary signaling components.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

The three members of the Grb7 family of signaling proteins, Grb7, Grb10 and Grb14, were first isolated through the ability of their SH21 domains to recognize phosphotyrosine-containing sequences on activated tyrosine kinase receptors. Grb7 has been shown to interact with the EGF, ErbB2/Her2, platelet-derived growth factor, and Fcepsilon RI receptors, the Ret proto-oncogene, the Syp phosphatase, and the SHC adapter protein (1-5). Grb10 has a much reduced affinity for the EGF receptor, but its interactions with the Ret proto-oncogene, ELK receptor, insulin-like growth factor-I receptor, and insulin receptor (IR) have been demonstrated in vivo (6-11). Grb14, the remaining member of this family, interacts in vitro with the platelet-derived growth factor receptor (12).

The function of the Grb7/10/14 proteins remains obscure. They are currently classified as adapter proteins, since they have no obvious enzymatic function and contain several conserved polypeptide-binding regions in addition to their carboxyl-terminal SH2 domain (see Fig. 1). A small proline-rich region in the NH2 terminus fits the consensus sequence for SH3-binding domains. In fact, in vitro binding by the Grb10 proline-rich domain to the cAbl SH3 domain has been demonstrated (10). Furthermore, all three of these proteins contain a central Pleckstrin homology domain. Similar elements have been shown to mediate protein-protein and/or protein-lipid interactions (for a review, see Ref. 13). The Grb7/10/14 genes are expressed in a tissue-specific pattern, and there is ample evidence of alternative splicing of the transcripts (1, 6, 7, 10-12, 14). Some of these splicing events result in the expression of proteins with altered properties; hGrb10beta 2 contains a deletion in the amino-terminal half of its Pleckstrin homology domain (6). Finally, the Grb7/10/14 proteins can be phosphorylated in vivo, and their levels of phosphorylation are regulated by hormone treatment (2, 4, 7, 12).

It is still unclear whether Grb10 acts as an inhibitor or an activator of signal transduction. Overexpression of hGrb10beta in Chinese hamster ovary-IR cells reduces insulin-dependent pp60 and insulin receptor substrate-1 phosphorylation and diminishes PI-3 kinase activation (6). Others have observed that microinjection of the Grb10 SH2 domain partially inhibits mitogenesis in insulin- or insulin-like growth factor-I-treated Rat1 fibroblasts but not in cells treated with EGF or serum (14). These results appear to be in contradiction with the work of Morrione et al. (15), whose cell lines overexpressing mGrb10alpha show growth reduction in the presence of insulin growth factor-I but not insulin. Unlike the microinjection experiments, these cells do not show an inhibition in S phase entry but rather an accumulation in the S and G2 phases. These contradictions might be explained by the use of different cell lines, experimental procedures, or Grb10 splicing variants. As for Grb7 and Grb14, both have been shown to be overexpressed in breast and prostate cancer tumors and cell lines (2, 12, 16).

The receptors that are recognized by the Grb7/10/14 family share the ability to activate the mitogenic MAP kinase signal transduction pathway (reviewed in Ref. 17). In this report, we present evidence that at least two members this pathway, Raf1 and MEK1, can interact with the SH2 domain of the Grb10 adapter protein. In some cultured cells, binding to Raf1 is constitutive, while the interaction of Grb10 with MEK1 is insulin-dependent. Interestingly, these interactions are phosphotyrosine-independent, and binding of MEK1 to Grb10 appears to be regulated through the retrophosphorylation of MEK1 by the MAP kinases. Finally, we have identified point mutations that affect the specificity of the Grb10 SH2 domain. Overexpression of these mutants was found to induce apoptosis in cultured cells.

    EXPERIMENTAL PROCEDURES
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Introduction
Procedures
Results
Discussion
References

Materials-- Most of our chemicals were purchased from Sigma. Restriction and modification enzymes as well as the plasmids and resins used for the expression and purification of GST and MBP fusions proteins were obtained from New England Biolabs or from Amersham Pharmacia Biotech. Sequencing reactions were performed with a DNA sequencing kit from Perkin-Elmer and separated on an Applied Biosystems DNA Sequencer model 370A. PCRs used the Expand High Fidelity PCR System (Boehringer Mannheim). Western blots were performed on Immobilon membranes (Millipore Corp.). The Grb10 (K-20), and Raf1 (C-12) antibodies were from Santa Cruz Biotechnology, the MEK1-NT antibody was from Upstate Biotechnology, and antibodies against the maltose-binding protein and activated ERK came from New England Biolabs. The anti-Flag (M2) monoclonal antibody was from Eastman Kodak Co. Horseradish peroxidase-conjugated secondary antibodies were purchased from Bio-Rad, and the ECL Western blotting detection reagents were from Amersham Pharmacia Biotech. Protein-A and Protein-G Sepharose were from Amersham Pharmacia Biotech. Finally, the HeLa cell cDNA library and all of our cell culture reagents were obtained from Life Technologies, Inc.

Two-hybrid Assays-- Following PCR amplification with the ANO-35 (GGGGGATCCAAATGCCCAAGAAGCCG) and ANO-36 (GCGCTCGAGGCTCTTTTGTTGCTTCCC) primers, the coding sequences of the full-length human MEK1 gene (a gift from S. Pelech) were introduced between the BamHI and XhoI sites of the pEG202 LexA fusion plasmid, resulting in the pAN104 construct. Two-hybrid screening of 2 × 106 primary transformants, from a human fetal brain cDNA library, was then performed exactly as described (18, 19).

EcoRI and XhoI sites were introduced around the coding sequence of the human Raf1 gene (a gift from S. Meloche) by PCR amplification. The fragment was then subcloned in the same sites of the pEG202 vector, yielding pAN130. A similar PCR amplification was also used to insert the regulatory (aa 1-330) or catalytic (aa 331-649) domains of Raf1, as well as the cytoplasmic domain of the insulin (aa 974-1370) and EGF (aa 672-1210) receptors, between the EcoRI and XhoI sites of pEG202. Subcloning of the partial MEK1 fusions in the pEG202 vector were performed as follows; N308, PCR amplification of the partial MEK1 gene with the ANO-35 and CTCGAGCCATGGGAGGTCGGCTGTCCTTCC primers; N293, subcloned 5' SmaI fragment of pAN104 plasmid; N220, removed 3' NcoI/XhoI fragments from pAN104; C304, subcloned the EcoRI/XhoI insert of the pMB6 cDNA (which encodes aa 304-392 of MEK1).

Quantitative beta -galactosidase assays were performed using a modification of the permeabilized cell assay of Guarente (20). Briefly, cells were grown in 1.5 ml of media to an approximate A600 of 0.5-1.0. They were then spun down for 2 min at 5000 rpm in a microcentrifuge and resuspended in 1.25 ml of Z-buffer (60 mM Na2HPO4·7H2O, 40 mM NaH2PO4·H2O, 10 mM KCl, 1 mM MgSO4·7H2O, 50 mM beta -mercaptoethanol, pH 7.0). We used 750 µl of the suspension to determine cell density by measuring the A600 (measuring cell density at this step reduced the introduction of errors due to loss of pelleted cells). We then added 25 µl of CHCl3 and 10 µl of 0.1% SDS to the rest of the cell suspension and vortexed at top speed for 10 s. The enzymatic reaction was started by the addition of 100 µl of ONPG solution (4 mg/ml o-nitrophenyl-beta -D-galactoside in Z-buffer) followed by incubation at 28 °C. Upon development of a pale yellow color, the reactions were stopped by the addition of 250 µl of 1 M Na2CO3. The samples were centrifuged at top speed for 2 min, and the amount of product was measured at A420. The activity, expressed as beta -galactosidase units, is calculated with the formula, (1000 × A420)/(A600 × reaction time (min)).

Amplification of a Full-length Grb10 Gene-- Two µg of DNA from a HeLa Cell cDNA library was used as a template in a PCR reaction with the GACGAATTCGAACCCATGGCTTTAGCCGGCTGCCCAG and GACCTCGAGCACAGACCGCTTCTTCACTCCAG primers. The resulting 2.2-kilobase pair fragment was fully sequenced on both strands and contains the coding sequences of the hGrb10zeta gene flanked by EcoRI and XhoI sites.

Production of Protein Fusions and Resin-binding Assay-- EcoRI/XhoI fragments containing the hGrb10zeta gene or the SH2 domain encoded by the pMB58 cDNA were subcloned in the pMAL-c2 plasmid. The MBP-hGrb10zeta and MBP-SH2 fusion proteins were then purified from Escherichia coli UT5600 cells by affinity chromatography on amylose resin columns as described by the manufacturer (New England Biolabs). Plasmids containing the GST-MEK1 and GST-ERK1 constructs were obtained from S. Pelech, while the SEK1 gene was obtained from Jim Woodgett. Expression and purification of GST-MEK1, GST-SEK1, and GST-ERK1 were performed as described (21). GST-Raf1 was constructed by inserting the EcoRI/XhoI insert of pAN130 in pGEX-5X-1 and purified as described by Zhang et al. (22).

For the resin-binding assays, one µg of the MBP fusion protein was incubated with 10-50 µl of glutathione-Sepharose resin, loaded with the GST-kinase fusions, in 1 ml of NETN buffer (20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5% Nonidet P-40, 5 mM dithiothreitol, 10 mg/ml bovine serum albumin) for 30 min at 4 °C. The resins were then spun down and washed three times with 1 ml of cold PBS containing 0.5% Nonidet P-40. Bound proteins were eluted by boiling in SDS-polyacrylamide gel electrophoresis buffer and detected by immunoblotting with an anti-MBP antibody. For the phosphatase assays, the GST-Raf1 and GST-MEK1 resins were first incubated with 0-4 units of potato acid phosphatase (Boehringer Mannheim) for 20 min at 30 °C in 40 mM Pipes, 50 mM NaCl, 1 mM beta -mercaptoethanol. The resins were then washed three times with 1 ml of NETN before being used in the resin-binding assays.

Grb10 Mutagenesis-- Site-directed mutagenesis of the Arg-beta B5 residue was done by overlap extension PCR (23). Random PCR mutagenesis of the pMB58 insert was performed according to Fromant et al. (24). The amplified fragment was then cleaved with EcoRI and XhoI and ligated back into pJG4-5. Following transformation in E. coli, colonies from >104 transformants were inoculated in 100 ml of 2YT plus 100 µg/ml ampicillin and grown for 4 h at 37 °C. Plasmids from this mutagenized library were then purified and used to transform Saccharomyces cerevisiae RFY206. One thousand yeast colonies were patched on glucose Trp- plates, which were subsequently replica-plated to generate four copies. One plate was kept as a master, while the other three were mated to lawns of S. cerevisiae EGY48 cells previously transformed with the pSH18-34 beta -galactosidase reporter plasmid as well as one of either the pAN129 (LexA-EGF receptor COOH terminus), pAN138 (LexA-IR COOH terminus), pAN168 (LexA-RAF NH2 terminus), or pAN104 (LexA-MEK1) bait plasmids. Following selection of the diploids on glucose His- Trp- Ura- plates, the cells were replica-plated to galactose/raffinose His- Leu- Trp- Ura- plates and scored for interaction. Plasmids from potentially interesting mutants were reisolated from the RFY206 master plates, sequenced, and retransformed in haploid EGY48 cells along with the pSH18-34 reporter and LexA fusion plasmids. Binding specificity was confirmed by quantitative beta -galactosidase assays on at least four independent transformants.

Cell Culture and Transfections-- 293 HEK, COS-7, and HTC-IR (25) cell lines were maintained in DMEM, supplemented with 10% fetal bovine serum (along with 400 µg/ml G418 for the HTC-IR), at 37 °C in a 5% CO2 atmosphere. The 8-aa Flag immunological tag was added at the carboxyl-terminal end of the hGrb10zeta polypeptide by PCR amplification followed by subcloning in the EcoRI/XhoI sites of the pcDNA3 expression vector (Invitrogen), yielding pAN185. Transient transfections with lipofectamine were performed according to the manufacturer's instructions (Life Technologies, Inc.). Incubation with the DNA-lipid complexes was carried on for 5 h followed by the addition of one volume of DMEM plus 20% fetal bovine serum. Twenty-four to forty-eight hours after transfection, the cells were starved overnight in DMEM plus 0.1% bovine serum albumin and then induced for 2-30 min with 100 nM insulin. Following hormone treatment, cells were washed with cold PBS, lysed for 10 min in lysis buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 100 mM NaF, 10 mM sodium pyrophosphate, 10% glycerol, 1.5% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 2 mM sodium vanadate) and spun down at maximum speed in a cold microcentrifuge for 10 min. Protein concentration was estimated with the Bio-Rad protein assay.

Production of Recombinant Adenovirus-- The adenovirus (AV) vector used in this report is a modification of the human strain 5 in which the E1a, E1b, and E3 regions are deleted (26). The insulin receptor cDNA was isolated from the CMVneo plasmid (a gift of A. Ullrich). The resulting 4.2-kilobase pair ClaI-SpeII fragment was blunted with Klenow and cloned in the EcoRV site of the pAdcmvpoly(A) transfer vector (a gift of B. Massie). The 1.6-kilobase pair EcoRI-XhoI fragment encoding the full-length hGrb10zeta was also blunted with Klenow and cloned in the EcoRV site of the transfer vector. The homologous recombination of the replication-defective human type 5 AV, large scale production, purification, and titration of the recombinant AV have been described in detail (27). All recombinant AV were stored at a concentration of 1-4 × 108 plaque-forming units/ml in DMEM supplemented with 10% fetal bovine serum. For the infections, 293 cells were transduced at a multiplicity of infection of 1 (1 plaque-forming unit/cell) for 2 h with stocks of either a control recombinant AV (Ad DE1/DE3) or recombinant AV (Ad-IR/Ad-Grb). Transduced cells were incubated for 24 h at 37 °C in 5% CO2 and then starved for 24 h in DMEM containing 0.1% bovine serum albumin. Cells were stimulated for 5 min with 100 nM insulin. The medium was then aspirated, and the cells were washed twice with PBS and solubilized in lysis buffer.

Immunoprecipitations-- Protein samples were diluted to equal concentration with lysis buffer and then incubated with the antibodies for 1 h at 4 °C. Protein A- or Protein G-Sepharose beads were then added, and the samples were incubated for another 1 h. The beads were then washed three times with PBS, and the bound proteins were released by boiling in SDS-polyacrylamide gel electrophoresis sample buffer.

Detection of Apoptosis by Microscopy and Flow Cytometry-- The RL (GCTTTTTCTCCTCCTTGACAGCCAGAG), TS (GCATTTGTACTCTCACTGTGTCATCACC) and SC (CTCCTCCGTGACTGCCAGAGTAATCC) primers were used to introduce SH2 domain mutations in the pAN185 plasmid using the Chameleon kit from Stratagene. The resulting pAN200, pAN208, and pAN209 plasmids express Grb10-R520L, Grb10-T531S, and Grb10-S522C, respectively. One million HTC-IR cells or 1-2 × 105 COS-7 cells were inoculated in 35-mm wells and co-transfected with 0.5 µg of pAdcMV5GFPQ (a gift from B. Massie) along with 1 µg of either the pcDNA3 vector alone or one of the Grb10 expression plasmids. Twenty-four hours later, the cells were observed in a fluorescence microscope. Cells that were small, round, and showed nuclear fragmentation or condensation following Hoecht staining were counted as apoptotic. HTC-IR cells were also washed in PBS, trypsinized, and analyzed by flow cytometry. For the detection of apoptotic cells by the TUNEL assay, we omitted the GFP plasmid and used the ApoBrdU kit and MPlus software (Phoenix Flow Systems).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Interaction of Grb10 with MEK1 in a Two-hybrid Screen-- The SH2 domain of Grb10 was isolated from the two-hybrid screen of a human fetal brain cDNA library using the full-length MAP kinase kinase MEK1. Our screen of 2 × 106 primary transformants resulted in the isolation of five independent genes. The pMB58 cDNA3 is almost identical to the 3'-end of the Grb10 gene (6, 10, 14) including the region that encodes the SH2 domain (Fig. 1A). A sequence comparison (Fig. 1B) between pMB58 and published Grb10 sequences suggests that this cDNA represents a novel splice variant with modifications in the extreme amino-terminal end of the SH2 domain. The borders between the regions of high and low sequence homology all contain sequences that are consistent with intron-exon borders. The variable region encoded by pMB58 still contains all the residues that have been shown to be important for SH2 domain function, namely a Trp-Phe-His motif in the first beta -sheet as well as a conserved arginine residue 2 amino acids into the first alpha -helix (Fig. 1C).


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Fig. 1.   Structure of the Grb10 proteins. A, three domains, conserved among Grb7, Grb10, and Grb14 are shown as boxes. The lines below represent the splicing variants identified in humans as well as the extent of the partial polypeptide encoded by the MEK1-binding cDNA (pMB58). Regions whose aa sequences differ significantly among the variants are illustrated as small boxes. B, DNA sequence alignment between the 5'-end of the pMB58 cDNA and the other hGrb10 cDNAs (base pair labeling is that of the hGrb10zeta variant). Nucleotide sequences from the linker are shown in lowercase type up to the 5' EcoRI site. Putative intron-exon borders in the hGrb10 sequence are underlined. C, amino acid sequence alignment of the beginning of the polypeptide encoded by the pMB58 cDNA with the SH2 domain of the hGrb10 proteins (aa labeling is that of the hGrb10zeta variant). Amino acids derived from vectorial sequences are shown in lowercase type, while dashes represent insertions. Identities are marked as a vertical dash. The cylinder and arrow define the position of major features in the SH2 domain secondary structure, while the asterisk denotes residues thought to be important for interaction with tyrosine-phosphorylated targets. The remaining pMB58 sequences are identical to the other hGrb10 genes.

Isolation of a Novel Grb10 Splice Variant-- Unfortunately, we were unable to isolate longer Grb10 cDNAs from the fetal brain library. Using primers based on published sequence data (6), we isolated by PCR the coding sequence of a full-length human Grb10 gene from a HeLa cell cDNA library. Sequencing of the 2.2-kilobase pair fragment revealed that this clone is a hybrid between hGrb10beta (6) and hGrb10gamma (10, 14) (see Fig. 1A). We named this new human splice variant, simultaneously isolated by Dong et al. (28), hGrb10zeta .2,4 It contains the longer amino-terminal sequences found in hGrb10beta as well as the complete Pleckstrin homology domain seen in the other human and mouse variants. Inclusion of the pMB58 SH2 domain variant into this nomenclature will have to await the isolation of a full-length cDNA. Two forms of Grb10 were thus used in our experiments. The full-length Grb10 protein encoded by the hGRB10zeta variant was used in most experiments, while for simplicity, experiments using the SH2 domain used the sequences of the pMB58 clone.

Grb10 Interacts with Phosphorylated Raf1 and MEK1-- Both the full-length hGrb10zeta protein and the SH2 domain initially isolated in the pMB58 cDNA were purified from bacterial extracts as fusions with the E. coli MBP. To confirm that the full-length hGrb10zeta could also bind to MEK1, we incubated the MBP-SH2 and the MBP-hGrb10zeta fusion proteins with glutathione-Sepharose resins loaded either with GST control protein or with GST fusions of various kinases. Interestingly, we found that MBP-SH2 was not only retained by the GST-MEK1 resin but also by GST-Raf1. We also observed binding by the full-length MBP-hGrb10zeta protein to the GST-MEK1 (Fig. 2C) and GST-Raf1 resins (Fig. 3), indicating that the modifications in the pMB58 SH2 domain are not solely responsible for this binding specificity.


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Fig. 2.   In vitro binding of Grb10 to Raf1 and MEK1. A, anti-MBP immunoblot showing the interaction of either MBP-LacZ or MBP-SH2 to resins loaded with GST alone or with GST-MEK1. The Control lanes contained 10 ng of the indicated MBP fusion. B, same experiment as in A except for the use of a resin loaded with GST-Raf1. C, glutathione-Sepharose resins, loaded with equal amounts of GST alone, GST-MEK1, or GST-SEK1 were used to pull down an MBP fusion of the full-length hGbr10zeta protein. The bound MBP fusion was detected by immunoblotting. Equivalent loading and autophosphorylation activity of the kinases on the resins was confirmed by Coomassie staining of SDS-polyacrylamide gels and autoradiography following autophosphorylation with [gamma -32P]ATP. D, anti-phosphotyrosine blot of 5 µg of resin-bound GST-Raf1, GST-MEK1, and GST-ERK1.


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Fig. 3.   Grb10 binds to phosphorylated Raf1 and MEK1. Glutathione-Sepharose resins, loaded with GST-Raf1 or GST-MEK1, were incubated with 0-4 units of potato acid phosphatase and then extensively washed. They were then used in a resin-binding assay on MBP-hGrb10zeta . The amount of precipitated MBP fusion was determined by an anti-MBP immunoblot (top panels), while the integrity of the resin-bound kinases was confirmed by Coomassie staining (bottom panels).

To determine if Grb10 will interact with any available kinases, we repeated the resin-binding assays using SEK1, a stress-activated kinase (also called JNKK, MEK4, or MKK4) (29-32) that is 44% identical and 63% homologous to MEK1. Although both the GST-MEK1 and the GST-SEK1 resins contained equivalent amounts of bound proteins and both kinases had roughly similar autophosphorylation activities, only the MEK1 resin could effectively retain the MBP-hGrb10zeta protein (Fig. 2C). Thus, binding by Grb10 shows some specificity to members of the mitogenic response pathway.

To confirm whether the SH2 domain recognizes a phosphotyrosine-containing sequence on the bacterially expressed kinases, we probed the resin-bound GST-Raf1, GST-MEK1, and a GST fusion to the MAP kinase ERK1 with an anti-phosphotyrosine antibody. Fig. 2D clearly shows that, as described previously (21, 33, 34), only the bacterially expressed GST-ERK1 contains phosphotyrosine residues. This suggests a novel mode of interaction between the Grb10 SH2 domain and the Raf1 and MEK1 kinases that does not depend on phosphotyrosine residues.

There is growing evidence that certain SH2 domains can recognize their targets through non-phosphotyrosine residues (35-40). To determine if the Grb10 SH2 domain recognizes another phosphorylated amino acid on Raf1 and MEK1, we incubated the appropriate GST resins with increasing concentrations of potato acid phosphatase. These resins were then washed and used in a binding assay with the full-length MBP-hGrb10zeta . As shown in Fig. 3, binding of MBP-hGrb10zeta to both Raf1 and MEK1 was greatly reduced when the resins were pretreated with the phosphatase. A Coomassie-stained gel of the same samples confirmed that equal amounts of resin-bound proteins were used and that the reduced binding is not the result of proteolytic degradation of the kinases. Thus, either the Grb10 SH2 domain recognizes a phosphothreonine- or phosphoserine-containing sequence, or the modified structures of the dephosphorylated kinases are less desirable targets for Grb10 binding.

Localization of the Grb10-binding Site on Raf1 and MEK1-- We returned to the two-hybrid assay both to confirm the observed Grb10-Raf1 interaction and to map the Grb10-binding sites on the Raf1 and MEK1 kinases. Neither the full-length Raf1 nor its carboxyl-terminal catalytic domain (aa 331-648) interacted with the SH2 domain in the two-hybrid assay. The SH2-binding domain in Raf1 was located in its NH2-terminal regulatory domain (aa 1-330), since a LexA fusion of this region was able to confer growth on media lacking leucine to cells containing the pMB58 cDNA (Table I). A deletion of the 88 amino acids in the carboxyl terminus of MEK1 abolished interaction with the SH2 domain. We established that this absence of interaction is not the result of a two-hybrid artifact, since two of the COOH-terminal mutants (1-308 and 1-293) were still capable of interacting with other MEK1-binding clones that were isolated in our initial screen (results not shown). Due to the high background, most of the results from the amino-terminal deletions were inconclusive except for the 304-393 construct in which growth with the Grb10 SH2 domain was clearly faster than with the vector alone. The carboxyl-terminal domain of MEK1 (aa 304-393) contains 9 Ser/Thr residues including Thr-386, which is phosphorylated by MAP kinases in vitro (41, 42). Another target for MAP kinase phosphorylation, Thr-292, lies very close to the end of the 1-308 mutant. After mutating either residue to alanine, a quantitative two-hybrid assay (Table II) demonstrated that Thr-292 is not involved in the interaction, while the T386A mutation reduced the affinity of MEK1 for Grb10 by almost half. These results thus locate the Grb10-binding site in the extreme COOH terminus of MEK1, a region that, incidentally, is not conserved in the sequence of the SEK1 kinase.

                              
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Table I
Mapping of the Grb10-binding sites by the two-hybrid assay
Full-length or partial Raf1 and MEK1 genes were fused to the LexA DNA-binding domain of pEG202 and tested in a two-hybrid assay against either the pMB58 cDNA, which encodes the Grb10 SH2 domain, or an empty pJG4-5 vector. Binding was detected by the appearance of colonies on leucine-deficient media after 2 (++) or 4 (+) days of growth.

                              
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Table II
Binding of MEK1 mutants to the Grb10 SH2 domain
Results of quantitative two-hybrid assays of LexA fusions of the wild type MEK1 or the T292A and T386A point mutants along with an acidic domain fusion of the Grb10 SH2 domain encoded in the pMB58 cDNA are shown. Results are given in beta -galactosidase units ± S.D. (n = 6). The p values represent the probability that the mutants have the same affinity for Grb10 as the wild type MEK1.

In Vivo Interaction among Grb10, Raf1, and MEK1-- To determine whether the interaction of Grb10 with Raf1 and MEK1 also occurs in vivo, we performed immunoprecipitations in Triton-soluble protein extracts taken from a variety of sources. Fig. 4A shows the results obtained from 293 HEK cells in which both hGrb10zeta and the insulin receptor were expressed using adenovirus vectors. In these cells, Grb10 was co-immunoprecipitated by an anti-Raf1 antibody in both starved and insulin-treated cells (lanes 3 and 4). We also used the anti-Flag monoclonal antibody to efficiently co-immunoprecipitate p74raf1 from extracts of 293 HEK cells transiently expressing a hGrb10zeta -Flag protein (Fig. 4B). As in the virus-infected 293 HEK cells, this interaction occurred irrespective of the presence of mitogenic agents. However, the Grb10-Raf1 interaction appears to be cell type-specific, since we were unable to co-immunoprecipitate these proteins from transfected NIH-3T3 or HTC-IR cells (not shown).


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Fig. 4.   Grb10 interacts with Raf1 and MEK1 in vivo. A, anti-Grb10 immunoblots from extracts of starved and insulin-treated 293 HEK cells that express both the insulin receptor and the hGrb10zeta proteins from an adenoviral vector. Lanes 1 and 2 contain 20 µg of Triton-soluble extracts; lanes 3 and 4 contain proteins immunoprecipitated with an anti-Raf1 antibody; and the extracts in lanes 5 and 6 were immunoprecipitated with a MEK1-specific antibody. B, Raf-1 immunoblot of anti-Flag (M2) immunoprecipitates of extracts from starved and serum-treated 293 HEK cells transfected with the empty pcDNA3 vector or one expressing a hGrb10zeta protein containing a Flag epitope on its carboxyl-terminal end. C, top, MEK1 immunoblot of anti-Flag (M2) immunoprecipitates of extracts from HTC-IR cells, transfected with the hGrb10zeta -Flag construct, that were starved or treated with 100 nM insulin for varying amounts of time. Middle and bottom, total soluble proteins probed with antibodies against either activated or total ERK MAP kinases.

The binding of Grb10 to MEK1 in adenovirus-infected 293 HEK cells was greatly increased following treatment with insulin (Fig. 4A, lanes 5 and 6). In vivo binding of Grb10 to MEK1 was also observed in NIH-3T3 cells transfected with an hGrb10zeta -Flag construct (not shown). Following the observation that the Thr-386 residue of MEK1 might be involved in interactions with Grb10 (Table II), we compared the kinetics of insulin-dependent MAP kinase activation with those of the Grb10-MEK1 interaction. HTC-IR cells were transfected with the hGrb10zeta -Flag construct, starved overnight, and treated with 100 nM insulin for 2, 5, 15, or 30 min. As seen in Fig. 4C, co-immunoprecipitation of MEK1 by the Flag antibody was first detectable after 5 min of treatment and peaked at 15 min. MAP kinase activation, as detected with an antibody that specifically recognizes phosphorylated ERK, was first observed after 2 min and peaked at 5 min. The maximum levels of Grb10-MEK1 interaction thus follow MAP kinase activation by insulin.

Mutagenesis of the Grb10 SH2 Domain-- The structure of several SH2 domains has revealed that an invariant arginine (Arg-520 in the case of hGrb10zeta ) interacts with the phosphate group of phosphotyrosine residues. We introduced an Arg-520 to leucine mutation in the pMB58 SH2 domain and observed that this mutant failed to interact, in a two-hybrid assay, with LexA fusions of the carboxyl-terminal domain of the insulin and EGF receptors, the regulatory domain of Raf1, or the full-length MEK1 kinase. Immunoblotting of total yeast cell extracts confirmed that expression of this mutant is not significantly different from that of the wild type SH2 domain (result not shown). This confirmed that a conserved residue, which is involved in the interaction of SH2 domains with the phosphate group of phosphotyrosine residues, is also necessary for the recognition of Raf1 and MEK1 by Grb10 although neither appears to be tyrosine-phosphorylated.

To identify additional residues involved in the interaction of Grb10 with the kinases, a library of mutagenized SH2 domains was generated by PCR amplification and screened for mutants defective in the interaction only with tyrosine kinase receptors, the amino-terminal domain of Raf1, or the full-length MEK1 kinase. Because of the high background resulting from expression of the LexA-IR fusion, we also tested our mutants with a LexA fusion of the carboxyl-terminal catalytic domain of the EGF receptor.

The positions of the point mutations are identified using the nomenclature for SH2 domain residues, first described by Waksman et al. (43), in which amino acids are labeled according to their position relative to major structural domains. Detailed structural data are available from three different SH2 domains, Src (43, 44), Lck (45), and the NH2-terminal SH2 domain of the mouse Syp phosphatase (46). The structural features of these domains being relatively well conserved, we used an alignment of the pMB58 SH2 domain with the NH2-terminal SH2 domain of the Syp phosphatase (47) (Fig. 5A) to better interpret our mutagenesis data.


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Fig. 5.   Characterization of SH2 domain mutants with altered binding specificities. A, alignment of the pMB58 SH2 domain with the amino-terminal SH2 domain of the mouse phosphatase Syp (47). Bars represent amino acid identities, while conservative substitutions are shown with semicolons. Arrows and cylinders show the positions of beta -sheets and alpha -helices observed in various SH2 domains and used in the labeling of residues. Hexagons show the positions of amino acids that had been shown, in the Src, Lck, and Syp structures, to be involved in interactions with the aromatic ring of the phosphotyrosine residue. The positions of the various point mutations are indicated. B, six plasmids, encoding point mutations in the SH2 domain, were retransformed in haploid yeast cells along with one of four LexA fusions, and their binding specificity was confirmed through quantitative beta -galactosidase assays. Values are given as percentages of the binding observed using the wild type SH2 domain, with error bars representing the S.D. (n = 4). Ventral (C) and side views (D) of the three-dimensional structure of the Syp amino-terminal SH2 domain complexed with a phosphotyrosine-containing high affinity peptide are shown (47). In D, some of the frontal residues were sliced off to give a better view of the phosphate-binding pocket. Amino acids from Syp are colored blue, while the atoms of the target molecule are colored yellow (phosphate group) and green (peptide). The atoms equivalent to the Grb10 SH2 domain mutations have been labeled according to their binding specificities (red, inactivating mutation; light blue, impaired binding to tyrosine kinase receptors; orange, impaired binding to all targets but especially Raf1; magenta, impaired binding to MEK1).

The binding specificities of the mutant SH2 domains are shown in Fig. 5B. In addition to the Arg-beta B5 right-arrow Leu mutant, the Arg-beta B5 right-arrow Cys and Asp-EF2 right-arrow Val mutants (shown in red in Fig. 5, C and D) also failed to interact with the LexA fusions. The Ser-beta B7 right-arrow Cys mutation (colored in light blue) shows a greatly reduced affinity for the EGF and insulin receptor, moderate effects on binding to MEK1, and an increased affinity for Raf1. We failed to isolate any mutant that unequivocally affected binding only to Raf1. The best of such mutants is the Phe-alpha B7 right-arrow Tyr mutation (colored in orange), which shows reduced affinity to the receptors and the MEK1 kinase along with its inability to recognize Raf1. In Syp, the terminal hydroxyl group of this tyrosine residue protrudes into the protein-binding groove (see Fig. 5C). Finally, the Thr-beta C5 right-arrow Ser mutant (colored magenta) shows a relatively normal affinity for the EGF receptor, greatly increased binding to Raf1 and the insulin receptor, and no affinity for MEK1. A serine at position beta C5 is normally seen in most of the other conventional SH2 domains, including those found in Syp, Grb7, and Grb14. Although this residue in Syp does not lie next to the tyrosine-phosphorylated peptide, it makes extensive contacts with, and is believed to orient, the Arg-beta B5 residue, a major constituent of the phosphate-binding pocket (Fig. 5D).

Induction of Apoptosis by Grb10 Mutants-- The Arg-beta B5 right-arrow Leu, Ser-beta B7 right-arrow Cys, and Thr-beta C5 right-arrow Ser mutations were introduced in the full-length hGrb10zeta gene. These constructs (named Grb10-RL, Grb10-SC, and Grb10-TS) were co-transfected in HTC-IR cells along with a reduced amount of a second plasmid containing the gene for the green fluorescent protein (GFP). Consistent with the results of Morrione et al. (15), overexpression of wild type Grb10 had little effect on cell morphology and survival, but initial observations by fluorescence microscopy, followed by more stringent analysis by flow cytometry, showed a strong reduction in the number of GFP-positive adherent cells cotransfected with either of the three SH2 domain mutants (Fig. 6A). Most of the GFP-positive cells were dead and could be found floating in the media, while the remaining adherent cells were smaller, rounder, and more refractive than normal (results not shown, but see Fig. 8). A TUNEL assay (49), which detects DNA strand breaks induced by programmed cell death, also showed an increase in the number of apoptotic cells following transfection with the Grb10-RL, Grb10-TS, and Grb10-SC plasmids (Fig. 6B). Because the small size and low transfection efficiencies of the HTC-IR cells made detailed analysis difficult, we repeated the transfections in COS-7. As seen in Fig. 7A, transfection of these normally large cells with SH2 domain mutants of Grb10 increased the proportion of small round cells from 17-23 to 42-53%. Co-staining of these cells with the DNA-binding dye Hoecht 33258 revealed the nuclear fragmentation that is one of the structural hallmarks of apoptosis (Fig. 8). Cells transfected with the pcDNA3 vector were similar to those expressing Grb10, while those transfected with Grb10-RL or Grb10-SC looked like cells transfected with Grb10-TS (results not shown). Finally, the effects of the Grb10-RL mutant can be reversed by co-expression of wild type Grb10 (Fig. 7B).


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Fig. 6.   Induction of apoptosis by Grb10 mutants in HTC-IR. A, detection of green fluorescent cells by flow cytometry 24 h following cotransfection of HTC-IR cells with pAdcMV5-GFPQ along with either the pcDNA3 vector alone or plasmids expressing various Grb10 constructs. B, TUNEL assay of HTC-IR cells transfected with plasmids containing the various Grb10 plasmids. These two-dimensional graphs shown the incorporation of bromodeoxyuridine in DNA strand breaks (anti-BrdU FITC) in relation to DNA content (as determined by propionium iodide). To account for untransfected cells and lipofectamine cytotoxicity, the data graph of cells transfected with the pcDNA3 control was subtracted from the results of the Grb10-transfected cells.


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Fig. 7.   Induction of apoptosis by Grb10 mutants in COS-7 cells. A, proportion of small round cells with condensed/fragmented nuclei in the GFP-positive population of COS-7 cells 24 h following transfection with either the pcDNA3 vector or one of the Grb10-expressing plasmids. Results were obtained by counting 400 cells in each of three independent transfections. B, same assay as in A except that the cells were transfected with the indicated ratio of the Grb10 and Grb10-RL expression plasmids. DNA content was kept constant through the addition of pcDNA3, and the error bars represent the S.D. from two independent transfections.


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Fig. 8.   Evidence of nuclear fragmentation in COS-7 cells transfected with Grb10 mutants. Fluorescence micrograph (magnification × 200) of COS-7 cells 24 h following transfection with plasmids expressing GFP along with either wild type Grb10 (A and B) or Grb10-TS (C and D). Panels A and C show GFP fluorescence, while panels B and D show Hoecht nuclear staining. The arrows indicate GFP-positive cells scored as normal (open) or apoptotic (filled).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We used the yeast two-hybrid assay, resin-binding assays, and co-immunoprecipitations to demonstrate that Grb10 interacts with at least two members of the mitogenic MAP kinase cascade: Raf1 and MEK1. The extreme 5'-end of the cDNA initially isolated in the two-hybrid screen differs slightly from other Grb10 clones, possibly the result of splicing variation. To confirm that the interactions with the kinases are not limited to this specific SH2 domain variant, we used PCR to isolate a full-length human Grb10 cDNA, hGrb10zeta , and observed, through resin-binding assays and immunoprecipitations, that it too can interact with Raf1 and MEK1. Phosphotyrosine blots, phosphatase treatment, and mutagenesis of the kinases have demonstrated that the recognition of Raf1 and MEK1 by the Grb10 SH2 domain is mediated by phosphothreonine or phosphoserine residues. Examples of phosphotyrosine-independent binding by SH2 domains have been described before. These include the recognition of Bcr by the SH2 domains of cAbl, phospholipase Cgamma , Src, and GTPase-activating protein (35, 36) and the binding of vAlb to Shc (40). It has also been reported that the SH2 domains of Fyn and Src interact with a phosphoserine residue on Raf (39), while a 62-kDa ubiquitin-binding protein is a phosphotyrosine-independent ligand of the p56lck SH2 domain (37). There is also evidence for the interaction of the SH2 domain of the Syp phosphatase with its own catalytic domain in the absence of phosphotyrosine (38).

In a two-hybrid assay, only the isolated amino-terminal domain of Raf1 binds to the Grb10 SH2 domain. The absence of interaction with the full-length kinase is either an indication that the binding site is masked in the context of the whole protein or simply an artifact (our laboratory and others have frequently observed in two-hybrid assays that, while individual domains will interact with a given protein, the full-length protein will not (49)). In vivo interaction between Raf1 and Grb10 was observed in resting 293 HEK cells, which suggests that binding is mediated via a constitutively phosphorylated residue. For example, in unstimulated Balb/3T3 cells, Ser-43 and Ser-259, both of which are located in the regulatory domain of Raf1, are phosphorylated by means of a mechanism other than autophosphorylation (34). Binding between Grb10 and Raf1 is cell-type specific, since we failed to detect this interaction in NIH-3T3 and HTC-IR cells. The Grb10-binding site on MEK1 appears to be located in its carboxyl-terminal tail. Threonine 386, a residue that is phosphorylated in vivo by MAK kinases (41, 42, 50, 51), was shown to play a partial role in Grb10 recognition. The residual binding of the SH2 domain to the MEK1-T386A mutant indicates that phosphorylation at this residue might not be as critical as it is for phosphotyrosine-containing targets. The difficulty in synthesizing threonine-phosphorylated peptides has greatly hampered further research into this area.

Based on the published structures of SH2 domains bound to phosphotyrosine targets, we know that Arg-beta B5 is positioned at the heart of the phosphate-binding pocket and makes hydrogen bonds with the phosphate residue. This interaction is also critical in the binding of the SH2 domain to Raf1 and MEK1, thus reinforcing our hypothesis for the recognition of a phosphoamino acid by Grb10. The equivalent of another SH2 domain residue necessary for interaction with the tyrosine kinase receptors and the two kinases, Asp-EF2, is positioned in Syp next to the polypeptide-binding groove and can theoretically make interactions with residues downstream of the phosphorylated amino acid. Structural analysis (43, 44, 46) and random mutagenesis of the phosphatidylinositol 3-kinase SH2 domain (52) have previously demonstrated the importance of the EF loop in binding specificity. The positions of the beta B5 and EF2 mutants therefore suggest that the tyrosine-phosphorylated receptors and the Raf1 and MEK1 kinases interact with the Grb10 SH2 domain in a very similar fashion, involving interactions with a phosphorylated amino acid as well as with residues located further downstream. These results also suggest that the Grb10 SH2 domain cannot interact with more than one of these targets simultaneously, although this hypothesis has not been tested experimentally. Other residues in the Grb10 SH2 domain were shown to be involved in interaction with only some of its targets. One of these is Ser-beta B7, which is conserved between Grb10 and Syp and, in the latter case, makes hydrogen bonds with the phosphotyrosine. Mutagenesis of this residue into a cysteine has much greater effects on interactions with the receptors. If binding of the Raf1 and MEK1 kinases to Grb10 is coordinated by a much shorter phosphoserine or phosphothreonine residue, the phosphate group might not be in a position to make significant interactions with Ser-beta B7. Another mutant, Thr-beta C5 right-arrow Ser, abrogates binding only to MEK1. Interestingly, almost all SH2 domains contain a serine at position beta C5, including those found in Grb7 and Grb14. Other elements of the Grb10 SH2 domain have not been studied in detail but might also play a role in its binding specificity. For example, two residues that are critical for phosphotyrosine interactions in most SH2 domains, His-beta D4 and Lys-beta D6, are not conserved in Grb10. It was suggested, based on the Syp three-dimensional structure, that the beta D-strand prevents shorter (3.5-Å) phosphothreonines and phosphoserines from coordinating with the Arg-beta B5 without an intervening water molecule, thus permitting interactions with only the 7.0-Å phosphotyrosine (46). In addition, the residue at position beta D6 was recently shown to be a major determinant in the recognition of the tyrosine-phosphorylated ErbB2 receptor by Grb7 (53).

Fath et al. (54) have shown that expression of an isoform of the Grb2 adapter protein that is missing part of its SH2 domain induces apoptosis in Swiss 3T3 cells. We observed a similar phenomenon when the expression of Grb10 point mutants, whose SH2 domains are unable to interact with either MEK1 or with two tyrosine kinase receptors, increased the number of apoptotic cells following lipofectamine-mediated transfection. Reversal of the cell death phenotype by concomitant expression of the wild type Grb10 suggests that these mutants are acting by sequestering necessary signaling components. It has already been demonstrated that some tyrosine kinase receptors and the RAF-MEK-ERK pathway inhibit apoptosis (56, 57) and that the equilibrium between the ERK and JNK pathways determines the choice between proliferation and programmed cell death (57, 58).

In conclusion, our work places Grb10 in the expanding family of kinase-anchoring (or scaffolding) proteins (for reviews, see Refs. 59 and 60). These include the budding yeast Ste5p (49, 61, 62), the Ksr kinase (63, 64), the phosphoserine-binding 14-3-3 protein (65, 66), AKAP79 (67), its close homologue Gravin (68), and the SH2/SH3 adapter proteins Nck (69, 70) and Grb2 (71). The specific role of Grb10 in the transmission of proliferative signals including the possibility that it, like many of these proteins, is involved in the subcellular localization of multiprotein complexes is still being investigated.

    ACKNOWLEDGEMENTS

We thank Roger Brent, Bernard Massie, Sylvain Meloche, Steve Pelech, Axel Ullrich, and Jim Woodgett for gifts of plasmids used in this study. We are also grateful to Csilla Csank, Louise Larose, Ekkerhard Leberer, and Malcolm Whiteway for comments and to Lucie Bourget for expertise in flow cytometry.

    FOOTNOTES

* This is National Research Council of Canada publication number 41397.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Recipient of a postdoctoral fellowship from the National Science and Engineering Research Council of Canada. To whom correspondence should be addressed: Tel.: 514-496-6145; Fax: 514-496-6213; E-mail: andre.nantel{at}bri.nrc.ca.

1 The abbreviations used are: SH2 and SH3, Src homology 2 and 3, respectively; AV, adenovirus(es); EGF, epidermal growth factor; GFP, green fluorescent protein; GST, glutathione S-transferase; IR, insulin receptor; MBP, maltose-binding protein; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; aa, amino acids; DMEM, Dulbecco's modified Eagle's medium, TUNEL, TdT-mediated dUTP nick end-labeling.

2 We use the nomenclature for Grb10 splice variants that was agreed upon by several researchers and is maintained on the World Wide Web at www.bri.nrc.ca/thomasweb/grb7.htm.

3 GenBankTM accession number AF000018.

4 GenBankTM accession number AF000017.

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Abstract
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Procedures
Results
Discussion
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