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Binding Specificity and Mutational Analysis of the Phosphotyrosine Binding Domain of the Brain-specific Adaptor Protein ShcC

1996; Elsevier BV; Volume: 271; Issue: 20 Linguagem: Inglês

10.1074/jbc.271.20.11787

1083-351X

John P. O’Bryan, Carol B. Martin, Zhou Songyang, Lewis C. Cantley, Channing J. Der,

Protein Tyrosine Phosphatases

Shc proteins (hereafter referred to as ShcA) represent major substrates of tyrosine phosphorylation by a wide variety of growth factors and cytokines. We have recently described a novel ShcA-like protein, ShcC, which like ShcA contains an NH2-terminal phosphotyrosine binding domain (PTB), a central effector region (CH1) and a COOH-terminal Src homology 2 domain (SH2). Both the SH2 and PTB domains of ShcC bind a similar profile of proteins as the comparable regions of ShcA. In an effort to define the functional differences or similarities between ShcA and ShcC, we have further characterized the PTB domain of ShcC. Using a degenerate phosphopeptide library screen, we show that the PTB domain of ShcC preferentially binds the sequence His-hydrophobic-Asn/hydrophobic-Asn-Pro-Ser/Thr-Tyr(P). This sequence is similar to the binding site for the ShcA PTB domain, suggesting that these two proteins may have overlapping specificities. In addition, random mutagenesis of the ShcC PTB domain has identified several amino acids important for PTB function (Gly32, Glu63, Ala136, Gly139, and Asp140). Mutation of these amino acids dramatically reduces the affinity of the ShcC PTB domain for the activated epidermal growth factor receptor in vitro. Shc proteins (hereafter referred to as ShcA) represent major substrates of tyrosine phosphorylation by a wide variety of growth factors and cytokines. We have recently described a novel ShcA-like protein, ShcC, which like ShcA contains an NH2-terminal phosphotyrosine binding domain (PTB), a central effector region (CH1) and a COOH-terminal Src homology 2 domain (SH2). Both the SH2 and PTB domains of ShcC bind a similar profile of proteins as the comparable regions of ShcA. In an effort to define the functional differences or similarities between ShcA and ShcC, we have further characterized the PTB domain of ShcC. Using a degenerate phosphopeptide library screen, we show that the PTB domain of ShcC preferentially binds the sequence His-hydrophobic-Asn/hydrophobic-Asn-Pro-Ser/Thr-Tyr(P). This sequence is similar to the binding site for the ShcA PTB domain, suggesting that these two proteins may have overlapping specificities. In addition, random mutagenesis of the ShcC PTB domain has identified several amino acids important for PTB function (Gly32, Glu63, Ala136, Gly139, and Asp140). Mutation of these amino acids dramatically reduces the affinity of the ShcC PTB domain for the activated epidermal growth factor receptor in vitro. INTRODUCTIONTyrosine phosphorylation represents a critical switch in the regulation of cell growth, differentiation, and development. Phosphorylation of cellular proteins on tyrosine residues creates high affinity binding sites for proteins containing Src homology 2 (SH2) 1The abbreviations used are: SH2Src homology 2PTBphosphotyrosine bindingEGF(R)epidermal growth factor (receptor)GSTglutathione S-transferasePCRpolymerase chain reactionkbpkilobase pair(s). domains. SH2 domains recognize tyrosine and the 3-6 amino acids COOH-terminal to the phosphotyrosine. The selectivity of a particular SH2 domain is dictated by these COOH-terminal amino acids. Recently another phosphotyrosine binding domain (PTB) has been described(1.Blaikie P. Immanuel D. Wu J. Li N. Yajnik V. Margolis B. J. Biol. Chem. 1994; 269: 32031-32034Abstract Full Text PDF PubMed Google Scholar, 2.Kavanaugh W.M. Williams L.T. Science. 1994; 266: 1862-1865Crossref PubMed Scopus (448) Google Scholar, 3.Gustafson T.A. He W. Craparo A. Schaub C.D. O'Neill T.J. Mol. Cell. Biol. 1995; 15: 2500-2508Crossref PubMed Scopus (321) Google Scholar). This domain, also known as PI (phosphotyrosine interaction domain) and SAIN (Shc and IRS-1 NPXY binding), recognizes phosphotyrosine in the context of amino acids NH2-terminal to the phosphotyrosine. Thus, PTB and SH2 domains represent distinct protein modules that recognize tyrosine-phosphorylated proteins, but under entirely different contexts.PTB domains were first described in the adaptor protein ShcA(1.Blaikie P. Immanuel D. Wu J. Li N. Yajnik V. Margolis B. J. Biol. Chem. 1994; 269: 32031-32034Abstract Full Text PDF PubMed Google Scholar, 2.Kavanaugh W.M. Williams L.T. Science. 1994; 266: 1862-1865Crossref PubMed Scopus (448) Google Scholar). ShcA represents a major target of tyrosine phosphorylation following stimulation by a variety of growth factors and cytokines(4.Pawson T. Nature. 1995; 373: 573-579Crossref PubMed Scopus (2216) Google Scholar). Upon activation of receptor tyrosine kinases, ShcA becomes physically associated with the receptor and phosphorylated on tyrosine. This association was initially believed to occur through the SH2 domain of ShcA(5.Pelicci G. Lanfrancone L. Grignani F. McGlade J. Cavallo F. Forni G. Nicoletti I. Grignani F. Pawson T. Pelicci P.G. Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1130) Google Scholar). Indeed, the ShcA SH2 binding site on the EGFR was mapped using a combination of in vitro binding and phosphopeptide competition assays(6.Batzer A.G. Rotin D. Urena J.M. Skolnick E.Y. Schlessinger J. Mol. Cell. Biol. 1994; 14: 5192-5201Crossref PubMed Google Scholar, 7.Okabayashi Y. Kido Y. Okutani T. Sugimoto Y. Sakaguchi K. Kasuga M. J. Biol. Chem. 1994; 269: 18674-18678Abstract Full Text PDF PubMed Google Scholar). The peptide selectivity of the ShcA SH2 domain was determined using a degenerate phosphopeptide library screen (8.Songyang Z. Shoelson S.E. McGlade J. Olivier P. Pawson T. Bustelo X.R. Barbacid M. Sabe H. Hanafusa H. Yi T. Ren R. Baltimore D. Ratnofsky A. Feldman R.A. Cantley L.C. Mol. Cell. Biol. 1994; 14: 2777-2785Crossref PubMed Scopus (829) Google Scholar). These results suggested that a number of receptors had putative ShcA binding sites. However, some confusion arose as to the true ShcA binding site due to the finding that ShcA association with the polyoma virus middle T antigen occurred through an Asn-Pro-Thr-Tyr sequence and not the consensus ShcA SH2 binding sequence(9.Campbell K.S. Ogris E. Burke B. Su W. Auger K.R. Druker B.J. Schaffhausen B.S. Roberts T.M. Pallas D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6344-6348Crossref PubMed Scopus (153) Google Scholar, 10.Dilworth S.M. Brewster C.E. Jones M.D. Lanfrancone L. Pelicci G. Pelicci P.G. Nature. 1994; 367: 87-90Crossref PubMed Scopus (175) Google Scholar). In addition, the association of ShcA with a 145-kDa phosphoprotein in platelet-derived growth factor-stimulated cells was shown to occur not through the SH2 domain, but rather through the NH2 terminus(2.Kavanaugh W.M. Williams L.T. Science. 1994; 266: 1862-1865Crossref PubMed Scopus (448) Google Scholar). The determination that ShcA contains two distinct phosphotyrosine binding motifs, a COOH-terminal SH2 and a NH2-terminal PTB, provided an explanation for these observations. PTB recognition sites are also present in the nerve growth factor receptor (TrkA), the insuln and insulin-related receptors, interleukin-2 receptor, and the EGFR.We have recently described the identification of two shc-like genes which we called shcB and shcC(11.O'Bryan J.P. Songyang Z. Cantley L. Der C. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2729-2734Crossref PubMed Scopus (95) Google Scholar). shcB is nearly identical in sequence to the partial human shc-like gene sck and most likely represents the mouse homolog of this gene(11.O'Bryan J.P. Songyang Z. Cantley L. Der C. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2729-2734Crossref PubMed Scopus (95) Google Scholar). shcC, however, has not yet been found in other organisms. In contrast to the wide expression of shcA, shcC is restricted in expression to tissues of neural origin, suggesting a role for this adaptor protein in brain-specific tyrosine kinase signaling. Like ShcA, ShcC contains an NH2-terminal PTB domain, a central proline-rich region (CH1) and a COOH-terminal SH2 domain. In addition, ShcC binds to activated growth factor receptors through both its SH2 and PTB domains. In this report, we have further characterized the PTB domain of ShcC. We have determined the phosphopeptide selectivity of the ShcC PTB domain as well as describe a number of point mutations that dramatically reduce the affinity of the ShcC PTB domain for activated growth factor receptors. These mutations occur in conserved regions of the PTB domain, suggesting an important role for these amino acids in phosphotyrosine recognition and binding.EXPERIMENTAL PROCEDURESPeptide Library ScreenThe peptide library used for these studies has the sequence Met-Ala-X-X-X-Asn-X-X-Tyr(P)-X-Ala-Lys-Lys-Lys, where X corresponds to any amino acid except for Trp and Cys. This library was synthesized as described previously(8.Songyang Z. Shoelson S.E. McGlade J. Olivier P. Pawson T. Bustelo X.R. Barbacid M. Sabe H. Hanafusa H. Yi T. Ren R. Baltimore D. Ratnofsky A. Feldman R.A. Cantley L.C. Mol. Cell. Biol. 1994; 14: 2777-2785Crossref PubMed Scopus (829) Google Scholar). The theoretical degeneracy of this library is 186 or approximately 3.4 × 107. Determination of the binding specificity was done as described previously(12.Songyang Z. Margolis B. Chaudhuri M. Shoelson S. Cantley L.C. J. Biol. Chem. 1996; 270: 14863-14866Abstract Full Text Full Text PDF Scopus (158) Google Scholar).Random Mutagenesis and Bacterial ExpressionThe PTB domain of ShcC (amino acids 28-212) was amplified by polymerase chain reaction (PCR) as described previously (11.O'Bryan J.P. Songyang Z. Cantley L. Der C. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2729-2734Crossref PubMed Scopus (95) Google Scholar) and then either directly subcloned into the pGEX bacterial expression plasmids or randomly mutagenized with hydroxylamine treatment(13.Feig L.A. Corbley M. Pan B.-T. Roberts T.M. Cooper G.M. Mol. Endocrinol. 1987; 1: 127-136Crossref PubMed Scopus (7) Google Scholar, 14.Quilliam L.A. Kato K. Rabun K. Hisaka M. Huff S. Campbell-Burk S. Der C.J. Mol. Cell. Biol. 1994; 14: 1113-1121Crossref PubMed Scopus (80) Google Scholar), subcloned into pCRII (Invitrogen) for sequence analysis, and then subcloned into pGEX. For hydroxylamine mutagenesis, 30 μl of a PCR fragment encoding the PTB domain was mixed with 150 μl of ethylene glycol, then heated to 70°C for 5 min. To this mixture was added 16 μl of hydroxylamine solution (0.5 M hydroxylamine, 0.2 M sodium pyrophosphate, pH 6.0) and then heated to 70°C for 20 min. After this incubation 80 μl of stop solution (0.6 M Tris, pH 8.0, 1.0 M NaCl, 20% acetone) was added and the mutagenized DNA purified over a G-50 Sephadex column equilibrated with TE (10 mM Tris, pH 7.5, 1 mM EDTA). The resulting DNA was subcloned into the pCRII vector using the TA Cloning Kit (Invitrogen). Point mutations were identified by dideoxy sequence analysis. All the PTB fragments were subcloned into pGEX vectors as BamHI-EcoRI fragments. Mutants 4a and 4b were constructed by digesting the pGEX-PTB constructs for the wild-type and mutant 4 PTB domains with BstEII, which produces two fragments of approximately 2 kbp (encoding amino acids 28-59 of the PTB) and 3.4 kbp (encoding the amino acids 60-213 of the PTB domain). Mutant 4a was constructed by ligating the 3.4-kbp wild-type fragment with the 2-kbp fragment from mutant 4. Mutant 4b was constructed by ligating the 3.4-kbp fragment from mutant 4 with the 2-kbp wild-type fragment. Plasmid constructs were sequenced to confirm the presence of each mutation. Mutant proteins were expressed in Escherichia coli strain DH5α at 37°C and purified on glutathione-agarose beads after induction with IPTG for 3 h. Beads containing GST-PTB were washed with phosphate-buffered saline containing 20% glycerol, 0.5% Tween 20, 1 mM dithiothreitol, 10 μg/ml aprotinin, and 10 μg/ml leupeptin, resuspended in a 50% slurry, and then stored at −70°C. Aliquots of each PTB domain were fractionated on SDS-polyacrylamide gel electrophoresis and the gel stained with Coomassie to assess concentrations.In Vitro Binding AssaysA431 cells (a human epidermoid carcinoma) were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, penicillin, and streptomycin in the presence of 5% CO2. Epidermal growth factor (EGF) was a kind gift of Dr. H. Shelton Earp and used at a concentration of 100 ng/ml. Briefly, lysates were prepared from A431 cells stimulated with EGF for 2 min at room temperature as described previously(11.O'Bryan J.P. Songyang Z. Cantley L. Der C. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2729-2734Crossref PubMed Scopus (95) Google Scholar). Approximately equal amounts of each GST-PTB were incubated with equivalent amounts of lysate from EGF stimulated A431 cells at 4°C for >90 min. These mixes were then centrifuged and pellets washed three times with phosphate-buffered saline containing 20% glycerol, 0.5% Tween 20, 1 mM dithiothreitol, 1 mM sodium vanadate, 10 μg/ml aprotinin, and 10 μg/ml leupeptin. The pellets were resuspended in 50 μl in 2 × SDS sample buffer, boiled for 5 min, fractionated on SDS-polyacrylamide gel electrophoresis, and transferred to Immobilon filters. Filters were probed with either anti-phosphotyrosine antibodies (PY20, Transduction Laboratories) to detect the phosphorylated EGFR or anti-GST antibodies (GST(Z-5)HRP, Santa Cruz Biotechnology) to assure equal amounts of fusion protein were used in the binding experiments. Blots were developed using ECL reagents (Amersham Corp.). Signals were quantitated using a Bio-Rad phosphorimager. Relative binding affinities were determined by dividing the anti-Tyr(P) signal by the anti-GST signal. The resulting ratio for each sample was divided by the ratio for the wild-type PTB domain to normalize the samples relative to the wild-type PTB.RESULTS AND DISCUSSIONA number of groups have defined the sequence requirements of a phosphopeptide for binding the ShcA PTB domain (12.Songyang Z. Margolis B. Chaudhuri M. Shoelson S. Cantley L.C. J. Biol. Chem. 1996; 270: 14863-14866Abstract Full Text Full Text PDF Scopus (158) Google Scholar, 15.Kavanaugh W.M. Turck C.W. Williams L.T. Science. 1995; 268: 1177-1179Crossref PubMed Scopus (222) Google Scholar, 16.Batzer A.G. Blaike P. Nelson K. Schlessinger J. Margolis B. Mol. Cell. Biol. 1995; 15: 4403-4409Crossref PubMed Scopus (116) Google Scholar, 17.Wolf G. Trub T. Ottinger E. Groninga L. Lynch A. White M. Miyazaki M. Lee J. Shoelson S. J. Biol. Chem. 1995; 270: 27407-27410Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar, 18.van der Geer P. Wiley S. Gish G. Lai V.K.-M. Stephens R. White M. Kaplan D. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 963-968Crossref PubMed Scopus (94) Google Scholar) (Table 1). The ShcA and ShcC PTB domains share a high degree of amino acid sequence homology (78% identity; Fig. 1). Given our interest in further defining functional differences or similarities between ShcA and ShcC, we have examined the peptide specificity of bacterially expressed ShcC PTB using a degenerate phosphopeptide library screen (see “Experimental Procedures”). Using this strategy, the ShcA PTB was shown to select phosphopeptides containing the sequence Asn-Pro-X-Tyr(P)-Phe-X-Arg with the strongest selectivity at positions −3 and −2 relative to the Tyr(P) (12.Songyang Z. Margolis B. Chaudhuri M. Shoelson S. Cantley L.C. J. Biol. Chem. 1996; 270: 14863-14866Abstract Full Text Full Text PDF Scopus (158) Google Scholar) (Table 1). Indeed, this motif (Asn-Pro-X-Tyr(P)) is present in a number of receptor tyrosine kinases, including TrkA, EGFR, and the insulin receptor as well as polyoma virus middle T-antigen.Tabled 1Figure 1:Alignment of PTB domains. Shown is an alignment of PTB domains from a number of proteins. This alignment does not include all of the potential PTB containing proteins described thus far(20.Yajnik V. Blaikie P. Bork P. Margolis B. J. Biol. Chem. 1996; 271: 1813-1816Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). The alignment was created using the Pileup program of the Genetics Computer Group software analysis package then imported into the Maligned multiple sequence alignment program and modified based on the reported NMR structure(21.Zhou M.-M. Meadows R.P. Logan T.M. Yoon H.S. Wade W.S. Ravichandran K.S. Burakoff S.J. Fesik S.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7784-7788Crossref PubMed Scopus (56) Google Scholar). Shown above the alignment is the predicted secondary structure of the PTB domains based on the NMR structure of ShcA. We have not included the α1 helix in this alignment, since our PTB constructs did not contain this region. Asterisks indicate the positions of amino acid changes. The arrow indicates the position of the Arg175 mutation of ShcA(18.van der Geer P. Wiley S. Gish G. Lai V.K.-M. Stephens R. White M. Kaplan D. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 963-968Crossref PubMed Scopus (94) Google Scholar, 21.Zhou M.-M. Meadows R.P. Logan T.M. Yoon H.S. Wade W.S. Ravichandran K.S. Burakoff S.J. Fesik S.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7784-7788Crossref PubMed Scopus (56) Google Scholar). ShcA and ShcC represent the predicted peptide sequences encoded by the respective mouse genes (GenBank(™) accession numbers U15784 and U46854, respectively). The ShcC sequence begins at amino acid 29. The ShcA sequence begins at amino acid 46. The accession numbers for the remaining sequences are listed in (20.Yajnik V. Blaikie P. Bork P. Margolis B. J. Biol. Chem. 1996; 271: 1813-1816Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Using a modification of the above phosphopeptide library screen, we determined the phosphopeptide selectivity of the ShcC PTB domain (Table 1). Previous experiments with ShcA indicated that Asn at the −3 position was absolutely required for efficient PTB binding(12.Songyang Z. Margolis B. Chaudhuri M. Shoelson S. Cantley L.C. J. Biol. Chem. 1996; 270: 14863-14866Abstract Full Text Full Text PDF Scopus (158) Google Scholar). In addition, most of the selectivity of PTB domains appears to be dictated by residues NH2-terminal to Tyr(P). Therefore, to increase the sensitivity of our experiments and to examine the importance of residues at −6 to −4, the −3 position of the phosphopeptide library was fixed as Asn. The library consisted of peptides containing the sequence Met-Ala-X-X-X-Asn-X-X-Tyr(P)-X-Ala-Lys-Lys-Lys, where X corresponds to any amino acid except for Trp and Cys. The ShcC PTB domain selects phosphopeptides containing the sequence His-hydrophobic-Asn/hydrophobic-Asn-Pro-Ser/Thr-Tyr(P) (Fig. 2 and Table 1). There also appears to be some selectivity at the +1 position for small chain amino acids (Table 1). These data suggest that the ShcC PTB may bind to similar phosphoproteins as the ShcA PTB. In agreement with these data, we have shown that the ShcA and ShcC PTB domains bind in vitro to the activated NGFR and EGFR in growth factor-stimulated cells with relatively equal affinities and this binding can be competed away with a phosphopeptide modeled on the Tyr490 juxtamembrane autophosphorylation site of TrkA(11.O'Bryan J.P. Songyang Z. Cantley L. Der C. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2729-2734Crossref PubMed Scopus (95) Google Scholar). In addition, both ShcC and ShcA PTB domains bind a 170-kDa phosphoprotein in EGF-stimulated A431 cell lysates (Fig. 3A). The identity of the protein is currently unknown. Thus, the ShcC PTB domain shares common recognition specificities with the corresponding PTB domain of ShcA.Figure 2:Phosphopeptide selectivity of the PTB domain of ShcC. A degenerate phosphopeptide library was used to examine the specificity of the ShcC PTB domain (see “Experimental Procedures”). The peptides that bound to immobilized GST-ShcC PTB were purified and sequenced, and the sequence of the purified peptides was compared with that of the starting peptide library. The data were normalized such that a value of 1 or less indicates no selectivity for a given amino acid(33.Songyang Z. Shoelson S.E. Chaudhuri M. Gish G. Pawson T. Haser W. King F. Roberts T. Ratnofsky S. Lechleider R.J. Neel B.G. Birge R.B. Fajardo J.E. Chou M.M. Hanafusa H. Schaffhausen B. Cantley L.C. Cell. 1993; 72: 767-778Abstract Full Text PDF PubMed Scopus (2372) Google Scholar). A, B, C, D, E and F show, respectively, the selectivity ot the Tyr(P)-6, Tyr(P)-5, Tyr(P)-4, Tyr(P)-2, Tyr(P)-1, and Tyr(P)+1 positions. Amino acids are given in their one-letter codes.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3:Mutations in the PTB domain of ShcC altered the binding to the activated EGFR. A, in vitro binding of the ShcC PTB domain to the activated EGFR. GST fusion proteins for each PTB were purified and used in an in vitro binding experiment, as described previously(11.O'Bryan J.P. Songyang Z. Cantley L. Der C. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2729-2734Crossref PubMed Scopus (95) Google Scholar). Western blots were probed with anti-phosphotyrosine and anti-GST antibodies. Signals were quantitated on a Bio-Rad phosphorimager. B, quantitation of PTB binding data shown in A. Shown are the binding affinities of the various ShcC PTB mutants relative to wild type. These results are the average of three independent binding assays with different preparations of GST-PTB protein. The graph on the right represents the results of binding experiments with the wild-type PTB, mutant 4 PTB, and the mutant 4 PTB derivatives 4a and 4b. Standard errors are indicated with bars.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To better understand the importance of particular amino acid residues in the PTB domain for recognition of target phosphoproteins, we set out to mutagenize the ShcC PTB domain (Fig. 1). In the absence of structural information regarding the importance of particular regions of the PTB domain, we employed a random mutagenesis approach with the aim of identifying amino acids important for PTB function. Using hydroxylamine mutagenesis and PCR, we isolated a number of PTB mutants, several of which are impaired in their ability to bind the phosphorylated EGFR (Table 2). Mutant 2, which contains a single point mutation (E63G), has dramatically reduced binding (>10-fold) to the EGFR as compared with the wild-type PTB (Fig. 3). This Glu residue at position 63 of ShcC (amino acid 80 of ShcA) is conserved in 14 of 22 PTB domains described thus far (Fig. 1 and (19.Bork P. Margolis B. Cell. 1995; 80: 693-694Abstract Full Text PDF PubMed Scopus (171) Google Scholar)). Four of the remaining eight PTB domains possess a conserved Asp. These findings suggest that a negatively charged side chain amino acid at this position plays a critical role in the recognition of tyrosine-phosphorylated substrates of the ShcC PTB domain.Tabled 1In addition to the E63G single mutation, PTB mutant 7, which possesses two tandem amino acid substitutions (G139E/D140N), also has dramatically reduced binding (>10-fold) to phosphotyrosine substrates (Fig. 3). These two amino acids occur in a region of the PTB domain which is highly conserved in Shc family members as well as other PTB containing proteins (Fig. 1). In particular, Asp is present in 15 of the 22 PTB domains(19.Bork P. Margolis B. Cell. 1995; 80: 693-694Abstract Full Text PDF PubMed Scopus (171) Google Scholar, 20.Yajnik V. Blaikie P. Bork P. Margolis B. J. Biol. Chem. 1996; 271: 1813-1816Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), suggesting an important role in PTB function. PTB mutant 4 also has dramatically reduced affinity (>9-fold) for the activated EGFR. This mutant contains three amino acid substitutions (G32R, A136T, C166R; Table 2). The Gly is conserved in 11 of 19 PTB domains, and the Ala is present in 6 of 22 PTB domains, suggesting a conserved role for these amino acids in PTB function. The Cys, however, is only present in 3 of 21 PTB domains, which suggests that this amino acid may not be critical for PTB function(19.Bork P. Margolis B. Cell. 1995; 80: 693-694Abstract Full Text PDF PubMed Scopus (171) Google Scholar, 20.Yajnik V. Blaikie P. Bork P. Margolis B. J. Biol. Chem. 1996; 271: 1813-1816Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). We have separated this triple mutant into a single mutant containing the G32R mutation and a double mutant containing the A132T and the C166R mutations. The relative binding affinities of these two mutants compared with the triple mutant and wild-type PTB domains were determined. Although the triple mutant had approximately a 90% decrease in binding the activated EGFR, neither the single nor double mutant were severely impaired in binding. The G32R mutation resulted in approximately a 20% decrease in binding, and the A136T/C166R double mutation resulted in approximately a 50% reduction in binding. We have not assessed the individual contributions of the A136T or C166R mutations. We believe the severity of the triple mutant is due to a synergistic effect of the three mutations on PTB binding function. We do not believe that the triple mutant is defective in binding due to a decrease in the stability of the protein, since equivalent amounts of fusion protein are obtained for mutants 4, 4a, and 4b (data not shown).During the course of this study, the NMR solution structure of the PTB domain of ShcA complexed to a TrkA phosphopeptide was described(21.Zhou M.-M. Meadows R.P. Logan T.M. Yoon H.S. Wade W.S. Ravichandran K.S. Burakoff S.J. Fesik S.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7784-7788Crossref PubMed Scopus (56) Google Scholar). The tertiary structure of the PTB domain is composed of two antiparallel β-sheets formed by a series of seven β strands and three α helices. The overall topology of the PTB domain bears a striking resemblance to that of another modular domain, the pleckstrin homology (PH) domain, although these two domains lack any sequence homology. The ShcA and ShcC PTB domains share a high degree of sequence identity. Overall these two domains are 78% identical particularly in the regions that form specific contacts with the phosphopeptide as determined by NMR(21.Zhou M.-M. Meadows R.P. Logan T.M. Yoon H.S. Wade W.S. Ravichandran K.S. Burakoff S.J. Fesik S.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7784-7788Crossref PubMed Scopus (56) Google Scholar). For example, the β5 strand is 100% identical in ShcA and ShcC and forms four contacts with the phosphopeptide ligand (21.Zhou M.-M. Meadows R.P. Logan T.M. Yoon H.S. Wade W.S. Ravichandran K.S. Burakoff S.J. Fesik S.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7784-7788Crossref PubMed Scopus (56) Google Scholar). These findings suggest that the solution structure of the ShcC PTB domain may be very similar to that of ShcA. Therefore, we have analyzed our mutations using the ShcA PTB structure as a framework for comparison. The E63G mutation occurs in the middle of α2 helix, which connects the β1 and β2 strands. These β strands comprise part of a β-sheet that forms a hydrophobic pocket into which the phosphopeptide binds. Thus, the E63G mutation likely disrupts important ionic interactions with the α2 helix, thereby abrogating phosphopeptide recognition. The G139E/D140N mutations occur in a loop between the β5 and β6 strands. The β5 strand forms several contacts with the phosphopeptide backbone(21.Zhou M.-M. Meadows R.P. Logan T.M. Yoon H.S. Wade W.S. Ravichandran K.S. Burakoff S.J. Fesik S.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7784-7788Crossref PubMed Scopus (56) Google Scholar). Gly139 appears important for forming a proper turn between these two β strands, which allows for their antiparallel arrangement. Thus, the G139E/D140N double mutant likely disrupts these contacts by restricting the ability of the loop to form a turn, thereby disrupting the alignment of β5 and β6 and diminishing the affinity of the PTB for phosphopeptide.The three mutations present in PTB mutant 4 occur in different regions of the PTB domain. Of particular interest is the fact that the A136T mutation occurs in the β5 strand, which forms part of the cleft into which the phosphotyrosine binds(21.Zhou M.-M. Meadows R.P. Logan T.M. Yoon H.S. Wade W.S. Ravichandran K.S. Burakoff S.J. Fesik S.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7784-7788Crossref PubMed Scopus (56) Google Scholar). Several amino acids in this β strand, including Ala139, are in close proximity with the phosphopeptide. Mutation of Ala139 likely disrupts these contacts, thereby abrogating binding to the activated EGFR. Thus, the A136T mutation likely accounts for the majority of the reduction in EGFR binding by mutant 4. The A136T/C166R double mutant does not appear to be as impaired in binding as the triple mutant (Fig. 3C). Although we have not assessed the individual effects of these two substitutions, we believe that the C166R mutation may have some compensatory effect in the context of the double mutant. This compensation in binding may not occur in the context of the triple mutant.In addition to mutations which affect phosphotyrosine binding, a number of PTB mutants are unaffected in their interaction with the activated EGFR (Fig. 3). Many of these mutations represent conservative substitutions that likely do not have a profound affect on the structure or interactions with other amino acids within the PTB domain itself or the phosphopeptide. Many of the nonconservative substitutions occur in loop regions that tend to be more resistant to mutational effects due to the ability of these regions to move freely in space.We have identified several mutations in the PTB domain of a novel adaptor protein, ShcC, which dramatically reduce the affinity of its PTB domain for the activated EGFR. Based on the predicted structure of the ShcA PTB domain, these mutations likely disrupt regions important in phosphopeptide recognition and binding. Several groups have identified additional mutants in the ShcA PTB domain that affect PTB binding to phosphotyrosine containing proteins(18.van der Geer P. Wiley S. Gish G. Lai V.K.-M. Stephens R. White M. Kaplan D. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 963-968Crossref PubMed Scopus (94) Google Scholar, 20.Yajnik V. Blaikie P. Bork P. Margolis B. J. Biol. Chem. 1996; 271: 1813-1816Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 21.Zhou M.-M. Meadows R.P. Logan T.M. Yoon H.S. Wade W.S. Ravichandran K.S. Burakoff S.J. Fesik S.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7784-7788Crossref PubMed Scopus (56) Google Scholar). Interestingly, Yajnik et. al. (20.Yajnik V. Blaikie P. Bork P. Margolis B. J. Biol. Chem. 1996; 271: 1813-1816Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) independently isolated an Ala to Thr mutation at amino acid 153 in ShcA, which is identical to the A136T mutation present in mutant 4 of ShcC. This mutation resulted in a 74% reduction in binding of the ShcA PTB to the activated EGFR, further supporting the notion that the A136T mutation of mutant 4 is indeed the critical amino acid mutation affecting binding.Mutation of Arg175 of ShcA to either Glu, Met, or Lys completely abolishes phosphotyrosine binding, suggesting a critical role for this Arg in substrate binding(18.van der Geer P. Wiley S. Gish G. Lai V.K.-M. Stephens R. White M. Kaplan D. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 963-968Crossref PubMed Scopus (94) Google Scholar, 21.Zhou M.-M. Meadows R.P. Logan T.M. Yoon H.S. Wade W.S. Ravichandran K.S. Burakoff S.J. Fesik S.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7784-7788Crossref PubMed Scopus (56) Google Scholar). Based on the recently described structure of the ShcA PTB domain, Arg175 directly participates in binding the phosphotyrosine residue of the phosphopeptide ligand. Interestingly, this Arg is not absolutely conserved in all PTB domains(19.Bork P. Margolis B. Cell. 1995; 80: 693-694Abstract Full Text PDF PubMed Scopus (171) Google Scholar). This finding is in contrast to SH2 domains, which contain an absolutely conserved Arg, mutation of which blocks SH2 binding to tyrosine-phosphorylated proteins(22.Marengere L.E. Pawson T. J. Biol. Chem. 1992; 267: 22779-22786Abstract Full Text PDF PubMed Google Scholar, 23.Waksman G. Shoelson S.E. Pant N. Cowburn D. Kuriyan J. Cell. 1993; 72: 779-790Abstract Full Text PDF PubMed Scopus (651) Google Scholar). In addition to Arg175, mutation of Phe198 drastically reduces binding (<1% of wild type)(20.Yajnik V. Blaikie P. Bork P. Margolis B. J. Biol. Chem. 1996; 271: 1813-1816Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Phe198, in contrast to Arg175, is conserved in the majority of PTB domains described thus far(19.Bork P. Margolis B. Cell. 1995; 80: 693-694Abstract Full Text PDF PubMed Scopus (171) Google Scholar, 20.Yajnik V. Blaikie P. Bork P. Margolis B. J. Biol. Chem. 1996; 271: 1813-1816Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Interestingly, the IRS-1 PTB domain binds to a similar sequence as the ShcA and ShcC PTB domains, yet shares no apparent sequence homology. These observations suggest that although different PTB domains may lack primary sequence homology, they may adopt similar three-dimensional structures. Indeed, the PTB domain of ShcA and the PH domain of pleckstrin share a similar three-dimensional structure in the absence of sequence homology(21.Zhou M.-M. Meadows R.P. Logan T.M. Yoon H.S. Wade W.S. Ravichandran K.S. Burakoff S.J. Fesik S.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7784-7788Crossref PubMed Scopus (56) Google Scholar). Alternatively, PTB domains lacking a comparable Arg175 as found in the PTB domains of Shc family members may adopt a different structure and, thus, employ a different mechanism for phosphopeptide recognition and binding. Determining the structures of other PTB domains will address these possibilities.We have examined the peptide selectivity of the ShcC PTB domain. This PTB domain has a similar selectivity as compared with the PTB domain of ShcA. Similar results were obtained with the SH2 domains of Shc family members(11.O'Bryan J.P. Songyang Z. Cantley L. Der C. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2729-2734Crossref PubMed Scopus (95) Google Scholar). The similarity in the sequence and the peptide selectivities of both ShcA and ShcC PTB and SH2 domains suggests that these two adaptor proteins may share overlapping functions, but in different cell types. Indeed, both proteins interact with similar receptors and tyrosine-phosphorylated proteins in vitro(11.O'Bryan J.P. Songyang Z. Cantley L. Der C. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2729-2734Crossref PubMed Scopus (95) Google Scholar). The identification of mutant PTB domains presents the possibility of designing ShcC dominant interfering mutants that may block signaling from tyrosine kinases as well as other proteins that signal through Shc family members.In addition to binding tyrosine-phosphorylated proteins, the PTB domain of ShcA has also been shown to bind phospholipids(21.Zhou M.-M. Meadows R.P. Logan T.M. Yoon H.S. Wade W.S. Ravichandran K.S. Burakoff S.J. Fesik S.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 7784-7788Crossref PubMed Scopus (56) Google Scholar). Furthermore, the SH2 domains of phosphatidylinositol 3′-kinase, as well as Src and Abl, have been shown to bind phospholipids(24.Rameh L.E. Chen C.-S. Cantley L.C. Cell. 1995; 83: 821-830Abstract Full Text PDF PubMed Scopus (289) Google Scholar). This interaction with lipids provides a possible explanation for how ShcA may translocate to the membrane to activate Ras in the absence of direct binding to activated growth factor receptors(25.Li N. Schlessinger J. Margolis B. Oncogene. 1994; 9: 3457-3465PubMed Google Scholar, 26.Gotoh N. Tojo A. Muroya K. Hashimoto Y. Hattori S. Nakamura S. Takenawa T. Yazaki Y. Shibuya M. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 167-171Crossref PubMed Scopus (107) Google Scholar, 27.Soler C. Alvarez C.V. Beguinot L. Carpenter G. Oncogene. 1994; 9: 2207-2215PubMed Google Scholar, 28.Fridell Y.-W. C. Jin Y. Quilliam L.A. Burchert A. McCloskey P. Spizz G. Varnum B. Der C. Liu E.T. Mol. Cell. Biol. 1996; 16: 135-145Crossref PubMed Scopus (143) Google Scholar). In addition, the interaction of ShcA with phospholipids may play a role in the regulation of phospholipid metabolism. INTRODUCTIONTyrosine phosphorylation represents a critical switch in the regulation of cell growth, differentiation, and development. Phosphorylation of cellular proteins on tyrosine residues creates high affinity binding sites for proteins containing Src homology 2 (SH2) 1The abbreviations used are: SH2Src homology 2PTBphosphotyrosine bindingEGF(R)epidermal growth factor (receptor)GSTglutathione S-transferasePCRpolymerase chain reactionkbpkilobase pair(s). domains. SH2 domains recognize tyrosine and the 3-6 amino acids COOH-terminal to the phosphotyrosine. The selectivity of a particular SH2 domain is dictated by these COOH-terminal amino acids. Recently another phosphotyrosine binding domain (PTB) has been described(1.Blaikie P. Immanuel D. Wu J. Li N. Yajnik V. Margolis B. J. Biol. Chem. 1994; 269: 32031-32034Abstract Full Text PDF PubMed Google Scholar, 2.Kavanaugh W.M. Williams L.T. Science. 1994; 266: 1862-1865Crossref PubMed Scopus (448) Google Scholar, 3.Gustafson T.A. He W. Craparo A. Schaub C.D. O'Neill T.J. Mol. Cell. Biol. 1995; 15: 2500-2508Crossref PubMed Scopus (321) Google Scholar). This domain, also known as PI (phosphotyrosine interaction domain) and SAIN (Shc and IRS-1 NPXY binding), recognizes phosphotyrosine in the context of amino acids NH2-terminal to the phosphotyrosine. Thus, PTB and SH2 domains represent distinct protein modules that recognize tyrosine-phosphorylated proteins, but under entirely different contexts.PTB domains were first described in the adaptor protein ShcA(1.Blaikie P. Immanuel D. Wu J. Li N. Yajnik V. Margolis B. J. Biol. Chem. 1994; 269: 32031-32034Abstract Full Text PDF PubMed Google Scholar, 2.Kavanaugh W.M. Williams L.T. Science. 1994; 266: 1862-1865Crossref PubMed Scopus (448) Google Scholar). ShcA represents a major target of tyrosine phosphorylation following stimulation by a variety of growth factors and cytokines(4.Pawson T. Nature. 1995; 373: 573-579Crossref PubMed Scopus (2216) Google Scholar). Upon activation of receptor tyrosine kinases, ShcA becomes physically associated with the receptor and phosphorylated on tyrosine. This association was initially believed to occur through the SH2 domain of ShcA(5.Pelicci G. Lanfrancone L. Grignani F. McGlade J. Cavallo F. Forni G. Nicoletti I. Grignani F. Pawson T. Pelicci P.G. Cell. 1992; 70: 93-104Abstract Full Text PDF PubMed Scopus (1130) Google Scholar). Indeed, the ShcA SH2 binding site on the EGFR was mapped using a combination of in vitro binding and phosphopeptide competition assays(6.Batzer A.G. Rotin D. Urena J.M. Skolnick E.Y. Schlessinger J. Mol. Cell. Biol. 1994; 14: 5192-5201Crossref PubMed Google Scholar, 7.Okabayashi Y. Kido Y. Okutani T. Sugimoto Y. Sakaguchi K. Kasuga M. J. Biol. Chem. 1994; 269: 18674-18678Abstract Full Text PDF PubMed Google Scholar). The peptide selectivity of the ShcA SH2 domain was determined using a degenerate phosphopeptide library screen (8.Songyang Z. Shoelson S.E. McGlade J. Olivier P. Pawson T. Bustelo X.R. Barbacid M. Sabe H. Hanafusa H. Yi T. Ren R. Baltimore D. Ratnofsky A. Feldman R.A. Cantley L.C. Mol. Cell. Biol. 1994; 14: 2777-2785Crossref PubMed Scopus (829) Google Scholar). These results suggested that a number of receptors had putative ShcA binding sites. However, some confusion arose as to the true ShcA binding site due to the finding that ShcA association with the polyoma virus middle T antigen occurred through an Asn-Pro-Thr-Tyr sequence and not the consensus ShcA SH2 binding sequence(9.Campbell K.S. Ogris E. Burke B. Su W. Auger K.R. Druker B.J. Schaffhausen B.S. Roberts T.M. Pallas D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6344-6348Crossref PubMed Scopus (153) Google Scholar, 10.Dilworth S.M. Brewster C.E. Jones M.D. Lanfrancone L. Pelicci G. Pelicci P.G. Nature. 1994; 367: 87-90Crossref PubMed Scopus (175) Google Scholar). In addition, the association of ShcA with a 145-kDa phosphoprotein in platelet-derived growth factor-stimulated cells was shown to occur not through the SH2 domain, but rather through the NH2 terminus(2.Kavanaugh W.M. Williams L.T. Science. 1994; 266: 1862-1865Crossref PubMed Scopus (448) Google Scholar). The determination that ShcA contains two distinct phosphotyrosine binding motifs, a COOH-terminal SH2 and a NH2-terminal PTB, provided an explanation for these observations. PTB recognition sites are also present in the nerve growth factor receptor (TrkA), the insuln and insulin-related receptors, interleukin-2 receptor, and the EGFR.We have recently described the identification of two shc-like genes which we called shcB and shcC(11.O'Bryan J.P. Songyang Z. Cantley L. Der C. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2729-2734Crossref PubMed Scopus (95) Google Scholar). shcB is nearly identical in sequence to the partial human shc-like gene sck and most likely represents the mouse homolog of this gene(11.O'Bryan J.P. Songyang Z. Cantley L. Der C. Pawson T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 2729-2734Crossref PubMed Scopus (95) Google Scholar). shcC, however, has not yet been found in other organisms. In contrast to the wide expression of shcA, shcC is restricted in expression to tissues of neural origin, suggesting a role for this adaptor protein in brain-specific tyrosine kinase signaling. Like ShcA, ShcC contains an NH2-terminal PTB domain, a central proline-rich region (CH1) and a COOH-terminal SH2 domain. In addition, ShcC binds to activated growth factor receptors through both its SH2 and PTB domains. In this report, we have further characterized the PTB domain of ShcC. We have determined the phosphopeptide selectivity of the ShcC PTB domain as well as describe a number of point mutations that dramatically reduce the affinity of the ShcC PTB domain for activated growth factor receptors. These mutations occur in conserved regions of the PTB domain, suggesting an important role for these amino acids in phosphotyrosine recognition and binding.