©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Specificity of the PTB Domain of Shc for Turn-forming Pentapeptide Motifs Amino-terminal to Phosphotyrosine (*)

(Received for publication, April 19, 1995; and in revised form, May 23, 1995)

Thomas Trb (1)(§) Wonjae E. Choi (1)(¶) Gert Wolf (1) Elizabeth Ottinger (1)(¶) YunJun Chen (2) Michael Weiss (2)(**) Steven E. Shoelson (1)(§§)

From the  (1)Joslin Diabetes Center and Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215 and the (2)Department of Molecular Oncology, The University of Chicago, Chicago Illinois 60637

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Shc phosphorylation in cells following growth factor, insulin, cytokine, and lymphocyte receptor activation leads to its association with Grb2 and activation of Ras. In addition to being a cytoplasmic substrate of tyrosine kinases, Shc contains an SH2 domain and a non-SH2 phosphotyrosine binding (PTB) domain. Here we show that the Shc PTB domain, but not the SH2 domain, binds with high affinity (ID ≅ 1 µM) to phosphopeptides corresponding to the sequence surrounding Tyr of the polyoma virus middle T (mT) antigen (LLSNPTpYSVMRSK). Truncation studies show that five residues amino-terminal to tyrosine are required for high affinity binding, whereas all residues carboxyl-terminal to tyrosine can be deleted without loss of affinity. Substitution studies show that tyrosine phosphorylation is required and residues at -5, -3, -2, and -1 positions relative to pTyr are important for this interaction. ^1H NMR studies demonstrate that the phosphorylated mT antigen-derived sequence forms a stable beta turn in solution, and correlations between structure and function indicate that the beta turn is important for PTB domain recognition. These results show that PTB domains are functionally distinct from SH2 domains. Whereas SH2 domain binding specificity derives from peptide sequences carboxyl-terminal to phosphotyrosine, the Shc PTB domain gains specificity by interacting with beta turn-forming sequences amino-terminal to phosphotyrosine.


INTRODUCTION

Many cell surface receptors involved in the regulation of growth, metabolism, and differentiation initiate their effects by catalyzing the phosphorylation of tyrosine residues within their own sequences and on additional cellular substrate proteins. The insulin, PDGF, (^1)EGF, and related receptors have intrinsic tyrosine kinase activity and catalyze these phosphorylations directly. Alternatively, lymphocyte, cytokine, and related receptors which lack intrinsic tyrosine kinase activity associate noncovalently with cytoplasmic tyrosine kinases to initiate their signaling events. In each case, phosphorylated tyrosine residues within the receptors and/or their substrates create docking sites for proteins with Src homology 2 (SH2) domains. SH2 domains are phosphotyrosine-binding modules associated with a wide variety of cytoplasmic proteins that participate in intracellular signal propagation.

Shc proteins contain an SH2 domain and serve as substrates for tyrosine kinase-linked receptors (e.g.(1, 2, 3) ). Following receptor activation, Shc is phosphorylated at Tyr and associates with the Grb2/Sos complex to activate Ras (e.g.(4, 5, 6, 7) ). (^2)However, in addition to serving as a Grb2 docking protein, Shc proteins also associate with phosphoproteins including activated EGF, PDGF, and insulin receptors, ErbB2, ErbB3, Trk, and the polyoma virus middle T antigen(1, 8, 9, 10, 11, 12, 13, 14) . Since many of these phosphoproteins contain Asn-Pro-Xaa sequences amino-terminal to phosphorylated tyrosines and Shc contains a carboxyl-terminal SH2 domain (Fig. 1A), it has been suggested that the Shc SH2 domain shows a preference for NPXY motifs(8, 10, 11) . This would be unusual in that alternative SH2 domains exhibit a high degree of specificity toward residues carboxyl-terminal to pTyr (e.g.(15, 16, 17, 18, 19) ). Although it has even been reported that the SH2 domain of Shc accounts for its interactions with NPXY-containing phosphoproteins (10, 20) , we have consistently failed to observe binding between the Shc SH2 domain and NPXY motifs. (^3)The realization that Shc contains an alternative phosphotyrosine binding (PTB) domain distinct from its SH2 domain (21, 22, 23) suggests that its PTB domain might be responsible for Shc interactions with NPXY motifs.


Figure 1: Expressed proteins and their capacity to bind with phosphopeptides. A, the domain structure of p52 and p46 Shc proteins shows the relative positions of its PTB, collagen homology (CH), and SH2 domains, the alternative start sites, the known phosphorylation site (Y318), and terminal positions of proteins used in this study. B, binding assays were conducted with I-radiolabeled peptide LLSNPTpYSVMRSK and GST/Shc fusion proteins 1-392, 1-238, 1-229, 1-215, 1-206, 1-196, 46-229, 46-215, and 371-474. The thick gray line represents the averaged initial slope for proteins 1-392, 1-238, 1-229, 1-215, and 1-206. C, competition assays using GST/Shc(1-238), [I]LLSNPTpYSVMRSK, and varying concentrations of unlabeled peptide LSNPTpYSV, the corresponding nonphosphorylated sequence, and a scrambled sequence (NTSVSpYPL).



We now show that the Shc PTB domain binds directly with a polyoma virus mT antigen-derived phosphopeptide, an LXNPTpY motif provides specificity for this interaction, and peptides corresponding to this sequence adopt a beta turn configuration in solution. Therefore, although PTB and SH2 domains appear to be similar in many respects, there are important functional distinctions between the interactions that they mediate.


MATERIALS AND METHODS

Recombinant Proteins and Synthetic Peptides

Fragments of the human Shc p52 cDNA generated by subcloning using the polymerase chain reaction were spliced into a pGEX4T vector. Escherichia coli DH5alpha cells were transformed with vectors encoding glutathione S-transferase/Shc fusion proteins GST/Shc(1-196), GST/Shc(1-206), GST/Shc(1-215), GST/Shc(1-229), GST/Shc(1-238), GST/Shc(46-215), GST/Shc(46-229), GST/Shc(1-392) (PTB + CH domains), and GST/Shc(371-474) (SH2 domain), where numbers in parentheses refer to residues of Shc p52(1) . Proteins were expressed following usual protocols and purified by affinity chromatography using glutathione-agarose. Proteins were eluted from the washed beads with glutathione or 8.0 M urea and dialyzed against 100 mM ammonium bicarbonate containing 1.0 mM dithiothreitol; no difference in function was noted following elution by the two methods. All isolated proteins were found to have >90% homogeneity and appropriate sizes following separation by SDS-polyacrylamide gel electrophoresis. Phosphopeptides were synthesized following a N-Fmoc protecting group strategy and purified by HPLC(17) .

Direct Binding Assays between Amino-terminal Shc Proteins and Phosphopeptides

Peptide LLSNPTpYSVMRSK was radiolabeled with I-labeled Bolton-Hunter reagent and purified by HPLC (24) . To determine which of the fusion proteins would bind to the radiolabeled peptide, 45 nCi (10^5 cpm) of I-phosphopeptide and varying concentrations of the fusion proteins were combined in 150 µl of assay buffer (20 mM Tris-HCl at pH 7.4, 250 mM NaCl, 0.1% bovine serum albumin, and 10 mM dithiothreitol). Glutathione-agarose (50 µl of a 1:10 slurry, was added, the solutions were vortexed, and the samples were incubated overnight at 22 °C with constant mixing. Following centrifugation for 5 min at 12,000 g, supernatant solutions were removed and I radioactivity associated with the pellets was determined. For competition assays, sufficient Shc fusion protein (typically 8-10 µg of GST/Shc(1-238)) to sequester geq7% of the radiolabeled peptide (45 nCi) and varying concentrations of unlabeled peptides were combined in 150 µl of assay buffer. Samples were otherwise treated as described above.

Solution Structures of mT NPXY Peptides

Peptides (1.5 mM) were dissolved in either 0.5 ml of D(2)O (pD 4.8, direct meter reading) or 0.7 ml of H(2)O (pH 4.5) containing 50 mM KCl. Spectra were observed at 400 MHz and 4 °C using a Varian Unity-Plus spectrometer Double-quantum-correlated spectroscopy (DQF-COSY), total correlation spectroscopy (TOCSY; mixing time 55 ms), nuclear Overhauser enhancement spectroscopy (NOESY; mixing times 200, 400, and 600 ms), and rotating-frame Overhauser enhancement spectroscopy (ROESY; mixing times 80 and 160 ms) experiments were performed. The water solvent resonance was attenuated by presaturation. In the NOESY spectra, no evidence of spin diffusion was observed. Resonance assignment was obtained by standard sequential methods. For quantitative comparisons NOESY and ROESY spectra at different mixing times were normalized relative to shared intraresidue cross-peaks (Asn NH(2) and Tyr ortho-meta).


RESULTS AND DISCUSSION

PTB Domain Delineation

Shc-related sequences were expressed as GST fusion proteins to determine the functional boundaries of the Shc PTB domain (Fig. 1, A and B). The fusion proteins were combined with glutathione-agarose beads and the radiolabeled phosphopeptide. Specific binding was observed for concentrations as low as 0.1-0.2 µM of Shc proteins 1-206, 1-215, 1-229, 1-238, and 1-392. Increasing levels of binding were seen with protein concentrations up to approx2 µM, and plateaus in binding were seen at higher concentrations, indicating that the mode of peptide binding was similar with each of the five proteins. In contrast, even the highest concentrations of Shc protein 1-196 showed no detectable peptide binding to demarcate the carboxyl terminus of the domain between residues 196 and 205 of Shc p52.

Since there are additional forms of Shc in mammalian tissues, and one (p46) has a shorter PTB domain due to alternative translational initiation at Met of p52, additional proteins initiated at Met were prepared (46-215 and 46-229). These proteins did exhibit peptide binding, although significantly higher protein concentrations were required for the effect (Fig. 1B). Similarly, the SH2 domain of Shc showed phosphopeptide binding, although even greater protein concentrations were required. By rearranging the equation used to describe bimolecular interactions, K = [RL]/[R][L], to [RL]/[L] = [R]K, the [RL]/[L] value can be represented by the ratio of bound/free ligand and the slope of a line defined by [RL]/[L] versus [R] represents K. Using this approach, initial slopes of the plots of bound/free versus [R] were used to compare relative PTB domain affinities. Analyses of the 9 Shc proteins reveal that (i) proteins 1-206, 1-215, 1-229, 1-238, and 1-392, having the same amino-terminal start site, bind to the mT peptide with equivalent affinities, (ii) proteins having the second start site (46-215 and 46-229) bind with 10-20-fold lower affinity, and (iii) the Shc SH2 domain(371-474) binds with about 50-fold lower relative affinity. These findings agree qualitatively with published studies suggesting that PTB domains from p52 and p46 have function(21, 22) . However, to our knowledge this is the first attempt to quantify relative binding affinities of PTB-related proteins and the first to show reduced affinities for Shc p46 related proteins.

PTB Domain Binding Specificity

Based on these findings, an assay method for analyzing the peptide binding specificity of the Shc PTB domain was developed using protein 1-238 (arbitrarily chosen). Mixtures of GST/Shc(1-238), radiolabeled peptide LLSNPTpYSVMRSK, glutathione-agarose beads, and varying concentrations of unlabeled peptides were equilibrated, and PTB-bound I-peptide was separated from free peptide by centrifugation. The amount of I-peptide associated with the glutathione-agarose pellet varied with concentration and relative affinity of competing ligand (Fig. 1C). In the example shown, the phosphopeptide having sequence LSNPTpYSV competes for PTB binding with an ID value of approx1 µM. In contrast, an identical but unphosphorylated sequence and a scrambled, phosphorylated sequence do not bind appreciably to the PTB domain at peptide concentrations up to 1 mM, indicating that the phosphorylation of tyrosine within a specific sequence context is required for high affinity interactions between the Shc PTB domain and cognate proteins or peptides.

Peptide truncations and substitutions were used to analyze determinants of Shc PTB domain binding specificity in detail (Table 1). The 13-residue peptide LLSNPTpYSVMRSK was designed to have six residues placed symmetrically on either side of pTyr. While one residue (Leu) could be removed from the amino terminus of this sequence with full retention of binding affinity,^4 removal of a second residue (Leu) resulted in a reduction in affinity below levels of assay detection. Results were obtained for a full series of peptides having progressively fewer residues amino-terminal to pTyr, although once the residue at the pTyr position had been deleted binding remained undetectable. A similar truncation series was analyzed for the carboxyl terminus of the peptide. However, in this case, all six residues could be removed from the peptide carboxyl terminus without decreasing affinity. Slightly higher affinities (0.6-1.3 µM) were consistently seen with the shorter sequences, although additional studies will be required to determine whether longer sequences have a negative influence on binding. The amino and carboxyl truncation series suggested that six residues from Leu to pTyr were needed for a high affinity interaction. This concept was tested further with the six-residue peptide LSNPTpY(NH(2)) to show that the hexapeptide motif is both necessary and sufficient for high affinity interactions.



Phosphotyrosine itself binds weakly with the Shc PTB domain as it does with SH2 domains. (^5)Neither phosphoserine nor phosphothreonine bound the PTB domain, as noted previously for SH2 domains.

These data indicate that peptide binding with the Shc p52 PTB domain is closely related to SH2 domain binding in that (i) phosphorylation serves as the on-off switch for recognition, (ii) the surrounding sequence provides specificity for the interaction, (iii) four to six or seven residues participate directly in the interaction, and (iv) overall binding energies and relative contributions for pTyr versus surrounding residues are similar for the interactions. However, the PTB domain shows a direct reversal in the orientation of required peptide interactions: residues amino- but not carboxyl-terminal to pTyr determine specificity. As a general feature of SH2 domain mediated interactions, residues carboxyl-, but not amino-, terminal to pTyr provide binding energy and thereby participate in conferring specificity to the interaction (e.g.(15, 16, 17, 18, 19) , and 24).

Results from the truncation studies show that six peptide residues spanning pTyr to pTyr participate in the interaction. An alanine scan through the LSNPTpYSV sequence was conducted to gather more information about the degree of involvement of each residue. The Leu substitution reduced binding affinity 50-fold, consistent with the role suggested for this residue by the truncation data. Substitution of Ser had no effect, indicating that this side chain may not participate directly. Replacements of Asn, Pro, and Thr within the NPXY motif all had dramatic effects on binding. Substitution of either Asn or Thr led to >300-fold reductions in affinity, while Pro Ala resulted in a 100-fold drop in affinity. Thus, substitutions of five of the six residues within the LSNPTpY sequence significantly reduced binding affinity. Surprisingly, substitution of Ser also reduced binding affinity 14-fold, even though deletion of the residue had no effect. The sequence specificity exhibited by the Shc PTB domain is underscored by binding studies with randomized (keeping pTyr at the same relative position) and inverted sequences, where affinity in each case was below levels of assay detection.

When substitutions do not perturb the structure, it is possible to assess side chain-specific contributions to binding energy from alanine scanning and related approaches. However, for PTB-binding ligands, this appears not to be the case. Since the K value is approximately 10M and DeltaG = -RTlnK(D), DeltaG ≅ -9.3 kcal/mol for the interaction between the parent mT peptide and the Shc PTB domain.^6 The effect of each substitution can be approximated by the expression Delta(DeltaG) = -RTln(ID^1/ID^2), such that a 100-fold drop in affinity translates to a loss of 2.6 kcal/mol in binding energy. The sum of the individual differences in free energy resulting from each substitution in the alanine scan is >16 kcal/mol. As this is much greater than the available binding energy (-9.3 kcal/mol), at least some of the replacements may have more global effects on peptide structure.

NPXpY Peptide Structure

We have analyzed the mT antigen-derived peptides for inherent structure. Algorithms for predicting secondary structure (25) suggest that the unphosphorylated NPTY sequence has a high probability of adopting a beta turn (data not shown). In fact, peptides corresponding to NPXY internalization motifs of such proteins as the LDL, transferrin, and insulin receptors do form relatively stable beta turn structures in solution(26, 27, 28) . The structure of the mT antigen-derived sequence has not been studied previously, nor has the general effect of phosphorylation on beta turn stability. The one-dimensional ^1H NMR spectra of peptide LSNPTpYSV exhibits chemical shift dispersion characteristic of ordered (non-random) structure (Fig. 2A). Each amide resonance is individually resolved and not attenuated by solvent presaturation. The spectra also exhibit evidence of a minor conformer (<10%) in slow exchange on the NMR time scale, presumably due to the cis-proline isomerization. The unphosphorylated sequence (LSNPTYSV) was analyzed under identical conditions (data not shown). Chemical shifts of the predominant conformer were similar, with two exceptions: (i) the ortho (2, 6) and meta (3, 5) aromatic resonances of tyrosine itself are shifted downfield as expected (Delta 0.10 and 0.33 ppm), and (ii) a beta-proton of Asn is perturbed. The latter, a transmitted effect, is consistent with the weakly polar amide-aromatic (Asn-Tyr) interaction as previously postulated in the unphosphorylated NPXY motif of the LDL receptor(26) .


Figure 2: ^1H NMR spectra of peptide LSNPTpYSV. A, one-dimensional spectrum, with amide resonance assignments: 1, Ser; 2, Asn; 3, Ser; 4, Thr; 5, pTyr; 6, Val; 7 and 7`, Asn NH(2); 8, CH pTyr; 8`, CH pTyr. Two-dimensional NOESY (B) and ROESY (C) spectra demonstrate inter-residue contacts. Cross-peaks are labeled: a or c, Thr/pTyr; b or d, pTyr/Ser; e, Ser/Val; and f, Ser/Asn. The NOESY and ROESY mixing times were 400 and 160 ms, respectively.



Two-dimensional NOESY and ROESY spectra obtained for the phosphorylated (Fig. 2, B and C) and unphosphorylated (data not shown) sequences reveal a strong contact between amide protons of Thr and pTyr or Tyr that is characteristic of a beta turn. Since the intensity of the NOE between Thr and Tyr is essentially the same, whether or not Tyr is phosphorylated, we conclude that the stability of the beta turn is unaffected by phosphorylation. Accentuation of this contact in the NOESY relative to the ROESY spectrum suggests that local correlation times () at the center of the turn are longer (i.e. more ordered) than at the ends of the peptide. This inference is supported by the temperature dependence of the amide proton resonances: with increasing temperature in the range 4-35 °C, amide resonances in unstructured peptides characteristically exhibit progressive broadening and attenuation due to more rapid exchange with solvent water. In the mT NPTY peptides, however, the amide resonances of the turn itself (Asn, Thr, pTyr, and Ser) remain sharp, whereas those of Ser and Val exhibit the expected attenuation. Protection from solvent exchange is associated with retention of the Thr/pTyr NOE throughout this temperature range. Together, these results demonstrate that the NPTY and NPTpY sequences nucleate a stable beta turn in solution. Alanine substitutions generally diminish the stability of beta turns (25) . Since Ala substitutions within the beta turn of the mT peptide have dramatic effects on binding, beta turn stability and PTB domain binding affinity appear to function in parallel.

Conclusion

Previous sequence alignments showed that the amino terminus of Shc represents a new class of phosphotyrosine binding domain structurally distinct from SH2 domains(21, 22, 23) . We now show that the Shc PTB domain binds a LXNPTpY sequence from the polyoma virus middle T antigen in a fashion that is functionally distinct from SH2 domains, that particular positions within this sequence are critical to the maintenance of a high affinity interaction, and that these sequences tend to form beta turn structures in solution. Since many proteins contain NPXY motifs, one might expect that each would be recognized by the Shc PTB domain. However, we have also shown that residues other than Asn and Pro are critical for high affinity interactions, particularly Leu and Thr, and the NPXY sequences from such proteins as the insulin and EGF receptors, ErbB2, ErbB3, ErbB4, TrkA, TrkB, and TrkC vary at these positions. In fact, in one case tested, an insulin receptor-derived NPEpY peptide binds the Shc PTB domain with low affinity (data not shown), in support of the notion that distinct NPXpY motifs are recognized differentially, perhaps by distinct PTB proteins yet to be described.


FOOTNOTES

*
These studies were funded by National Science Foundation Grant MCB 93-04469 and a grant from the Juvenile Diabetes Foundation, International. The Biochemistry Facility at the Joslin Diabetes Center is supported by National Institutes of Health grant DK36836. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Supported by a fellowship from the Swiss Cancer League.

Funded by institutional and individual fellowships from National Institutes of Health Grants DK07260 and DK09146, respectively.

**
Established Investigator of the American Heart Association.

§§
Recipient of a Burroughs Wellcome Fund Scholar Award in Experimental Therapeutics. To whom correspondence and reprint requests should be addressed: Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. Tel.: 617-732-2528; Fax: 617-732-2593; Shoelson{at}Joslab.Harvard.edu.

^1
The abbreviations used are: PDGF, platelet-derived growth factor; EGF, epidermal growth factor; GST, glutathione S-transferase; pTyr, phosphotyrosine; PTB, phosphotyrosine binding; SH2, Src homology 2; NOESY, nuclear Overhauser enhancement spectroscopy; ROESY, rotating-frame Overhauser enhancement spectroscopy.

^2
Numbering of the Shc sequence takes into account the presence of a GCA triplet encoding Ala between residues 307 and 308 that is not present in the published sequence.

^3
G. Wolf, R. D. Case, and S. E. Shoelson, unpublished observations.

^4
The positions of peptide residues are referred to relative to the position of pTyr.

^5
G. Wolf and S. E. Shoelson, submitted for publication.

^6
G. Wolf and S. E. Shoelson, unpublished observations.


ACKNOWLEDGEMENTS

We thank D. N. M. Jones, Q. X. Hua, and K. Hallenga for advice on NMR methods.


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