©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Phosphotyrosine Interaction Domain of SHC Recognizes Tyrosine-phosphorylated NPXY Motif (*)

Zhou Songyang (1) (2), Benjamin Margolis (3), Manas Chaudhuri (4), Steve E. Shoelson (4), Lewis C. Cantley (1)(§)

From the (1)Department of Cell Biology, Harvard Medical School and Department of Medicine, Beth Israel Hospital, Boston, Massachusetts 02115, (2)Department of Physiology, Tufts University School of Medicine, Boston, Massachusetts 02111, (3)Department of Pharmacology, New York University Medical School, New York, New York 10016, and (4)Research Division, Joslin Diabetes Center, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Reversible assembly of intracellular signaling complexes is, in some cases, mediated by direct binding of a Src homology 2 (SH2) domain of one protein to a phosphotyrosine moiety of another protein (Cantley, L. C., Auger, K. R., Carpenter, C. L., Duckworth, B., Graziani, A., Kapeller, R., and Soltoff, S.(1991) Cell 64, 281-302). Using a degenerate phosphotyrosine-containing peptide library, we showed that individual SH2 domains recognize phosphotyrosine in a specific sequence context to provide fidelity in signaling (Songyang, Z., Shoelson, S. E., Chaudhuri, M., Gish, G., Pawson, T., Haser, W. G., 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., and Cantley, L. C.(1993) Cell 72, 767-778). Recently a second type of phosphotyrosine interaction domain (PID) or phosphotyrosine-binding domain (PTB) was discovered in the amino terminus of the SHC proto-oncoprotein (Kavanaugh, W. M., and Williams, L.(1994) Science 266, 1862-1865; Blaikie, P., Immanuel, D., Wu, J., Li, N., Yajnik, V., and Margolis, B.(1994) J. Biol. Chem. 269, 32031-32034). Here we demonstrate, using a phosphotyrosine peptide library, that the SHC PID domain preferentially binds to the sequence Asn-Pro-Xaa-phosphotyrosine. This motif is in agreement with sequences at sites implicated in in vivo SHC binding. These results indicate that while SH2 domains predominantly interact with specific residues carboxyl-terminal of phosphotyrosine, the PID domain has high specificity for residues amino-terminal of phosphotyrosine.


INTRODUCTION

Protein-tyrosine kinases (PTKs)()play a crucial role in cellular proliferation and differentiation. Tyrosine phosphorylation by PTKs creates binding sites for SH2 domains that specifically recognize the phosphotyrosine moiety. Therefore, proteins containing SH2 domains can be recruited to the site of phosphorylation and signaling complexes can be formed in a phosphotyrosine-dependent manner(1, 2) .

Primary sequences adjacent to the phosphotyrosine are required for specific SH2 domain recognition(2, 7) . Using the oriented phosphopeptide library technique, we showed that residues COOH-terminal to the phosphotyrosine are critical for high affinity and specificity in SH2 domain binding(3, 4) . In many cases, the specificities predicted by the peptide library technique have been confirmed by mapping in vivo binding sites of SH2 domains, by affinity analysis, and by structural studies(8, 9, 10, 11, 12, 13, 14) . While the original phosphopeptide libraries only varied amino acids COOH-terminal of phosphotyrosine, studies from other laboratories indicated that residues NH-terminal of phosphotyrosine are critical for specific binding to certain SH2-containing proteins(15) . For example, many groups have reported that SHC, a SH2 domain-containing protein, binds to tyrosine-phosphorylated NPXY motifs on several signal molecules(16, 17, 18) . It was thought that the SH2 domain of SHC mediated these interactions(16, 17, 18) . In fact, many of the sites mapped for in vivo SHC binding were in poor agreement with the optimal motif we predicted for the SH2 domain of SHC using the original peptide library(4) . A more degenerate library was constructed in order to explore the relative importance of residues both NH- and COOH-terminal of the phosphotyrosine moiety for SH2 domain recognition. While this work was in progress, a second type of phosphotyrosine-binding domain, structurally distinct from SH2 domains, was discovered in the NH terminus of SHC (PID or PTB domain)(5, 6, 26) . Using the new peptide library we show here that the SHC SH2 domain specificity is determined mainly by residues COOH-terminal of the phosphotyrosine while the SHC PID domain specificity is determined primarily by residues NH-terminal of phosphotyrosine.


MATERIALS AND METHODS

GST Fusion Proteins

SH2 domains of p85, Nck, SHC, and Crk were constructed as described previously(3, 4) . GST-SHC PID is a GST fusion containing the NH-terminal (PID) region of SHC (1-209)(6) .

Peptide Libraries

The peptide library used for these studies has the sequence GAXXXpYXXXKKK, where pY indicates phosphotyrosine and X indicates any amino acid except for Trp and Cys. This library was synthesized as described previously(3) . The theoretical degeneracy of this peptide library is 1834 million.

Peptide Affinity Purification and Data Analysis

After incubating the degenerate peptide library (1 mg) with 200-300 µg of bead-immobilized GST-SH2 or PID fusion proteins, the beads were washed twice with phosphate-buffered saline. The phosphopeptides bound were eluted using 20 mM phenyl phosphate(3) . Peptide sequencing and data analysis were as described previously(3) .


RESULTS AND DISCUSSION

The Importance of Amino Acids NH-terminal to the Phosphotyrosine for SH2 Domain Specificity

The specificities of SH2 domains from SHC, Crk, Nck, and p85 were studied using a peptide library with residues degenerate at positions both N- and COOH-terminal to the phosphotyrosine. The GST-SH2 domains were used to affinity-purify a subgroup of high affinity peptides. To determine the preference of amino acids, the bound phosphopeptides were sequenced and compared with the sequence of the peptides nonspecifically retained on the GST beads and the sequence of the complete peptide library. The data were normalized such that the sum of the quantity of all amino acids at a degenerate position is 18 (the number of different amino acids). Thus, a selectivity value greater than 1 indicates preference for a given amino acid. The results are presented in . These results were also compared with the selectivities obtained using a peptide library in which only three amino acids COOH-terminal to the phosphotyrosine are degenerate(3, 4) . As shown in , although the selectivities determined with the more highly degenerate library are lower than those observed with the original library, the optimal amino acids at positions COOH-terminal of phosphotyrosine were similar with the two different peptide libraries. However, in contrast to expectations from mutational analysis of SHC and Nck binding sites(16, 17, 18, 19) , the SH2 domains of these proteins did not exhibit strong selectivity for amino acids NH-terminal of phosphotyrosine. Interestingly, the Crk, Nck, and SHC SH2 domains showed a weak selection for Tyr at the Tyr(P)-1 position. The p85, Crk, and SHC domains also exhibited a weak selection for acidic amino acids at the Tyr(P) and Tyr(P) positions.

NH-terminal PID Domain of SHC Selects for Peptides with a NPXpY Motif

The fact that the SH2 domain of SHC shows little selectivity for amino acids NH-terminal of phosphotyrosine raised the possibility that a different region of SHC mediates binding of SHC to proteins with the NPXpY motif. Recent studies have indicated the presence of a second domain (PID domain) in the NH terminus of SHC that can interact with tyrosine-phosphorylated proteins(5, 6, 26) . The PID domain appears to have no significant structural similarity to SH2 domains. We tested the specificity of the SHC PID domain using the degenerate peptide library. This domain showed a strong selection for peptides with Asn 3 residues NH-terminal to the phosphotyrosine (Fig. 1). The optimal motif selected is Asn-Pro-X-pY-Phe-X-Arg. In contrast to SH2 domains, the PID domain selectivity appears to be dominated by contact with residues NH-terminal of the phosphotyrosine moiety. Since the sequence NPXY is found in the sites where SHC binds to tyrosine-phosphorylated TrkA, ErbB3, and polyoma middle t antigen(16, 17, 18) , binding to these proteins is likely to be mediated by the PID domain rather than the SH2 domain of SHC (). Consistent with our peptide library prediction that Asn at Tyr(P) and Pro at Tyr(P) are important for binding to the PID domain, mutation of these 2 residues in polyoma middle t antigen and ErbB3 inhibited in vivo SHC association(16, 17) .


Figure 1: The specificity of the PID domain of SHC. A degenerate peptide library was used to study the specificity of the SHC PID domain (see ``Materials and Methods''). The peptides that bound to GST-SHC PID were purified and sequenced, and the sequence of purified peptides was compared with that of the starting peptide library. The data were normalized such that a value 1 or less indicates no selectivity for a given amino acid (3). A, B, C, D, E, and F show, respectively, the selectivity (average of two experiments) at the Tyr(P), Tyr(P), Tyr(P), Tyr(P), Tyr(P), and Tyr(P) positions. Amino acids are presented in one-letter codes.



In addition to the conserved Asn at the Tyr(P) and Pro at the Tyr(P) positions, some of SHC-binding sites also have phenylalanine or hydrophobic amino acids at the Tyr(P) position (), in agreement with the weak selection for these residues in the peptide library result (Fig. 1). However, in contrast to SH2 domains, which generally select for peptides with hydrophobic amino acids at the Tyr(P) position(3, 4) , the PID domain weakly selected for hydrophilic amino acids at the 3-position with Arg being preferred. Although none of the known SHC binding sites has Arg at the 3-position, most have hydrophilic amino acids (). The sites on TrkA, EGF receptor, and ErbB3 also have acidic residues at the Tyr(P) position. In polyoma middle t antigen, this residue is replaced by Thr. In our peptide library experiments, we didn't detect any selection at the Tyr(P) position, indicating that this position is not critical for SHC binding. Instead, the Tyr(P) residue could be a signal for tyrosine phosphorylation. We have recently determined the substrate specificities of several protein-tyrosine kinases (PTKs)(20) . All the receptor PTKs we studied were shown to prefer acidic residues at the -1 position, while cytosolic PTKs selected -branch amino acids at the same position (20). Consistently, all the NPXY sequences on the receptor PTKs, TrkA, EGF receptor, and ErbB3, have acidic residues at the -1 positions and are thought to be autophosphorylation sites. In contrast, the NPTY site on middle t antigen contains a -branch amino acid at the -1 position and is phosphorylated by cytosolic Src family PTKs.

A search of protein data bases for other NPXY sequences revealed many potential candidates for SHC binding. The NPXY motif was originally discovered as a code for protein internalization (21). NMR studies have determined that this sequence could form a tight turn structure(22) . Unfortunately, little work has been done to tell how many of these sequences are tyrosine-phosphorylated in vivo. An NPXY site on the insulin receptor (Tyr-960) is known to be phosphorylated and is critical for signaling(23) . The NPXY sites on the Trk PTK family are very likely recognized by SHC, because they are similar to the TrkA NPXY sequence (). While this paper was in review, two groups reported that the PID domain rather than SH2 domain of SHC is responsible for binding the NPXY sites on the EGF, nerve growth factor, and insulin receptors(24, 25) .

Our finding that the PID domain of SHC preferentially recognizes sequences NH-terminal to the phosphotyrosine provides a new insight into protein-protein interactions mediated by tyrosine phosphorylation. It is clear now that two different classes of domains are responsible for binding to phosphotyrosine. While one class (the SH2 domains) primarily contacts amino acids COOH-terminal to the phosphotyrosine, the other class (PID domain family) contacts sequences NH-terminal to the phosphotyrosine. Several homologues of the SHC PID domain have been identified, and it is likely that more will be found(5, 25, 26) . The SHC protein now becomes a very interesting adapter molecule. It is able to trap tyrosine-phosphorylated proteins in two distinct ways, either through its SH2 domain or its PID domain. In addition, phosphorylation of SHC on tyrosine results in recruitment of GRB2 into the complex via the GRB2 SH2 domain. Thus, the distinct specificities of the SH2 and PID domains of SHC increase the number of proteins with which SHC can interact in signaling.

  
Table: Comparison of phosphopeptide specificity of the SHC PID domain to specificities of SH2 domains


  
Table: Sequences at SHC binding sites



FOOTNOTES

*
This work was supported by the American Cancer Society and the Lucille P. Markey Charitable Trust. 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.

§
To whom correspondence should be addressed. Tel.: 617-278-3040; Fax: 617-278-3131.

The abbreviations used are: PTK, protein-tyrosine kinase; GST, glutathione S-transferase; PID, phosphotyrosine interaction domain; PTB, phosphotyrosine-binding domain; EGF, epidermal growth factor.


ACKNOWLEDGEMENTS

We thank Michael Berne (Tufts University) for peptide sequencing.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.