A Novel Phosphotyrosine Motif with a Critical Amino Acid at Position −2 for the SH2 Domain-mediated Activation of the Tyrosine Phosphatase SHP-1*

SHP-1 is a protein-tyrosine phosphatase associated with inhibition of activation pathways in hematopoietic cells. The catalytic activity of SHP-1 is regulated by its two SH2 (Src homology 2) domains; phosphotyrosine peptides that bind to the SH2 domains activate SHP-1. The consensus sequence (I/V)XYXX(L/V) is present in the cytoplasmic tails of several lymphocyte receptors that interact with the second SH2 domain of SHP-1. In several of these receptors, there are two or three occurrences of the motif. Here we show that the conserved hydrophobic amino acid preceding the phosphotyrosine is critical for binding to and activation of SHP-1 by peptides corresponding to sequences from killer cell inhibitory receptors. The interaction of most SH2 domains with phosphopeptides requires only the phosphotyrosine and the three residues downstream of the tyrosine. In contrast, the shortest peptide able to bind or activate SHP-1 also included the two residues upstream of the phosphotyrosine. A biphosphopeptide corresponding to the cytoplasmic tail of a killer cell inhibitory receptor with the potential to interact simultaneously with both SH2 domains of SHP-1 was the most potent activator of SHP-1. The hydrophobic residue upstream of the tyrosine was also critical in the context of the biphosphopeptide. The contribution of a hydrophobic amino acid two residues upstream of the tyrosine in the SHP-1-binding motif may be an important feature that distinguishes inhibitory receptors from those that provide activation signals.

Receptor-mediated activation of cellular responses is often initiated by the activation of tyrosine kinases. The signal is propagated by the sequential recruitment of proteins to the phosphorylated targets of the kinases. SH2 (Src homology 2) domains are protein modules that specifically bind to phosphotyrosine residues and are found in a variety of proteins such as protein kinases and adapter molecules. A general feature of all SH2 domains is a conserved pocket that binds the phosphotyrosine moiety. The specificity of the interaction between individual SH2 domains and particular phosphoproteins is generally determined by three to six residues following the phosphotyrosine and often incorporates a hydrophobic residue at the third position (reviewed in Ref. 1). This specificity is provided by specific pockets that bind the phosphotyrosine and ϩ3 residues as revealed by structural studies of several SH2 domains complexed with phosphopeptides (1).
A small family of protein-tyrosine phosphatases contain SH2 domains. This family is composed of mammalian SHP-1 and SHP-2 and the Drosophila homologue of SHP-2, corkscrew. These protein-tyrosine phosphatases are important regulators of many cellular signaling processes (reviewed in Ref. 2). SHP-2 is broadly expressed and is important for activation signals through several different growth factor receptors. In contrast, SHP-1 is expressed predominantly in hematopoietic cells and has been implicated in inhibition of signaling through growth factor, cytokine, and antigen receptors. These protein-tyrosine phosphatases contain two SH2 domains in tandem and a single catalytic domain. The protein-tyrosine phosphatase activity is negatively regulated by the SH2 domains in that their removal or occupancy by phosphopeptides increases the phosphatase activity of the catalytic domain (3)(4)(5)(6)(7)(8). The SH2 domains of SHP-1 and SHP-2 are more closely related to each other than to any other SH2 domains known, with their next closest relatives being the SH2 domains of phosphatidylinositol 3-kinase and phospholipase C-␥1 (9). It is of particular interest to understand the specificity of these SH2 domains because they control both the localization and activation of these phosphatases.
The optimal binding sequences for SH2 domains have been defined with degenerate phosphopeptide libraries and pooled sequence analysis. The NH 2 -terminal SH2 domain (SH2N domain) 1 of SHP-2 binds preferably to pY(I/V)X(V/I) (9), and the SH2N domain of SHP-1 binds preferably to pYFXF (10). The predictive value of these motifs was supported by the presence of appropriate sequences in several receptors that bind to the SH2N domain of SHP-1, such as c-Kit, and erythropoietin and interleukin-3 receptors. Due to a strong sequence similarity to the SH2N domains of both SHP-1 and SHP-2, the second SH2 domain (SH2C domain) of SHP-1 is predicted to have a similar preference for hydrophobic residues at positions ϩ1 and ϩ3 (10).
In contrast to the binding studies that considered only residues downstream of the phosphotyrosine, we identified the motif (I/V)XYXXL by sequence alignment of inhibitory receptors known to recruit SHP-1 (11). These include the B lymphocyte receptors CD22 (12) and Fc␥RIIB (13) and the family of killer cell inhibitory receptors (KIR) expressed in natural killer and T lymphocytes (11). Peptides derived from KIR and Fc␥RIIB have been reported to bind to the SH2C domain of SHP-1 (11,13), whereas intact CD22 has been reported to interact with the SH2N domain (14). The motif is also found in NKG2A (11,15), a molecule associated with a receptor complex recently shown to be involved in inhibition of human natural killer cells (16), and in gp49, a molecule with inhibitory potential expressed in mouse mast cells (17) and in natural killer cells (18). A related motif, VXYXXV, was found in Ly-49 molecules (11), another family of inhibitory receptors that are expressed in mouse natural killer cells (19). Phosphopeptides derived from Ly-49 interact with the SH2 domains of both SHP-1 and SHP-2 (20). The inhibitory receptors expressed in natural killer cells, including human KIR and NKG2, as well as the mouse inhibitory receptors Ly-49 and gp49 also share the sequence QEVT just upstream of the tyrosine.
Several recent observations have raised questions about the specificity of the interaction of receptors with the (I/ V)XYXX(L/V) motif and SHP-1. SHP-1 was implicated in the inhibitory signals of Fc␥RIIB because of a defect in the receptor's signaling in B cells from SHP-1-deficient mice (13). Recently, it has been reported that Fc␥RIIB functions in mast cells independently of SHP-1 and associates with the inositol phosphatase SHIP, which also contains an SH2 domain (21). Catalytically inactive SHP-1 acts as a dominant-interfering molecule for the inhibitory signals delivered by KIR (11), and SHP-1 associates with the receptor upon tyrosine phosphorylation (11,20,22,23). However, it remained possible that the dominant-interfering mutant of SHP-1 exerted its effect by preventing the association of a different protein with tyrosinephosphorylated KIR, which was itself important in providing the inhibitory signal. In addition, studies with metabolically labeled cells show the association of KIR with a protein that corresponded in size to SHP-1 as well as several unidentified proteins (23). To address this issue, we have analyzed proteins that bind to synthetic phosphotyrosine peptides corresponding to cytoplasmic tail sequences of KIR and Fc␥RIIB.
To determine whether the amino acids identified in the consensus sequence (I/V)XYXX(L/V) are important for interaction with SHP-1, we have examined the specificity of the interaction of sequences containing this motif with SHP-1 and the contribution of these residues to the activation and binding of SHP-1. The data established a critical role for the amino acid two residues upstream of the tyrosine in SHP-1 binding and activation.

EXPERIMENTAL PROCEDURES
Peptides-All peptides were purchased from Quality Controlled Biochemicals, Inc. (Hopkinton, MA) and supplied at a purity of Ͼ98%. The phosphotyrosyl residue was incorporated during peptide synthesis. The peptides were synthesized by Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry, purified by reversed-phase high pressure liquid chromatography, and analyzed for purity by ion spray mass spectrometry and 31 P NMR. Phosphopeptides were dissolved at 0.057 mM and coupled to Affi-Gel-10 beads as instructed by the manufacturer (Bio-Rad) at a ratio of 1 ml of peptide to 1 ml of settled beads. The sequence of peptide Y1Y2 is EQDPQEVTYAQLNHSVFTQRKITRPSQRPKTPPTDIIVYTELPNA. pY1Y2, Y1pY2, and pY1pY2 have the same sequence as Y1Y2 with the first, second, or both tyrosines phosphorylated, respectively. The sequence of pY1pY2-2A is EQDPQEAT(pY)AQLNHSVFTQRKITRP-SQRPKTPPTDIAV(pY)TELPNA.
Silver Stain--Cell lysates were prepared from bulk human T cell populations cultured in the presence of 100 units/ml recombinant interleukin-2 (gift of Hoffmann-La Roche). The cells were lysed at 4 ϫ 10 7 /ml in lysis buffer (1% Triton X-100, 0.15 M NaCl, and 20 mM Tris, pH 8, with 1 mM NaVO 3 , 1 mM iodoacetamide, 5 g/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride). For each sample, a suspension of beads corresponding to 10 g of peptide was added to 1 ml of lysate and mixed at 4°C for 5 h. The samples were washed six times with lysis buffer, separated by 9% SDS-polyacrylamide gel electrophoresis under reducing conditions, and stained with Silver Stain Plus according to the manufacturer's instructions (Bio-Rad).
GST Fusion Proteins-The fusion protein of GST and mouse SHP-1 or the SH2C domain of SHP-1 has been described (24). To generate a construct of GST and the catalytic domain of SHP-1, GST-catSHP-1, a cDNA fragment encoding amino acids 247-595 of SHP-1 was derived from a murine SHP-1 cDNA in the pGEX vector (Pharmacia Biotech Inc.) using polymerase chain reaction primers (forward primer, GAAT-TCGAGATGGAGTTTGAGAGTCTACAA; and reverse primer, TGTCA-GAGGTGGGCACGTCA). The amplified cDNA fragment was digested with the restriction enzyme EcoRI and cloned into the EcoRI site of the pGEX vector. The fusion proteins were purified on glutathione-Sepharose 4B (Pharmacia) and eluted with 15 mM reduced glutathione. Purified GST-SHP-1 was dialyzed into phosphate-buffered saline, pH 7.4; GST-catSHP-1 was dialyzed into 0.15 M NaCl and 10 mM Tris-HCl, pH 8. The concentration of the fusion proteins was determined with the micro-BCA assay kit (Pierce).
Phosphatase Assay-Assays using soluble GST-SHP-1 and the substrate p-NPP were performed essentially as described (11). Peptides were dissolved in a small volume of Me 2 SO; diluted in phosphatase buffer (100 mM NaCl, 1 mM dithiothreitol, 0.5 mM EDTA, and 100 mM Hepes, pH 7.4) to a final concentration of 285 M peptide and 5% Me 2 SO; and then serially diluted in a microtiter plate. 25 l of 20 mM p-NPP (Sigma) and 5 l of GST-SHP-1 in phosphatase buffer were added and incubated at 37°C for 10 min. The reaction was stopped by addition of 100 l of 2 N NaOH.
The assay to measure release of inorganic phosphate from phosphopeptides was based on a malachite green detection system (25). The standard curve was determined with KH 2 PO 4 (dried overnight at 80°C prior to weighing). A stock solution for the standard was made by first dissolving in water and then diluting in assay buffer (100 mM Tris-HCl, pH 7.5, and 5 mM 2-mercaptoethanol). The peptides were dissolved in a small volume of Me 2 SO and then diluted in assay buffer to 5% Me 2 SO and 0.057 mM peptide. 11.4 nmol of peptide were incubated in the absence or presence of 0.1 g of GST-catSHP-1 (in a final volume of 25 l) at 22°C for 10 min. 50 l of the malachite green detection solution was added, and the absorbance at 650 nm was measured after 5 min.
SHP-1 Binding Assay-Cell lysate was prepared at a concentration of 10 7 Jurkat cells in 1 ml of lysis buffer (1% Triton X-100, 0.15 M NaCl, 2 mM NaVO 3 , 5 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, and 20 mM Tris-HCl, pH 8). Peptide stocks were prepared at 0.57 mM peptide in 10% Me 2 SO and were diluted 1:1 in 2 ϫ 0.3 M NaCl and 40 mM Tris-HCl, pH 8, and then serially diluted in 0.15 M NaCl and 20 mM Tris-HCl, pH 8. A 100-l volume of peptide was added to 100 l of lysate or GST-SH2C(SHP-1) diluted in lysis buffer. Beads bearing KIR-pY1 were added, and the mixture was incubated at 4°C for 90 min. Samples were washed three times in lysis buffer and analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting with anti-SHP-1 antibodies or with anti-GST antibodies (Upstate Biotechnology, Inc., Lake Placid, NY) using the chemiluminescence detection system (Pierce).

RESULTS
Binding of SHP-1 to Phosphotyrosine Peptides Derived from KIR-To assess the relative specificity of the cytoplasmic tail sequences of KIR and Fc␥RIIB for SHP-1, proteins that associate with phosphopeptides coupled to beads were analyzed by silver staining after separation by SDS-polyacrylamide gel electrophoresis. The phosphopeptide derived from the Fc␥RIIB sequence bound two major proteins from T cell lysates, corresponding in size to SHIP and SHP-1, respectively (Fig. 1). In contrast, phosphopeptides derived from the first tyrosine in the KIR cytoplasmic tail or corresponding to the biphosphorylated KIR sequence each bound to a single major protein that corresponded in size to SHP-1. Western blotting confirmed the presence of SHIP at 155 kDa in the Fc␥RIIB peptide-associated proteins and of SHP-1 at 62 kDa in both the Fc␥RIIB and KIR peptide-associated proteins (data not shown). A control tyrosine-phosphorylated peptide corresponding to a sequence in the T cell receptor -chain did not detectably bind to proteins in cell lysates (Fig. 1). By this method, the tyrosine-phosphorylated sequence from Ly-49 did not appear to bind to any proteins. Similar data were obtained with lysate prepared from human natural killer cells or from a human B cell line (data not shown). In experiments using mouse cell lines as a source of lysate, weak bands were detected for Ly-49 at 62 and 66 kDa (data not shown). Therefore, whereas all sequences with the motif (I/V)XYXX(L/V) interact with SHP-1, only Fc␥RIIB was able to interact with both SHP-1 and SHIP.
Activation of SHP-1 by Phosphotyrosine Peptides Derived from Lymphocyte Receptors That Bind SHP-1-To evaluate the importance of the (I/V)XYXX(L/V) motif in the interaction with SHP-1, synthetic phosphopeptides derived from the primary sequence of several lymphocyte receptors (Table I) were tested for their ability to activate SHP-1 in vitro. The peptides activated SHP-1 with the hierarchy CD22-pY5 Ͼ KIR-pY1 Ϸ Fc␥RIIB Ͼ KIR-pY2 Ϸ Ly-49-pY1 ( Fig. 2A). The -pY5 peptide derived from the human T cell receptor-associated -chain corresponds to part of the immune receptor tyrosine-based activation motif that binds the SH2 domains of the ZAP-70 kinase (26). This peptide, containing the sequence GLpYQGL, failed to activate SHP-1 ( Fig. 2A), suggesting that pYXXL is not sufficient for SHP-1 activation. These data correlate well with reported binding measurements that indicated a higher affinity of SHP-1 for KIR-pY1 than for KIR-pY2 and Ly-49-pY1 and a lack of binding to another -chain-derived peptide (20).
To establish the core sequence necessary for activation of SHP-1 by the KIR-pY1 peptide, a set of shorter peptides based on the sequence of KIR-pY1 were tested for their ability to activate SHP-1. Whereas the four-residue peptide pYAQL was insufficient to activate SHP-1, the two amino acids upstream of the tyrosine in VTpYAQL conferred almost all of the activity of the original KIR-pY1 peptide (Fig. 2B).
Residues Upstream of Phosphotyrosine Are Important for Activation of SHP-1-To directly test the role of residues at positions Ϫ1 and Ϫ2 in SHP-1 activation, a set of KIR-pY1 analogues were synthesized with substitutions at these positions (Table II). Substitutions of threonine at position Ϫ1 with histidine (found in CD22-pY5) or leucine (found in -pY5) did not substantially affect SHP-1 activity (Fig. 2C). However, an alanine in this position partially compromised the peptide's activity. The effect of substitutions at position Ϫ2 provided clear evidence for the importance of this residue in SHP-1 activation (Fig. 2B). Substitution of valine with isoleucine, the residue found at this position in the CD22-pY5, Fc␥RIIB, and KIR-pY2 peptides, increased the activation of SHP-1. A conservative substitution with leucine did not significantly alter the activity of the peptide. All other substitutions tested (alanine, serine, aspartate, and arginine) seriously compromised the ability of the peptide to activate SHP-1 (Fig. 2D).
To control for the possibility that position Ϫ2 had an influ-ence on dephosphorylation of the peptide by SHP-1, we tested the various peptides in an assay that compared them as substrates of the isolated phosphatase domain of SHP-1 (Fig. 3).
Although differences in the ability of SHP-1 to dephosphorylate the various peptides were obvious, there was no correlation between resistance to dephosphorylation by GST-catSHP-1 and the ability to activate full-length SHP-1. For example, pY1-2A, which failed to activate SHP-1, is not a better substrate than KIR-pY1. Also of interest, pYAQL was a worse substrate than KIR-pY1 (data not shown). Therefore, the influence of position Ϫ2 is most likely at the level of interaction with the SH2 domains of SHP-1.
The hierarchy in the ability of peptides from different receptors to activate SHP-1 and the conservation of the sequence QEVTYXXL in the receptors expressed in natural killer cells suggested that positions other than position Ϫ2 could be important for achieving optimal activation of SHP-1. Therefore, another series of peptides with various substitutions in the KIR-pY1 sequence were generated (Table II) and tested for their ability to activate SHP-1. Substitutions with alanine at position Ϫ4 caused a partial reduction of SHP-1 activity (data not shown), but not below levels obtained with the KIR-pY2 or Ly-49 peptide. Individual substitution at position Ϫ6 and double substitutions at positions Ϫ4 and Ϫ3 did not modify the ability of the peptide to activate SHP-1 (data not shown).
Role of Residues Downstream of Phosphotyrosine in Activation of SHP-1-The reduced activation of SHP-1 by Ly-49 as compared with that by KIR-pY1 can be explained by their difference at position ϩ3. Substitution of leucine at position ϩ3 in KIR-pY1 with valine (found in Ly-49) diminished the activation of SHP-1 to the same level as that obtained with the Ly-49 peptide (Table III). These data indicate that the SHP-1 SH2 domains prefer leucine over valine at position ϩ3. Two chimeric peptides, initially designed to test whether residues upstream of the YXXL motif were sufficient to dictate recognition by SHP-1, revealed that residues ϩ1 and ϩ2 and perhaps residues ϩ4 and ϩ5 may also contribute to the interaction with SHP-1. The chimeric peptides pY1/ and pY1/Y2 (Table II) behaved similarly to -pY5 and KIR-pY2, respectively (Table  III). Therefore, residues at positions ϩ1, ϩ2, ϩ4, or ϩ5 could interfere with the interaction of SHP-1 with the motif VXYXXL.
Binding of Phosphopeptides to SHP-1 Correlates with Their Ability to Activate SHP-1-The relationship between activation of SHP-1 and peptide binding to SHP-1 was examined. The majority of the peptides listed in Table II were also tested for their ability to compete with the binding of SHP-1 to KIR-pY1 coupled to beads. Representative experiments are shown in Fig. 4. The nonactivating peptide -pY5 did not compete with KIR-pY1, whereas soluble KIR-pY1 competed efficiently (Fig.  4A, top and middle panels). Similar to the weaker activation of SHP-1 by KIR-pY2 relative to that by KIR-pY1, the ability of KIR-pY2 to compete with KIR-pY1 for binding to SHP-1 was  RGKGHDGLpYQGLST -pY5 a This indicates the position of the tyrosine in the mature protein.
b The CD22 peptide was chosen because it corresponds to the sequence from the CD22 cytoplasmic tail reported to have the strongest interaction with SHP-1 (12). c TCR-, T cell receptor -chain.
below that of KIR-pY1 (Fig. 4A, middle panel). The ability of KIR-pY1 peptides with substitutions at position Ϫ2 to compete for binding also correlated with their ability to activate SHP-1. These results reiterated the importance of position Ϫ2 in the consensus motif (I/V/L)XYXX(L/V). The effect of position Ϫ2 was also observed for interaction with the isolated SH2C domain of SHP-1 (Fig. 4B). The only peptide for which the binding and activation data did not correlate perfectly was CD22-pY5. CD22-pY5 was the most potent monophosphopeptide for activation of SHP-1, whereas its efficiency in the competitive binding assay was equivalent to that of KIR-pY1. Given that KIR-pY1 binding was detectable with the isolated SH2C domain and not with the SH2N domain (11), the competition assay likely measures the relative affinity of peptides for the SH2C domain. However, CD22 has been reported to interact with the SH2N domain (14). An interaction of CD22-pY5 with both SH2C and SH2N domains of SHP-1 could explain why it is a better activator of SHP-1 because activation of SHP-1 by phosphopeptides has been reported to be more efficient through the SH2N domain (8,27). On the other hand, the relative resistance of the CD22 peptide to the phosphatase activity of SHP-1 (see Fig. 3) may explain its higher performance in the protein-tyrosine phosphatase assay with p-NPP as a substrate.
Interaction of the KIR-derived peptides and the isolated SH2N domain of SHP-1 was not readily detectable (11). However, biphosphopeptides spanning both tyrosines in the KIR sequence bind SHP-1 with a greater affinity than the corre- sponding monophosphopeptides (22), suggesting that these biphosphopeptides may interact simultaneously with the SH2C and SH2N domains. The ability of such long biphosphopeptides to activate SHP-1 in vitro as compared with the mixture of the individual phosphopeptides was measured (Fig. 5). The left-ward shift of the curve demonstrated that the biphosphopeptide pY1pY2 is the best activator of SHP-1. The much lower level of activity of the peptide pY1pY2-2A confirmed that position Ϫ2 is also important in the context of the biphosphopeptide. The activity of pY1Y2 and Y1pY2 was similar to that of the corresponding 14-amino amino peptides pY1 and pY2, respectively. Therefore, the high activity of pY1pY2 is due to the phosphate moiety on both tyrosines and not to the intervening sequence.

DISCUSSION
Phosphopeptides corresponding to KIR-pY1 or KIR-pY1pY2 bound a single major protein in lymphocyte lysates that comigrated with SHP-1. These observations support the conclusion that a dominant-interfering form of SHP-1 prevents KIR function by specifically competing with SHP-1 (11,28). In contrast, a phosphopeptide corresponding to the Fc␥RIIB cytoplasmic tail bound two major proteins, one of which comigrated with SHP-1 and one with the inositol phosphatase SHIP. These results support the reports of Fc␥RIIB-mediated inhibition by either SHP-1 or SHIP (13,21). More important, these results indicate that the (I/V)XYXXL consensus motif contains elements that are important for binding and activation of SHP-1, but not SHIP.
Short peptides corresponding to the KIR-pY1 sequence showed that the minimal consensus sequence (I/V)XYXXL was sufficient to confer most of the activity of the KIR-pY1 peptide. The short peptide VTpYAQL activated SHP-1, whereas pYAQL  Ϫ8  Ϫ7  Ϫ6  Ϫ5  Ϫ4  Ϫ3  Ϫ2  Ϫ1  ϩ1  ϩ2  ϩ3  ϩ4  ϩ5  KIR-pY1  E  Q  D  P  Q  E  V  T  pY  A  Q  L  N  H   pYAQL  Ac----NH 2  VTpYAQL Ac- a Dashes indicate identity to KIR-pY1. Ac indicates acetylation and NH 2 indicates amidation at the amino and carboxyl termini of the peptide, respectively.  did not, indicating a need for the upstream residues in the peptide to interact with SHP-1. Studies with analogues of KIR-pY1 indicated that position Ϫ2 is the most influential position upstream as only KIR-pY1 analogues with Val/Ile/Leu hydrophobic residues at this position were able to activate the SHP-1 protein-tyrosine phosphatase. Position Ϫ2 also influences binding of the KIR-pY1 sequence to the isolated SH2C domain, indicating that it is involved in the peptide binding to this SH2 domain. The utilization of upstream residues in a phosphotyrosine-binding motif is reminiscent of phosphotyrosine-binding domains, which are not related to SH2 domains, but similarly bind phosphotyrosine in a pocket created by conserved amino acids. The fine specificity of phosphotyrosine-binding domain binding is controlled by amino acids exclusively upstream of the phosphotyrosine, e.g. the motif PNXpY (reviewed in Ref. 1). The (I/V/L)XYXXL motif for activation of SHP-1 is unique in that it involves residues on both sides of the tyrosine.
The importance of residue Ϫ2 was clear in the context of the highly active biphosphopeptide pY1pY2. This result is particularly relevant because a long peptide is more likely than a short one to adopt the conformation of the actual protein.
In fact, the peptide tested included 45 of the 76 amino acids of the KIR cytoplasmic tail. Although short peptides rarely exhibit a fixed conformation in aqueous environments (29), it is possible that the upstream residues invoke a conformation favorable for the interaction of the peptide and the SH2 domain. However, such an explanation is unlikely because pYAQL was not able to activate SHP-1.
The crystal structure of the SH2 domains of the related protein-tyrosine phosphatase SHP-2 complexed with phos-phopeptides suggest how position Ϫ2 may be involved in peptide binding to the SH2 domains of SHP-1. These structures revealed that the peptide-binding groove accommodates residues Ϫ2 to ϩ5 relative to the phosphotyrosine (30,31). In contrast to a peptide complexed with the SH2 domain of Src (32), residue Ϫ2 is in contact with the SH2 domains of SHP-2 (30,31). Based on their sequence similarity to SHP-2, the SH2 domains of SHP-1 are likely to share the feature of an extended peptide-binding groove relative to prototypic SH2 domains. The peptide from the platelet-derived growth factor receptor crystallized with SHP-2 has a valine at position Ϫ2. In the structure, the side chain of the valine caps the phosphotyrosinebinding pocket. Although not all the peptides that bind to SHP-2 contain a similar residue at position Ϫ2, the valine at position Ϫ2 in the platelet-derived growth factor receptor peptide has been shown to be important for binding and activation of SHP-2 (33). It is possible that the interaction with the tyrosine ring and the side chain of residue Ϫ2 provides an entropic contribution for binding. Such an interaction may be required in general for the binding of peptides to the SH2C domain of SHP-1. The phosphotyrosine position in the SH2 domains of SHP-2 is different than in typical SH2 domains (30) and may contribute to the need for the extra interactions. Alternatively, a hydrophobic pocket in the SH2C domain of SHP-1, but not present in SHP-2, could explain how Ile/Val/Leu at position Ϫ2 contributes to the binding of peptides to the SHP-1 SH2C domain. Perhaps these interactions compensate for a lack of interaction with position ϩ1 and become necessary for the sequences from the lymphocyte receptors that lack a hydrophobic residue at position ϩ1. Of note, there are interactions reported for KIR-and Ly-49-derived peptides with SHP-2, but these interactions are quite weak (20).
A long groove for peptide binding can impose stringent re- Protein degradation during preparation of the lysate is likely to account for the double band observed in some of the Jurkat cell lysates. B, shown is the binding of GST-SH2C(SHP-1). Competition for binding to beads bearing KIR-pY1 was performed essentially as described above, except that the source of the protein was purified GST-SH2C(SHP-1); detection was by Western blotting with anti-GST antibody; and the peptide titration corresponds to 140, 47, 15.5, and 5.2 M. The portion of the gel shown is between 35 and 50 kDa.
FIG. 5. Activation of SHP-1 by biphosphopeptides. The hydrolysis of p-NPP by GST-SHP-1 was measured as described in the legend to Fig. 1 in the presence of the indicated peptides or a combination of peptides KIR-pY1 and KIR-pY2 at the dose indicated on the x axis. The activity of GST-SHP-1 alone is plotted for each peptide dose, but indicates the constitutive activity for corresponding concentrations of Me 2 SO in the peptide titrations. quirements for many of the residues downstream of a phosphotyrosine. In the structural studies of SHP-2, interactions of both crystallized peptides with the protein occurred for side chains ϩ3 and ϩ5 (30). Such a long groove may exclude the majority of peptides due to steric interference. In support of this notion, the upstream portion of pY1 did not confer activity to the downstream portions of KIR-pY2 and -pY5 in terms of SHP-1 activation. This suggests that -pY5 and KIR-pY2 could lack other necessary residues downstream of the tyrosine. The lack of consensus at any of these positions in the sequences that do bind suggest that -pY5 and KIR-pY2 possess residues that are detrimental to the interaction. Whereas the two residues between the tyrosine and the leucine are likely culprits, residues beyond position ϩ3 have also been implicated for high affinity binding of phosphopeptides to the related SH2N domain of SHP-2 (34).
The issue of which SH2 domain is involved with recognition of residue Ϫ2 is intriguing. A specific interaction of the peptides with the SH2N domain alone was not observed. Thus, it is tempting to presume that the SH2C domain is the only relevant domain. However, the ability of the biphosphopeptide to produce such strong activation as compared with the monophosphopeptides suggests that both tyrosines and both SH2 domains are involved in the interaction. The interaction with the SH2N domain may be of low affinity and therefore is not detected in isolation. However, when the two phosphotyrosines are linked, a high avidity interaction is generated. An even more dramatic example of enhanced activation by biphosphopeptides has been observed for SHP-2 (7). More important, substitution with alanine at position Ϫ2 drastically decreased the ability of the biphosphopeptide to activate SHP-1.
We have provided evidence that the consensus residues (I/V/ L)XYXX(L/V) are critical amino acids for binding and activation of SHP-1. Ile/Val/Leu at position Ϫ2 differentiates the YXXL sequences of inhibitory receptors from the YXXL sequences in the immune receptor tyrosine-based activation motifs of lymphocyte-activating receptors. This motif differs from the usual binding motifs of SH2 domains by the inclusion of upstream residues.