(Received for publication, September 27, 1995; and in revised form, November 27, 1995)
From the
Multipin peptide synthesis has been employed to produce biotinylated 11-mer phosphopeptides that account for every tyrosine residue in insulin receptor substrate-1 (IRS-1) and the cytoplasmic domains of the insulin-, epidermal growth factor-, platelet-derived growth factor- and basic fibroblast growth factor receptors. These phosphopeptides have been screened for their capacity to bind to the SH2 domains of Shc and Grb in a solution phase enzyme-linked immunosorbent assay. The data revealed new potential Grb2 binding sites at Tyr-1114 (epidermal growth factor receptor (EGFR) C-tail); Tyr-743 (platelet-derived growth factor receptor (PDGFR) insert region), Tyr-1110 from the E-helix of the catalytic domain of insulin receptor (IR), and Tyr-47, Tyr-939, and Tyr-727 in IRS-1. None of the phosphopeptides from the juxtamembrane or C-tail regions of IR bound Grb2 significantly, and only one phosphopeptide from the basic fibroblast growth factor receptor (Tyr-556) bound Grb2 but with medium strength. Tyr-1068 and -1086 from the C-tail of EGFR, Tyr-684 from the kinase insert region of PDGFR, and Tyr-895 from IRS-1 were confirmed as major binding sites for the Grb2 SH2 domain. With regard to Shc binding, the data revealed new potential binding sites at Tyr-703 and Tyr-789 from the catalytic domain of EGFR and at Tyr-557 in the juxtamembrane region of PDGFR. It also identified new potential Shc binding sites at Tyr-764, in the C-tail of basic fibroblast growth factor receptor, and Tyr-960, in the juxtamembrane of IR, a residue previously known to be required for Shc phosphorylation in response to insulin. The study confirmed the previous identification of Tyr-992 and Tyr-1173 in the C-tail of EGFR and several phosphopeptides from the PDGFR as medium strength binding sites for the SH2 domain of Shc. None of the 34 phosphopeptides from IRS-1 bound Shc strongly, although Tyr-690 showed medium strength binding. The specificity characteristics of the SH2 domains of Grb2 and Shc are discussed. This systematic peptide mapping strategy provides a way of rapidly scanning candidate proteins for potential SH2 binding sites as a first step to establishing their involvement in kinase-mediated signaling pathways.
SH2 domains are one of the conserved protein modules that regulate signal transduction pathways involving phospholipid metabolism, protein phosphorylation and dephosphorylation, activation of Ras-like GTPases, gene expression, protein trafficking, and cytoskeletal architecture (1) . SH2 domains promote protein-protein interactions by binding short, specific, phosphotyrosine peptide sequences on activated receptors and cytoplasmic proteins(1, 2) . The three-dimensional structure of several SH2 domains have now been determined(3, 4, 5, 6, 7) , and the nature of the phosphopeptide binding site has been described.
Individual SH2 domain binding sites have been identified and their specificity requirements determined by (i) isolation of specific phosphopeptides after receptor activation, (ii) site-specific mutagenesis, (iii) synthetic peptide binding studies (see Refs. 2 and 8), or (iv) analysis of degenerate phosphopeptide libraries(9, 10) . It is generally accepted that the SH2 binding sites of receptor tyrosine kinases are located outside the kinase domain proper in the juxtamembrane region, the kinase insert region, or the C-terminal tail (1) . However, there are some data (11, 12, 13) that suggest that Tyr(P) residues within the catalytic domain proper can act as binding sites for SH2 domain or other signaling proteins. The phosphatidylinositol 3`-kinase binding site in the tyrosine kinase receptor Trk-A is Tyr-751 (11) . This Tyr residue is located in the H-helix (see (14) ), 24 residues upstream of the C-terminal residue of the catalytic domain as defined by the alignments of Hanks(15) . Similarly, the highly conserved activation loop residue Tyr-809 of the CSF-1 receptor (see (14) ) has been shown to be required for induction and sustained transcription of c-myc and the control of ligand-dependent cell growth without significantly affecting receptor tyrosine kinase activity or immediate early gene expression(12, 13) .
In this paper we describe a
systematic approach to identify potential binding sites for SH2 domains
on cytoplasmic proteins or transmembrane receptors that includes an
analysis of every tyrosine-containing peptide. This approach is rapid
and sensitive and complements the studies on direct identification of
binding sites by mutagenesis and competition binding or specificity
determination with degenerate peptide
libraries(9, 10) . It confirms many of the sites
established by alternative methods and reveals new potential binding
sites for Grb2 at Tyr-1110 in IR, ()Tyr-1114 in EGFR,
Tyr-743 in PDGFR, and Tyr-47, Tyr-727, and Tyr-939 in IRS-1 and for Shc
at Tyr-960 in IR, Tyr-703 and Tyr-789 in EGFR, and Tyr-557 in PDGFR.
Figure 1: Aligned sequences of human IR, human EGFR, mouse PDGFR, and chicken bFGFR cytoplasmic domains showing the relative location of the Tyr residues and their positions in the protein structure (see (14) ). The sequence of human TrkA is also shown to indicate the relative position of the phosphatidylinositol 3`-kinase kinase binding site at Tyr-751, which is located in the catalytic domain proper(11) .
Saturation binding curves for the Grb2-fusion protein with
100 µM of phosphopeptide EGFR-1068 (27) and the
Shc-SH2-fusion protein with 100 µM PDGFR-708 (28) were carried out and showed linear binding within the
range 1-20 µg/ml (data not shown). A concentration of 10
µg/ml of fusion protein was chosen for all screening assays.
Ca ions (2 mM), which have been shown to
stabilize the conformation of the p85N-SH2 domain and enhance its
binding to activated
-PDGFR (29) , were found to be
inhibitory to Grb2-SH2 binding to EGFR-1068 and Shc-SH2 binding to
PDGFR-708 and were not included in the binding screens with Grb2- and
Shc-SH2-fusion proteins.
Figure 2: Grb2- and Shc-SH2 domain binding to phosphopeptides from human EGFR. Binding was carried out at as described under ``Materials and Methods.'' The net optical density values obtained at 1.0 µM are shown for each Tyr residue in human EGFR. Peptides EGFR-777, -803, -845, -867, -891, -920, -954, -974, and -1173 showed no net binding by the SH2 domain of Grb2. Peptides EGFR-777, -803, -845, -867, -891, -920, -1045, -1068, -1086, -1101, -1114, and -1148 showed negligible binding to the Shc-SH2 domain. A schematic representation of the EGFR is shown to indicate the approximate location of each of the positively binding peptides in the sequence.
Figure 3: Grb2- and Shc-SH2 domain binding to phosphopeptides from mouse PDGFR. Binding was carried out at as described under ``Materials and Methods.'' The net optical density values obtained at 1.0 µM are shown for each Tyr residue in mouse PDGFR. Peptides PDGFR-660, -719, -731, -746, -755, -832, and -889 showed no net binding by the SH2 domain of Grb2. Peptides PDGFR-530, -549, -651, -660, -684, -731, -746, -755, -776, -825, -872, -882, -889, -934, -938, and -977 showed negligible binding to the Shc-SH2 domain. A schematic representation of the PDGFR is shown to indicate the approximate location of each of the positively binding peptides in the sequence.
Figure 4: Grb2- and Shc-SH2 domain binding to phosphopeptides from chicken basic FGFR. Binding was carried out at as described under ``Materials and Methods.'' The net optical density values obtained at 1.0 µM are shown for each Tyr residue in chicken bFGFR. Peptides PDGFR-461, -561, -570, -581, -583, -603, -611, -651, -652, -675, -699, -728, -764, and -774 showed negligible binding by the SH2 domain of Grb2. Peptides bFGFR-461, -561, -570, -581, -583, -603, -611, -651, -652, -675, -699, and -774 showed negligible binding to the Shc-SH2 domain. A schematic representation of the bFGFR is shown to indicate the approximate location of each of the positively binding peptides in the sequence.
Figure 5: Grb2- and Shc-SH2 domain binding to phosphopeptides from human IR. Binding was carried out at as described under ``Materials and Methods.'' The net optical density values obtained at 1.0 µM are shown for each Tyr residue in IR. Peptides IR-960, -999, -1075, -1150, -1151, 1198, -1215, -1316, and -1322 showed negligible binding by the SH2 domain of Grb2. Peptides IR-953, -972, -999, -1075, -1110, -1146, -1150, -1151, -1215, -1316, and -1322 showed negligible binding to the Shc-SH2 domain. A schematic representation of the IR is shown to indicate the approximate location of each of the positively binding peptides in the sequence.
Figure 6: Grb2- and Shc-SH2 domain binding to phosphopeptides from rat IRS-1. Binding was carried out at as described under ``Materials and Methods.'' The net optical density values obtained at 1.0 µM are shown for each Tyr residue in rat IRS-1. Peptides IRS1-18, -87, -107, -147, -178, -426, -478, -489, -546, -567, -578, -608, -628, -690, -745, -746, -815, -907, and -1222 showed negligible binding to the Grb2-SH2 domain. Peptides IRS1-18, -46, -47, -87, -147, -178, -426, -478, -489, -546, -555, -567, -578, -608, -628, -746, -759, -760, -795, -815, -907, -987, -999, -1010 and -1172 showed negligible binding to the Shc-SH2 domain. A schematic representation of the IRS-1 is shown to indicate the approximate location of each of the positively binding peptides in the sequence.
Songyang and co-workers (9, 10) have used degenerate phosphopeptide or peptide libraries to systematically search for optimal sequences that serve as binding sites for SH2 domains or substrates for protein-tyrosine kinases(30) . In this paper we describe a complementary approach in which all tyrosine-containing sequences in signaling proteins are synthesized and screened for their capacity to be bound by specific SH2 domains. It is obvious that the method could be extended to include a systematic analysis of the potential for these sites to be phosphorylated by any specific tyrosine kinase.
To date most known tyrosine autophosphorylation sites are reported to be located within noncatalytic regions of receptors(2) . However, the demonstration that some phosphorylation/dephosphorylation sites occur within the catalytic domain of tyrosine kinase proteins and that at least one SH2-containing protein has been shown to bind to a Tyr(P) sequence within the catalytic domain of TrkA (11) highlights the need to examine all Tyr-containing sequences for their potential to bind SH2 (1, 2) or PID domains (31, 32) if phosphorylated. Such studies do not establish that these sites are used in vivo but merely indicate their potential to be phosphorylated and participate in SH2-mediated signaling if they were exposed and accessible. Additional data are required to establish their in vivo significance and must take account of the fact that the stoichiometry of phosphorylation at specific sites at any given time in living cells is not known(33) . In addition the net signaling outcomes will be influenced by direct competition between different SH2- or PID-containing proteins for the same or closely overlapping phosphorylation sites and by the effects of secondary phosphorylation of Ser or Thr residues on these phosphotyrosinemediated interactions.
In this paper we have synthesized and screened all the tyrosine-containing sequences in IRS-1 and the cytoplasmic domains of IR, EGFR, PDGFR, and bFGFR for their capacity to bind to the SH2 domains of the signaling proteins Grb2 and Shc.
The major potential binding site for Shc on EGFR (Tyr-703) and the two medium binding peptides (Tyr-740, Tyr-789) are located in the catalytic domain, while the weak to medium binding peptides (Tyr-876, -954, -974, and -1173) are all in the C-tail region ( Fig. 1and Fig. 2). The physiological significance of these multiple Shc binding sites within the catalytic domain and the C-tail is uncertain. Competition binding and dephosphorylation protection experiments showed that Tyr-1173 was a major and Tyr-992 a minor binding site for the SH2 domain of Shc on EGFR(33) . However, other sites have been shown to be able to compensate for the loss of these C-tail sites for Shc, and stable association of Shc with EGFR may not be necessary for in vivo function(37) . Phosphorylation of Shc, complex formation of Shc-Grb2 and activation of mitogen-activated protein kinase were found to be normal in an EGFR truncated at residue 1011 and mutated to Phe at Tyr-992, even though the association between the receptor and Shc was hardly detectable(37) . Our findings suggest that any one of six additional potential binding sites could be responsible for Shc binding and activation in such an EGFR construct (Fig. 2).
With Shc binding, we show that Tyr-557 at the end of the juxtamembrane region binds the Shc-SH2 domain strongly, while 12 other sites are medium to weak binders (Fig. 3). Yokote et al.(28) , using synthetic peptide analyses, showed that multiple autophosphorylation sites (equivalent to 547, 708, 719, and 739) were able to mediate binding to the Shc-SH2 domain but suggested that additional unidentified autophosphorylation sites in PDGFR may also bind Shc since mutant receptors, in which each of these Shc binding site tyrosines were changed to phenylalanine, showed normal (Tyr-708, -719, -739 mutants) or partially (40%) reduced (Tyr-547 mutant) Shc binding and were still capable of phosphorylating Shc at close to normal levels. Our data suggest Tyr-557 could be the unidentified mediator of this Shc binding. It is interesting to note that Tyr-571 in the CSF-1 receptor (homologous to Tyr-557 in mouse PDGFR) occurs in a sequence where the amino acid residues at the +1, +2, and +3 positions are different from those found in PDGFR(40) . This may explain the absence of demonstrable Shc binding to the CSF-1 receptor in Rat2 cells stimulated with CSF-1, even though Shc was phosphorylated and shown to be associated with Grb2 (39) .
Fibroblast growth factor-induced mitogenesis is mediated by
activation of the mitogen-activated protein kinase
cascade(44) , presumably through the activation of Shc (see (42) ), although the site of Shc binding has not been reported.
In this report the C-tail Tyr-764, (equivalent to Tyr-766, the
phospholipase C site in human FGFR-1), shows medium binding to the
SH2 domain of Shc. Mutation of Tyr-766 in FGFR-1 had no effect on
mitogenesis but inhibited phosphatidylinositol hydrolysis,
Ca
influx(45, 46) , and
internalization(47) . The lack of effect on mitogenesis
suggests that Shc activation with the FGFR-1 may be mediated by SH2
binding to the minor sites (Tyr-556 and Tyr-728) in the catalytic
domain identified here or by receptor association through its
PID(31, 32) . No sites exist in chick bFGFR (24) that match the specificity requirements of the Shc
PID(1, 48, 49) .
In contrast to IR, IRS-1 showed strong potential to bind Grb2 at several sites: Tyr-47, Tyr-895, Tyr-939, and to a lesser extent Tyr-727 (Fig. 6). The physical association of phosphorylated IRS-1 with Grb2 has been reported(52, 53) . One of these sites (Tyr-895) is known to be phosphorylated in vivo and to bind to a fusion protein containing the SH2 domain of Grb2(20) . Two other phosphopeptide fractions were shown to be bound by the Grb2-SH2 fusion protein but were not identified and did not include the Tyr-939 phosphopeptide (20) . The reason for this discrepancy between their findings with the Tyr-939 phosphopeptide and ours is not known. The additional Grb2 binding sites may come from the region 530-768 in IRS-1, since a GST fusion protein containing these IRS-1 residues, but not amino acids 3-67, bound Grb2 when phosphorylated by the IR(53) . The region of IRS-1 from 530 to 768 contains 13 tyrosines including the strong binder Tyr-727 (Fig. 6).
Shc can be phosphorylated by purified IR and IGF-1R in vitro, suggesting it is a direct substrate(54) . In
addition, in 32-D myeloid progenitor cells (which lack IRS-1 and have
low levels of IR), expression of IR alone was sufficient for Shc
phosphorylation and mitogen-activated protein kinase
activation(55) . In this report we have identified a potential
Shc-SH2 binding site at Tyr-960 in the juxtamembrane region of the IR.
In addition, immunoprecipitation and Western blotting has shown that
the Shc-SH2 domain fusion protein can bind to autophosphorylated IR in vitro. ()The direct binding of Shc-SH2 domain to
IR has not been reported previously(52, 56) . Tyr-960
is known to be an autophosphorylation site in IR, although the
detection of this phosphorylation was
difficult(57, 58) . It has also been shown recently to
be the site at which the PID of Shc interacts(59) . Removal of
Tyr-960 by mutagenesis severely impaired insulin-dependent
phosphorylation of Shc(60) . However, it also impaired the
phosphorylation of IRS-1(57, 61) , consistent with the
fact that both Shc and IRS-1 compete for this residue in their
association with the phosphorylated IR(59) . Residues in the
C-tail of the IR are also required for Shc
phosphorylation(60) , but neither of the C-tail phosphopeptides
exhibited significant binding to the Shc-SH2 domain (Fig. 6).
As shown in Fig. 6, none of the 34-phosphotyrosine peptides from IRS-1 bound Shc strongly, although Tyr-690 showed moderate binding and Tyr-107 weak to moderate binding. The significance of this potential binding is not known. Neither of these sites has been positively identified as a phosphorylation target for the IR kinase, although additional phosphorylation sites in IRS-1 do exist(20) . While some groups have failed to detect Shc binding to IRS-1(52, 56) , Shc has been detected in IRS-1 immunoprecipitates by others(54) .
The top 29 phosphopeptides bound by the Shc-SH2 domain are also shown in Table 1. The results reveal a strong tendency for hydrophobic residues, particularly Leu and Met, at the +3 position and for Trp and Ser at the +4 position. There is also a high frequency of small hydrophilic residues at the -3 and -2 positions. Almost any residue can occur at the -1 and +2 positions. While there is a higher occurrence of hydrophobic residues at the +1 position in the peptides examined, almost any residue can be accommodated here, and the two strongest binding peptides have Asp or Lys (Table 1).
These results are
in good agreement with the findings obtained with degenerate peptide
libraries(9, 10) . The method complements their
approach and allows all tyrosine-containing sequences from any tyrosine
kinase or tyrosine kinase substrate to be rapidly screened for their
potential to bind specific SH2 domains. Replacement net analyses using
the multipin synthesis methodology employed here (26) could
also be used to map the effects, on SH2 domain specificity, of amino
acid substitutions at each position. This information would allow a
more comprehensive search of protein data bases for potential SH2
domain binding sites. Recent studies with phospholipase C have
revealed that SH2 domain binding specificity is more complex than at
first thought and that residues at the +4 and +5 positions
can have an inhibitory effect on the binding of some SH2
domains(62) .