Profiling the global tyrosine phosphorylation state

Protein tyrosine kinases and protein tyrosine phosphatases play a key role in cell signaling, and the recent success of specific tyrosine kinase inhibitors in cancer treatment strongly validates the clinical relevance of basic research on tyrosine phosphorylation. Functional profiling of the tyrosine phosphoproteome is likely to lead to the identification of novel targets for drug discovery and provide a basis for novel molecular diagnostic approaches. The ultimate aim of current mass spectrometry-based phosphoproteomic approaches is the comprehensive characterization of the phosphoproteome. However, current methods are not yet sensitive enough for routine detection of a large percentage of tyrosine-phosphorylated proteins, which are generally of low abundance. In this article, we discuss alternative methods that exploit Src homology 2 (SH2) domains for profiling the tyrosine phosphoproteome. SH2 domains are small protein modules that bind specifically to tyrosine-phosphorylated peptides; there are more than 100 SH2 domains in the human genome, and different SH2 domains bind to different classes of tyrosine-phosphorylated ligands. These domains play a critical role in the propagation of signals in the cell, mediating the relocalization and complex formation of proteins in response to changes in tyrosine phosphorylation. We have developed an SH2 profiling method based on far-Western blotting, in which a battery of SH2 domains is used to probe the global state of tyrosine phosphorylation. Application to the classification of human malignancies suggests that this approach has potential as a molecular diagnostic tool. We also describe ongoing efforts to modify and improve SH2 profiling, including the development of a multiplexed assay system that will allow high-throughput functional profiling of the tyrosine phosphoproteome.     

Tandem SH2 binding sites mediate the RasGAP-RhoGAP interaction: a conformational mechanism for SH3 domain regulation.

Many cellular signaling proteins contain SH3 (Src homology 3) domains that mediate protein interactions via specific proline-containing peptides. Unlike SH2 domains, whose interactions with tyrosine-containing peptides are promoted by phosphorylation of the SH2 binding site, the regulatory mechanism for SH3 interactions is unclear. p120 RasGAP (GTPase-activating protein), which contains an SH3 domain flanked by two SH2 domains, forms an abundant SH2-mediated complex with p190 RhoGAP in cells expressing activated tyrosine kinases. We have identified two closely linked tyrosine-containing peptides in p190 that bind simultaneously to the RasGAP SH2 domains upon p190 phosphorylation. This interaction is expected to bring the two SH2 domains into close proximity. Consequently, RasGAP undergoes a conformational change that results in a 100-fold increase in the accessibility of the target binding surface of its SH3 domain. These results indicate that the tandem arrangement of SH2 and SH3 domains found in a variety of cellular signaling proteins can provide a conformational mechanism for regulating SH3-dependent interactions through tyrosine phosphorylation. In addition, it appears that the role of p190 in the RasGAP signaling complex is to promote additional protein interactions with RasGAP via its SH3 domain.     

Simultaneous assay of Src SH3 and SH2 domain binding using different wavelength fluorescence polarization probes.

pp60(c-src) is a prototypical nonreceptor tyrosine kinase and may play a role in diseases as diverse as cancer and osteoporosis. In Src, the SH3 domain (Src homology 3) binds proteins at specific, proline-rich sequences, while the SH2 domain (Src homology 2) binds phosphotyrosine-containing sequences. Inhibition of Src SH3 and SH2 domain function is of potential therapeutic value because of their importance in signaling pathways involved in disease states. We have developed dual-wavelength fluorescent peptide probes for both the Src SH3 and the Src SH2 domains, which allow the simultaneous measurement of compounds binding to each domain in assays based on the technique of fluorescence polarization. We demonstrate the utility of these probes in a dual-binding assay (suitable for high-throughput screening) to study the interactions of various peptides with these domains, including a sequence from the rat protein p130(CAS) which has been reported to bind simultaneously to both Src SH3 and SH2 domains. Utilizing this dual-binding assay, we confirm that sequences from p130(CAS) can simultaneously bind Src via both its SH3 and its SH2 domains. We also use the dual-binding assay as an internal control to identify substances which inhibit SH3 and SH2 binding via nonspecific mechanisms.     

Multiplexed molecular interactions of nuclear receptors using fluorescent microspheres

BACKGROUND: We describe a novel microsphere-based system to identify and characterize multiplexed interactions of nuclear receptors with peptides that represent the LXXLL binding region of coactivator proteins. METHODS: In this system, individual microsphere populations with unique red and orange fluorescent profiles are coupled to specific coactivator peptides. The coactivator peptide-coupled microsphere populations are combined and incubated with a nuclear receptor that has been coupled to a green fluorochrome. Flow cytometric analysis of the microspheres simultaneously decodes each population and detects the binding of receptor to respective coactivator peptides by the acquisition of green fluorescence. RESULTS: We have used this system to determine the binding affinities of human estrogen receptor beta ligand binding domain (ERbeta LBD) and human peroxisome proliferator activated receptor gamma ligand binding domain (PPARgamma LBD) to a set of 34 coactivator peptides. Binding of ERbeta LBD to a coactivator peptide sequence containing the second LXXLL motif of steroid receptor coactivator-1 (SRC-1(2) (676-700) is shown to be specific and saturable. Analysis of receptor binding to a multiplexed set of coactivator peptides shows PPARgamma LBD binds with high affinity to cAMP response element binding protein (CBP) peptides and to the related P300 peptide while ERbeta LBD exibits little binding to these peptides. Using the microsphere-based assay we demonstrate that ERbeta LBD and PPARgamma LBD binding affinities for the coactivator peptides are increased in the presence of agonist (estradiol or GW1929, respectively) and that ERbeta LBD binding is decreased in the presence of antagonist (raloxifene or tamoxifen). CONCLUSIONS: This unique microsphere-based system is a sensitive and efficient method to simultaneously evaluate many receptor-coactivator interactions in a single assay volume. In addition, the system offers a powerful approach to study small molecule modulation of nuclear receptor binding.     

High-throughput phosphotyrosine profiling using SH2 domains

Protein tyrosine phosphorylation controls many aspects of signaling in multicellular organisms. One of the major consequences of tyrosine phosphorylation is the creation of binding sites for proteins containing Src homology 2 (SH2) domains. To profile the global tyrosine phosphorylation state of the cell, we have developed proteomic binding assays encompassing nearly the full complement of human SH2 domains. Here we provide a global view of SH2 domain binding to cellular proteins based on large-scale far-western analyses. We also use reverse-phase protein arrays to generate comprehensive, quantitative SH2 binding profiles for phosphopeptides, recombinant proteins, and entire proteomes. As an example, we profiled the adhesion-dependent SH2 binding interactions in fibroblasts and identified specific focal adhesion complex proteins whose tyrosine phosphorylation and binding to SH2 domains are modulated by adhesion. These results demonstrate that high-throughput comprehensive SH2 profiling provides valuable mechanistic insights into tyrosine kinase signaling pathways.     

Molecular Dynamics Simulations Reveal that Tyr-317 Phosphorylation Reduces Shc Binding Affinity for Phosphotyrosyl Residues of Epidermal Growth Factor Receptor

The Src homology 2 (SH2) and collagen domain protein Shc plays a pivotal role in signaling via tyrosine kinase receptors, including epidermal growth factor receptor (EGFR). Shc binding to phospho-tyrosine residues on activated receptors is mediated by the SH2 and phospho-tyrosine binding (PTB) domains. Subsequent phosphorylation on Tyr-317 within the Shc linker region induces Shc interactions with Grb2-Son of Sevenless that initiate Ras-mitogen-activated protein kinase signaling. We use molecular dynamics simulations of full-length Shc to examine how Tyr-317 phosphorylation controls Shc conformation and interactions with EGFR. Our simulations reveal that Shc tyrosine phosphorylation results in a significant rearrangement of the relative position of its domains, suggesting a key conformational change. Importantly, computational estimations of binding affinities show that EGFR-derived phosphotyrosyl peptides bind with significantly more strength to unphosphorylated than to phosphorylated Shc. Our results unveil what we believe is a novel structural phenomenon, i.e., tyrosine phosphorylation of Shc within its linker region regulates the binding affinity of SH2 and PTB domains for phosphorylated Shc partners, with important implications for signaling dynamics.     

Identification of Fyn-binding proteins in MC/9 mast cells using mass spectrometry

Fyn is a Src kinase known to have an essential role in mast cell degranulation induced following aggregation of the high affinity IgE-receptor. Although Fyn possesses SH2 and SH3 protein binding domains, the molecules that interact with Fyn have not been characterized in mast cells. We thus analyzed Fyn-binding proteins in MC/9 mast cells to explore the Fyn-mediated signaling pathway. On mass spectrometric analysis of proteins binding to the SH2 and SH3 domains of Fyn, we identified six proteins that bind to Fyn including vimentin, pyruvate kinase, p62 ras-GAP associated phosphoprotein, SLP-76, HS-1, and FYB. Among these proteins, vimentin and pyruvate kinase have not been shown to bind to Fyn. After IgE-receptor mediated stimulation, binding of vimentin to Fyn was increased; and this interaction was via binding to the SH2, but not the SH3, domain of Fyn. Mast cells from vimentin-deficient mice showed enhanced mediator release and tyrosine phosphorylation of intracellular proteins including NTAL and LAT. The observation that vimentin and pyruvate kinase bind to Fyn provides additional insight into Fyn-mediated signaling pathways, and suggests a critical role for Fyn in mast cell degranulation in interacting with both cytosolic and structural proteins.     

The N-Methyl-d-Aspartate Receptor Subunits NR2A and NR2B Bind to the SH2 Domains of Phospholipase C-γ

Abstract: The NMDA receptor has recently been found to be phosphorylated on tyrosine. To assess the possible connection between tyrosine phosphorylation of the NMDA receptor and signaling pathways in the postsynaptic cell, we have investigated the relationship between tyrosine phosphorylation and the binding of NMDA receptor subunits to the SH2 domains of phospholipase C-γ (PLC-γ). A glutathione S -transferase (GST) fusion protein containing both the N- and the C-proximal SH2 domains of PLC-γ was bound to glutathione-agarose and reacted with synaptic junctional proteins and glycoproteins. Tyrosine-phosphorylated PSD-GP180, which has been identified as the NR2B subunit of the NMDA receptor, bound to the SH2-agarose beads in a phosphorylation-dependent fashion. Immunoblot analysis with antibodies specific for individual NMDA receptor subunits showed that both NR2A and NR2B subunits bound to the SH2-agarose. No binding occurred to GST-agarose lacking an associated SH2 domain, indicating that binding was specific for the SH2 domains. The binding of receptor subunits increased after the incubation of synaptic junctions with ATP and decreased after treatment of synaptic junctions with exogenous protein tyrosine phosphatase. Immunoprecipitation experiments confirmed that NR2A and NR2B were phosphorylated on tyrosine and further that tyrosine phosphorylation of each of the subunits was increased after incubation with ATP. The results demonstrate that NMDA receptor subunits NR2A and NR2B will bind to the SH2 domains of PLC-γ and that isolated synaptic junctions contain endogenous protein tyrosine kinase(s) that can phosphorylate both NR2A and NR2B receptor subunits, and suggest that interaction of the tyrosine-phosphorylated NMDA receptor with proteins that contain SH2 domains may serve to link it to signaling pathways in the postsynaptic cell.     

Phosphorylation regulates tau interactions with Src homology 3 domains of phosphatidylinositol 3-kinase, phospholipase Cgamma1, Grb2, and Src family kinases

The microtubule-associated protein tau can associate with various other proteins in addition to tubulin, including the SH3 domains of Src family tyrosine kinases. Tau is well known to aggregate to form hyperphosphorylated filamentous deposits in several neurodegenerative diseases (tauopathies) including Alzheimer disease. We now report that tau can bind to SH3 domains derived from the p85alpha subunit of phosphatidylinositol 3-kinase, phospholipase Cgamma1, and the N-terminal (but not the C-terminal) SH3 of Grb2 as well as to the kinases Fyn, cSrc, and Fgr. However, the short inserts found in neuron-specific isoforms of Src prevented the binding of tau. The experimentally determined binding of tau peptides is well accounted for when modeled into the peptide binding cleft in the SH3 domain of Fyn. After phosphorylation in vitro or in transfected cells, tau showed reduced binding to SH3 domains; no binding was detected with hyperphosphorylated tau isolated from Alzheimer brain, but SH3 binding was restored by phosphatase treatment. Tau mutants with serines and threonines replaced by glutamate, to mimic phosphorylation, showed reduced SH3 binding. These results strongly suggest that tau has a potential role in cell signaling in addition to its accepted role in cytoskeletal assembly, with regulation by phosphorylation that may be disrupted in the tauopathies including Alzheimer disease.     

SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma.

Several cytoplasmic tyrosine kinases contain a conserved, non-catalytic stretch of approximately 100 amino acids called the src homology 2 (SH2) domain, and a region of approximately 50 amino acids called the SH3 domain. SH2/SH3 domains are also found in several other proteins, including phospholipase C-gamma (PLC gamma). Recent studies indicate that SH2 domains promote association between autophosphorylated growth factor receptors such as the epidermal growth factor (EGF) receptor and signal transducing molecules such as PLC gamma. Because SH2 domains bind specifically to protein sequences containing phosphotyrosine, we examined their capacity to prevent tyrosine dephosphorylation of the EGF and other receptors with tyrosine kinase activity. For this purpose, various SH2/SH3 constructs of PLC gamma were expressed in Escherichia coli as glutathione-S-transferase fusion proteins. Our results show that purified SH2 domains of PLC gamma are able to prevent tyrosine dephosphorylation of the EGF receptor and other receptors with tyrosine activity. The inhibition of tyrosine dephosphorylation paralleled the capacity of various SH2-containing constructs to bind to the EGF receptor, suggesting that the tyrosine phosphatase and the SH2 domain compete for the same tyrosine phosphorylation sites in the carboxy-terminal tail of the EGF receptor. Analysis of the phosphorylation sites protected from dephosphorylation by PLC gamma-SH2 revealed substantial inhibition of dephosphorylation of Tyr992 at 1 microM SH2. This indicates that Tyr992 and its flanking sequence is the high-affinity binding site for SH2 domains of PLC gamma.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural basis for the requirement of two phosphotyrosine residues in signaling mediated by Syk tyrosine kinase

The protein-tyrosine kinase Syk couples immune recognition receptors to multiple signal transduction pathways, including the mobilization of calcium and the activation of NFAT. The ability of Syk to regulate signaling is influenced by its phosphorylation on tyrosine residues within the linker B region. The phosphorylation of both Y342 and Y346 is necessary for optimal signaling from the B cell receptor for antigen. The SH2 domains of multiple signaling proteins share the ability to bind this doubly phosphorylated site. The NMR structure of the C-terminal SH2 domain of PLCgamma (PLCC) bound to a doubly phosphorylated Syk peptide reveals a novel mode of phosphotyrosine recognition. PLCC undergoes extensive conformational changes upon binding to form a second phosphotyrosine-binding pocket in which pY346 is largely desolvated and stabilized through electrostatic interactions. The formation of the second binding pocket is distinct from other modes of phosphotyrosine recognition in SH2-protein association. The dependence of signaling on simultaneous phosphorylation of these two tyrosine residues offers a new mechanism to fine-tune the cellular response to external stimulation.     

Single phosphorylation of Tyr304 in the cytoplasmic tail of ephrin B2 confers high-affinity and bifunctional binding to both the SH2 domain of Grb4 and the PDZ domain of the PDZ-RGS3 protein.

The B class cell-attached ephrins mediate contact-dependent cell-cell communications and transduce the contact signals to the host cells through the binding interactions of their cytoplasmic domains. Two classes of intracellular effectors of B ephrins have been identified: one contains the PSD-95/Dlg/ZO-1 (PDZ) domain (for example PDZ-RGS3), and the second the Src homology 2 (SH2) domain (e.g. the Grb4 adaptor protein). The interaction with Grb4 requires phosphorylation of tyrosine residues on the conserved cytoplasmic C-terminal region of B ephrins, while binding to the PDZ domain is independent of tyrosine phosphorylation. However, the exact phosphorylation site(s) required for signaling remained obscure and it is also unknown whether the two classes of effectors can bind to B ephrins simultaneously or if the binding of one affects the binding of the other. We report here that phosphorylation of Tyr304 in the functional C-terminal region (residues 301-333) of ephrin B2 confers high-affinity binding to the SH2 domain of the Grb4 protein. Tyrosine phosphorylation at other candidate sites resulted in only minor change of the binding of Tyr304-phosphorylated ephrin B peptide (i.e. ephrinB2(301-333)-pY304) with the SH2 domain. (1)H-(15)N NMR HSQC experiments show that only the ephrinB2(301-333)-pY304 peptide forms a stable and specific binding complex with the SH2 domain of Grb4. The SH2 and PDZ domains were found to bind to the Tyr304 phosphopeptide both independently and at the same time, forming a three-component molecular complex. Taken together, our studies identify a novel SH2 domain binding motif, PHpY304EKV, on the cytoplasmic domains of B ephrins that may be essential for reverse signaling via the Grb4 adaptor protein alone or in concert with proteins containing PDZ domains.     

A quantitative study of the recruitment potential of all intracellular tyrosine residues on EGFR, FGFR1 and IGF1R

Receptor tyrosine kinases transmit and process extracellular cues by recruiting intracellular signaling proteins to sites of tyrosine phosphorylation. Using protein microarrays comprising virtually every human SH2 and PTB domain, we generated quantitative protein interaction maps for three well-studied receptors--EGFR, FGFR1 and IGF1R--using phosphopeptides derived from every intracellular tyrosine residue on each receptor, regardless of whether or not they are phosphorylated in vivo. We found that, in general, peptides derived from physiological sites of tyrosine phosphorylation bind to substantially more SH2 or PTB domains than do peptides derived from nonphysiological sites, supporting the idea that kinases and interaction domains co-evolve and suggesting that new sites arise predominantly through selection favoring advantageous interactions, rather than through selection disfavoring unwanted interactions. We also found substantial qualitative overlap in the recruitment profiles of these three receptors, suggesting that their different biological effects arise, at least in part, from quantitative differences in their affinities for the proteins they recruit.     

The N-terminal SH2 domains of Syk and ZAP-70 mediate phosphotyrosine-independent binding to integrin beta cytoplasmic domains

Syk and ZAP-70 form a subfamily of nonreceptor tyrosine kinases that contain tandem SH2 domains at their N termini. Engagement of these SH2 domains by tyrosine-phosphorylated immunoreceptor tyrosine-based activation motifs leads to kinase activation and downstream signaling. These kinases are also regulated by beta3 integrin-dependent cell adhesion via a phosphorylation-independent interaction with the beta3 integrin cytoplasmic domain. Here, we report that the interaction of integrins with Syk and ZAP-70 depends on the N-terminal SH2 domain and the interdomain A region of the kinase. The N-terminal SH2 domain alone is sufficient for weak binding, and this interaction is independent of tyrosine phosphorylation of the integrin tail. Indeed, phosphorylation of tyrosines within the two conserved NXXY motifs in the integrin beta3 cytoplasmic domain blocks Syk binding. The tandem SH2 domains of these kinases bind to multiple integrin beta cytoplasmic domains with varying affinities (beta3 (Kd = 24 nm) > beta2 (Kd = 38 nm) > beta1 (Kd = 71 nm)) as judged by both affinity chromatography and surface plasmon resonance. Thus, the binding of Syk and ZAP-70 to integrin beta cytoplasmic domains represents a novel phosphotyrosine-independent interaction mediated by their N-terminal SH2 domains.     

Structural and biochemical evaluation of the interaction of the phosphatidylinositol 3-kinase p85alpha Src homology 2 domains with phosphoinositides and inositol polyphosphates.

Src homology 2 (SH2) domains exist in many intracellular proteins and have well characterized roles in signal transduction. SH2 domains bind to phosphotyrosine (Tyr(P))-containing proteins. Although tyrosine phosphorylation is essential for protein-SH2 domain interactions, the binding specificity also derives from sequences C-terminal to the Tyr(P) residue. The high affinity and specificity of this interaction is critical for precluding aberrant cross-talk between signaling pathways. The p85alpha subunit of phosphoinositide 3-kinase (PI 3-kinase) contains two SH2 domains, and it has been proposed that in competition with Tyr(P) binding they may also mediate membrane attachment via interactions with phosphoinositide products of PI 3-kinase. We used nuclear magnetic resonance spectroscopy and biosensor experiments to investigate interactions between the p85alpha SH2 domains and phosphoinositides or inositol polyphosphates. We reported previously a similar approach when demonstrating that some pleckstrin homology domains show binding specificity for distinct phosphoinositides (Salim, K., Bottomley, M. J., Querfurth, E., Zvelebil, M. J., Gout, I., Scaife, R., Margolis, R. L., Gigg, R., Smith, C. I., Driscoll, P. C., Waterfield, M. D., and Panayotou, G. (1996) EMBO J. 15, 6241-6250). However, neither SH2 domain exhibited binding specificity for phosphoinositides in phospholipid bilayers. We show that the p85alpha SH2 domain Tyr(P) binding pockets indiscriminately accommodate phosphoinositides and inositol polyphosphates. Binding of the SH2 domains to Tyr(P) peptides was only poorly competed for by phosphoinositides or inositol polyphosphates. We conclude that these ligands do not bind p85alpha SH2 domains with high affinity or specificity. Moreover, we observed that although wortmannin blocks PI 3-kinase activity in vivo, it does not affect the ability of tyrosine-phosphorylated proteins to bind to p85alpha. Consequently phosphoinositide products of PI 3-kinase are unlikely to regulate signaling through p85alpha SH2 domains.     

FRS2 proteins recruit intracellular signaling pathways by binding to diverse targets on fibroblast growth factor and nerve growth factor receptors

The docking protein FRS2 was implicated in the transmission of extracellular signals from the fibroblast growth factor (FGF) or nerve growth factor (NGF) receptors to the Ras/mitogen-activated protein kinase signaling cascade. The two members of the FRS2 family, FRS2alpha and FRS2beta, are structurally very similar. Each is composed of an N-terminal myristylation signal, a phosphotyrosine-binding (PTB) domain, and a C-terminal tail containing multiple binding sites for the SH2 domains of the adapter protein Grb2 and the protein tyrosine phosphatase Shp2. Here we show that the PTB domains of both the alpha and beta isoforms of FRS2 bind directly to the FGF or NGF receptors. The PTB domains of the FRS2 proteins bind to a highly conserved sequence in the juxtamembrane region of FGFR1. While FGFR1 interacts with FRS2 constitutively, independent of ligand stimulation and tyrosine phosphorylation, NGF receptor (TrkA) binding to FRS2 is strongly dependent on receptor activation. Complex formation with TrkA is dependent on phosphorylation of Y490, a canonical PTB domain binding site that also functions as a binding site for Shc (NPXpY). Using deletion and alanine scanning mutagenesis as well as peptide competition assays, we demonstrate that the PTB domains of the FRS2 proteins specifically recognize two different primary structures in two different receptors in a phosphorylation-dependent or -independent manner. In addition, NGF-induced tyrosine phosphorylation of FRS2alpha is diminished in cells that overexpress a kinase-inactive mutant of FGFR1. This experiment suggests that FGFR1 may regulate signaling via NGF receptors by sequestering a common key element which both receptors utilize for transmitting their signals. The multiple interactions mediated by FRS2 appear to play an important role in target selection and in defining the specificity of several families of receptor tyrosine kinases.     

Reciprocal regulation of SH3 and SH2 domain binding via tyrosine phosphorylation of a common site in CD3epsilon

Recruitment of cellular signaling proteins by the CD3 polypeptides of the TCR complex mediates T cell activation. We have screened a human Src homology 3 (SH3) domain phage display library for proteins that can bind to the proline-rich region of CD3epsilon. This screening identified Eps8L1 (epidermal growth factor receptor pathway substrate 8-like 1) together with the N-terminal SH3 domain of Nck1 and Nck2 as its preferred SH3 partners. Studies with recombinant proteins confirmed strong binding of CD3epsilon to Eps8L1 and Nck SH3 domains. CD3epsilon bound well also to Eps8 and Eps8L3, and modestly to Eps8L2, but not detectably to other SH3 domains tested. Interestingly, binding of Nck and Eps8L1 SH3 domains was mapped to a PxxDY motif that shared its tyrosine residue (Y166) with the ITAM of CD3epsilon. Phosphorylation of this residue abolished binding of Eps/Nck SH3 domains in peptide spot filter assays, as well as in cells cotransfected with a dominantly active Lck kinase. TCR ligation-induced binding and phosphorylation-dependent loss of binding were also demonstrated between Eps8L1 and endogenous CD3epsilon in Jurkat T cells. Thus, phosphorylation of Y166 serves as a molecular switch during T cell activation that determines the capacity of CD3epsilon to interact with either SH3 or SH2 domain-containing proteins.     

Alternative mode of binding to phosphotyrosyl peptides by Src homology-2 domains

Src homology-2 (SH2) domains recognize specific phosphotyrosyl (pY) proteins and promote protein-protein interactions. In their classical binding mode, the SH2 domain makes specific contacts with the pY residue and the three residues immediately C-terminal to the pY, although for a few SH2 domains, residues N-terminal to pY have recently been shown to also contribute to the overall binding affinity and specificity. In this work, the ability of an SH2 domain to bind to the N-terminal side of pY has been systematically examined. A pY peptide library containing completely randomized residues at positions -5 to -1 (relative to pY, which is position 0) was synthesized on TentaGel resin and screened against the four SH2 domains of phosphatases SHP-1 and SHP-2. Positive beads that carry high-affinity ligands of the SH2 domains were identified using an enzyme-linked assay, and the peptides were sequenced by partial Edman degradation and matrix-assisted laser desorption ionization mass spectrometry. The N-terminal SH2 domain of SHP-2 binds specifically to peptides of the consensus sequence (H/F)XVX(T/S/A)pY. Further binding studies with individually synthesized pY peptides show that pY and the five residues N-terminal to pY, but not any of the C-terminal residues, are important for binding. The other three SH2 domains also bound to the library beads, albeit more weakly, and the selected peptides did not show any clear consensus. These results demonstrate that at least some SH2 domains can bind to pY peptides in an alternative mode by recognizing only the residues N-terminal to pY.     

Potent inhibitory ligands of the GRB2 SH2 domain from recombinant peptide libraries.

We cloned and expressed the SH2 domain of human GRB2 as glutathione S-transferase and maltose binding protein fusion proteins. We screened three phagemid-based fd pVIII-protein phage display libraries against SH2 domain fusion proteins. Sequence analysis of the peptide extensions yielded a variety of related peptides. By examining the ability of the phage clones to bind other SH2 domains, we demonstrated that the phage were specific for the SH2 domain of GRB2. Based on the sequence motif identified in the "random" library screening experiment, we also built and screened a phage display library based on a Tyr-X-Asn motif (X5-Tyr-X-Asn-X8). To examine the affinity of the phage derived peptides for GRB2, we set up a radioligand competition binding assay based on immobilized GRB2 and radiolabelled autophosphorylated EGFR ICD as the radioligand. Results obtained with peptide competitors derived from the phage sequences demonstrated that nonphosphotyrosine-containing peptides identified with the phage display technology had an affinity for the receptor similar to tyrosine-phosphorylated peptides derived from the EGFR natural substrate. Interestingly, when the phage display peptides were then phosphorylated on tyrosine, their affinity for GRB2 increased dramatically. We also demonstrated the ability of the peptides to block the binding of the GRB2 SH2 domain to EGFR in a mammalian cell-based binding assay.     

Quantitative analysis of SH2 domain binding. Evidence for specificity and competition.

We report the development of a quantitative assay for measuring SH2 domain binding in vitro. Using this assay we have analyzed the binding of purified recombinant SH2 domains from ras GTPase activating protein (GAP) and the 85-kDa subunit of phosphatidylinositol 3-kinase (p85) to proteins from epidermal growth factor-stimulated and v-src-transformed cells. The purified recombinant SH2 domains from GAP and p85 bind to the tyrosine phosphorylated epidermal growth factor receptor with nanomolar affinities. Moreover, competition studies suggest that these two proteins bind to equivalent or overlapping sites on this receptor. In v-src-transformed cells the purified recombinant SH2 domains from GAP and p85 bind to distinct but overlapping sets of proteins.