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.     

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.     

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.     

Src homology 2 domain-based high throughput assays for profiling downstream molecules in receptor tyrosine kinase pathways

Src homology 2 (SH2) domains are evolutionary conserved small protein modules that bind specifically to tyrosine-phosphorylated peptides. More than 100 SH2 domains have been identified in proteins encoded by the human genome. The binding specificity of these domains plays a critical role in signaling within the cell, mediating the relocalization and interaction of proteins in response to changes in tyrosine phosphorylation states. Here we developed an SH2 domain profiling method based on a multiplexed fluorescent microsphere assay in which various SH2 domains are used to probe the global state of tyrosine phosphorylation within a cell and to screen synthetic peptides that specifically bind to each SH2 domain. The multiplexed, fluorescent microsphere-based assay is a recently developed technology that can potentially detect a wide variety of interactions between biological molecules. We constructed 25-plex SH2 domain-GST fusion protein-conjugated fluorescent microsphere sets to investigate phosphorylation-mediated cell signaling through the specific binding of SH2 domains to activated target proteins. The response of HeLa, COS-1, A431, and 293 cells and four breast cancer cell lines to epidermal growth factor and insulin were quantitatively profiled using this novel microsphere-based, multiplexed, high throughput assay system.     

Identification of phosphorylation sites within the SH3 domains of Tec family tyrosine kinases

Tec family protein tyrosine kinases (TFKs) play a central role in hematopoietic cellular signaling. Initial activation takes place through specific tyrosine phosphorylation situated in the activation loop. Further activation occurs within the SH3 domain via a transphosphorylation mechanism, which for Bruton's tyrosine kinase (Btk) affects tyrosine 223. We found that TFKs phosphorylate preferentially their own SH3 domains, but differentially phosphorylate other member family SH3 domains, whereas non-related SH3 domains are not phosphorylated. We demonstrate that SH3 domains are good and reliable substrates. We observe that transphosphorylation is selective not only for SH3 domains, but also for dual SH3SH2 domains. However, the dual domain is phosphorylated more effectively. The major phosphorylation sites were identified as conserved tyrosines, for Itk Y180 and for Bmx Y215, both sites being homologous to the Y223 site in Btk. There is, however, one exception because the Tec-SH3 domain is phosphorylated at a non-homologous site, nevertheless a conserved tyrosine, Y206. Consistent with these findings, the 3D structures for SH3 domains point out that these phosphorylated tyrosines are located on the ligand-binding surface. Because a number of Tec family kinases are coexpressed in cells, it is possible that they could regulate the activity of each other through transphosphorylation.     

Mac-1-dependent tyrosine phosphorylation during neutrophil adhesion

Activated neutrophils display an array of physiological responses, including initiation of the oxidative burst, phagocytosis, and cell migration, that are associated with cellular adhesion. Under conditions that lead to cellular adhesion, we observed rapid tyrosine phosphorylation of an intracellular protein with an approximate relative molecular mass of 92 kDa (p92). Phosphorylation of p92 was inducible when Mac-1 was activated by phorbol 12-myristate 13-acetate, the beta(2)-specific activating antibody CBR LFA-1/2, or interleukin-8 (77 amino acids). In addition, tyrosine phosphorylation of p92 was dependent on engagement of Mac-1 with ligand. Several observations suggest that this event may be an important step in the signaling pathway initiated by Mac-1 binding. p92 phosphorylation was specifically blocked with antibodies to CD11b, the alpha-subunit of Mac-1, and was rapidly reversible on disengagement of the integrin ligand interaction. Integrin-stimulated phosphorylation of p92 created binding sites that were recognized in vitro by the SH2 domains of c-CrkII and Src. Our observations suggest that neutrophil adhesion mediated through the binding of the beta(2)-integrin Mac-1 initiates a signaling cascade that involves the activation of protein tyrosine kinases and leads to the regulation of protein-protein interactions via SH2 domains, a key process shared with growth factor 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.     

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.     

Structure-function relationships in Src family and related protein tyrosine kinases

There is increasing evidence to suggest that cytoplasmic tyrosine kinases of the Src family have a pivotal role in the regulation of a number of cellular processes. Members of this family have been implicated in cellular responses to a variety of extracellular signals, such as those arising from growth factors and cell-cell interactions, as well as in differentiative and developmental processes in both vertebrates and invertebrates. A better understanding of the regulation and of the structure-function relationships of these enzymes might aid in the development of specific ways to interfere with their action, as well as serving as a paradigm for regulation of other protein tyrosine kinases that have SH2 and SH3 domains. In this review we will first discuss the regulation of Src family protein tyrosine kinases, with particular emphasis on their SH2 and SH3 domains. We will then briefly review other non-receptor protein tyrosine kinases that have SH2 and SH3 domains.     

Activation of the high-affinity immunoglobulin E receptor Fc epsilon RI in RBL-2H3 cells is inhibited by Syk SH2 domains.

Antigen-mediated aggregation of the high-affinity receptor for immunoglobulin E, Fc epsilon RI, results in the activation of multiple signaling pathways, leading to the release of mediators of the allergic response. One of the earliest responses to receptor stimulation is the tyrosine phosphorylation of the beta and gamma subunits of Fc epsilon RI and the association of the tyrosine kinase Syk with the phosphorylated receptor. This association is mediated by the SH2 domains of Syk and is believed to be critical for activating signaling pathways resulting in mediator release. To examine the importance of the interaction of Syk with Fc epsilon RI in signaling events following receptor activation, we introduced a protein containing the SH2 domains of Syk into streptolysin O-permeabilized RBL-2H3 cells. The Syk SH2 domains completely inhibited degranulation and leukotriene production following receptor aggregation, and they blocked the increase in protein tyrosine phosphorylation observed after receptor activation. Inhibition was specific for Fc epsilon RI-mediated signaling, since degranulation of cells activated by alternative stimuli was not blocked by the Syk SH2 domains. A protein containing a point mutation in the carboxy-terminal SH2 domain which abolishes phosphotyrosine binding was not inhibitory. In addition, inhibition of degranulation was reversed by pretreatment of the SH2 domains with a tyrosine phosphorylated peptide corresponding to the tyrosine-based activation motif found in the gamma subunit of Fc epsilon RI, the nonphosphorylated peptide had no effect. The association of Syk with the tyrosine-phosphorylated gamma subunit of the activated receptor was blocked by the Syk SH2 domains, and deregulation in cells activated by clustering of Syk directly without Fc epsilon RI aggregation was not affected by the Syk SH2 domains. These results demonstrate that the association of Syk with the activated Fc epsilon RI is critical for both early and late events following receptor activation and confirm the key role Syk plays in signaling through the high-affinity IgE receptor.     

Identification of CrkL-SH3 binding proteins from embryonic murine brain: implications for Reelin signaling during brain development

The Crk and Crk-like (CrkL) adaptor proteins play important roles in numerous signaling pathways, bridging tyrosine kinase substrates to downstream signaling effectors by virtue of their phosphotyrosine-binding SH2 domains and their effector-binding SH3 domains. Critical to understanding the diverse roles of Crk/CrkL is the identification of tissue- and signal-specific tyrosine phosphorylated substrates to which they are recruited and the tissue-specific effector proteins they chaperone into signaling complexes. Crk and CrkL are known biochemically and genetically to be essential mediators of Reelin/Disabled-1 (Dab1) signaling, which governs proper mammalian brain development. Multimeric Reelin clusters its receptors as well as the receptor-bound intracellular scaffolding protein Dab1. Clustering induces Fyn/Src-dependent Dab1 tyrosine phosphorylation, which recruits Crk/CrkL and SH3-bound effectors. Previously, 21 Crk/CrkL-SH3 binding proteins were identified from diverse cell types. We present here the proteomic identification of 101 CrkL-SH3 binding proteins from embryonic murine brain. The identified proteins are enriched in the Crk/CrkL-SH3 binding motif and signaling activities regulating cell adhesion and motility. These results suggest Reelin-induced Dab1 tyrosine phosphorylation may generate a multifaceted signaling scaffold containing a rich array of Crk/CrkL-SH3 binding effectors and may explain a growing diversity of cellular activities suggested to be influenced by Reelin/Dab1 signaling.     

Modulation of STAT5 interaction with LMW-PTP during early megakaryocyte differentiation

STATs are involved in a variety of cellular processes, including cell proliferation and differentiation. They are activated through tyrosine phosphorylation, which promotes their dimerization via SH2 domains. We have demonstrated previously that in DAMI megakaryoblastic cells LMW-PTP dephosphorylates STAT5, interacting with an essential sequence of nine amino acids in its C-terminal region. Here we characterize STAT5 tyrosine phosphorylation and its interaction with LMW-PTP during early phorbol-12-myristate-13-acetate-induced megakaryocyte differentiation; these processes show clear dependence on STAT5 threonine phosphorylation. Since protein kinase C inhibition prevents phorbol-12-myristate-13-acetate-induced STAT5 threonine phosphorylation and association with LMW-PTP, it follows that these processes depend on protein kinase C activity. By using a Thr757/Val mutant of STAT5 we also demonstrate that the 757 serine/threonine conserved residue, which is in the STAT5A region involved in the interaction with LMW-PTP, is essential for such an association, though its phosphorylation is not necessary.     

Dissecting the interaction of SHP-2 with PZR, an immunoglobulin family protein containing immunoreceptor tyrosine-based inhibitory motifs

Tyrosine phosphorylation of membrane proteins plays a crucial role in cell signaling by recruiting Src homology 2 (SH2) domain-containing signaling molecules. Recently, we have isolated a transmembrane protein designated PZR that specifically binds tyrosine phosphatase SHP-2, which has two SH2 domains (Zhao, Z. J., and Zhao, R. (1998) J. Biol. Chem. 273, 29367-29372). PZR belongs to the immunoglobulin superfamily. Its intracellular segment contains four putative sites of tyrosine phosphorylation. By site-specific mutagenesis, we found that the tyrosine 241 and 263 embedded in the consensus immunoreceptor tyrosine-based inhibitory motifs VIYAQL and VVYADI, respectively, accounted for the entire tyrosine phosphorylation of PZR. The interaction between PZR and SHP-2 requires involvement of both tyrosyl residues of the former and both SH2 domains of the latter, since its was disrupted by mutating a single tyrosyl residue or an SH2 domain. Overexpression of catalytically inactive but not active forms of SHP-2 bearing intact SH2 domains in cells caused hyperphosphorylation of PZR. In vitro, tyrosine-phosphorylated PZR was efficiently dephosphorylated by the full-length form of SHP-2 but not by its SH2 domain-truncated form. Together, the data indicate that PZR serves not only as a specific anchor protein of SHP-2 on the plasma membrane but also as a physiological substrate of the enzyme. The coexisting binding and dephosphorylation of PZR by SHP-2 may function to terminate signal transduction initiated by PZR and SHP-2 and to set a threshold for the signal transduction to be initiated.     

The JAK-binding protein JAB inhibits Janus tyrosine kinase activity through binding in the activation loop

The Janus family of protein tyrosine kinases (JAKs) regulate cellular processes involved in cell growth, differentiation and transformation through their association with cytokine receptors. However, compared with other kinases, little is known about cellular regulators of the JAKs. We have recently identified a JAK-binding protein (JAB) that inhibits JAK signaling in cells. In the studies presented here we demonstrate that JAB specifically binds to the tyrosine residue (Y1007) in the activation loop of JAK2, whose phosphorylation is required for activation of kinase activity. Binding to the phosphorylated activation loop requires the JAB SH2 domain and an additional N-terminal 12 amino acids (extended SH2 subdomain) containing two residues (Ile68 and Leu75) that are conserved in JAB-related proteins. An additional N-terminal 12-amino-acid region (kinase inhibitory region) of JAB also contributes to high-affinity binding to the JAK2 tyrosine kinase domain and is required for inhibition of JAK2 signaling and kinase activity. Our studies define a novel type of regulation of tyrosine kinases and might provide a basis for the design of specific tyrosine kinase inhibitors.     

Direct Regulation of Nephrin Tyrosine Phosphorylation by Nck Adaptor Proteins

The transmembrane protein nephrin is a key component of the kidney slit diaphragm that contributes to the morphology of podocyte foot processes through signaling to the underlying actin cytoskeleton. We have recently reported that tyrosine phosphorylation of the cytoplasmic tail of nephrin facilitates recruitment of Nck SH2/SH3 adaptor proteins and subsequent actin remodeling and that phosphorylation of the Nck binding sites on nephrin is decreased during podocyte injury. We now demonstrate that Nck directly modulates nephrin phosphorylation through formation of a signaling complex with the Src family kinase Fyn. The ability of Nck to enhance nephrin phosphorylation is compromised in the presence of a Src family kinase inhibitor and when the SH3 domains of Nck are mutated. Furthermore, induced loss of Nck expression in podocytes in vivo is associated with a rapid reduction in nephrin tyrosine phosphorylation. Our results suggest that Nck may facilitate dynamic signaling events at the slit diaphragm by promoting Fyn-dependent phosphorylation of nephrin, which may be important in the regulation of foot process morphology and response to podocyte injury.     

Tyrosine-phosphorylated SOCS3 interacts with the Nck and Crk-L adapter proteins and regulates Nck activation

Suppressors of cytokine signaling (SOCS) are negative feedback inhibitors of cytokine and growth factor signal transduction. Although the affect of SOCS proteins on the Jak-STAT pathway has been well characterized, their role in the regulation of other signaling modules is not well understood. In the present study, we demonstrate that SOCS3 physically interacts with the SH2/SH3-containing adapter proteins Nck and Crk-L, which are known to couple activated receptors to multiple downstream signaling pathways and the actin cytoskeleton. Our data show that the SOCS3/Nck and SOCS3/Crk-L interactions depend on tyrosine phosphorylation of SOCS3 Tyr(221) within the conserved SOCS box motif and intact SH2 domains of Nck and Crk-L. Furthermore, SOCS3 Tyr(221) forms a YXXP motif, which is a consensus binding site for the Nck and Crk-L SH2 domains. Expression of SOCS3 in NIH3T3 cells induces constitutive recruitment of a Nck-GFP fusion protein to the plasma membrane and constitutive tyrosine phosphorylation of endogenous Nck. Our findings suggest that SOCS3 regulates multiple cytokine and growth factor-activated signaling pathways by acting as a recruitment factor for adapter proteins.     

The LMP2A protein of Epstein–Barr virus regulates phosphorylation of ITSN1 and Shb adaptors by tyrosine kinases

Latent Membrane Protein 2A (LMP2A) is an Epstein–Barr virus-encoded protein that is important for the maintenance of latent infection. Its activity affects cellular differentiation, migration, proliferation and B cell survival. LMP2A resembles a constitutively activated B cell antigen receptor and exploits host kinases to activate a set of downstream signaling pathways. In the current study we demonstrate the interaction of LMP2A with intersectin 1 (ITSN1), a key endocytic adaptor protein. This interaction occurs via both the N- and C-tails of LMP2A and is mediated by the SH3 domains of ITSN1. Additionally, we identified the Shb adaptor and the Syk kinase as novel binding ligands of ITSN1. The Shb adaptor interacts simultaneously with the phosphorylated tyrosines of LMP2A and the SH3 domains of ITSN1 and mediates indirect interaction of ITSN1 to LMP2A. Syk kinase promotes phosphorylation of both ITSN1 and Shb adaptors in LMP2A-expressing cells. In contrast to ITSN1, Shb phosphorylation depends additionally on Lyn kinase activity.Considering that Shb and ITSN1 are implicated in various receptor tyrosine kinase signaling, our results indicate that LMP2A can affect a number of signaling pathways by regulating the phosphorylation of the ITSN1 and Shb adaptors.     

Characterizing tyrosine phosphorylation signaling in lung cancer using SH2 profiling

Background

Tyrosine kinases drive the proliferation and survival of many human cancers. Thus profiling the global state of tyrosine phosphorylation of a tumor is likely to provide a wealth of information that can be used to classify tumors for prognosis and prediction. However, the comprehensive analysis of tyrosine phosphorylation of large numbers of human cancer specimens is technically challenging using current methods.

Methodology/Principal Findings

We used a phosphoproteomic method termed SH2 profiling to characterize the global state of phosphotyrosine (pTyr) signaling in human lung cancer cell lines. This method quantifies the phosphorylated binding sites for SH2 domains, which are used by cells to respond to changes in pTyr during signaling. Cells could be grouped based on SH2 binding patterns, with some clusters correlated with EGF receptor (EGFR) or K-RAS mutation status. Binding of specific SH2 domains, most prominently RAS pathway activators Grb2 and ShcA, correlated with EGFR mutation and sensitivity to the EGFR inhibitor erlotinib. SH2 binding patterns also reflected MET activation and could identify cells driven by multiple kinases. The pTyr responses of cells treated with kinase inhibitors provided evidence of distinct mechanisms of inhibition.

Conclusions/Significance

This study illustrates the potential of modular protein domains and their proteomic binding profiles as powerful molecular diagnostic tools for tumor classification and biomarker identification.