High-Throughput Phosphotyrosine Profiling Using SH2 Domains

Raymond and Beverly Sackler Laboratory of Genetics and Molecular Medicine, Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT 06030, USA.
Molecular Cell (Impact Factor: 14.02). 07/2007; 26(6):899-915. DOI: 10.1016/j.molcel.2007.05.031
Source: PubMed


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.

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    • "As a consequence, signaling pathways are increasingly viewed as being embedded within multiprotein networks. This complexity is typified by phosphotyrosine (pY)-based signaling networks, which utilize SH2 domains to engage pYmotifs with modest levels of specificity and affinity (Liu et al., 2006; Machida et al., 2007); for example, the Grb2 adaptor binds numerous distinct pYXNX motifs on activated receptor tyrosine kinases and scaffolds through its SH2 domain, and effectors through its flanking SH3 domains (Bisson et al., 2011). Motifbased analyses of a proteome reveal hundreds of potential binding sites for Grb2 SH2 (Obenauer et al., 2003); of these, dozens may be present in a particular cellular context. "
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    ABSTRACT: Cell signaling depends on dynamic protein-protein interaction (PPI) networks, often assembled through modular domains each interacting with multiple peptide motifs. This complexity raises a conceptual challenge, namely to define whether a particular cellular response requires assembly of the complete PPI network of interest or can be driven by a specific interaction. To address this issue, we designed variants of the Grb2 SH2 domain ("pY-clamps") whose specificity is highly biased toward a single phosphotyrosine (pY) motif among many potential pYXNX Grb2-binding sites. Surprisingly, directing Grb2 predominantly to a single pY site of the Ptpn11/Shp2 phosphatase, but not other sites tested, was sufficient for differentiation of the essential primitive endoderm lineage from embryonic stem cells. Our data suggest that discrete connections within complex PPI networks can underpin regulation of particular biological events. We propose that this directed wiring approach will be of general utility in functionally annotating specific PPIs.
    Full-text · Article · Jun 2014 · Molecular Cell
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    • "). In the case of SH2 domain specificity, this approach has been extended by using arrays of P.Tyr-containing peptides derived from physiological Tyr phosphorylation sites and recombinant SH2 domains (Machida et al. 2007; Tinti et al. 2013). "
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    ABSTRACT: Tyrosine phosphorylation of proteins was discovered in 1979, but this posttranslational modification had been "invented" by evolution more than a billion years ago in single-celled eukaryotic organisms that were the antecedents of the first multicellular animals. Because sophisticated cell-cell communication is a sine qua non for the existence of multicellular organisms, the development of cell-surface receptor systems that use tyrosine phosphorylation for transmembrane signal transduction and intracellular signaling seems likely to have been a crucial event in the evolution of metazoans. Like all types of protein phosphorylation, tyrosine phosphorylation serves to regulate proteins in multiple ways, including causing electrostatic repulsion and inducing allosteric transitions, but the most important function of phosphotyrosine (P.Tyr) is to serve as a docking site that promotes a specific interaction between a tyrosine phosphorylated protein and another protein that contains a P.Tyr-binding domain, such as an SH2 or PTB domain. Such docking interactions are essential for signal transduction downstream from receptor tyrosine kinases (RTKs) on the cell surface, which are activated on binding a cognate extracellular ligand, and, as a consequence, elicit specific cellular outcomes.
    Full-text · Article · May 2014 · Cold Spring Harbor perspectives in biology
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    • "Having found that TANGO could predict increased solubility of the TSAd-SH2 domain, we then asked whether beta-aggregation propensity is a common characteristic of SH2 domains. Most of the 120 SH2 domains encoded by the human genome have been expressed as recombinant His- [17] or GST-fusion proteins [18,19]. All available human SH2 domain sequences [2] were analysed by TANGO (Additional file 2: Table S1) and results were correlated to the recently published information on SH2 solubility. "
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    ABSTRACT: Signalling proteins often contain several well defined and conserved protein domains. Structural analyses of such domains by nuclear magnetic spectroscopy or X-ray crystallography may greatly inform the function of proteins. A limiting step is often the production of sufficient amounts of the recombinant protein. However, there is no particular way to predict whether a protein will be soluble when expressed in E.coli. Here we report our experience with expression of a Src homology 2 (SH2) domain. The SH2 domain of the SH2D2A protein (or T cell specific adapter protein, TSAd) forms insoluble aggregates when expressed as various GST-fusion proteins in Escherichia coli (E. coli). Alteration of the flanking sequences, or growth temperature influenced expression and solubility of TSAd-SH2, however overall yield of soluble protein remained low. The algorithm TANGO, which predicts amyloid fibril formation in eukaryotic cells, identified a hydrophobic sequence within the TSAd-SH2 domain with high propensity for beta-aggregation. Mutation to the corresponding amino acids of the related HSH2- (or ALX) SH2 domain increased the yield of soluble TSAd-SH2 domains. High beta-aggregation values predicted by TANGO correlated with low solubility of recombinant SH2 domains as reported in the literature. Solubility of recombinant proteins expressed in E.coli can be predicted by TANGO, an algorithm developed to determine the aggregation propensity of peptides. Targeted mutations representing corresponding amino acids in similar protein domains may increase solubility of recombinant proteins.
    Full-text · Article · Jan 2014 · BMC Biotechnology
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