High-Throughput Phosphotyrosine Proﬁling Using SH2 Domains
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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 ofhuman 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.
Animals; Cancer; Cell adhesion; Cell Adhesion/physiology; Cellular signal transduction; Fibroblasts; Fibroblasts/cytology; Fibroblasts/metabolism; Focal adhesions; Focal adhesions—Formation; Focal Adhesions/physiology; Humans; Mice; Multiprotein Complexes/metabolism; NIH 3T3 Cells; Peptides; Peptides/metabolism; Phosphorylation; Phosphotyrosine/metabolism; Protein Array Analysis; Protein Processing; Post-Translational/physiology; Protein-Tyrosine Kinases/metabolism; Proteome/metabolism; Recombinant proteins; Recombinant Proteins/metabolism; Signal Transduction/physiology; src Homology Domains/physiology
Cancer Biology | Life Sciences | Molecular Biology | Structural Biology | Virology
Thompson, C. M.,
Nash, P. D.,
Newman, D. K.,
Renkema, G. H.,
Schiller, M. R.,
Mayer, B. J.
High-Throughput Phosphotyrosine Proﬁling Using SH2 Domains.
Molecular Cell, 26(6),