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 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),