title: High-throughput phosphotyrosine profiling using SH2 domains.
abstract: 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.
authors.affiliation: 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.
authors.forename: Kazuya
authors.initials: K
authors.lastname: Machida
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Thompson
authors.forename: Kevin
authors.initials: K
authors.lastname: Dierck
authors.forename: Karl
authors.initials: K
authors.lastname: Jablonowski
authors.forename: Satu
authors.initials: S
authors.lastname: Kärkkäinen
authors.forename: Bernard
authors.initials: B
authors.lastname: Liu
authors.forename: Haimin
authors.initials: H
authors.lastname: Zhang
authors.forename: Piers D
authors.initials: PD
authors.lastname: Nash
authors.forename: Debra K
authors.initials: DK
authors.lastname: Newman
authors.forename: Peter
authors.initials: P
authors.lastname: Nollau
authors.forename: Tony
authors.initials: T
authors.lastname: Pawson
authors.forename: G Herma
authors.initials: GH
authors.lastname: Renkema
authors.forename: Kalle
authors.initials: K
authors.lastname: Saksela
authors.forename: Martin R
authors.initials: MR
authors.lastname: Schiller
authors.forename: Dong-Guk
authors.initials: DG
authors.lastname: Shin
authors.forename: Bruce J
authors.initials: BJ
authors.lastname: Mayer
mesh_terms: D000818:Animals; D002448:Cell Adhesion / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D022001:Focal Adhesions / Q000502:physiology*; D006801:Humans; D051379:Mice; D046912:Multiprotein Complexes / Q000378:metabolism; D041681:NIH 3T3 Cells; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D040081:Protein Array Analysis; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D020543:Proteome / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000502:physiology*; D018909:src Homology Domains / Q000502:physiology*
chemical_list: D046912:Multiprotein Complexes; D010455:Peptides; D020543:Proteome; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases