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.
Machida
Kazuya
K
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.
Thompson
Christopher M
CM
Dierck
Kevin
K
Jablonowski
Karl
K
Kärkkäinen
Satu
S
Liu
Bernard
B
Zhang
Haimin
H
Nash
Piers D
PD
Newman
Debra K
DK
Nollau
Peter
P
Pawson
Tony
T
Renkema
G Herma
GH
Saksela
Kalle
K
Schiller
Martin R
MR
Shin
Dong-Guk
DG
Mayer
Bruce J
BJ
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*
D046912:Multiprotein Complexes; D010455:Peptides; D020543:Proteome; D011994:Recombinant Proteins; D019000:Phosphotyrosine; D011505:Protein-Tyrosine Kinases