High-Throughput Phosphotyrosine Profiling Using SH2 Domains

Authors

Kazuya Machida, University of Connecticut Health Center
Christopher M. Thompson, University of Connecticut Health Center
Kevin Dierck, University Medical Center Hamburg-Eppendorf
Kar Jablonowski, University of Chicago
Satu Karkkainen, University of Tampere and Tampere University Hospita
Bernard Liu, University of Chicago
Haimin Zhang, University of Connecticut - Storrs
Piers D. Nash, University of Chicago
Debra K. Newman, The BloodCenter of Southeastern Wisconsin
Peter Nollau, University Medical Center Hamburg-Eppendorf
Tony Pawson, 8Samuel Lunenfeld Research Institute
G. Herma Renkema, University of Connecticut - Storrs
Kalle Saksela, University of Tampere and Tampere University Hospital
Martin R. Schiller, University of Nevada, Las VegasFollow
Dong-Guk Shin, University of Connecticut - StorrsFollow
Bruce J. Mayer, University of Connecticut Health CenterFollow

Document Type

Article

Publication Date

6-22-2007

Publication Title

Molecular Cell

Volume

26

Issue

6

First page number:

899

Last page number:

915

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 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.

Keywords

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

Disciplines

Cancer Biology | Life Sciences | Molecular Biology | Structural Biology | Virology

Language

English

Repository Citation

Machida, K., Thompson, C. M., Dierck, K., Jablonowski, K., Karkkainen, S., Liu, B., Zhang, H., Nash, P. D., Newman, D. K., Nollau, P., Pawson, T., Renkema, G. H., Saksela, K., Schiller, M. R., Shin, D., Mayer, B. J. (2007). High-Throughput Phosphotyrosine Profiling Using SH2 Domains. Molecular Cell, 26(6), 899-915.
http://dx.doi.org/10.1016/j.molcel.2007.05.031