Profiling the global tyrosine phosphorylation state by Src homology 2 domain binding

doi:10.1073/pnas.241215998

Profiling the global tyrosine phosphorylation state by Src homology 2 domain binding

Peter Nollau*,† and
Bruce J. Mayer*,‡,§

^*Laboratory of Molecular Medicine, Children's Hospital, and Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115; and ^‡Department of Genetics and Developmental Biology, University of Connecticut Health Center, Farmington, CT 06030-3301

Edited by Tony Hunter, The Salk Institute for Biological Studies, La Jolla, CA, and approved September 27, 2001 (received for review June 4, 2001)

Abstract

Reversible tyrosine phosphorylation plays a crucial role in signal transduction, regulating many biological functions including proliferation, differentiation, and motility. The comprehensive characterization of the tyrosine phosphorylation state of a cell is of great interest for understanding the mechanisms that underlie signaling; however, current methods for analyzing tyrosine-phosphorylated proteins in crude protein extracts provide limited information, or are laborious and require relatively large amounts of protein. We have developed a simple, rapid, and flexible competitive binding assay based on the far-Western blot technique, in which a battery of Src homology 2 domain probes is used to detect patterns of specific tyrosine-phosphorylated sites. We demonstrate that distinct profiles of tyrosine phosphorylation can be detected with high sensitivity and specificity and low background. This proteomic approach can be used to rapidly profile the global tyrosine phosphorylation state of any cell of interest and has obvious applications as a molecular diagnostic tool, for example in the classification of tumors. The general strategy we describe here is not limited to Src homology 2 domains and could be used to profile the binding sites for any class of protein interaction domain.

Footnotes

↵ † Present address: Universitätskrankenhaus Eppendorf, Abteilung für Klinische Chemie, Martinistrasse 52, 20246 Hamburg, Germany.
↵ § To whom reprint requests should be addressed. E-mail: bmayer{at}neuron.uchc.edu.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations:

SH2,

Src homology 2;

PTyr,

phosphotyrosine;

GST,

glutathione S-transferase;

GSH-HRP,

glutathione-conjugated horseradish peroxidase;

PDGFRβ,

platelet-derived growth factor β receptor;

PI3-kinase,

phosphatidylinositol 3-kinase;

PLC-γ,

phospholipase C-γ