Profiling the global tyrosine phosphorylation state by Src homology 2 domain binding.
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.
Nollau
P
P
Laboratory of Molecular Medicine, Children's Hospital, Boston, MA 02115, USA.
Mayer
B J
BJ
D000818:Animals; D001402:B-Lymphocytes; D006801:Humans; D019169:Jurkat Cells; D020014:K562 Cells; D051379:Mice; D016296:Mutagenesis; D010766:Phosphorylation; D011485:Protein Binding; D016315:Proto-Oncogene Proteins c-abl / Q000235:genetics / Q000378:metabolism; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000378:metabolism; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*; D018909:src Homology Domains / Q000235:genetics
D014443:Tyrosine; D020797:Receptor, Platelet-Derived Growth Factor beta; D016315:Proto-Oncogene Proteins c-abl