Time-resolved multimodal analysis of Src Homology 2 (SH2) domain binding in signaling by receptor tyrosine kinases

While the affinities and specificities of SH2 domain-phosphotyrosine interactions have been well characterized, spatio-temporal changes in phosphosite availability in response to signals, and their impact on recruitment of SH2-containing proteins in vivo, are not well understood. To address this iss...

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Bibliographic Details
Published in:eLife 2016-04, Vol.5, p.e11835-e11835
Main Authors: Jadwin, Joshua A, Oh, Dongmyung, Curran, Timothy G, Ogiue-Ikeda, Mari, Jia, Lin, White, Forest M, Machida, Kazuya, Yu, Ji, Mayer, Bruce J
Format: Article
Language:English
Subjects:
Binding sites
Biochemistry
Biophysics and Structural Biology
Blotting, Far-Western
Cancer
Cell Line, Tumor
Cell Membrane - metabolism
Epidermal growth factor
Epidermal Growth Factor - metabolism
Epithelial Cells - drug effects
Epithelial Cells - physiology
Glass substrates
Health aspects
Homology
Homology (Biology)
Humans
Kinases
Localization
Mass Spectrometry
Mass spectroscopy
Medical research
Microscopy
Optical Imaging
Phosphorylation
Phosphotransferases
Phosphotyrosine
Protein Binding
Proteins
Receptor Protein-Tyrosine Kinases - metabolism
receptor tyrosine kinase
Scientific imaging
SH2 domain
Signal Transduction
src Homology Domains
Tyrosine
Western blotting
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Summary:While the affinities and specificities of SH2 domain-phosphotyrosine interactions have been well characterized, spatio-temporal changes in phosphosite availability in response to signals, and their impact on recruitment of SH2-containing proteins in vivo, are not well understood. To address this issue, we used three complementary experimental approaches to monitor phosphorylation and SH2 binding in human A431 cells stimulated with epidermal growth factor (EGF): 1) phospho-specific mass spectrometry; 2) far-Western blotting; and 3) live cell single-molecule imaging of SH2 membrane recruitment. Far-Western and MS analyses identified both well-established and previously undocumented EGF-dependent tyrosine phosphorylation and binding events, as well as dynamic changes in binding patterns over time. In comparing SH2 binding site phosphorylation with SH2 domain membrane recruitment in living cells, we found in vivo binding to be much slower. Delayed SH2 domain recruitment correlated with clustering of SH2 domain binding sites on the membrane, consistent with membrane retention via SH2 rebinding.
ISSN:2050-084X
2050-084X
DOI:10.7554/elife.11835