Extracellular stimuli are transduced inside the cell by posttranslational modifications (PTMs), such as phosphorylation, of proteins in signaling networks. Insight into the structure of these networks requires quantification of PTM levels in individual cells. Fluorescence resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to image PTM levels in situ. FLIM on cell arrays that express fluorescent protein fusions can quantify tyrosine phosphorylation patterns in large networks in individual cells. We identified tyrosine kinase substrates by imaging their phosphorylation levels after inhibition of protein tyrosine phosphatases. Analysis of the correlation between protein phosphorylation and expression levels at single cell resolution allowed us to identify positive feedback motifs. Using FLIM on cell arrays (CA-FLIM), we uncovered components that transduce signals from epidermal growth factor receptor.
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P.R.-N. was supported by a Marie Curie Intra-European fellowship for career development (FP7-PEOPLE 2007-2-1-EIF). This work was supported by Interaction Proteome (Integrated Project from FP6) and the Centre for Systems Biology in Dortmund (cofinanced by the European Regional Development Fund and the State of North Rhine-Westfalia). S. Dhe-Paganon (University of Toronto) provided the pET28a-LIC-EPHA3c plasmid.
The authors declare no competing financial interests.
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Grecco, H., Roda-Navarro, P., Girod, A. et al. In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays. Nat Methods 7, 467–472 (2010) doi:10.1038/nmeth.1458
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