In situ analysis of tyrosine phosphorylation networks by FLIM on cell arrays.
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.
Grecco
Hernán E
HE
Max Planck Institute for Molecular Physiology, Department of Systemic Cell Biology, Dortmund, Germany.
Roda-Navarro
Pedro
P
Girod
Andreas
A
Hou
Jian
J
Frahm
Thomas
T
Truxius
Dina C
DC
Pepperkok
Rainer
R
Squire
Anthony
A
Bastiaens
Philippe I H
PI
D045744:Cell Line, Tumor; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D010750:Phosphoproteins / Q000032:analysis; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*
D010750:Phosphoproteins; D014443:Tyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D017027:Protein Tyrosine Phosphatases
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