Imaging phosphorylation dynamics of the epidermal growth factor receptor.
Epidermal growth factor receptor (EGFR) signaling is initiated by ligand binding followed by homodimerization and rapid receptor autophosphorylation. Monitoring EGFR phosphorylation was achieved by measuring translocation and binding of an enhanced yellow fluorescent protein (EYFP)-labeled phosphotyrosine-binding domain (PTB) to enhanced cyan fluorescent protein (ECFP)-tagged EGFR using fluorescence lifetime imaging microscopy or sensitized emission measurements. To simplify dynamic phosphorylation pattern measurements in cells, FLAME, a ratiometric sensor containing both EGFR-ECFP and PTB-EYFP in one molecule, was designed and examined in COS7 cells. Epidermal growth factor (EGF) treatment demonstrated rapid and reversible changes in the EYFP/ECFP fluorescence emission ratios, due to binding of the PTB domain to its consensus binding sites upon phosphorylation at the cell periphery, whereas perinuclear regions failed to respond to EGF but were responsive to tyrosine kinase inhibition. Long-term EGF treatment resulted in accumulation of dephosphorylated receptor in the perinuclear region due to active dephosphorylation occurring at intracellular sites. This indicates that the sensor closely approaches the true dynamics of tyrosine kinase autophosphorylation and dephosphorylation. Phosphatase inhibition by pervanadate resulted in an irreversible response in all cellular compartments. These data show that EGFR is under tonic phosphatase suppression maintaining the receptor in an unphosphorylated (silent) state and is dephosphorylated at endomembranes after ligand-mediated endocytosis.
Offterdinger
Martin
M
EMBL-Heidelberg, Cell Biology and Cell Biophysics Programme, Meyerhofstrasse 1, D-69117 Heidelberg, Germany.
Georget
Virginie
V
Girod
Andreas
A
Bastiaens
Philippe I H
PI
D000818:Animals; D019556:COS Cells; D045744:Cell Line, Tumor; D003599:Cytoskeleton / Q000378:metabolism; D019281:Dimerization; D004705:Endocytosis; D066246:ErbB Receptors / Q000737:chemistry* / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006801:Humans; D015151:Immunoblotting; D008024:Ligands; D008164:Luminescent Proteins / Q000378:metabolism; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D013997:Time Factors; D014162:Transfection
D008024:Ligands; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D019000:Phosphotyrosine; D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases