ERK2 signalling from internalised epidermal growth factor receptor in broken A431 cells.
Activation of extracellular signal-regulated kinase 2 (ERK2) signalling from epidermal growth factor receptors (EGFRs) is widely assumed to originate in the plasma membrane. Using an in vitro assay, we investigated whether EGF/EGFR complexes internalised by endocytosis in A431 cells can initiate signalling in the ERK2 pathway. At 0 degrees C, binding of EGF induced tyrosine phosphorlyation of EGFR and, when the cells were subsequently broken by scraping and warmed in the presence of exogenous cytosol, activation of ERK2 occurred. At 0 degrees C, washes with pH 4.5 media reversed EGF binding, tyrosine phosphorylation and ERK-2 activation in exogenous cytosol, providing conditions in which signalling from the cell surface and internalised EGFRs could be distinguished. When cells containing internalised EGF/EGFR complexes were first washed in low pH media at 0 degrees C and then broken and incubated in exogenous cytosol, substantial activation of ERK2 occurred. This activation reached a maximum after a 5-min internalisation and was almost completely prevented by incubation in 0.45 M sucrose, a known inhibitor of receptor-mediated endocytosis. These data are consistent with activation of the ERK2 signalling pathway by internalised EGRFs situated in endosomal compartments. Our observation that EGFR tyrosine dephosphorylation is incomplete above pH 5.5 suggests that signalling is initiated in early endosomes.
Department of Anatomy and Physiology, University of Dundee, Scotland.
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D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000378:metabolism; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D019950:Mitogen-Activated Protein Kinase 1; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D019000:Phosphotyrosine; D004815:Epidermal Growth Factor; D066246:ErbB Receptors; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019950:Mitogen-Activated Protein Kinase 1