ABSTRACT
Epidermal growth factor (EGF)-induced signaling was investigated in cells conditionally defective in clathrin-dependent endocytosis by overexpression of K44A dynamin in HeLa cells and potassium depletion in Hep2 cells. Overexpression of mutant dynamin disrupts high-affinity EGF-EGF receptor (EGFR) interaction (T. Ringerike, E. Stang, L. E. Johannessen, D. Sandnes, F. O. Levy, and I. H. Madshus, 1998, J. Biol. Chem. 273, 16639-16642). However, the EGFR substrates Shc and c-Cbl were as efficiently tyrosine phosphorylated in endocytosis-deficient HeLa cells exhibiting only low-affinity EGFRs as in HeLa cells with intact endocytosis and with both high- and low-affinity EGFRs. Both Raf and mitogen-activated protein kinase (MAPK) were activated to the same extent and with the same kinetics. HeLa cells distributed equally in the cell cycle regardless of EGFR internalization. Upon potassium depletion of Hep2 cells, EGF-induced EGFR endocytosis was inhibited. However, the EGFR and MAPK were efficiently activated by EGF in both the absence and the presence of clathrin-dependent endocytosis. The EGFR was weakly tyrosine phosphorylated by potassium depletion even in the absence of EGF, and this activation resulted in detectable activation of MAPK. Our results demonstrate that internalization of EGFR by clathrin-dependent endocytosis is not required for activation of MAPK.
Subject(s)
ErbB Receptors/metabolism , Mitogen-Activated Protein Kinases/metabolism , Cell Cycle , Cell Line , Clathrin/metabolism , Dynamins , Endocytosis , Enzyme Activation , Epidermal Growth Factor/pharmacology , GTP Phosphohydrolases/genetics , GTP Phosphohydrolases/metabolism , HeLa Cells , Humans , Mutagenesis, Site-Directed , Phosphorylation , Potassium/metabolism , Proto-Oncogene Proteins c-raf/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , TransfectionABSTRACT
Activation of the epidermal growth factor receptor (EGFR) kinase was analyzed in cells conditionally defective for clathrin-dependent endocytosis by overexpression of mutant dynamin (K44A). EGF-induced autophosphorylation of the EGFR on ice was strongly reduced in cells overexpressing mutant dynamin, and consistently, binding analyses showed that high-affinity EGFRs were lost. In the absence of mutant dynamin the cells displayed both high- and low-affinity EGFR. At 4 degreesC EGF-EGFR localized mainly outside coated pits regardless of expression of mutant dynamin. However, also low-affinity EGFR efficiently moved to coated pits upon incubating cells at 37 degreesC. Thus, expression of mutant dynamin disrupts high-affinity binding of EGF, but not ligand-induced recruitment of EGFR to clathrin-coated pits.
