Epidermal growth factor receptor efficiently activates mitogen-activated protein kinase in HeLa cells and Hep2 cells conditionally defective in clathrin-dependent endocytosis

Exp Cell Res. 2000 Oct 10;260(1):136-45. doi: 10.1006/excr.2000.5004.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Cycle
  • Cell Line
  • Clathrin / metabolism
  • Dynamins
  • Endocytosis
  • Enzyme Activation
  • Epidermal Growth Factor / pharmacology
  • ErbB Receptors / metabolism*
  • GTP Phosphohydrolases / genetics
  • GTP Phosphohydrolases / metabolism
  • HeLa Cells
  • Humans
  • Mitogen-Activated Protein Kinases / metabolism*
  • Mutagenesis, Site-Directed
  • Phosphorylation
  • Potassium / metabolism
  • Proto-Oncogene Proteins c-raf / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Transfection

Substances

  • Clathrin
  • Recombinant Proteins
  • Epidermal Growth Factor
  • ErbB Receptors
  • Proto-Oncogene Proteins c-raf
  • Mitogen-Activated Protein Kinases
  • GTP Phosphohydrolases
  • Dynamins
  • Potassium