Endosomal dynamics of Met determine signaling output

Mol Biol Cell. 2003 Apr;14(4):1346-54. doi: 10.1091/mbc.e02-09-0578.

Abstract

Proteasomal activity is required for Met receptor degradation after acute stimulation with hepatocyte growth factor (HGF). Inhibition of proteasomal activity with lactacystin leads to a block in the endocytic trafficking of Met such that the receptor fails to reach late endosomes/lysosomes, where degradation by acid-dependent proteases takes place (). In this article, we have biochemically determined Met internalization rates from the cell surface and shown that lactacystin does not inhibit the initial HGF-dependent internalization step of Met. Instead, it promotes the recycling pathway from early endosomes at the expense of sorting to late endosomes, thereby ensuring rapid return of internalized Met to the cell surface. We have used this perturbation of Met endosomal sorting by lactacystin to examine the consequences for HGF-dependent signaling outputs. In control cells HGF-dependent receptor autophosphorylation reaches a maximal level over 5-10 min but then attenuates over the ensuing 50 min. Furthermore, Met dephosphorylation can be kinetically dissociated from Met degradation. In lactacystin-treated cells, we observe a failure of Met dephosphorylation as well as Met degradation. Elements of the mitogen-activated protein kinase cascade, downstream of receptor activation, show a normal kinetic profile of phosphorylation, indicating that the mitogen-activated protein kinase pathway can attenuate in the face of sustained receptor activation. The HGF-dependent phosphorylation of a receptor substrate that is localized to clathrin-coated regions of sorting endosomes, Hrs, is dramatically reduced by lactacystin treatment. Reduction of cellular Hrs levels by short interfering RNA modestly retards Met degradation and markedly prevents the attenuation of Met phosphorylation. HGF-dependent Hrs phosphorylation and Met dephosphorylation may provide signatures for retention of the receptor in coated regions of the endosome implicated in sorting to lysosomes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcysteine / analogs & derivatives*
  • Acetylcysteine / pharmacology
  • Cell Line
  • Cysteine Endopeptidases / metabolism
  • Endosomal Sorting Complexes Required for Transport
  • Endosomes / metabolism*
  • HeLa Cells
  • Hepatocyte Growth Factor / metabolism
  • Humans
  • Mitogen-Activated Protein Kinases / metabolism
  • Multienzyme Complexes / metabolism
  • Phosphoproteins / metabolism
  • Phosphorylation
  • Proteasome Endopeptidase Complex
  • Proto-Oncogene Proteins c-met / metabolism*
  • Signal Transduction
  • Ubiquitin / metabolism

Substances

  • Endosomal Sorting Complexes Required for Transport
  • Multienzyme Complexes
  • Phosphoproteins
  • Ubiquitin
  • hepatocyte growth factor-regulated tyrosine kinase substrate
  • lactacystin
  • Hepatocyte Growth Factor
  • Proto-Oncogene Proteins c-met
  • Mitogen-Activated Protein Kinases
  • Cysteine Endopeptidases
  • Proteasome Endopeptidase Complex
  • Acetylcysteine