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Preubiquitinated chimeric ErbB2 is constitutively endocytosed and subsequently degraded in lysosomes

Tram Thu Vuong1, Christian Berger, Vibeke Bertelsen

  • 1Institute of Clinical Medicine, University of Oslo, Rikshospitalet, 0027 Oslo, Norway.

Experimental Cell Research
|November 7, 2012
PubMed

Insights

Ubiquitination triggers the internalization and lysosomal degradation of the oncoprotein ErbB2, which is normally resistant to endocytosis. This process is clathrin-dependent and does not require C-terminal cleavage.

Area of Science:

  • Cell Biology
  • Molecular Oncology
  • Protein Trafficking

Background:

  • The oncoprotein ErbB2 typically resists endocytosis due to Heat shock protein 90 interaction.
  • Previous studies showed Ansamycin derivatives induce clathrin-dependent ErbB2 endocytosis.
  • Pre-ubiquitinated EGFR (EGFR-Ub(4)) undergoes constitutive clathrin-dependent endocytosis.

Purpose of the Study:

  • To investigate if ubiquitination is sufficient to induce clathrin-mediated endocytosis of ErbB2.
  • To determine the lysosomal trafficking and degradation pathway of ubiquitinated ErbB2.
  • To explore the role of ubiquitination in ErbB2 internalization.

Main Methods:

  • Construction and expression of an ErbB2-Ub(4) chimera.
  • Western blotting to detect polyubiquitin chain formation (Lys48 and Lys63).
  • Analysis of ErbB2-Ub(4) internalization, trafficking, and degradation using cell-based assays.
  • Over-expression of dominant-negative clathrin adaptor protein fragments.

Main Results:

  • ErbB2-Ub(4) chimera exhibited constitutive, clathrin-dependent endocytosis.
  • ErbB2-Ub(4) formed Lys48- and Lys63-linked polyubiquitin chains.
  • Internalized ErbB2-Ub(4) was sorted to late endosomes and lysosomes for degradation.
  • Dominant-negative epsin1 and Eps15 fragments inhibited ErbB2-Ub(4) endocytosis.
  • C-terminal cleavage was not required for ErbB2-Ub(4) endocytosis.

Conclusions:

  • Ubiquitination is sufficient to induce clathrin-mediated endocytosis of plasma membrane-localized ErbB2.
  • Ubiquitination targets ErbB2 for lysosomal degradation.
  • Clathrin-dependent endocytosis and lysosomal sorting of ErbB2 can be mediated by ubiquitination alone.

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