Identification of an ubiquitin-ligation system for the epidermal-growth-factor receptor--herbimycin A induces in vitro ubiquitination in rabbit-reticulocyte lysate

Eur J Biochem. 1997 Aug 1;247(3):1190-6. doi: 10.1111/j.1432-1033.1997.01190.x.


Some receptor tyrosine kinases such as the receptors for epidermal-growth factor (EGF) and platelet-derived growth factor undergo polyubiquitination as a consequence of ligand binding. The EGF receptor is also ubiquitinated by treatment with herbimycin A, an ansamycin antibiotic widely used as a tyrosine kinase inhibitor. To investigate the mechanism of the receptor ubiquitination, we have established an assay system in which herbimycin-A-induced ubiquitination processes can be analyzed in vitro. We now show that herbimycin A treatment of the purified EGF receptor induces polyubiquitination of the receptor in rabbit-reticulocyte lysate. Both DEAE unadsorbed material (fraction I) and high salt eluate (fraction II) of the reticulocyte lysate are involved cooperatively in the ubiquitination process, where the ubiquitin-conjugating enzyme UBC4 can functionally substitute for fraction I. A ubiquitin-protein ligase-like activity, partially purified from fraction II by DEAE anion-exchange chromatography, also functions in concert with UBC4. The precise mechanism of herbimycin A-induced ubiquitination of the EGF receptor is not fully understood, however, our present findings suggest that direct interaction with herbimycin A results in some modification of the receptor which is recognized by the ubiquitin-conjugating system in rabbit-reticulocyte lysate.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Benzoquinones
  • Chromatography, DEAE-Cellulose
  • ErbB Receptors / metabolism*
  • Humans
  • Lactams, Macrocyclic
  • Quinones / pharmacology*
  • Rabbits
  • Reticulocytes / metabolism
  • Rifabutin / analogs & derivatives
  • Tumor Cells, Cultured
  • Ubiquitins / metabolism*


  • Benzoquinones
  • Lactams, Macrocyclic
  • Quinones
  • Ubiquitins
  • Rifabutin
  • herbimycin
  • ErbB Receptors