Stepwise multipolyubiquitination of p53 by the E6AP-E6 ubiquitin ligase complex

J Biol Chem. 2019 Oct 11;294(41):14860-14875. doi: 10.1074/jbc.RA119.008374. Epub 2019 Sep 6.


The human papillomavirus (HPV) oncoprotein E6 specifically binds to E6AP (E6-associated protein), a HECT (homologous to the E6AP C terminus)-type ubiquitin ligase, and directs its ligase activity toward the tumor suppressor p53. To examine the biochemical reaction in vitro, we established an efficient reconstitution system for the polyubiquitination of p53 by the E6AP-E6 complex. We demonstrate that E6AP-E6 formed a stable ternary complex with p53, which underwent extensive polyubiquitination when the isolated ternary complex was incubated with E1, E2, and ubiquitin. Mass spectrometry and biochemical analysis of the reaction products identified lysine residues as p53 ubiquitination sites. A p53 mutant with arginine substitutions of its 18 lysine residues was not ubiquitinated. Analysis of additional p53 mutants retaining only one or two intact ubiquitination sites revealed that chain elongation at each of these sites was limited to 5-6-mers. We also determined the size distribution of ubiquitin chains released by en bloc cleavage from polyubiquitinated p53 to be 2-6-mers. Taken together, these results strongly suggest that p53 is multipolyubiquitinated with short chains by E6AP-E6. In addition, analysis of growing chains provided strong evidence for step-by-step chain elongation. Thus, we hypothesize that p53 is polyubiquitinated in a stepwise manner through the back-and-forth movement of the C-lobe, and the permissive distance for the movement of the C-lobe restricts the length of the chains in the E6AP-E6-p53 ternary complex. Finally, we show that multipolyubiquitination at different sites provides a signal for proteasomal degradation.

Keywords: E3 ubiquitin ligase; E6-associated protein; Lys48-linked ubiquitin chain; enzyme mechanism; human papillomavirus (HPV) oncoprotein E6; in vitro reconstitution; linkage specificity; mass spectrometry (MS); p53; polyubiquitin chain.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Cell Line
  • Humans
  • Kinetics
  • Mutation
  • Oncogene Proteins, Viral / metabolism*
  • Protein Stability
  • Tumor Suppressor Protein p53 / chemistry
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism*
  • Ubiquitin-Protein Ligases / metabolism*
  • Ubiquitination*


  • Oncogene Proteins, Viral
  • Tumor Suppressor Protein p53
  • UBE3A protein, human
  • Ubiquitin-Protein Ligases