Abstract
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.
Highlights
The human papillomavirus (HPV) oncoprotein E6 binds to E6AP (E6-associated protein), a HECT-type ubiquitin ligase, and directs its ligase activity toward the tumor suppressor p53
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
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
Summary
A reconstitution system for the polyubiquitination of p53 by the E6AP-E6 complex in vitro. The amounts of polyubiquitinated products increased in a manner dependent on E6AP-E6 concentration, relatively large products were detectable even in the presence of low E6AP-E6 concentrations This result suggested that once E6AP-E6 binds to hisp, it successively transfers many Ub molecules without dissociation (i.e. E6AP-E6 might form a stable complex with p53). The ligase activity of the ternary complex was confirmed by a ubiquitination assay with the additional introduction of E1, UBCH5c, and Ub (Fig. 2C) These results suggest that the stable binding of E6AP-E6 to p53 ensures that each p53 molecule is ubiquitinated processively. The results showed that the concentration of E2 strongly affected the size of the products during a 10-min incubation Both UBCH5c and UBCH7 supported the ubiquitination of hisp at similar concentrations (Fig. 3, A and B). We tested two specific mutants of UBCH5c, S22R
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