COMPLETE RECONSTITUTION OF CONJUGATION AND SUBSEQUENT DEGRADATION OF THE TUMOR-SUPPRESSOR PROTEIN P53 BY PURIFIED COMPONENTS OF THE UBIQUITIN PROTEOLYTIC SYSTEM
D. Shkedy et al., COMPLETE RECONSTITUTION OF CONJUGATION AND SUBSEQUENT DEGRADATION OF THE TUMOR-SUPPRESSOR PROTEIN P53 BY PURIFIED COMPONENTS OF THE UBIQUITIN PROTEOLYTIC SYSTEM, FEBS letters, 348(2), 1994, pp. 126-130
The wild-type tumor suppressor protein p53 is a short-lived protein th
at plays important roles in regulation of cell cycle, differentiation,
and survival. Mutations that inactivate or alter the tumor suppressor
activity of the protein seem to be the most common genetic change in
human cancer and are frequently associated with changes in its stabili
ty. The ubiquitin system has been implicated in the degradation of p53
both in vivo and in vitro. A mutant cell line that harbors a thermola
bile ubiquitin-activating enzyme, El, fails to degrade p53 at the nonp
ermissive temperature. Studies in cell-free extracts have shown that c
ovalent attachment of ubiquitin to the protein requires the three conj
ugating enzymes: E1, a novel species of ubiquitin-carrier protein (ubi
quitin-conjugating enzyme; UBC),E2-F1, and an ubiquitin-protein ligase
, E3. Recognition of p53 by the ligase is facilitated by formation of
a complex between the protein and the human papillomavirus (HPV) oncop
rotein E6. Therefore, the ligase has been designated E6-associated pro
tein (EB-AP). However, these in vitro studies have not demonstrated th
at the conjugates serve as essential intermediates in the proteolytic
process. In fact, in many cases, conjugation of ubiquitin to the targe
t protein does not signal its degradation. Thus, it is essential to de
monstrate that p53-ubiquitin adducts serve as essential proteolytic in
termediates and are recognized and degraded by the 26S protease comple
x, the proteolytic arm of the ubiquitin pathway. In this study, we dem
onstrate that conjugates of p53 generated in the presence of purified,
E1, E2, E6-AP, E6, ubiquitin and ATP, are specifically recognized by
the 26S protease complex and degraded. In contrast, unconjugated p53 r
emains stable. The ability to reconstitute the system from purified co
mponents will enable detailed analysis of the recognition process and
the structural motifs involved in targeting the protein for degradatio
n.