COMPLETE RECONSTITUTION OF CONJUGATION AND SUBSEQUENT DEGRADATION OF THE TUMOR-SUPPRESSOR PROTEIN P53 BY PURIFIED COMPONENTS OF THE UBIQUITIN PROTEOLYTIC SYSTEM

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.