Abstract
The N-terminal transcription activation domain of p53 is intrinsically unstructured. We show in vitro and in vivo that this domain initiates p53 degradation by the 20 S proteasome in a ubiquitin-independent fashion. The decay of metabolically labeled p53 follows biphasic kinetics with an immediate fast phase that is ubiquitin-independent and a second slower phase that is ubiquitin-dependent. The 20 S proteasome executes the first phase by default, whereas the second phase requires the 26 S proteasome. p53 N-terminal binding proteins, such as Hdmx, can selectively block the first phase of degradation. Remarkably, gamma-irradiation inhibits both p53 decay phases, whereas UV selectively negates the second phase, giving rise to discrete levels of p53 accumulation. Our data of a single protein experiencing double mode degradation mechanisms each with unique kinetics provide the mechanistic basis for programmable protein homeostasis (proteostasis).
Highlights
Supporting this possibility is that at least a fraction of certain proteins is subjected to UI degradation, a process that might be executed by the 20 S proteasome
UI degradation of p53 can be blocked by NQO1, an NADH-regulated enzyme [24], which is in association with the 20 S proteasomes [7]
We demonstrate that p53 undergoes biphasic decay kinetics and further correlate the biphasic kinetics curve with the UI (20 S proteasome) and UD degradation processes
Summary
Plasmids and Transfections—The plasmids used are as follows: pRc/CMV p53 encoding wild-type human p53, pcDNA3.1 WT p53, 1–363, 1–312, 40 –393(⌬40), 97393(⌬97), all with an HA tag at the C terminus pcDNA3.1 Hdmx-HA and Mdm. The degradation experiments were performed at least three times, and the results indicated that only the ⌬N97 is resistant to degradation by the 20 S proteasome (Fig. 1H), suggesting that an unstructured domain at the N terminus of p53 is crucial for its susceptibility to. After 30 min, cells were col- proteasomal degradation may suggest that p53 degradation is lected, pelleted, and resuspended in MϪ containing [35S]Met initiated at the N terminus. To test this possibility, we examined (10 mCi/ml; Amersham Biosciences) and incubated at 37 °C for the p53 partial degradation products by the 20 S proteasome the indicated times.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
