MDM2 promotes p21waf1/cip1 proteasomal turnover independently of ubiquitylation

Abstract

The CDK inhibitor p21waf1/cip1 is degraded by a ubiquitin-independent proteolytic pathway. Here, we show that MDM2 mediates this degradation process. Overexpression of wild-type or ring finger-deleted, but not nuclear localization signal (NLS)-deleted, MDM2 decreased p21waf1/cip1 levels without ubiquitylating this protein and affecting its mRNA level in p53(-/-) cells. This decrease was reversed by the proteasome inhibitors MG132 and lactacystin, by p19(arf), and by small interfering RNA (siRNA) against MDM2. p21waf1/cip1 bound to MDM2 in vitro and in cells. The p21waf1/cip1-binding-defective mutant of MDM2 was unable to degrade p21waf1/cip1. MDM2 shortened the half-life of both exogenous and endogenous p21waf1/cip1 by 50% and led to the degradation of its lysine-free mutant. Consequently, MDM2 suppressed p21waf1/cip1-induced cell growth arrest of human p53(-/-) and p53(-/-)/Rb(-/-)cells. These results demonstrate that MDM2 directly inhibits p21waf1/cip1 function by reducing p21waf1/cip1 stability in a ubiquitin-independent fashion.

hAda3 regulates p14ARF-induced p53 acetylation and senescence

“…p21cip1 is regulated by complex p53-dependent and p53-independent mechanisms. Interestingly, MDM2 targets p21cip1 for degradation and this reaction is inhibited by p19ARF (Jin et al, 2003;Zhang et al, 2004). Since both p53 and MDM2 are acetylated by p300, and acetylation inhibits MDM2 ubiquitin ligase function , the hAda3 may affect p21cip1 levels by activating p53-dependent p21cip1 transcription and inhibiting MDM2-dependent p21cip1 protein degradation.…”

Antizyme1 mediates AURKAIP1-dependent degradation of Aurora-A

“…Studies carried out using the A-box mutant of Aurora-A and temperature-sensitive cell line defective in ubiquitination at the restrictive temperature, suggest that Az1-mediated degradation of Aurora-A is mechanistically different from the cdh1-dependent degradation of Aurora-A and is Ub-independent. Despite the dismal knowledge we have on the significance and the physiological relevance of this alternative pathway, the growing list of protein substrates (Jin et al, 2003;Asher et al, 2005;Sdek et al, 2005) targeted to the proteasome in the absence of ubiquitination suggests that the Ub-independent route to the proteasome is as important as the Ub-dependent pathway. Az-mediated degradation of ODC, the first demonstrated prototype example of Ub-independent degradation, is essential for the maintenance of Thirty-six hours post-transfection, cells were harvested for western blot analysis of the endogenous Aurora-A.…”

Ceramide-induced G2 arrest in rhabdomyosarcoma (RMS) cells requires p21Cip1/Waf1 induction and is prevented by MDM2 overexpression

“…23 Additionally, MDM2 can function in a p53-independent manner, interacting directly with a number of other proteins including p21 Cip1/Waf115 , p19/14 Arf24 , E2F1 25 and p73. 26 Further, MDM2 overexpression suppresses p21 Cip1/Waf1 -induced growth arrest of human p53 À/À and p53 À/À /Rb À/À cells 12 by promoting its degradation by the proteasome independent of ubiquitination and also p53/Rb status. 12,15 RMS is the most common soft tissue sarcoma in pediatrics arising from cells committed to skeletal muscle lineage.…”

“…Proteasomal activity is essential in maintaining low-level basal p21 Cip1/Waf1 protein expression by regulating its degradation. [12][13][14][15] The murine double minute 2 (MDM2) oncogene is amplified and overexpressed in numerous human cancers [16][17][18][19] including Rhabdomyosarcoma (RMS) tumors and cell lines. 20,21 MDM2 negatively regulates p53 transcriptional activity 18,22 and promotes p53 degradation by the proteasome.…”