Targeted Inactivation of Mdm2 RING Finger E3 Ubiquitin Ligase Activity in the Mouse Reveals Mechanistic Insights into p53 Regulation

Itahana, Koji; Mao, Hua; Jin, Aiwen; Itahana, Yoko; Clegg, Hilary V.; Lindström, Mikael S.; Bhat, Krishna; Godfrey, Virginia; Evan, Gérard I.; Zhang, Yanping

doi:10.1016/j.ccr.2007.09.007

Cited by 224 publications

(239 citation statements)

References 35 publications

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“…However, this occurs without an apparent increase in the specific activity of p53 Toledo et al, 2006). This is consistent with a predominant role of Mdm2 in normal cells of promoting p53-degradation rather than transcriptional repression by direct binding to p53 (Itahana et al, 2007). MdmX does not possess intrinsic ubiquitin E3 ligase activity towards p53 in vivo, but it binds to p53 and inhibits its transcriptional activity (Shvarts et al, 1996;Bottger et al, 1999).…”

Section: Introductionsupporting

confidence: 71%

MdmX is a substrate for the deubiquitinating enzyme USP2a

et al. 2009

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It has previously been shown that ubiquitin-specific protease 2a (USP2a) is a regulator of the Mdm2/p53 pathway. USP2a binds to Mdm2 and can deubiquitinate Mdm2 without reversing Mdm2-mediated p53 ubiquitination. Overexpression of USP2a causes accumulation of Mdm2 and promotes p53 degradation. We now show that MdmX is also a substrate for USP2a. MdmX associates with USP2a independently of Mdm2. Ectopic expression of wild-type USP2a but not a catalytic mutant prevents Mdm2-mediated degradation of MdmX. This correlates with the ability of wild-type USP2a to deubiquitinate MdmX. siRNA-mediated knockdown of USP2a in NTERA-2 testicular embryonal carcinoma cells and MCF7 breast cancer cells causes destabilization of MdmX and results in a decrease in MdmX protein levels, showing that endogenous USP2a participates in the regulation of MdmX stability. The therapeutic drug, cisplatin decreases MdmX protein expression. USP2a mRNA and protein levels were also reduced after cisplatin exposure. The magnitude and time course of USP2a downregulation suggests that the reduction in USP2a levels could contribute to the decrease in MdmX expression following treatment with cisplatin. Knockdown of USP2a increases the sensitivity of NTERA-2 cells to cisplatin, raising the possibility that suppression of USP2a in combination with cisplatin may be an approach for cancer therapy.

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Section: Introductionsupporting

confidence: 71%

MdmX is a substrate for the deubiquitinating enzyme USP2a

et al. 2009

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“…24 It should be noted however that in embryonic fibroblasts derived from knock-in mice carrying a RING-inactivating mutation in Mdm2, no difference was observed in proteasome-dependent degradation rates of the mutant protein compared with that of the WT ligase. 26 In the same cells, however, overexpressed HDM2 did undergo proteasomal degradation, which was dependent on its own RING activity, suggesting that the mechanisms of Mdm2 degradation in cells are dependent on its level. It appears therefore that under physiological levels, Mdm2 is targeted for degradation by an external ligase.…”

Section: Degradation Of Ligases Via Self-catalyzed Ubiquitinationmentioning

confidence: 99%

Ubiquitination of E3 ligases: self-regulation of the ubiquitin system via proteolytic and non-proteolytic mechanisms

2011

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Ubiquitin modification of many cellular proteins targets them for proteasomal degradation, but in addition can also serve non-proteolytic functions. Over the last years, a significant progress has been made in our understanding of how modification of the substrates of the ubiquitin system is regulated. However, little is known on how the ubiquitin system that is comprised of B1500 components is regulated. Here, we discuss how the biggest subfamily within the system, that of the E3 ubiquitin ligases that endow the system with its high specificity towards the numerous substrates, is regulated and in particular via self-regulation mediated by ubiquitin modification. Ligases can be targeted for degradation in a self-catalyzed manner, or through modification mediated by an external ligase(s). In addition, non-proteolytic functions of self-ubiquitination, for example activation of the ligase, of E3s are discussed. Cell Death and Differentiation (2011) 18, 1393-1402 doi:10.1038/cdd.2011; published online 4 March 2011One of the major roles of the covalent modification of cellular proteins by ubiquitin is signaling them for proteasomal degradation ( Figure 1). The first step of the modification is catalyzed by the ubiquitin-activating enzyme, E1, which generates a high-energy thiol ester intermediate that is subsequently transferred to the second enzyme, a ubiquitinconjugating enzyme, E2. The third step ascertains substrate specificity, and is catalyzed by one of the numerous (B650) ubiquitin ligases, E3s. Typically, it results in the formation of an isopeptide bond between the C-terminal Gly of ubiquitin and an e-NH 2 group of an internal Lys of the substrate. Less frequently, it can generate a linear peptide bond with the a-NH 2 group, a thiol ester bond with an internal Cys, or an ester bond with a Thr or Ser. The three-step cascade of reactions is repeated, where additional ubiquitin moieties are attached sequentially to one another in an isopeptide bond involving one of the seven internal Lys residues in the ubiquitin moiety, thus generating a polyubiquitin chain. Lys48-based chains serve as a signal for proteasomal degradation, whereas chains based on other internal Lys residues, or modification by single moiety(ies) can serve non-proteolytic functions.Ligases fall into two main families: RING (really interesting new gene) and HECT (homologous to the E6-AP carboxy terminus) domain-containing E3s. RING ligases serve as scaffolds that facilitate direct transfer of ubiquitin from the E2 to the target protein. HECT E3s contain an active Cys residue to which ubiquitin binds prior to its transfer to the substrate (Figure 1). There are B600 RING finger and B30 HECT ligases in humans. Smaller families of ligases (U-box, plant homology domain, and zinc finger) have also been described.An important problem relates to regulation of the ubiquitin system components, and in particular to that of the ligases that are the specific substrate-recognizing elements. 1,2 Phosphorylation of an E3 can activate the protein, such as the ca...

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“…More recently, homozygous mice bearing a single-residue substitution (C462A) abolishing the E3 function without affecting p53-binding have been generated. These mice die before E7.5, and deletion of p53 rescued this lethality 40 suggesting that the Mdm2-p53 physical interaction, without Mdm2-mediated p53 ubiquitylation, cannot control p53 activity sufficiently to allow early mouse embryonic development. Together, these genetic data Figure 1 The p53-Mdm2 negative feedback loop…”

Section: Genetic Evidence For a Key Role Of Mdm2 In Regulating P53 Lementioning

confidence: 99%

Mdm2-mediated ubiquitylation: p53 and beyond

2009

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The really interesting genes (RING)-finger-containing oncoprotein, Mdm2, is a promising drug target for cancer therapy. A key Mdm2 function is to promote ubiquitylation and proteasomal-dependent degradation of the tumor suppressor protein p53. Recent reports provide novel important insights into Mdm2-mediated regulation of p53 and how the physical and functional interactions between these two proteins are regulated. Moreover, a p53-independent role of Mdm2 has recently been confirmed by genetic data. These advances and their potential implications for the development of new cancer therapeutic strategies form the focus of this review.

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Targeted Inactivation of Mdm2 RING Finger E3 Ubiquitin Ligase Activity in the Mouse Reveals Mechanistic Insights into p53 Regulation

Cited by 224 publications

References 35 publications

MdmX is a substrate for the deubiquitinating enzyme USP2a

MdmX is a substrate for the deubiquitinating enzyme USP2a

Ubiquitination of E3 ligases: self-regulation of the ubiquitin system via proteolytic and non-proteolytic mechanisms

Mdm2-mediated ubiquitylation: p53 and beyond

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