Regulation of p53 and MDM2 Activity by MTBP

Abstract

p53 is a critical coordinator of a wide range of stress responses. To facilitate a rapid response to stress, p53 is produced constitutively but is negatively regulated by MDM2. MDM2 can inhibit p53 in multiple independent ways: by binding to its transcription activation domain, inhibiting p53 acetylation, promoting nuclear export, and probably most importantly by promoting proteasomal degradation of p53. The latter is achieved via MDM2's E3 ubiquitin ligase activity harbored within the MDM2 RING finger domain. We have discovered that MTBP promotes MDM2-mediated ubiquitination and degradation of p53 and also MDM2 stabilization in an MDM2 RING finger-dependent manner. Moreover, using small interfering RNA to down-regulate endogenous MTBP in unstressed cells, we have found that MTBP significantly contributes to MDM2-mediated regulation of p53 levels and activity. However, following exposure of cells to UV, but not ␥-irradiation, MTBP is destabilized as part of the coordinated cellular response. Our findings suggest that MTBP differentially regulates the E3 ubiquitin ligase activity of MDM2 towards two of its most critical targets (itself and p53) and in doing so significantly contributes to MDM2-dependent p53 homeostasis in unstressed cells.p53 is a critical coordinator of a wide range of cellular stresses ranging from myocyte stretch-induced apoptosis to increased global DNA repair in fibroblasts exposed to UV (30, 44). To facilitate a rapid response to stress, cells have evolved a mechanism that relies upon stabilization and activation, by posttranslational modification, of existing constitutively expressed p53 protein. In normal cells it has been found that p53 is both functionally inhibited and, moreover, maintained in an unstable state by the action of MDM2 (35).Originally discovered as one of three genes amplified on double minute chromosomes in a tumorigenic derivative of NIH 3T3 cells (5), MDM2 was later shown to possess oncogenic potential when overexpressed (10, 12). High-level expression of MDM2 has also been shown to confer tumorigenic potential upon nontransformed rodent fibroblasts in athymic nude mice (10, 12). MDM2 can immortalize rat embryo fibroblasts and can cooperate with activated RAS to transform these cells (12). Elevated levels of MDM2 protein have been found in a variety of human tumors, most notably in soft tissue sarcomas where up to 30% of primary tumors contain multiple copies of the MDM2 gene (27). One mechanism by which MDM2 overexpression promotes tumor development is through its ability to bind to the p53 tumor suppressor (36, 38), thereby blocking the transactivation (36, 39), cell cycle arrest (6), and apoptotic functions of p53 (17). MDM2 can inhibit p53 activity in a number of ways including preventing p53 from recruiting TAFs (45), promoting nuclear export (13), inhibiting p53 acetylation (20), and perhaps most importantly by virtue of its function as an E3 ubiquitin ligase with specificity for, among others, p53 (19). In addition to regulating p53 levels by targeting p53 for p...

Mtbp haploinsufficiency in mice increases tumor metastasis

“…Because the Mtbp-null lethality was not rescued by loss of p53, and because Mtbp heterozygosity did not show any obvious effects on p53 activity in tumor development, we hypothesized that p53 activity was not increased in Mtbp þ /À heterozygous cells, in contrast to previously published in vitro data (Brady et al, 2005). To test this hypothesis, we compared p53 and Mdm2 protein levels in thymocytes between wild-type and Mtbp þ /À mice (Figure 2e).…”

“…One study suggests that overexpression of MTBP causes G 1 arrest of cell cycle independent of p53 (Boyd et al, 2000). The other reported that MTBP decreases p53 activity via the stabilization of Mdm2 by protecting it from self-ubiquitination (Brady et al, 2005). Thus, MTBP is downstream of Mdm2 in a p53-independent manner, but is also an upstream regulator of Mdm2, leading to decreased p53 activity.…”

“…The MTBP-Mdm2 interaction was observed using purified proteins in vitro and by overexpression in cells. Overexpression of MTBP protects Mdm2 from self-ubiquitination, leading to Mdm2 stabilization and p53 degradation (Brady et al, 2005). We therefore hypothesized that MTBP affects tumor development through modulation of p53 activity.…”

MTBP plays a crucial role in mitotic progression and chromosome segregation

“…1 Overexpression of MTBP was shown to suppress cell proliferation and colony formation of several human cancer cell lines independent of their p53 status. 1 Brady et al 2 reported that overexpression of MTBP in MCF7 cells resulted in MDM2 stabilization and subsequent p53 degradation. However, our previous findings demonstrated that complete deletion of MTBP in mice resulted in an early embryonic lethal phenotype independent of p53.…”

PLAGL2 controls the stability of Pirh2, an E3 ubiquitin ligase for p53

“…There are various regulatory mechanisms to modulate Mdm2 functions. Mdm2 enzymatic activity is inhibited by its association with p19 ARF [13], ribosomal protein L11 [14] or TSG101 [15], while enhanced by interacting with MTBP [16] or YY1 [17]. Post-translational modification, such as phosphorylation by Ataxia Telangiectasia-mutated (ATM) in response to DNA damage, also regulates Mdm2-p53 interaction [18].…”