ASPP1 and ASPP2: common activators of p53 family members

We recently showed that ASPP1 and ASPP2 stimulate the apoptotic function of p53. We show here that ASPP1 and ASPP2 also induce apoptosis independently of p53. By binding to p63 and p73 in vitro and in vivo, ASPP1 and ASPP2 stimulate the transactivation function of p63 and p73 on the promoters of Bax, PIG3, and PUMA but not mdm2 or p21(WAF-1/CIP1). The expression of ASPP1 and ASPP2 also enhances the apoptotic function of p63 and p73 by selectively inducing the expression of endogenous p53 target genes, such as PIG3 and PUMA, but not mdm2 or p21(WAF-1/CIP1). Removal of endogenous p63 or p73 with RNA interference demonstrated that (16) the p53-independent apoptotic function of ASPP1 and ASPP2 is mediated mainly by p63 and p73. Hence, ASPP1 and ASPP2 are the first two identified common activators of all p53 family members. All these results suggest that ASPP1 and ASPP2 could suppress tumor growth even in tumors expressing mutant p53.     

MDM2 Promotes Proteasomal Degradation of p21Waf1 via a Conformation Change

MDM2 plays a major role in cancer development and progression via both p53-dependent and -independent functions. One of its p53-independent functions is the induction of the ubiquitin-independent proteasomal degradation of p21^Waf1. The present study was designed to characterize the mechanism(s) by which MDM2 induces p21^Waf1 degradation. We first determined the regions of MDM2 required for p21^Waf1 degradation using pulldown assays and Western blotting and then examined the mechanisms using limited proteolysis and fluorescence resonance energy transfer assays. We found that the MDM2-p21^Waf1 interaction depended on the central domain of MDM2 and that nuclear localization of both proteins was necessary for p21^Waf1 degradation. Specifically, amino acids 226–250 of MDM2 were required for p21^Waf1 binding and degradation, and amino acids 251–260 were necessary for p21^Waf1 degradation. The latter region induced a conformation change in p21^Waf1, increasing its interaction with the C8 subunit of the proteasome, leading to its degradation. When MDM2 lacked either segment (aa 226–250 or aa 251–260), its capacity to promote p21^Waf1 degradation and cell cycle progression was significantly reduced. In summary, the present study elucidated a previously unknown mechanism by which MDM2 promotes the degradation of an intact protein (p21^Waf1) through an ubiquitin-independent proteasomal degradation pathway. Because MDM2 also increases the degradation of other proteins in a ubiquitin-independent manner, this mechanism may underlie part of its tumorigenic properties.     

Searching for target sequences by p53 protein is influenced by DNA length

One of the most important functions of the tumor suppressor p53 protein is its sequence-specific binding to DNA. Using a competition assay on agarose gels we found that the p53 consensus sequences in longer DNA fragments are better targets than the same sequences in shorter DNAs. Semi-quantitative evaluation of the competition experiments showed a correlation between the relative p53-DNA binding and the DNA lengths. Our results are consistent with a model of the p53-DNA interactions involving one-dimensional migration of the p53 protein along the DNA for distances of about 1000 bp while searching for its target sites. Positioning of the p53 target in the DNA fragment did not substantially affect the apparent p53-DNA binding, suggesting that p53 can slide along the DNA in a bi-directional manner. In contrast to full-length p53, the isolated core domain did not show any significant correlation between sequence-specific DNA binding and fragment length.     

The conformationally flexible S9-S10 linker region in the core domain of p53 contains a novel MDM2 binding site whose mutation increases ubiquitination of p53 in vivo

Although the N-terminal BOX-I domain of the tumor suppressor protein p53 contains the primary docking site for MDM2, previous studies demonstrated that RNA stabilizes the MDM2.p53 complex using a p53 mutant lacking the BOX-I motif. In vitro assays measuring the specific activity of MDM2 in the ligand-free and RNA-bound state identified a novel MDM2 interaction site in the core domain of p53. As defined using phage-peptide display, the RNA.MDM2 isoform exhibited a notable switch in peptide binding specificity, with enhanced affinity for novel peptide sequences in either p53 or small nuclear ribonucleoprotein-U (snRNP-U) and substantially reduced affinity for the primary p53 binding site in the BOX-I domain. The consensus binding site for the RNA.MDM2 complex within p53 is SGXLLGESXF, which links the S9-S10 beta-sheets flanking the BOX-IV and BOX-V motifs in the core domain and which is a site of reversible conformational flexibility in p53. Mutation of conserved amino acids in the linker at Ser(261) and Leu(264), which bridges the S9-S10 beta-sheets, stimulated p53 activity from reporter templates and increased MDM2-dependent ubiquitination of p53. Furthermore, mutation of the conserved Phe(270) within the S10 beta-sheet resulted in a mutant p53, which binds more stably to RNA.MDM2 complexes in vitro and which is strikingly hyper-ubiquitinated in vivo. Introducing an Ala(19) mutation into the p53(F270A) protein abolished both RNA.MDM2 complex binding and hyper-ubiquitination in vivo, thus indicating that p53(F270A) protein hyper-ubiquitination depends upon MDM2 binding to its primary site in the BOX-I domain. Together, these data identify a novel MDM2 binding interface within the S9-S10 beta-sheet region of p53 that plays a regulatory role in modulating the rate of MDM2-dependent ubiquitination of p53 in cells.     

Mechanisms of differential activation of target gene promoters by p53 hinge domain mutants with impaired apoptotic function

Suppression of tumor cell growth by p53 results from the activation of both apoptosis and cell cycle arrest functions that have been shown to be separable activities of p53. We report here that some mutants in the p53 hinge domain, a short linker between the DNA binding and tetramerization domains, differentially activated the promoters of p53 target genes and possessed an impaired apoptotic function. Our results indicate that the hinge domain may play an important role in differentially regulating p53 cell cycle arrest and apoptotic functions. However, the mechanisms by which p53 hinge domain mutants differentially activate its target genes, e.g. p21(WAF1/CIP1) and Bax, remain unknown. To investigate the possible mechanisms, recombinant p21(WAF1/CIP1) and Bax promoters were constructed, resulting in rearrangement of the existing p53 binding sites within a given promoter or actually swapping p53 binding sites between the two promoters. Our results suggest that multiple mechanisms of differential transactivation occur, depending on the molecular nature of the relevant hinge domain mutant, such as the possibility that dual separate DNA binding sites in the p21(WAF1/CIP1) promoter are responsible for the selective transactivation activity of p53 hinge domain mutant del300-327, which has a large deletion in the hinge domain. Lack of ideal p53 binding sites in the Bax promoter results in less potent activation than that seen with the p21(WAF1/CIP1) promoter when it is transactivated by hinge domain point mutant mutR306P or short deletion mutant del300-308 proteins. How the single mutation or the short deletion affect the conformation of p53 and consequently the transactivation of the Bax promoter will require further investigation of the relevant p53 protein: DNA-binding domain by NMR and x-ray crystallographic techniques.     

MDM2 is a negative regulator of p21WAF1/CIP1, independent of p53

The MDM2 oncogene has both p53-dependent and p53-independent activities. We have previously reported that antisense MDM2 inhibitors have significant anti-tumor activity in multiple human cancer models with various p53 statuses (Zhang, Z., Li, M., Wang, H., Agrawal, S., and Zhang, R. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 11636-11641). We have also provided evidence that MDM2 has a direct role in the regulation of p21, a cyclin-dependent kinase inhibitor. Here we provide evidence supporting functional interaction between MDM2 and p21 in vitro and in vivo. The inhibition of MDM2 with anti-MDM2 antisense oligonucleotide or Short Interference RNA targeting MDM2 significantly elevated p21 protein levels in PC3 cells (p53 null). In contrast, overexpression of MDM2 diminished the p21 level in the same cells by shortening the p21 half-life, an effect reversed by MDM2 antisense inhibition. MDM2 facilitates p21 degradation independent of ubiquitination and the E3 ligase function of MDM2. Instead, MDM2 promotes p21 degradation by facilitating binding of p21 with the proteasomal C8 subunit. The physical interaction between p21 and MDM2 was demonstrated both in vitro and in vivo with the binding region in amino acids 180-298 of the MDM2 protein. In summary, we provide evidence supporting a physical interaction between MDM2 and p21. We also demonstrate that, by reducing p21 protein stability via proteasome-mediated degradation, MDM2 functions as a negative regulator of p21, an effect independent of both p53 and ubiquitination.