The alternative translated MDMX(p60) isoform regulates MDM2 activity.

Isoforms derived from alternative splicing, mRNA translation initiation or promoter usage extend the functional repertoire of the p53, p63 and p73 genes family and of their regulators MDM2 and MDMX. Here we show cap-independent translation of an N-terminal truncated isoform of hMDMX, hMDMX(p60), which is initiated at the 7th AUG codon downstream of the initiation site for full length hMDMX(FL) at position +384. hMDMX(p60) lacks the p53 binding motif but retains the RING domain and interacts with hMDM2 and hMDMX(FL). hMDMX(p60) shows higher affinity for hMDM2, as compared to hMDMX(FL). In vitro data reveal a positive cooperative interaction between hMDMX(p60) and hMDM2 and in cellulo data show that low levels of hMDMX(p60) promote degradation of hMDM2 whereas higher levels stabilize hMDM2 and prevent hMDM2-mediated degradation of hMDMX(FL). These results describe a novel alternatively translated hMDMX isoform that exhibits unique regulatory activity toward hMDM2 autoubiquitination. The data illustrate how the N-terminus of hMDMX regulates its C-terminal RING domain and the hMDM2 activity.

HDMX folds the nascent p53 mRNA following activation by the ATM kinase.

Regulated protein synthesis via changes in mRNA structures forms an important part of how prokaryotic cells adapt protein expression in response to changes in the environment. Little is known regarding how this concept has adapted to regulate mRNA translation via signaling pathways in mammalian cells. Here, we show that following phosphorylation by the ataxia telangiectasia mutated (ATM) kinase at serine 403, the C-terminal RING domain of HDMX binds the nascent p53 mRNA to promote a conformation that supports the p53 mRNA-HDM2 interaction and the induction of p53 synthesis. HDMX and its homolog HDM2 bind the same p53 internal ribosome entry sequences (IRES) structure but with different specificity and function. The results show how HDMX and HDM2 act as nonredundant IRES trans-acting factors (ITAFs) to bring a positive synergistic effect on p53 expression during genotoxic stress by first altering the structure of the newly synthesized p53 mRNA followed by stimulation of translation.

Endoplasmic reticulum stress induces G2 cell-cycle arrest via mRNA translation of the p53 isoform p53/47.

p53 downstream pathways control G1 and G2 cell-cycle arrest, DNA repair, or apoptosis. However, it is still not clear how cells differentiate the cell-biological outcome of p53 activation in response to different types of stresses. The p53/47 isoform lacks the first 39 amino acids of full-length p53 including the Mdm2 binding site and the first trans-activation domain, and tetramers including p53/47 exhibit altered activity and biochemical properties. Here we show that endoplasmic reticulum stress promotes PERK-dependent induction of p53/47 mRNA translation and p53/47 homo-oligomerization. p53/47 induces 14-3-3sigma and G2 arrest but does not affect G1 progression. This is contrary to p53FL, which promotes G1 arrest but has no effect on the G2. These results show a unique role for p53/47 in the p53 pathway and illustrate how a cellular stress leads to a defined cell-biological outcome through expression of a p53 isoform.

The p53 mRNA-Mdm2 interaction.

The E3 ligase Mdm2 is a key regulator of p53 activity via a complex regulatory feedback system that involves all levels of expression control including transcription, mRNA translation and protein degradation. Best known is the effect of p53 on Mdm2 transcription and the capacity of Mdm2 to target p53 for degradation, but more recently the role of Mdm2 as a positive regulator of p53 activity has also started to emerge. Mdm2 stimulates p53 mRNA translation by binding the p53 mRNA and, interestingly, this interaction also suppresses Mdm2's capacity to promote p53 polyubiquitination and degradation. Another interesting aspect of the p53 mRNA-Mdm2 interaction is that the p53 mRNA sequence encoding the amino acids which bind the N-terminus of Mdm2 is the same that interacts with the Mdm2 RING domain. Indeed, the regulatory elements for controlling Mdm2-dependent expression of p53 are derived from the same p53 genomic sequence. In addition, the RNA binding and the E3 ligase domain of Mdm2 overlap, indicati that the two functions of Mdm2 to control p53 synthesis and degradation have co-evolved in parallel in both p53 and Mdm2. Here we illustrate how the p53-Mdm2 protein-protein and p53 mRNA-Mdm2 interactions affect Mdm2-mediated control of p53 expression using the Phe19Ala p53 mutant. We discuss how the new insights into the regulation of p53 expression levels can help to shed light on the origin of this elegant feedback system and on the function of Mdm2 isoforms.

Gly-Ala repeats induce position- and substrate-specific regulation of 26 S proteasome-dependent partial processing.

Partial degradation or regulated ubiquitin proteasome-dependent processing by the 26 S proteasome has been demonstrated, but the underlying molecular mechanisms and the prevalence of this phenomenon remain obscure. Here we show that the Gly-Ala repeat (GAr) sequence of EBNA1 affects processing of substrates via the ubiquitin-dependent degradation pathway in a substrate- and position-specific fashion. GAr-mediated increase in stability of proteins targeted for degradation via the 26 S proteasome was associated with a fraction of the substrates being partially processed and the release of the free GAr. The GAr did not cause a problem for the proteolytic activity of the proteasome, and its fusion to the N terminus of p53 resulted in an increase in the rate of degradation of the entire chimera. Interestingly the GAr had little effect on the stability of EBNA1 protein itself, and targeting EBNA1 for 26 S proteasome-dependent degradation led to its complete degradation. Taken together, our data suggest a model in which the GAr prevents degradation or promotes endoproteolytic processing of substrates targeted for the 26 S proteasome by interfering with the initiation step of substrate unfolding. These results will help to further understand the underlying mechanisms for partial proteasome-dependent degradation.

P53 mRNA controls p53 activity by managing Mdm2 functions.

The E3 ubiquitin ligase Mdm2 is a focal regulator of p53 tumour suppressor activity. It binds p53, promoting its polyubiquitination and degradation, and also controls p53 synthesis. However, it is not known how this dual function of Mdm2 on p53 synthesis and degradation is achieved. Here we show that the p53 mRNA region encoding the Mdm2-binding site interacts directly with the RING domain of Mdm2. This impairs the E3 ligase activity of Mdm2 and promotes p53 mRNA translation. We also show that introduction of cancer-derived single silent point-mutations in the p53 mRNA weakens its binding to Mdm2 and results in reduced p53 activity. These data are consistent with a mechanism by which changes in silent nucleotides can affect the function of the encoded protein, and indicate that Mdm2-mediated control of p53 synthesis and degradation has evolved in the p53 mRNA sequence and its encoded amino acids.