The recently discovered p53-related genes, p73 and p63, express multiple splice variants and N-terminally truncated forms initiated from an alternative promoter in intron 3. To date, no alternative promoter and multiple splice variants have been described for the p53 gene. In this study, we show that p53 has a gene structure similar to the p73 and p63 genes. The human p53 gene contains an alternative promoter and transcribes multiple splice variants. We show that p53 variants are expressed in normal human tissue in a tissue-dependent manner. We determine that the alternative promoter is conserved through evolution from Drosophila to man, suggesting that the p53 family gene structure plays an essential role in the multiple activities of the p53 family members. Consistent with this hypothesis, p53 variants are differentially expressed in human breast tumors compared with normal breast tissue. We establish that p53β can bind differentially to promoters and can enhance p53 target gene expression in a promoter-dependent manner, while Δ133p53 is dominant-negative toward full-length p53, inhibiting p53-mediated apoptosis. The differential expression of the p53 isoforms in human tumors may explain the difficulties in linking p53 status to the biological properties and drug sensitivity of human cancer.

Mdm2 is an E3 ubiquitin ligase that promotes its own ubiquitination and also ubiquitination of the p53 tumour suppressor. In a bacterial two‐hybrid screen, using Mdm2 as bait, we identified an Mdm2‐interacting peptide that bears sequence similarity to the deubiquitinating enzyme USP2a. We have established that full‐length USP2a associates with Mdm2 in cells where it can deubiquitinate Mdm2 while demonstrating no deubiquitinating activity towards p53. Ectopic expression of USP2a causes accumulation of Mdm2 in a dose‐dependent manner and consequently promotes Mdm2‐mediated p53 degradation. This differs from the behaviour of HAUSP, which deubiquitinates p53 in addition to Mdm2 and thus protects p53 from Mdm2‐mediated degradation. We further demonstrate that suppression of endogenous USP2a destabilises Mdm2 and causes accumulation of p53 protein and activation of p53. Our data identify the deubiquitinating enzyme USP2a as a novel regulator of the p53 pathway that acts through its ability to selectively target Mdm2.

NF-κB activation is a critical component in the transcriptional response to hypoxia. However, the underlying mechanisms that control its activity under these conditions are unknown. Here we report that under hypoxic conditions, IκB kinase (IKK) activity is induced through a calcium/calmodulin-dependent kinase 2 (CaMK2)-dependent pathway distinct from that for other common inducers of NF-κB. This process still requires IKK and the IKK kinase TAK1, like that for inflammatory inducers of NF-κB, but the TAK1-associated proteins TAB1 and TAB2 are not essential. IKK complex activation following hypoxia requires Ubc13 but not the recently identified LUBAC (linear ubiquitin chain assembly complex) ubiquitin conjugation system. In contrast to the action of other NF-κB inducers, IKK-mediated phosphorylation of IκBα does not result in its degradation. We show that this results from IκBα sumoylation by Sumo-2/3 on critical lysine residues, normally required for K-48-linked polyubiquitination. Furthermore, inhibition of specific Sumo proteases is sufficient to release RelA from IκBα and activate NF-κB target genes. These results define a novel pathway regulating NF-κB activation, important to its physiological role in human health and disease.

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