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p73 is a p53-related transcription factor with fundamental roles in development and tumor suppression, Transcription from two different promoters on the p73 gene results in generation of transcriptionally active TAp73 isoforms and dominant negative ANp73 isoforms with opposing pro- and anti-apoptotic functions. Therefore, the relative ratio of each isoform is an important determinant of the cell fate. Proteasomal degradation of p73 is mediated by polyubiquitination-dependent and -independent processes both of which appear, thus far, to lack selectivity for the TAp73 and ANp73 isoforms. Here, we describe the characterization of another transcriptional target of TAp73; a ring finger domain ubiquitin ligase p73 Induced RING 2 protein (PIR2). Although PIR2 was initially identified a p53-induced gene (p53RFP), low abundance of PIR2 transcript in mouse embryonic fibroblasts of TAp73 KO mice compared with WT mice and comparison of PIR2 mRNA and protein levels following TAp73 or p53 overexpression substantiate TAp73 isoforms as strong inducers of PIR2. Although PIR2 expression was induced by DNA damage, its expression did not alter apoptotic response or cell cycle profile per se. However, coexpression of PIR2 with TAp73 or ANp73 resulted in an increase of the TA/ANp73 ratio, due to preferential degradation of ANp73. Finally, PIR2 was able to relieve the inhibitory effect of ANp73 on TAp73 induced apoptosis following DNA damage. These results suggest that PIR2, by being induced by TAp73 and degrading ANp73, differentially regulates TAp73/ANp73 stability, and, hence, it may offer a therapeutic approach to enhance the chemosensitivity of tumor cells. p73 | RNF144b | ubiquitylation | DNA damage doi/ 10.1073/pnas.0911828107

p73 belongs to a family of transcription factors, including p53 and p63, that mediate response to DNA damage and cellular stress by inducing DNA repair, cell cycle arrest, and apoptosis. TP73 gene contains two promotors and several splice variants resulting in up to 24 possible permutations of p73 proteins which underlies the complexity of the family and its regulatory mechanisms. p73 variants lacking the N-terminal, denoted as DeltaTAp73, are not transcriptionally competent and they act in a dominant negative fashion over TAp73. DeltaTAp73 isoforms can be generated by alternative promotor usage, giving rise to DeltaNp73, or alternative splicing of exons 2, 3 or 2, and 3 together. Such transcript isoforms potentially produce oncogenic proteins and they were shown to be present in primary tumors and tumor-derived cell lines. We investigated the possibility of additional mechanisms by which p73 protein could be regulated and discovered a putative internal ribosome entry site (IRES) in exon 2. Translation initiation of TAp73 mRNA results in a DeltaNp73-like peptide, thus demonstrating an additional mechanism whereby a DeltaTA p73 protein is produced from a transcript originally generated from the P1 promotor of the p73 gene.

The epidermis, the outer layer of the skin composed of keratinocytes, develops following the action of the transcription factor p63. The mouse Trp63 gene contains two promoters, driving the production of distinct proteins, one with an N-terminal trans-activation domain (TAp63) and one without (DeltaNp63), although their relative contribution to epidermal development is not clearly established. To identify the relative role of p63 isoforms in relation to IKKalpha, also known to be essential for epithelial development, we performed both molecular and in vivo analyses using genetic complementation in mice. We found that the action of TAp63 is mediated at the molecular level by direct and indirect transactivation of IKKalpha and Ets-1, respectively. We also found that DeltaNp63 upregulates IKKalpha indirectly, through GATA-3. Our data are consistent with a role for p63 directly upstream of IKKalpha in epithelial development.