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p53 is a central hub in controlling cell proliferation. To maintain genome integrity in response to cellular stress, p53 directly regulates the transcription of genes involved in cell cycle arrest, DNA repair, apoptosis and/or senescence. An array of post-translational modifications and protein-protein interactions modulates its stability and activities in order to avoid malignant transformation. However, to date it is still not clear how cells decide their own fate in response to different types of stress. We described here that the human TRIM8 protein, a member of the TRIM family, is a new modulator of the p53-mediated tumor suppression mechanism. We showed that under stress conditions, such as UV exposure, p53 induced the expression of TRIM8, which in turn stabilized p53 leading to cell cycle arrest and reduction of cell proliferation through enhancement of CDKN1A (p21) and GADD45 expression. TRIM8 silencing reduced the capacity of p53 to activate genes involved in cell cycle arrest and DNA repair, in response to cellular stress. Concurrently, TRIM8 overexpression induced the degradation of the MDM2 protein, the principal regulator of p53 stability. Co-immunoprecipitation experiments showed that TRIM8 physically interacted with p53, impairing its interaction with MDM2. Altogether, our results reveal a previously unknown regulatory pathway controlling p53 activity and suggest TRIM8 as a novel therapeutic target to enhance p53 tumor suppressor activity.

The epidermis is a stratified epithelium which develops depending on the transcription factor p63, a member of the p53 family of transcription factors. p63 is strongly expressed in the innermost basal layer where highly proliferative epithelial cells reside. p63 functions as a molecular switch that initiates epithelial stratification or cell fate determination while regulating proliferation and differentiation of developmentally mature keratinocytes. p63 acts upstream of Dlx3 homeobox gene in a transcriptional regulatory pathway relevant to ectodermal dysplasia. Here we show that Dlx3 triggers p63 protein degradation by a proteasome-dependent pathway. Mutant DeltaNp63alpha in which Threonine397 and Serine383 were replaced with Alanine as well as C-terminal truncated versions of DeltaNp63alpha are resistant to Dlx3-mediated degradation. Transient expression of Dlx3 is associated with Raf1 phosphorylation. Dlx3 is unable to promote p63 degradation in Raf1 depleted MEF cells or upon pharmacological knockdown of Raf1. Our data support a previously unrecognized role for Dlx3 in posttranslational regulation of DeltaNp63alpha protein level, a mechanism that may contribute to reduce the abundance of DeltaNp63alpha during differentiation of stratified epithelia.

The p53 family of transcription factors plays a pivotal role in the control of the cellular response to DNA damaging agents. In addition to pro-apoptotic molecules such as p53, TAp73 and TAp63, this gene family also encodes for the anti-apoptotic molecules deltaNp73, deltaNp63, deltaNp53, and p53 mutants are often found in tumor cells, that have the role to limit and to modulate the pro-apoptotic side of the family. The ratio between the different members of the family is critical to make the life or death decision following DNA damage and is tightly regulated by post-translational and transcriptional mechanisms. In this study we have uncovered a novel positive feedback that involves the transcriptional activation of the anti-apoptotic molecule deltaNp63 by the anti-apoptotic molecules deltaNp73 and mutant p53, and that is put into motion upon treatment with a subset of DNA damaging agents such as Doxorubicin and 5-FU. DeltaNp73 and mutant p53 associate with the deltaNp63 promoter inducing its transcription and this is enhanced by doxorubicin treatment. Furthermore we have observed that deltaNp73- and mutp53-mediated activation of the deltaNp63 promoter requires the functionality of the proximal CCAAT boxes of this promoter, being hampered by mutation of CCAAT boxes or by dominant negative NFYA expression. This mechanism may serve as an additional control of the response of a normal cell to DNA damage or as an anti-apoptotic barrier of cancer cells subjected to DNA damage.

p63, a protein related to the tumor suppressor p53, is a transcription factor that plays an important role in epidermal differentiation and limb development. The gene has two distinct promoters that allow the formation of proteins that either contain (TA) or lack (DeltaN) a transactivation domain. In addition, alternative splicing at the 3' end generates proteins with different C-termini, denoted alpha, beta and gamma for a total of six isoforms. DeltaNp63alpha isoform is the main isoform expressed at all stages of development, however the relative contribution of individual p63 isoform during ectodermal differentiation and organogenesis is still far from understood. Overexpression of DeltaNp63 led to increased growth of transformed cells in vitro and in vivo while treatment of keratinocytes with ultraviolet irradiation causes downregulation of DeltaNp63 proteins and their corresponding mRNA. The p63 gene locus is often amplified in squamous cell carcinomas while alterations in the relative levels of TA and DeltaNp63 correlate with prognosis in several human cancers suggesting that fine regulation of p63 intracellular levels must be of pivotal importance in controlling cell proliferation, death and differentiation. Despite its relevance little is known on the mechanisms controlling p63 protein levels. Here we show that Itch/AIP4, a HECT E3-ubiquitin ligase, promotes p63 degradation. Using a set of p63 deletion mutants, we have identified a region and two critical lysine residues of p63, associated to human Split-Hand and Foot Malformation-4 (SHFM-4) syndrome, which are involved in the mechanism of Itch-mediated p63 degradation.

The cyclin-dependent kinase (CDK) inhibitor p57Kip2 is a negative regulator of cell proliferation, binding to a variety of cyclin-CDK complexes and inhibiting their kinase activities. The p57Kip2 gene was recognized as a target gene for p73beta, one member of the p53 family. In spite of this, the phenotypes of p73 and p57Kip2 knockout mice do not resemble each other while there is a phenotypic overlap between the p57Kip2 null mice, the p63 null mice and patients affected by p63 associated syndromes, suggesting that p57Kip2 could be indeed a downstream target of p63. By ChIP we determined that in the HaCaT cell line the DeltaNp63alpha protein is associated to three different regions of the p57Kip2 gene. DeltaNp63 can activate both the endogenous p57Kip2 gene and a reporter vector containing a -2191 promoter fragment of the p57Kip2 gene. Natural p63 mutants, associated to the AEC syndrome, show a partial or complete lack of transactivation potential of the p57Kip2 promoter, while three other natural p63 mutants, associated to the EEC, LMS and SHFM-4 syndromes, were less affected. These data suggests that p63 play an important role in the regulation of p57Kip2 expression and that this regulation is subverted in AEC p63 mutants.

Post-translational modification of proteins by the ubiquitin-like molecule SUMO-1 regulates their stability and activity with crucial implications for many cellular processes. Here we show that p63alpha, but not p63beta and gamma, is sumoylated in vitro and in vivo at a single lysine residue, K637, in the post-SAM domain. SUMO-1 attachment targets DeltaNp63alpha for proteasome mediated degradation while it does not influence p63alpha intracellular localization, as wild-type protein and a mutant carring the K637 mutated into arginine (K637R), have the same nuclear localization. Four natural p63 mutations, falling within the SAM and post-SAM domain of p63alpha, were found to be altered in their sumoylation capacity. The transcriptional activities of the natural mutants and of K637R were strongly increased compared to that of wild type p63, suggesting that sumoylation has a negative effect on p63 driven transcription. The findings that DeltaNp63alpha protein levels are regulated by SUMO-1 and that this regulation is altered in natural p63 mutants, suggest that SUMO conjugation to p63 plays a critical role in regulating the biological activity of p63.