SummaryTight control of p63 protein levels must be achieved under differentiation or apoptotic conditions. Here, we describe a new regulatory pathway for the Np63 protein. We found that MDM2 binds Np63 in the nucleus promoting its translocation to the cytoplasm. The MDM2 nuclear localization signal is required for Np63 nuclear export and subsequent degradation, whereas the MDM2 ringfinger domain is dispensable. Once exported to the cytoplasm by MDM2, p63 is targeted for degradation by the Fbw7 E3-ubiquitin ligase. Efficient degradation of Np63 by Fbw7 (also known as FBXW7) requires GSK3 kinase activity. By deletion and point mutations analysis we have identified a phosphodegron located in the  and  tail of p63 that is required for degradation. Furthermore, we show that MDM2 or Fbw7 depletion inhibits degradation of endogenous Np63 in cells exposed to UV irradiation, adriamycin and upon keratinocyte differentiation. Our findings suggest that following DNA damage and cellular differentiation MDM2 and Fbw7 can cooperate to regulate the levels of the pro-proliferative Np63 protein.

ABSTRACTp63, 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 (∆N) a transactivation domain. In addition, alternative splicing at the 3' end generates proteins with different C-termini, denoted α, β and γ for a total of six isoforms. ∆Np63α 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 ∆Np63 led to increased growth of transformed cells in vitro and in vivo while treatment of keratinocytes with ultraviolet irradiation causes downregulation of ∆Np63 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 ∆Np63 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.

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Heterogeneity in the use of G-CSF and, in particular, biosimilar filgrastim across different Italian centres was observed, probably due to different regional healthcare policy interventions. During the first year of treatment, switching between different G-CSFs was frequent. Considering the impact of biological drugs on pharmaceutical expenses, it is necessary to harmonize healthcare policies promoting the use of biological drugs with the lowest cost.