The ΔNp63α Phosphoprotein Binds the p21 and 14-3-3σ Promoters In Vivo and Has Transcriptional Repressor Activity That Is Reduced by Hay-Wells Syndrome-Derived Mutations

Recently, p63 and p73, two p53 homologues, were identified (2, 27, 39, 54, 57). These proteins exhibit a high sequence and structural homology to the p53 protein. Each gene encodes an amino-transactivating domain, a core DNA-binding domain, and a carboxy-oligomerization domain. However, there are significant differences between these homologues and p53. Both p63 and p73 genes contain two transcriptional start sites that are used to generate transcripts that encode proteins with or without an amino-transactivating domain. Proteins with the transactivating domain are termed TAp63 or TAp73, and proteins lacking the transactivation domain are termed ΔNp63 or ΔNp73. In addition, both genes can be alternatively spliced to generate proteins with different carboxy termini. For example, six splice variants can be generated from the two promoters of the p63 gene with three different C termini termed α, β, and γ (7, 57). The p63α and p73α proteins also contain an additional region not found in p53 known as a sterile alpha motif (SAM) domain. This domain is found in the α form of p63 and p73 (27, 57) and is a protein-protein interaction domain implicated in developmental processes (46, 53). In addition to the structural differences within the p53 gene family, differing functional properties were discovered. These differences became apparent after analysis of p63−/− and p73−/− mice. Whereas p53−/− mice are developmentally normal but prone to neoplastic disease (14), the p63−/− and p73−/− mice have severe developmental abnormalities. The p63−/− mice are born but die shortly after birth and are deficient in the development of limbs and several epithelial tissues such as skin, prostate, mammary gland, and urothelia (36, 58). The p73−/− mice exhibit neurological, pheromonal, and inflammatory defects (59). The p63 protein is localized to the nucleus of basal cells of stratified epithelia such as skin, oral mucosa, cervix, vaginal epithelium, urothelium, prostate, breast, and other tissues (12, 13, 57). The ΔNp63α splice variant is the predominant, if not the only, form expressed in these basal epithelial cells (13, 41, 57). Ectopic expression of the ΔN splice variants can decrease p53 target gene promoter activity, suggesting a role for ΔNp63α in maintaining the proliferative capacity of cells by repressing p53 target genes involved in growth arrest (27, 57). This hypothesis is supported by the data of Parsa et al. and Pellegrini et al. showing that a decrease in p63 was associated with a reduced proliferative potential and subsequent terminal differentiation of skin keratinocytes (41, 42). We analyzed here the role of p63 in primary human epidermal keratinocyte (HEK) differentiation. Our results indicate that ΔNp63α is the predominant form of p63 protein expressed in primary cultures of HEKs and is downregulated during differentiation. We also show that ΔNp63α is a phosphoprotein that can function as a transcriptional repressor and bind consensus p53-binding sites in the p21waf1 (p21) and 14-3-3σ promoters in vivo.