DNA-binding and transactivation activities are essential for TAp63 protein degradation

The p53-related p63 gene encodes six isoforms with differing N and C termini. TAp63 isoforms possess a transactivation domain at the N terminus and are able to transactivate a set of genes, including some targets downstream of p53. Accumulating evidence indicates that TAp63 plays an important role in regulation of cell proliferation, differentiation, and apoptosis, whereas transactivation-inert deltaNp63 functions to inhibit p63 and other p53 family members. Mutations in the p63 gene that abolish p63 DNA-binding and transactivation activities cause human diseases, including ectrodactyly ectodermal dysplasia and facial clefting (EEC) syndrome. In this study, we show that mutant p63 proteins with a single amino acid substitution found in EEC syndrome are DNA binding deficient, transactivation inert, and highly stable. We demonstrate that TAp63 protein expression is tightly controlled by its specific DNA-binding and transactivation activities and that p63 is degraded in a proteasome-dependent, MDM2-independent pathway. In addition, the N-terminal transactivation domain of p63 is indispensable for its protein degradation. Furthermore, the wild-type TAp63gamma can act in trans to promote degradation of mutant TAp63gamma defective in DNA binding, and the TA domain deletion mutant of TAp63gamma inhibits transactivation activity and stabilizes the wild-type TAp63 protein. Taken together, these data suggest a feedback loop for p63 regulation, analogous to the p53-MDM2 feedback loop.     

Ionizing radiation-induced TAp63α phosphorylation at C-terminal S/TQ motifs requires the N-terminal transactivation (TA) domain

TAp63α, a homolog of p53 and one of six alternatively spliced p63 isoforms, is a critical mediator of the ionizing radiation (IR)-induced DNA damage response in female germ cells and also tumor suppression in somatic cells. The ΔNp63α isoform, lacking the N-terminal transactivation (TA) domain, is associated with oncogenic potential. The mechanism of p63 functional regulation is not well understood. TAp63α is phosphorylated by ionizing radiation (IR)-induced DNA damage and gene transactivation is likely to be involved. Based on information gleaned from studies on p53, we explored the possibility that TAp63α S/TQ sites may be phosphorylated by IR-induced DNA damage. Our findings show a wortmanin-sensitive kinase phosphorylates TAp63α at C-terminal Ser-Gln and Thr-Gln (S/TQ) sites but not N-terminal S/TQ sites. ΔNp63α, lacking the TA domain, and TAp63γ, lacking C-terminal domains, including S/TQ sites, fail to undergo IR-induced phosphorylation. We propose a model for TA domain-dependent C-terminal phosphorylation drawing from previously described self-inactivating intramolecular interaction between N-terminal TA domain and C-terminal Transactivation Inhibitory Domain (TID) of TAp63α. A specific topology adopted only by TAp63α, but not possible for ΔNp63α or TAp63γ, may lead to TAp63α-specific kinase recruitment, phosphorylation and self-inactivation release. TID-lacking TAp63γ, like p53, is constitutively active and thus may forgo phosphorylation-dependent activation. Thus, p53 is regulated by protein stabilization and TAp63α by protein activation but both appear to involve S/TQ phosphorylation. The difference in phosphorylation potential of TAp63α and ΔNp63α may in part help explain why the two similar isoforms have diametrically opposite tumor suppression and oncogene functions, respectively.     

p63 is necessary for the activation of human papillomavirus late viral functions upon epithelial differentiation

The late phase of the human papillomavirus (HPV) life cycle is linked to epithelial differentiation, and we investigated the factors that regulate this process. One potential regulator is p63, a member of the p53 family of proteins, which modulates epithelial development, as well as proliferation capability, in stem cells. In this study, we examined the role of p63 in the HPV life cycle using a lentiviral knockdown system for p63. In epithelial cells, the ΔN truncated isoforms of p63 predominate, while the full-length TA isoforms are present at very low levels. Upon the differentiation of normal keratinocytes, p63 levels rapidly decreased while higher levels were retained in HPV-positive cells. Our studies indicate that reducing p63 levels in differentiated HPV-positive cells resulted in the loss of viral genome amplification and late gene expression. p63 regulates the expression of cell cycle regulators, and we determined that cyclin A, cyclin B1, cdk1, and cdc25c were reduced in p63-deficient, HPV-positive keratinocytes, which suggests a possible mechanism of action. In addition, activation of the DNA repair pathway is necessary for genome amplification, and the expression of two members, BRCA2 and RAD51, was altered in the absence of p63 in HPV-positive cells. Our studies indicate that p63 is necessary for the activation of differentiation-dependent HPV late viral functions and provide insights into relevant cellular targets.     

p73 in apoptosis

The TP53 tumour-suppressor gene belongs to a family that includes the two recently identified homologues TP63 and TP73. Overexpression of p73 can activate typical p53-responsive genes and induce apoptosis like p53. In addition, activation of p73 has been implicated in apoptotic cell death induced by aberrant cell proliferation and some forms of DNA-damage. These data together with the localization of TP73 on chromosome 1p36, a region frequently deleted in a variety of human cancers, led to the hypothesis that p73 has tumour suppressor activity just like p53. However, despite its proapoptotic activity in vitro, the lack of tumour-formation in p73 knock-out mice and primary human tumour data demonstrating overexpression of wild-type p73 currently argue against p73 being a classical tumour suppressor. Interestingly, in contrast to TP53, TP73 gives rise to a complex pattern of pro- and antiapoptotic p73 isoforms generated by differential splicing and alternative promoter usage. Therefore further insight into the function and regulation of these structurally and functionally diverse p73 proteins is needed to elucidate the role of TP73 for apoptosis and human tumorigenesis.