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.     

Activation of c-myc Gene Expression by Tumor-Derived p53 Mutants Requires a Discrete C-Terminal Domain

Mutation of the p53 tumor suppressor gene is the most common genetic alteration in human cancer, and tumors that express mutant p53 may be more aggressive and have a worse prognosis than p53-null cancers. Mutant p53 enhances tumorigenicity in the absence of a transdominant negative mechanism, and this tumor-promoting activity correlates with its ability to transactivate reporter genes in transient transfection assays. However, the mechanism by which mutant p53 functions in transactivation and its endogenous cellular targets that promote tumorigenicity are unknown. Here we report that (i) mutant p53 can regulate the expression of the endogenous c-myc gene and is a potent activator of the c-myc promoter; (ii) the region of mutant p53 responsiveness in the c-myc gene has been mapped to the 3′ end of exon 1; (iii) the mutant p53 response region is position and orientation dependent and therefore does not function as an enhancer; and (iv) transactivation by mutant p53 requires the C terminus, which is not essential for wild-type p53 transactivation. These data suggest that it may be possible to selectively inhibit mutant p53 gain of function and consequently reduce the tumorigenic potential of cancer cells. A possible mechanism for transactivation of the c-myc gene by mutant p53 is proposed.     

Abrogation of the transactivation activity of p53 by BCCIP down-regulation

The tumor suppression function of p53 is mostly conferred by its transactivation activity, which is inactivated by p53 mutations in approximately 50% of human cancers. In cancers harboring wild type p53, the p53 transactivation activity may be compromised by other mechanisms. Identifying the mechanisms by which wild type p53 transactivation activity can be abrogated may provide insights into the molecular etiology of cancers harboring wild type p53. In this report, we show that BCCIP, a BRCA2 and CDKN1A-interacting protein, is required for the transactivation activity of wild type p53. In p53 wild type cells, BCCIP knock down by RNA interference diminishes the transactivation activity of p53 without reducing the p53 protein level, inhibits the binding of p53 to the promoters of p53 target genes p21 and HDM2, and reduces the tetrameric formation of p53. These data demonstrate a critical role of BCCIP in maintaining the transactivation activity of wild type p53 and further suggest down-regulation of BCCIP as a novel mechanism to impair the p53 function in cells harboring wild type p53.     

Mdm2 inhibits the apoptotic function of p53 mainly by targeting it for degradation

The ability of Mdm2 to inhibit the activities of a C-terminal truncated p53 mutant, p53-Delta30, which can bind Mdm2 but is resistant to Mdm2-mediated protein degradation was investigated. The inhibitory function of an Mdm2 mutant, Mdm2-Delta(222-437), which can bind p53 but is defective in targeting p53 for degradation was also studied. We have demonstrated that targeting p53 for degradation is the most effective way for Mdm2 to inhibit the apoptotic function of p53. However, we have also shown that Mdm2 can inhibit the transactivation function of p53 without targeting it for degradation, although Mdm2 releases the transrepression ability of p53 mainly by targeting it for degradation. The ability of Mdm2 to inhibit the apoptotic function of p53 was linked to its ability to inhibit the transrepression but not the transactivation function of p53. Furthermore, we have demonstrated that the transrepression function of p53 was specific to p53-induced apoptosis and was not simply a result of cell death.