TMS web ubiquitination. NMR and fluorescence anisotropy measurements have also suggested that a second MDM2 binding site is located within p53 core domain. On the other hand, Bech-Otschir et al. observed that phoshorylation of T155, a threonine residue also situated in the core domain, by the COP9 signalosome appeared to be important for p53 degradation by the Ub-26S proteasome system and that replacement of T155 by valine stabilized p53 in HeLa cells. Remarkably, when the above-mentioned mutations are plotted on “9353416 the structural model of p53, it can be observed that they are all focused on the same region of the p53 core domain. A fragment of the region defined by Gu et al. in 2001, spanning approximately residues 99107, forms an extended N-terminal tail structure that runs on the surface of the core domain. This “9226994 NOctober 2011 | Volume 6 | Issue 10 | e25981 p53 Degradation Mediated by HPV E6 terminal tail is anchored to the rest of the core structure via aromatic residues Y103 and Y107, which interact with residues L264 and L265 that belong to the loop connecting b-strands S9 and S10. Moreover, the amino acid T155 highlighted by Bech-Otschir et al. in 2001 is also located in the vicinity of these residues. In the present work, we generated p53 constructs bearing various mutations on key residues situated within the above mentioned region of p53 core domain. E6 and MDM2 dependent degradation and transactivation activity of the mutated p53 proteins were analysed both in vitro within cell extracts and in vivo within a p53-null cell line. In addition, the incidence of the mutations on the structural integrity of p53 was studied by circular dichroism and NMR 1H-15N correlation spectroscopies. While mutants T155A, T155V and L264A were still degraded by E6, the mutants Y103G, Y107G, T155D and L265A became resistant to E6 mediated degradation. By contrast, all mutants were still degraded by MDM2. Among the E6-resistant mutants, Y107G, T155D and L265A appeared to have completely lost the native p53 fold, whereas Y103G displayed a folded yet significantly altered conformation. vitro and in vivo. We then generated single point mutants in which Y103 and Y107 were substituted by glycine. Both p53-Y103G and p53-Y107G were resistant to HPV16 E6mediated degradation in vivo. Mutation T155D but not T155A or T155V, protects p53 from degradation by HPV16 E6 The phoshorylation of T155 can modulate p53 stability via the COP9 signalosome, which participates in p53 ubiquitination. The phoshorylation state of T155 was also described as important in high-risk E6 induced degradation of p53. We generated mutants p53-T155V, p53-T155A and p53-T155D. The replacement of T155 by alanine or valine aimed 
to inactivate the putative phosphorylation site, whereas its replacement by aspartate aimed to mimic a constitutively phosphorylated threonine. The mutant proteins p53-T155A and p53-T155V were degraded in presence of HPV16 E6 in transfected H1299 cells, whereas p53-T155D was resistant to degradation mediated by E6. All p53 core mutants are still degraded by MDM2 We also examined whether the various mutations altered the MDM2-dependent degradation of p53 in H1299 cells. The major binding site of MDM2 on p53 is located in the N-terminal transactivation domain . A secondary putative MDM2-binding site has been reported in the p53 core domain which seems to play a regulatory role in modulating p53 ubiquitination. A vector expressing MDM2 was cotransfected with vectors expressing the
Posted inUncategorized