Figure 6

ΔNp63α mutants alter ATM transcription and ATM-dependent phosphorylation. (A) (I) Sequence alignment of the amino-termini of TAp63 and ΔNp63, indicating conserved residues. (II) Domain structure of ΔNp63α, indicating sites of point mutations used in this study, and an expansion of the TA2 transactivation domain showing ΔN-specific residues deleted in the CUT-1 mutant, and residues common to both ΔN and TA isoforms that are deleted in the CUT-2 mutant. (B) Effect of TA2 transactivation domain mutation on ΔNp63α-stimulated ATM promoter activity. H1299 cells were co-transfected with 1 μg either wild-type or the indicated mutant ΔNp63α plasmids, and 1 μg ATM-LUC and 0.2 μg pRL-CMV reporter plasmids. Cells were lysed and processed after 24 hrs, and specific ATM reporter activity was determined as in Figure 4 and is normalised to wild-type values. (C) DNA binding domain mutants have opposing effects on ΔNp63α-stimulated ATM promoter activity. H1299 cells were co-transfected with 1 μg either wild-type or the indicated mutant ΔNp63α plasmids, and 1 μg ATM-LUC and 0.2 μg pRL-CMV reporter plasmids. Cells were lysed and processed after 24 hrs, and specific ATM reporter activity was determined as in Figure 4 and is normalised to wild-type values. (D) Mutation of the CCAAT sequence blocks R298Q ΔNp63α-mediated stimulation of ATM transcription. H1299 cells were co-transfected with 1 μg R298Q ΔNp63α, 1 μg ATM-Luc (wt or mutant) and 0.2 μg pRL-CMV control plasmids, then lysed and processed after 24 hrs. Specific ATM reporter activity was determined as in Figure 4 and is normalised to wild-type values. (E) SAM domain mutants attenuate ΔNp63α-stimulated ATM promoter activity. H1299 cells were co-transfected with 1 μg either wild-type or the indicated mutant ΔNp63α plasmids, and 1 μg ATM-LUC and 0.2 μg pRL-CMV reporter plasmids. Cells were lysed and processed after 24 hrs, and specific ATM reporter activity was determined as in Figure 4 and is normalised to wild-type values. (F) Hay-Wells/AEC syndrome-associated ΔNp63α mutants inhibit ATM-dependent p53 Serine-15 phosphorylation. H1299 cells were cotransfected with 0.2 μg p53 and 1 μg of either wild-type or mutant ΔNp63α as indicated, and harvested after 48hrs. Lysates were blotted with phosphoSerine-15 p53, total p53 and p63 protein.