Figure 1

SPRR2A blocks p300 induced acetylation of K382-p53. (A)Western blotting of nuclear and cytoplasmic protein obtained from HuCCT-1 cells 48 hours post-transfection with V5-SPRR2A, and FLAG-p53 plasmids. SPRR2A and p53 were detected in both the cytoplasm and nucleus. SPRR2A expression did not change the distribution of p53. Halo-EGFP vector was used to adjust vector amounts. Lamin A/C and Tubulin were used to verify nuclear and cytosolic separation, respectively. (B) Western blot analysis of the distribution of acetyltransferases p300 and PCAF protein 48 hours after transfection with the indicated vectors. P300 and its cysteine/histidine rich region 3 (ΔCH3) deletion construct distribute primarily to the nucleus, with some protein detected in the cytoplasm. PCAF was only detected in the nucleus. Lamin A/C and tubulin were used to verify separation of nuclear and cytoplasmic fractions. (C) HuCCT-1 cells transfected with a DsRed-SPRR2A plasmid and SPRR2A protein visualized via Axiovision, an advanced, automated imaging system (Carl Zeiss, Gottingen, Germany). Axiovision software enables Z-stack capture (10 stack levels/0.35 micron spacing per level). In the vertical cut surface, the cytoplasm is positive for DsRed-SPRR2A expression. The horizontal cut surface shows DsRed-SPRR2A co-localized with DAPI staining in the nucleus. (D) Upper panel: Western blotting of Ac-K382-p53 in cells transfected with indicated vectors shows that SPRR2A reduces Ac-K382-p53, both with and without p300 over-expression. Lower panel, left: A SPRR2A stable transfectant also shows deacetylation of p53 without p53 or p300 over-expression. Lower panel, right: EP300 siRNA further reduced Ac-K382-p53 levels in the SPRR2A stable transfectant, suggesting acetylation of p53 occurs through both p300 and non-p300 mediated pathways. (E) Immunoprecipitation of p300 shows that the presence of SPRR2A reduces p300/p53 binding.