Arsenic Trioxide Reactivates Proteasome-Dependent Degradation of Mutant p53 Protein in Cancer Cells in Part via Enhanced Expression of Pirh2 E3 Ligase
Figure 5
Pirh2 physically associates with mutant p53 protein for polyubiquitination.
(A) HaCaT and MIA PaCa-2 cells were treated with 5 or 7.5 µM ATO for 6 h. Cell extracts from HaCaT (left) and MIA PaCa-2 (right) cells were immunoprecipitated with anti-p53 or control IgG. The immunocomplexes were then used to detect mutant p53 and Pirh2 along with whole-cell lysates as input control. (B) The experiment was performed as described in (A), except that anti-Pirh2 antibody was used in immunoprecipitation. (C–E) In vitro synthesized 35S-labeled wild-type p53 (C), R175H (D), and R273H (E) were mixed with GST, GST-tagged Pirh2, Pirh2-DN, or Pirh2-ΔRING. The complexes were then mixed with a buffer containing E1, E2 (UbcH5b), and Ub and then incubated at 30°C for 2 h. Ubiquitinated p53 was analyzed by SDS-PAGE and detected by autoradiography. (F) A model of Pirh2-mediated degradation of mutant p53 induced by ATO.