Fig. 7. Wip1 inhibits phosphorylation of p53 induced by UV radiation. (A) In vitro phosphorylation of p53 by p38. COS-7 cells were cotransfected with 0.1 µg of the expression plasmid encoding Flag–p38, with 0.3 µg of the empty vector or the pWip1 expression vector. Transfected cells were stimulated with UV, and kinase activity of Flag–p38 was assayed in an immunocomplex kinase assay as in Figure 3A, except that GST–p53 was used as a substrate. (B) Wip1 blocks phosphorylation of p53 at Ser33 and Ser46 induced by UV radiation. The p53-deficient H1299 cells were transiently transfected with 0.1 µg of an expression vector encoding wild-type p53, together with 0.3 µg of either the pWip1 expression vector or the empty vector pcDNA3 (Vector). After 18 h, cells were either treated with UV (25 J/m²) (+) or left untreated (–). Seven hours after UV treatment, cell extracts were prepared and analyzed for p53 phosphorylation by immunoblot analysis using anti-p53 antibodies specific for phosphorylation at Ser33, Ser46 or Ser392 as indicated. The levels of p53 expression in the samples were monitored using a p53-specific mAb, DO-1. (C) Wip1 does not dephosphorylate p53 at Ser33 and Ser46 in vitro. Phosphorylated p53 was immunopurified from UV-treated H1299 cells transfected with wild-type p53 expression vector. Following incubation with purified GST–Wip1, in the presence or absence of Mg²⁺, the phosphorylation state of p53 was examined by immunoblotting using antibodies specific for the phosphorylated Ser33 or Ser46 sites of p53. (D) Time-course of p53 phosphorylation at Ser33 and Ser46 in A549 cells. Phosphorylation status of p53 was determined in cell extracts from A549 cells treated with UV (30 J/m²) by immunoblotting using antibodies specific for p53 Ser33 (upper panel) and Ser46 (middle panel) phosphorylation sites, respectively. The levels of p53 expression in the samples were indicated (lower panel). (E) Effect of SB203580 on p53 phosphorylation at Ser33 and Ser46 in response to UV radiation. p38-specific inhibitor SB203580 were added to A549 cells 1 h before irradiation with UV (30 J/m²), and extracts were prepared 12 h later. Endogenous wt-p53 was analyzed for phosphorylation by immunoblotting as in (D). The amount of p53 in cell lysates was indicated (lower panel).