Overexpression of ATR^ki inhibits DNA damage-induced phosphorylation of p53 on Ser-15. (A) Inhibition of γ-radiation-induced Ser-15 phosphorylation. GM847/ATR^ki cells were cultured either in the absence (−) or presence (+) of Dox for 48 hr to induce ATR^ki. Cells were either left untreated or exposed to 20 Gy of γ-radiation, and cell lysates were prepared after 4 hr. Immunoblots were performed with the indicated antibodies. (B) Time course of Ser-15 phosphorylation. Cells were treated as in A, except that cells were harvested at the indicated times after irradiation.

ATR phosphorylates p53 on Ser-15 and Ser-37. (A) Wild-type GST–p53, and p53 mutants bearing single (S15A and S37A) or double (S15A, S37A) Ser → Ala amino acid substitutions were tested as ATR substrates. ATR was immunoprecipitated from testis extracts using either preimmune rabbit serum (P) or α-ATR (A). Numbers below each lane indicate the incorporation of [³²P]phosphate into each substrate normalized to the value obtained with wild-type GST–p53. (B) Immunoreactivity of the phosphorylated wild-type and mutant GST–p53 fusion proteins with antibodies specific for phospho-Ser-15 and phospho-Ser-37, respectively. (C) Phosphorylation of p53 by ATR requires a functional kinase domain. K562 cells were transiently transfected with either pcDNA3, or plasmid constructs encoding Flag-tagged ATR (ATR^wt), or Flag-tagged ATR^ki. Cell extracts were immunoprecipitated with Flag mAb, and immune complex kinase assays were performed with GST–p53 as the substrate. (D) ATR kinase activity in AT cells. Detergent extracts prepared from AT3BI cells or bovine testis extract were immunoprecipitated as in A, and kinase assays performed as described.

γ-Radiation-induced ATM activation in ATR^ki overexpressing cells. GM847/ATR^ki cells were cultured and cells treated as in Fig. 1A. After 1 hr, ATM was immunoprecipitated and ATM kinase assays were performed as in Fig. 2A. Numbers below sample lanes are as in Fig. 2A. Levels of ATM protein were determined by immunoblotting with α-ATM (bottom).

Effects of ATR^ki overexpression on Ser-15 phosphorylation induced by UV light. GM847/ATR^ki cells were cultured as in Fig. 1A and were either left untreated or exposed to 50 J/m² UV light; cell lysates were prepared at the indicated time intervals. Detergent-soluble proteins were separated by SDS-PAGE and were immunoblotted with the indicated antibodies.