p53R2 is a substrate of ATM kinase. (A) UV induced an increased ³²P-labeling of p53R2. HEK293 cells were transiently transfected with Myc-tagged p53R2 or empty vector. Eighteen hours after transfection, cells were metabolically labeled with ³²P-orthophosphate and later stimulated with (+) or without (−) UV irradiation (20 J/m²). After 30 min, radiolabeled p53R2 was immunoprecipitated with anti-Myc antibody and evaluated by Phosphoimager (Upper) or Western blot (Lower) by anti-p53R2 antibody. (B) Bacterially expressed p53R2 recombinant proteins were subjected to in vitro ATM immunocomplex kinase assay (KA) using the ATM immunoprecipitated from KB cells at the indicated times after UV (20 J/m²) treatment in the presence of [γ-³²P]ATP. The resulting mixture was resolved by a NuPAGE gel and visualized by autoradiography and Coomassie blue staining (CB). (C) Identification of individual ATM phosphorylation site. WT p53R2 and Ala substitution mutants at Ser⁷² (S72A), Ser¹¹² (S112A), or both residues (S72A/S112A) were subjected to in vitro ATM kinase assay, and visualized by autoradiography and Coomassie staining as described earlier.

In vivo Ser⁷² phosphorylation stabilizes p53R2 against UV. (A) 293 cells were transfected with Myc-tagged WT-p53R2, S72A-p53R2, or ATM expressing plasmids 30 min after UV exposure (20 J/m²) and subjected to immunoblotting analysis using anti-pS72-p53R2 or anti-Myc antibodies. (B) KB cells treated without or with UV (20 or 80 J/m²), cisplatin (20 μM), or IR were immunoprecipitated with anti-p53R2 antibodies and immunoblotted with anti-pS72-p53R2 antibodies. (C) UV induced destabilization of p53R2 proteins in ATM-deficient cells. A-T cells complemented without (pEBS7 vector) or with ATM expressing plasmids (pEBS7-YZ5) were stimulated by UV (20 J/m²) and subjected to immunoblotting analysis. (D) Ser⁷² phosphorylation is required for p53R2 protein stabilization to UV. 293 cells were transfected with Myc-tagged WT-p53R2 or S72-p53R2 together with or without ATM expression plasmids. After 24 h, cells were stimulated by UV (20 J/m²) and subjected to immunoblotting analysis using anti-p53R2. (E) S72A-p53R2 has a shorter half-life after exposure to UV. 293 cells were transfected with Myc-tagged WT- or S72A-p53R2, and protein half-life was determined by pulse-chase analysis. (F) S72A-p53R2 mutant is polyubiquitinated. 293 cells were transfected with HA-ubiquitin plus Myc-tagged WT-, S72A-p53R2, or empty vector. After 24 h, cells were exposed to UV and ubiquitination was examined by immunoprecipitation with anti-Myc antibodies and immunoblotting with HA antibodies.

MDM2 ubiquitinates and interacts with p53R2. (A) MDM2 induced S72A mutant p53R2 ubiquitination in response to UV. 293 cells were transiently transfected with the plasmids encoding Myc-tagged WT- or S72A- p53R2 along with ATM and MDM2. The cells were preincubated with or without MG-132 for 30 min, followed by stimulation with or without UV (20 J/m²) for 6 h. After treatment with UV, the cell lysates were immunoprecipitated with anti-Myc. Ubiquitination of WT- or S72A-p53R2 was examined by immunoblotting with a specific ubiquitin antibody. p53R2 protein levels were examined by anti-p53R2 antibody. (B) Enhanced interaction between endogenous MDM2 and unphosphorylated p53R2. Cell lysates isolated either from pEBS7-YZ5 (ATM proficient) or MG132-pretreated pEBS7 (ATM deficient) cells at the indicated times after UV stress (20 J/m²) were immunoprecipitated with anti-p53R2 antibody, followed by immunoblotting with anti-MDM2 antibodies. (C) Enhanced interaction between exogenous MDM2 and S72A-p53R2 mutant. 293 cells, after transient transfection with plasmids encoding Myc-tagged WT- or S72A-p53R2 together with ATM and MDM2, were preincubated with MG-132 for 30 min before exposure to UV (20J/m²). At the indicated times, cells were lysed and immunoprecipitated with Myc-tagged antibody, followed by immunoblotting with MDM2 or Myc-tagged antibodies.

Interaction between ATM and p53R2. (A) Co-IP of endogenous ATM by anti-p53R2 antibody. Cell lysates were isolated from KB cells at the indicated times after UV stress (20 J/m²) and immunoprecipitated by anti-p53R2 antibody, followed by immunoblotting with anti-ATM or anti-p53R2 antibodies. (B) Same as in A except that ATM antibody was used for co-IP, followed by immunoblotting with anti-ATM, anti-p53R2, and anti-RRM1 antibodies. (C) Same as in A except that ATR antibody was used for co-IP, followed by immunoblotting with anti-ATR or anti-p53R2 antibodies. (D) Same as in A except that DNA-PKcs antibody was used for co-IP, followed by immunoblotting with anti- DNA-PKcs or anti-p53R2 antibodies.

Cells overexpressing S72A-p53R2 are more sensitive to genotoxic stress. Overexpressing S72A-p53R2 increased cellular sensitivity to genotoxic stress. (A) KB cells stably expressing WT-p53R2, S72A, or control vector were exposed to UV (20 J/m²) and their cell cycle profiles were determined by propidium iodide staining and FACS analysis at the indicated time points. Only surviving cells that remained attached to the plates were analyzed. (B) KB cells stably expressing WT-p53R2, S72A, or control vector were analyzed for cell viability by MTT assay 72 h after exposure to UV, IR, or cisplatin at the indicated doses.