FIG. 8.

Caspase 3-mediated cleavage of ATM disrupts its p53 kinase activity. (A) Control immunoprecipitations to show the specificity of the monoclonal antibody raised against amino acid residues 819 to 844 of ATM (SYR 10G31). Full-length ATM was immunoprecipitated from HeLa nuclear extract (300 μg) or nuclear extracts (300 μg) prepared from control (wt) or A-T lymphoblastoid cells with monoclonal antibody SYR 10G31. A control immunoprecipitation with an isotype-matched antibody (anti-PARP) and HeLa nuclear extract (300 μg) was also carried out. ATM was visualized by probing a Western blot of the immunoprecipitated materials with the polyclonal antiserum ATM-B. Samples of the immunoprecipitates were also incubated with recombinant p53 and [γ-³²P]ATP in an in vitro kinase assay, and radiolabelled p53 was detected by autoradiography (lower panel). (B) Full-length ATM or caspase 3-cleaved ATM was immunoprecipitated from HeLa nuclear extract (N.E.) (300 μg) incubated at 37°C for 15 min in the absence or presence of recombinant caspase 3 (3 μg). ATM and ΔATM were visualized as described for panel A. Immunoprecipitated ATM samples were analyzed for p53 kinase function as described for panel A. (C) ATM and ΔATM were immunoprecipitated, as described for panel A, from nuclear extracts prepared from untreated HL60 cells or HL60 cells treated with 68 μM etoposide for 4 h (left-hand panel). Immunoprecipitated ATM samples were analyzed for p53 kinase function as described for panel A.