FIG. 6.

Identification of caspase 3 cleavage sites in ATM. (A) The ATM region containing the motif DYPD/S, but not the region containing the motif DIAD/I, is cleaved by caspase 3 in vitro. As shown in the top panel, the region of ATM comprising amino acid (a.a.) residues 727 to 859 (region X) or the region comprising residues 819 to 939 (region Y) was transcribed and translated in vitro in the presence of ³⁵S-radiolabelled methionine before being incubated for 30 min at 37°C in the absence or presence of 300 ng of caspase 3, as indicated. Reaction products were detected by SDS-PAGE (18%) followed by autoradiography. The bottom panel is a schematic representation of the regions of ATM utilized in the above-mentioned studies together with potential caspase 3 cleavage sites. (B) Cleavage of the ATM caspase 3 cleavage site as a GST fusion. Bacterially expressed and purified GST-ATM-CS fusion protein (2 μg) was incubated with no caspase 3 (−) or with increasing amounts (33, 100, or 300 ng) of caspase 3 for 30 min at 37°C. Products were analyzed by SDS-PAGE (12% gel) followed by Western immunoblotting with an anti-GST monoclonal antibody.