Fig. 4: ATM binds with and phosphorylates TOP2β at Ser1134 to promote its degradation by VM-26.

a Inhibition of ATM, but not ATR or DNA-PK, blocks VM-26-induced TOP2β degradation. Cells were pretreated with KU60019, AZD6738, or LTURM34 for 1 h and then treated with VM-26 for an additional 2 h. Cells were then harvested for IB with the indicated Abs. b–d Inhibition of ATM extends the protein half-life of TOP2β. SK-BR3 and MDA-MB231 cells were pretreated with DMSO or KU60019 (5 μM) for 1 h (b) or transfected with the indicated siRNA (c), followed by treatment with CHX and VM-26. Atm WT or KO MEFs (d) were also treated with CHX and VM-26 for various time periods, and then, IB was undertaken with the indicated Abs. Densitometry quantification was performed with ImageJ, and the decay curves are shown (bottom, b; right, c and d). e TOP2β binds to endogenous ATM. HEK293 cells were transfected with the indicated plasmids, and then, IP was conducted with anti-FLAG beads (top), and direct IB was undertaken with the indicated Abs (bottom). f Evolutionary conservation of two putative ATM phosphorylation sites (in red) on TOP2β is shown. g ATM phosphorylates TOP2β at Ser1134. HEK293 cells transfected with the indicated plasmids were left untreated or treated with VM-26, AZD6738, and LTURM34 for 5 h. Cells were harvested after MG132 treatment for 5 h, and then, IP was conducted using anti-FLAG Abs (top), and direct IB was undertaken with the indicated Abs (bottom).