Cleavage and Inactivation of ATM during Apoptosis

Abstract

The activation of the cysteine proteases with aspartate specificity, termed caspases, is of fundamental importance for the execution of programmed cell death. These proteases are highly specific in their action and activate or inhibit a variety of key protein molecules in the cell. Here, we study the effect of apoptosis on the integrity of two proteins that have critical roles in DNA damage signalling, cell cycle checkpoint controls, and genome maintenance—the product of the gene defective in ataxia telangiectasia, ATM, and the related protein ATR. We find that ATM but not ATR is specifically cleaved in cells induced to undergo apoptosis by a variety of stimuli. We establish that ATM cleavage in vivo is dependent on caspases, reveal that ATM is an efficient substrate for caspase 3 but not caspase 6 in vitro, and show that the in vitro caspase 3 cleavage pattern mirrors that in cells undergoing apoptosis. Strikingly, apoptotic cleavage of ATM in vivo abrogates its protein kinase activity against p53 but has no apparent effect on the DNA binding properties of ATM. These data suggest that the cleavage of ATM during apoptosis generates a kinase-inactive protein that acts, through its DNA binding ability, in a trans-dominant-negative fashion to prevent DNA repair and DNA damage signalling.

ACKNOWLEDGMENTS

This work was funded by grants SP2143/0103 and SP2143/0501 from the Cancer Research Campaign and by grants from the Kay Kendall Leukaemia Fund and the A-T Childrens’ Project.

We thank members of the SPJ laboratory for their advice and support. We thank Y. Shiloh for generously providing us with the anti-ATM monoclonal antibody used for the ATM kinase assay and Eva Lee for provision of the anti-ATM monoclonal antibody 2C1. The caspase-3 expression construct was a kind gift from G. Cohen at the University of Leicester and the p53 protein was provided by Byron Hann, UCSF. Thanks also to Mike Waldon at the Protein Nucleic Acid Chemistry facility, Biochemistry Department, University of Cambridge, for technical assistance with N-terminal sequence analysis.