PARP4 ADP-ribosylates PIDD1 to complete a phospho/SUMO/PAR-ylation cascade that orchestrates PIDDosome assembly.

Adenosine diphosphate (ADP) ribosylation (ADPr) regulates multiple stress responses, yet substrates in the apoptotic machinery remain elusive. We show that a single, DNA damage-induced ADPr event controls proapoptotic PIDDosome (PIDD1/RAIDD/caspase-2) formation in response to unresolved interstrand DNA cross-links (ICL). ADPr targets conserved E783 in the PIDD1 death domain (DD); is catalyzed by poly(ADP-ribose) polymerase 4 (PARP4), a phylogenetically orphan PARP of previously unknown function; is reversed by the ribosylhydrolase activity of PARP14; and is triggered by Ataxia Telangiectasia and RAD3-related (ATR) phosphorylation-induced, PIAS1-mediated SUMOylation of the PIDD1 DD, which enables PARP4 docking. PIDD1 ADPr is dispensable for the recruitments of RAIDD and caspase-2 but essential for the dimerization of the caspase. Hence, denying E783 ADPr spares the onset of PIDDosome assembly but blocks its completion, thus eliminating caspase-2 activation and ensuing apoptosis. Conversely, removal of PARP14 forces apoptosis, even in cells with tolerable damage. The data identify PARP4 as an ICL response effector and illuminate a three-step modification sequence of the PIDD1 DD that conducts PIDDosome assembly from initiation to completion.