ATR Signalling mediates the prosurvival function of phospho-NPM against PIDDosome mediated cell death

Abstract Background: ATR kinase is studied largely in the context of externally imposed genotoxic stress, even though its endogenous basal level plays important homeostatic cellular functions. Results: We show that pNPM triggers the dissociation of NPM from P1DD thus preventing the cell from undergoing caspase 2 mediated cell death via PIDDosome, thereby acting as an endogenous negative regulator keeping the PIDDosome activation under check. pChk1 interacts with NPM before phosphorylating it, which is abrogated following ATR kinase inhibition, dropping nucleoplasmic/chromatin pNPM level, thereby inducing PIDD. Expectedly, phosphodead mutants of Chk1 and NPM are not viable as the pNPM regulatory axis is abrogated, whereas the phosphomimic mutants of Chk1 and NPM render cells refractory to ATR/pChk1 kinase inhibition induced cell death. Interestingly, cells expressing phosphomimic mutant of Chk1(S345E) withstand ATRi, but not Chk1i mediated PIDDosome activation implying that the Chk1 kinase plays a direct role in pNPM homeostasis, even in the absence of replication stress. Conclusion: In the current study, we uncover the non-canonical role of ATR kinase in the control of PIDDosome activation, and show that under normal cellular conditions involving no replication stress, ATR kinase controls the phosphorylation of NPM via pChk1 and the two phosphatases regulating pNPM, namely, PPM1D and PP1β.