Temporal phosphoproteomics reveals WEE1-dependent control of 53BP1 pathway

Wee1-like protein kinase (WEE1) restrains activities of cyclin-dependent kinases (CDKs) in S and G2 phase. Inhibition of WEE1 evokes drastic increase in CDK activity, which perturbs replication dynamics and compromises cell cycle check-points. Notably, WEE1 inhibitors such as adavosertib are tested in cancer treat-ment trials; however, WEE1-regulated phosphoproteomes and their dynamics have not been systematically investigated. In this study, we identified acute time-resolved alterations in the cellular phosphoproteome following WEE1 inhi-bition with adavosertib. These treatments acutely elevated CDK activities with distinct phosphorylation dynamics revealing more than 600 potential uncharac-terized CDK sites. Moreover, we identified a major role for WEE1 in controlling CDK-dependent phosphorylation of multiple clustered sites in the key DNA repair factors MDC1, 53BP1, and RIF1. Functional analysis revealed that WEE1 fine-tunes CDK activities to permit recruitment of 53BP1 to chromatin. Thus, our findings uncover WEE1-controlled targets and pathways with translational potential for the clinical application of WEE1 inhibitors.