FBXL12 degrades FANCD2 to regulate replication recovery and promote cancer cell survival under conditions of replication stress

Oncogene-induced replication stress constitutes an early obstacle for pre-cancerous cells to overcome to progress towards malignancy. Fanconi anaemia signalling represents a major genomic maintenance pathway that is activated in response to replication stress, impinging on stalled replication fork stability and recovery. We report that upon replication stress, phosphorylation of the FANCD2 N-terminus by CHK1 triggers FBXL12-dependent proteasomal degradation of FANCD2, facilitating clearance of FANCD2 at stalled replication forks and promoting replication recovery. Monoubiquitination of FANCD2 at K561 by the Fanconi anaemia core complex regulates clamping of the FANCD2-FANCI heterodimer onto DNA. To investigate if K561-monoubiquitination influences FANCD2 phosphorylation and/or polyubiquitination by FBXL12, we performed MS-based proteomics using HEK293 cells co-transfected with the monoubiquitin-deficient FANCD2 mutant (K561R) and FANCI, or a CHK1 phosphorylation-deficient FANCD2-AA (S8A/S10A) mutant as control. To induce rapid activation of CHK1, we inhibited WEE1 kinase using AZD1775, immunoprecipitated FANCD2 from chromatin extracts and performed MS analysis. The MS data revealed several phosphorylated N-terminal peptides in the K561R-FANCD2 sample, including S8, S10 and also S15 demonstrating that K561R is phosphorylated in response to replication stress induced by AZD1775.