Abstract
FBXO31 was originally identified as a putative tumor suppressor gene in breast, ovarian, hepatocellular, and prostate cancers. By screening a set of cell cycle-regulated proteins as potential FBXO31 interaction partners, we have now identified Cdt1 as a novel substrate. Cdt1 DNA replication licensing factor is part of the pre-replication complex and essential for the maintenance of genomic integrity. We show that FBXO31 specifically interacts with Cdt1 and regulates its abundance by ubiquitylation leading to subsequent degradation. We also show that Cdt1 regulation by FBXO31 is limited to the G2 phase of the cell cycle and is independent of the pathways previously described for Cdt1 proteolysis in S and G2 phase. FBXO31 targeting of Cdt1 is mediated through the N terminus of Cdt1, a region previously shown to be responsible for its cell cycle regulation. Finally, we show that Cdt1 stabilization due to FBXO31 depletion results in re-replication. Our data present an additional pathway that contributes to the FBXO31 function as a tumor suppressor.
Highlights
SCF E3 ligases regulate degradation of proteins involved in many processes including cell cycle progression and DNA repair
We show that Cdt1 regulation by FBXO31 is limited to the G2 phase of the cell cycle and is independent of the pathways previously described for Cdt1 proteolysis in S and G2 phase
Protein extracts from HEK293T cells transiently expressing Myc-tagged Cdt1 were subjected to immunoprecipitation with either anti-Myc antibody or IgG control and Western blotted with an anti-FBXO31 antibody
Summary
SCF E3 ligases regulate degradation of proteins involved in many processes including cell cycle progression and DNA repair. By screening a set of cell cycle-regulated proteins as potential FBXO31 interaction partners, we have identified Cdt as a novel substrate. The activity and protein levels of Cdt are tightly controlled in S and G2 phases of the cell cycle through ubiquitylation-dependent proteolysis by SCF-Skp ubiquitin ligase and the proliferating cell nuclear antigen-dependent Cul4-DDB1 and Cdt ubiquitin ligases as well as the inhibitory geminin binding to prevent rereplication and genomic instability (18, 19). Cdt regulation by FBXO31 is limited to the G2 and the early mitotic phase of the cell cycle and is independent of the pathways previously described for Cdt proteolysis in S and G2 phase. Our data present a novel pathway that regulates Cdt in the G2 phase of the cell cycle and is likely to contribute to FBXO31 tumor suppressor activity
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