F-Box Protein FBXO31 Mediates Cyclin D1 Degradation to Induce G1 Arrest Following DNA Damage

In response to DNA damage eukaryotic cells initiate a complex signalling pathway, termed the DNA damage response (DDR), which coordinates cell cycle arrest with DNA repair. Studies have shown that oncogene-induced senescence, which provides a barrier to tumour development, involves activation of the DDR1–3. Using a genome-wide RNA interference (RNAi) screen, we have identified 17 factors required for oncogenic BRAF to induce senescence in primary fibroblasts and melanocytes4. One of these factors is an F-box protein, FBXO31, a candidate tumour suppressor encoded in 16q24.3, a region in which there is loss of heterozygosity in breast, ovarian, hepatocellular and prostate cancers5–9. Here we study the cellular role of FBXO31, identify its target substrate and elucidate the basis for its growth inhibitory activity. We show that ectopic expression of FBXO31 acts through a proteasome-directed pathway to mediate degradation of cyclin D1, an important regulator of G1 to S phase progression, resulting in G1 arrest. Cyclin D1 degradation results from a direct interaction with FBXO31 and is dependent upon the F-box motif of FBXO31 and phosphorylation of cyclin D1 at threonine-286, which is known to be required for cyclin D1 proteolysis. The involvement of the DDR in oncogene-induced senescence prompted us to investigate the role of FBXO31 in DNA repair. We find that DNA damage induced by γ-irradiation results in increased FBXO31 levels, which requires phosphorylation of FBXO31 by the DDR-initiating kinase, ATM. Significantly, RNAi-mediated knockdown of FBXO31 prevents cells from undergoing efficient G1 arrest following γ-irradiation and dramatically increases sensitivity to DNA damage. Finally, we show that diverse DNA damaging agents all result in a large increase in FBXO31 levels, indicating that induction of FBXO31 is a general response to genotoxic stress. Our results reveal FBXO31 as a regulator of the G1/S transition that is specifically required for DNA damage-induced growth arrest.