F-box protein FBXO31 mediates cyclin D1 degradation to induce G1 arrest after DNA damage

Open Access
Publisher Website
Google Scholar
Abstract
During the cellular response to DNA damage in eukaryotes, cell-cycle arrest is coordinated with DNA repair. Here the human F-box protein FBXO31 is shown to regulate the cell-cycle transition from G1 to S phase in response to DNA damage by mediating degradation of cyclin D1. Diverse DNA damaging agents result in increased FBXO31 levels, indicating that FBXO31 is a general mediator required to arrest cell growth after DNA damage. Upon DNA damage, eukaryotic cells initiate the DNA damage response, a complex signalling pathway which coordinates cell cycle arrest with DNA repair. Using a genome-wide RNA interference screen, the human F-box protein FBXO31 is shown to regulate the G1/S cell cycle transition in response to DNA damage by mediating degradation of cyclin D1, an important regulator of progression from G1 to S phase. 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 DDR1,2,3. 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 cancers5,6,7,8,9. Here we study the cellular role of FBXO31, identify its target substrate and determine the basis for its growth inhibitory activity. We show that ectopic expression of FBXO31 acts through a proteasome-directed pathway to mediate the degradation of cyclin D1, an important regulator of progression from G1 to S phase, resulting in arrest in G1. Cyclin D1 degradation results from a direct interaction with FBXO31 and is dependent on the F-box motif of FBXO31 and phosphorylation of cyclin D1 at Thr 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. RNAi-mediated knockdown of FBXO31 prevents cells from undergoing efficient arrest in G1 after γ-irradiation and markedly increases sensitivity to DNA damage. Finally, we show that a variety of 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.