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

Abstract

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 DDR. Using a genome-wide RNA interference (RNAi) screen, we have identified 17 factors required for oncogenic BRAF to induce senescence in primary fibroblasts and melanocytes. 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 cancers. 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 gamma-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 gamma-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.

Figure 1

Ectopic expression of FBXO31 induces G1 arrest and selective degradation of cyclin D1. a, FACS analysis in SK-MEL-28 cells transduced with a retrovirus expressing empty vector or FBXO31 in the absence or presence of nocodazole. b, DNA replication assay, monitored by bromodeoxyuridine (BrdU) incorporation (error bars, s.d., n=3). c–e, Immunoblot analysis showing levels of cyclins (c), CDKs (d) and CDK inhibitors (e) at various time points following transduction with an FBXO31 retrovirus. Tubulin was monitored as a loading control. Ectopic expression of FBXO31 did not induce a DDR (Supplementary Figure 17).

Figure 2

FBXO31-mediated cyclin D1 degradation occurs through the proteasomal pathway. a, Cyclin D1 levels in SK-MEL-28 cells transduced with a retrovirus expressing FBXO31 or empty vector and treated in the presence or absence of lactacystin. b, Quantitative real-time RT-PCR monitoring cyclin D1 mRNA levels (error bars, s.d., n=3). c, Cyclin D1 levels in SK-MEL-28 cells stably transfected with a plasmid expressing either HA-tagged cyclin D1 or cyclin D1(T286A) and transduced with an FBXO31 retrovirus. d, FACS analysis in cells described in (d). e, DNA replication assay (error bars, s.d., n=3).

Figure 3

FBXO31 interacts with and directs ubiquitination of cyclin D1. a, Co-immunoprecipitation of FBXO31 with cyclin D1 and SCF complex components. The FBXO31-cyclin D1 interaction was specific (Supplementary Fig. 18) and predominantly cytoplasmic (Supplementary Fig. 19). b, Co-immunoprecipitation of endogenous FBXO31 and cyclin D1. c, Cyclin D1 levels in SK-MEL-28 cells ectopically expressing vector, FBXO31 or FBXO31ΔF. d, In vivo ubiquitination assay. Polyubiquitinated cyclin D1 was detected by immunoprecipitation of Flag-tagged ubiquitin followed by immunoblotting for HA-cyclin D1. e, In vitro ubiquitination assay. Immunopurified SCF complexes were incubated with GST-cyclin D1, Erk2, E1, E2, ATP and ubiquitin.

Figure 4

Cell cycle arrest following DNA damage requires ATM-mediated induction of FBXO31. a, Immunoblot analysis of FBXO31 following γ-irradiation. For comparison to ectopically expressed FBXO31 levels see Supplementary Fig. 20. b, Cyclin D1 levels in cells expressing a NS or FBXO31 shRNA following γ-irradiation. c, FACS analysis. d, (Top) Putative ATM sites in FBXO31. (Bottom) In vitro phosphorylation of GST-FBXO31 fusion proteins harbouring wild type (WT) or mutated (SDM) SQ sites. ATM-WT, wild type ATM; ATM-KD, kinase dead ATM mutant. e–f, Levels of ectopically expressed WT or mutant FBXO31 following γ-irradiation (e) or ectopic ATM expression (f). g, FBXO31, cyclin D1, ATM and phosphorylated ATM in γ-irradiated cells expressing a NS or ATM shRNA. h, Colony formation assay in untreated or γ-irradiated cells. i, FBXO31 and cyclin D1 levels following treatment with DNA damaging agents. j, Model.