We studied FBXO31 function in human SK-MEL-28 melanoma cells. We first tested whether, as in breast cancer cell lines9
, ectopic expression of FBXO31 could induce G1 arrest in SK-MEL-28 cells. Fluorescence-activated cell sorting (FACS) analysis revealed, as expected, that nocodazole treatment of SK-MEL-28 cells resulted in G2/M arrest (). Ectopic expression of FBXO31 prevented the nocodazole-induced G2/M block and instead substantially increased the fraction of cells in G1. Identical results were obtained in cells ectopically expressing FBXO31 in the absence of nocodazole (Supplementary Fig. 1
). Moreover, ectopic expression of FBXO31 blocked DNA synthesis (). Notably, ectopic expression of FBXO31 also markedly inhibited the growth of SK-MEL-28 cells in culture (Supplementary Fig. 2a
) and SK-MEL-28 mouse xenografts (Supplementary Fig. 2b
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, (more ...)
F-box proteins are best known for their role as the substrate-recognition components of the SCF (SKP/Cullin/F-box protein) class of E3 ubiquitin ligases10
. To investigate the basis by which FBXO31 induces G1 arrest, we analyzed a panel of cell cycle regulatory proteins by immunoblot analysis following ectopic expression of FBXO31. shows that following expression of FBXO31, but not vector alone (Supplementary Fig. 3
), the level of cyclin D1, a key regulator of the G1/S phase transition11
, markedly declined. By contrast, the levels of other G1 cyclins and the G2/M regulator cyclin B1 were unaffected. Ectopic FBXO31 expression also had no effect on the levels of all cyclin-dependent kinases (CDKs) () and CDK inhibitors () examined. As expected, the reduction in cyclin D1 levels was accompanied by an increase in the fraction of cells in G1 (Supplementary Fig. 4
). RNAi-mediated knockdown of cyclin D1 in SK-MEL-28 cells resulted in a similar G1 arrest (Supplementary Fig. 5
). Collectively, these results suggest that FBXO31 induces G1 arrest through selective degradation of cyclin D1.
We next sought to determine whether the FBXO31-mediated decrease in cyclin D1 resulted from proteasomal degradation. shows that addition of the proteasome inhibitor lactacystin blocked the ability of ectopically expressed FBXO31 to decrease cyclin D1 levels. Consistent with this result, cyclin D1 mRNA levels were unaffected by ectopic FBXO31 expression (). Moreover, following ectopic expression of FBXO31 the half-life of cyclin D1 was substantially decreased (Supplementary Fig. 6
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 (more ...)
Proteasomal-mediated degradation of cyclin D1 requires phosphorylation on threonine-28612–14
. We therefore tested whether FBXO31-mediated degradation of cyclin D1 was dependent upon threonine-286 phosphorylation. SK-MEL-28 cells were stably transfected with a plasmid expressing HA-tagged cyclin D1 or a cyclin D1 derivative bearing a threonine-to-alanine substitution at position 286 [cyclin D1(T286A)]12
and then transduced with a retrovirus expressing FBXO31. shows that ectopic expression of FBXO31 resulted in degradation of wild type cyclin D1 but not the cyclin D1(T286A) mutant. Importantly, following expression of cyclin D1(T286A), ectopically expressed FBXO31 failed to induce efficient G1 arrest ( and Supplementary Fig. 7a
) or block DNA synthesis ().
To test whether FBXO31 and cyclin D1 interact, we performed co-immunoprecipitation experiments. SK-MEL-28 cells transduced with a retrovirus expressing myc-tagged FBXO31 were stably transfected with a plasmid expressing HA-tagged cyclin D1 and treated with lactacystin to prevent cyclin D1 degradation (see ). The results of show the presence of cyclin D1 in the FBXO31 immunoprecipitate, which was most evident when lactacystin was added. A similar result was obtained in the reciprocal co-immunoprecipitation. By contrast, FBXO31 failed to interact with cyclin D1(T286A) (Supplementary Fig. 7b
). also shows, consistent with previous reports9
, that FBXO31 was part of the SCF complex as evidenced by its association with SKP1 and CUL1. The co-immunoprecipitation experiment of shows that there was also an interaction between the endogenous FBXO31 and cyclin D1 proteins.
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 (more ...)
To determine whether the F-box motif of FBXO31 was required for cyclin D1 proteolysis, we used an FBXO31 derivative in which the F-box had been deleted (FBXO31ΔF)9
. In contrast to wild type FBXO31, ectopic expression of FBXO31ΔF did not result in decreased levels of cyclin D1 (). Consistent with this finding, ectopic expression of FBXO31 but not FBXO31ΔF resulted in polyubquitination of cyclin D1 (). As expected, cyclin D1(T286A) was not polyubiquitinated. shows that FBXO31 directed polyubiquitination of cyclin D1 in vitro
in the absence of any other F-box protein.
As noted above, it has been previously shown that oncogene-induced senescence involves activation of the DDR1–3
. We therefore considered the possibility that in addition to its role in BRAF-induced senescence4
, FBXO31 might also be involved in mediating G1 arrest following DNA damage. shows that following induction of DNA damage by γ-irradiation, the levels of FBXO31 increased and, consistent with previous reports15
, this was accompanied by a decrease in cyclin D1 levels. Quantitative real-time RT-PCR confirmed that following γ-irradiation, FBXO31 and cyclin D1 mRNA levels were unchanged (Supplementary Fig. 8
). Consistent with this finding, in untreated SK-MEL-28 cells the low levels of FBXO31 resulted, at least in part, from proteasomal degradation (Supplementary Fig. 9
). Significantly, RNAi-mediated knockdown of FBXO31 prevented the decrease in cyclin D1 following γ-irradiation ( and Supplementary Fig. 10
). In support of this conclusion, γ-irradiation decreased the half-life of cyclin D1 in SK-MEL-28 cells expressing a control non-silencing (NS) shRNA but not an FBXO31 shRNA (Supplementary Fig. 11
). Most importantly, in FBXO31 knockdown (KD) SK-MEL-28 cells γ-irradiation failed to induce G1 arrest ( and Supplementary Fig. 12
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 (more ...)
Phosphorylation of cyclin D1 at threonine-286 can be mediated by glycogen synthase kinase 3β (GSK3β)12,13
and through a MAP kinase pathway14
. We found that GSK3β was not required for cyclin D1 degradation following γ-irradiation, whereas blocking MAP kinase signalling using a chemical inhibitor of MEK prevented cyclin D1 degradation following γ-irradiation (Supplementary Fig. 13
ATM is a serine-threonine protein kinase that plays a critical role in the DDR16–18
. In response to DNA double-strand breaks, ATM is autophosphorylated on several residues including serine-1981, and functions by phosphorylating, and in many cases stabilizing, a number of downstream protein targets that function in the DDR16,19
. FBXO31 contains two putative ATM phosphorylation sites at amino acids 278/279 and 400/401, the former of which is conserved (, top). Co-immunoprecipitation experiments revealed that FBXO31 and ATM interacted (Supplementary Fig. 14
). A GST-fusion protein containing the 278/279serine-glutamine site was phosphorylated by ATM in vitro
(, bottom), and an FBXO31 derivative bearing a serine-to-alanine mutation at position 278 failed to accumulate following γ-irradiation () or upon co-transfection with ATM (). In ATM KD SK-MEL-28 cells, FBXO31 was not induced following γ-irradiation and, accordingly, cyclin D1 levels remained unchanged ( and Supplementary Fig. 15
). Significantly, knockdown of FBXO31 substantially increased sensitivity of SK-MEL-28 cells to γ-irradiation (). Collectively, these results indicate that following γ-irradiation ATM directly phosphorylates FBXO31, which results in FBXO31 stabilization and subsequent cyclin D1 degradation.
Finally, we tested whether other DNA damaging agents also induced FBXO31. shows that FBXO31 levels greatly increased and cyclin D1 levels declined to varying extents following UV irradiation, X-ray irradiation, oxidative stress (H2O2), or addition of the chemotherapeutic DNA damaging agents etoposide, adriamycin, cisplatin or fluorouracil. Based upon these results we conclude that induction of FBXO31 is a general response to genotoxic stress.
Collectively, the results reported here show that FBXO31 is induced by DNA damage and, once stabilized, targets cyclin D1 for degradation by ubiquitin-mediated proteolysis, leading to G1 arrest (). Our results address a long-standing problem in the DNA repair field. It has been previously shown that G1 arrest following DNA damage occurs in a two-phase response, referred to as initiation and maintenance15
. The maintenance phase is a slow response that is primarily due to p53-dependent transcription induction of the cell cycle inhibitor p21. By contrast, initiation is a rapid, p53-independent response that primarily results from cyclin D1 degradation. However, the mechanistic basis by which cyclin D1 becomes rapidly degraded after genotoxic stress remained to be determined. Our results indicate that initiation of G1 arrest following genotoxic stress is due to induction of FBXO31, which then interacts with cyclin D1 and mediates its degradation.
Two other F-box proteins, FBXO4 (also called FBX4) and FBXW8, have been previously reported to mediate cyclin D1 degradation14,20
. FBXO31, FBXO4 and FBXW8 likely function through a common mechanism as evidenced by the observations that all three proteins are incorporated into the SCF complex and require phosphorylation of cyclin D1 at threonine-286 to direct cyclin D1 degradation. However, an important distinction between the three proteins is that FBXO4 and FBXW8 appear to be required for normal cell cycle progression14,20
, whereas we find no evidence for a cell cycle defect or decreased growth rate following knockdown of FBXO31 (ref. 4
; see above and data not shown). Moreover, in contrast to FBXO31, the levels of FBXO4 and FBXW8 do not increase following γ-irradiation (Supplementary Figure 16a
), and knockdown of FBXO4 or FBXW8 does not affect cyclin D1 degradation in γ-irradiated SK-MEL-28 cells (Supplementary Figure 16b
). The results presented here reveal that FBXO31 is a dedicated checkpoint protein whose function is to arrest cells following genotoxic stress.