Oncogene (2008) 27, 1716–1725 & 2008 Nature Publishing Group All rights reserved 0950-9232/08 $30.00 www.nature.com/onc ORIGINAL ARTICLE Prohibitin interacts with RNF2 and regulates function via dual pathways

D Choi, S-J Lee, S Hong, I-H Kim and S Kang

Graduate School of Life Sciences and Biotechnology, Korea University, Seoul, Republic of Korea

Prohibitin, a tumor suppresser , plays an important The transcriptional activator E2F1 has been shown to role in the transcriptional regulation of various upregulate the expression of many genes involved in involved in cell-cycle control and proliferation. Recent differentiation, proliferation and apoptosis, and it plays studies have reported that the growth-suppressive property a major role in the G1/S transition and DNA synthesis of the prohibitin protein is exhibited in its physical (Bell and Ryan, 2004).Prohibitin represses the tran- interaction with E2F family and its subsequent scriptional activity of E2Fs1-5 by recruiting co-repres- repression of their transcriptional activity. Herein, we sors, including histone deacetylase 1, N-CoR and BrG1/ report that prohibitin interacts with RING finger protein 2 Brm (Wang et al., 2002), whereas Rb represses the (RNF2), a member of the PcG (polycomb-group) family transcriptional activity of E2Fs1-3 by recruiting histone of proteins, and that the two proteins regulate the activity deacetylase 1, DNA methyltransferase and BrG1/Brm of E2F1 via dual pathways: the direct, prohibitin-mediated (Luo et al., 1998; Magnaghi-Jaulin et al., 1998; Zhu, pathway and the indirect, p16-mediated pathway of E2F1 2005).Prohibitin and Rb interact with distinct regions transcriptional regulation. Co-immunoprecipitation experi- of E2F1 and respond to different upstream signals ments showed that endogenous prohibitin interacts with (Wang et al., 1999b). endogenous RNF2. Interestingly, the expressed amounts of The Rb protein negatively controls the G1/S phase RNF2 and prohibitin were interdependently affected at the transition of the cell cycle, thus acting as a tumor post-translational level. Furthermore, the depletion of either suppressor.At the beginning of G1, Rb proteins exist in endogenous RNF2 or prohibitin using the RNA interference an active, non-phosphorylated state that allows them to technique increased the level of p16 protein expression, bind to a large number of proteins, including E2F1 resulting in a decrease in the transcriptional activity of E2F1 (Harbour and Dean, 2000).E2F1 is functionally inactive via the p16–CDK4–Rb pathway. In addition, chromatin when bound to Rb.The Rb proteins are inactivated by immunoprecipitation assays showed that RNF2 was recruited phosphorylation mediated by various cyclin-dependent to E2F1-response promoters along with prohibitin to inhibit kinases (Cdks).D-type cyclins synthesized in the early G1 the transcriptional activity of E2F1. Cell proliferation was phase confer catalytic activity to Cdk4 and Cdk6.The also regulated by the prohibitin–RNF2 interaction. These activated Cdk4/6–cyclin D complexes phosphorylate Rb, results suggest that the RNF2–prohibitin complex regulates which dissociates Rb from the activator the activity of E2F1 via dual pathways. E2F1, thus activating E2F1.The p16 protein binds to the Oncogene (2008) 27, 1716–1725; doi:10.1038/sj.onc.1210806; non-catalytic side of Cdk4 and Cdk6 and blocks the published online 17 September 2007 interaction between Cdk4/6 and cyclin D (Sherr, 2000). RING finger protein 2 (RNF2), also known as Keywords: prohibitin; RNF2; E2F1; p16; cell cycle dinG/RING1b, was previously identified as a member of the PcG family (polycomb group).RNF2 acts as a transcriptional repressor and contains a conserved RING finger domain in its N-terminal region.We Introduction previously found that the RNF2 protein plays the role of an E3 ligase and that RNF2 interacts with the S6’ Prohibitin is an evolutionarily conserved and ubiquitously protein (Lee et al., 2001, 2005). It was recently found expressed protein.Prohibitin has the potential to act as a that the RNF2-containing polycomb-group complexes tumor suppressor, antiproliferative protein and regulator participate in histone H2A ubiquitination (Wang et al., of cell-cycle progression in the nucleus.The roles of 2004).Interestingly, Voncken et al.(2003) reported that prohibitin are accomplished by its interaction with E2F, the RNF2-null mouse embryo arrests at the early pRb and (Wang et al., 1999a; Fusaro et al., 2003). developmental stage, but the arrest is bypassed by the genetic inactivation of the p16 locus, suggesting that RNF2 is involved in the p16–CDK4–Rb pathway. Correspondence: Professor S Kang, Graduate School of Life Sciences and Herein, our in vivo binding experiment shows that the Biotechnology, Korea University, Seoul 136-701, Republic of Korea. E-mail: [email protected] RNF2 protein interacts with prohibitin.Furthermore, Received 9 May 2007; revised 16 August 2007; accepted 16 August 2007; our results demonstrate that the two proteins regulate published online 17 September 2007 the function of E2F1 by dual pathways: the direct, Interaction between prohibitin and RNF2 D Choi et al 1717 prohibitin-mediated pathway and the indirect, p16- analysed in HEK293 cells transfected with HA-tagged mediated pathway of E2F1 translational regulation. RNF2 and/or the RNF2 shRNA (short interfering hairpin RNA) expression vector.Interestingly, the level of the prohibitin protein was increased when RNF2 was Results overexpressed, while the level of prohibitin was not increased when HA-tagged RNF2 and RNF2 shRNA Interaction between RNF2 and prohibitin in mammalian expression vectors were co-transfected (Figure 2a).To cells further investigate, we performed a knockdown of We used a yeast two-hybrid screen to identify the proteins that interacted with RNF2 and found that prohibitin interacted with RNF2 (data not shown).We 4 performed co-immunoprecipitation experiments in HEK293 cells and confirmed the RNF2–prohibitin + shRNF2- interaction in mammalian cells (Supplementary Figure S1a).To investigate whether endogenous prohibitin Control HA-RNF2HA-RNF2 interacts with endogenous RNF2, we performed co- kDa immunoprecipitation experiments in HEK293 cells 43 Anti-RNF2 using antibodies against prohibitin (Figure 1, right panel) or RNF2 (Figure 1, left panel).The precipitated 30 Anti-prohibitin proteins were then immunoblotted for the RNF2 antibody and prohibitin antibody, respectively.Endo- Anti -actin genous cellular RNF2 was found in immunocomplexes with prohibitin, while endogenous prohibitin was found in immunocomplexes with RNF2.The RNF–prohibitin interaction was also found in HeLa and MCF7 cells Control shRNF2-4 (Supplementary Figure S1b).Furthermore, we used prohi- kDa bitin deletion mutants to define the interaction domain and 43 Anti-RNF2 found that RNF2 interacts with the N-terminus of prohibitin (Supplementary Figure S1c).Taken together, 30 Anti-prohibitin these results demonstrate that endogenous protein complexes containing prohibitin and RNF2 exist in 37 Anti D-prohibitin various cell lines. 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The stability of RNF2 and prohibitin was regulated Figure 2 The protein levels of prohibitin and RNF2 are interdependently at the post-translational level interdependently regulated.( a) HEK293 cells were transfected We investigated the expression levels of the two with HA empty, HA-tagged RNF2 or both the RNF2 shRNA expression vector and HA-tagged RNF2.( b) HEK293 cells were proteins, since one protein might influence the stability transfected with an empty or RNF2 shRNA expression vector. of the other protein if they interact each other.The HA, hemagglutinin; RNF2, RING finger protein 2; shRNA, short effect of RNF2 on prohibitin stability was initially interfering hairpin RNA.

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IB: Prohibitin IB: RNF2 (lysate) (lysate) (HEK 293) (HEK 293) Figure 1 RNF2 interacts with prohibitin in mammalian cells.We performed co-immunoprecipitation experiments in HEK293 cells to investigate whether endogenous prohibitin interacts with endogenous RNF2.Lysates were immunoprecipitated with anti-prohibitin (right panel) or anti-RNF2 (left panel) antibodies and then subjected to western blot analysis.RNF2, RING finger protein 2.

Oncogene Interaction between prohibitin and RNF2 D Choi et al 1718 endogenous RNF2 using a RNF2 shRNA expression Taken together, these results showed that RNF2 and vector and found that the knockdown of RNF2 decreased prohibitin interdependently regulate the stability of the the levels of the prohibitin protein (Figure 2b).However, other proteins. the stability of D-prohibitin, a member of prohibitin We used a variety of experimental approaches in an superfamily, was not influenced by the RNF2 knockdown. effort to gain insight into the mechanism responsible for We also investigated the effect of prohibitin on RNF2 the interdependent regulation of prohibitin and RNF2 expression.As shown in Supplementary Figure S2a, the stability.We performed real-time reverse transcription– level of RNF2 was increased by the overexpression of PCR experiments to determine whether the interdepen- prohibitin.We also tested the effect of knockdown of dent regulation of prohibitin and RNF2 was carried out endogenous prohibitin by using prohibitin shRNA expres- at the transcriptional or post-transcriptional level.As sion vectors (Supplementary Figure S2b).Prohibitin knock- shown in Figure 3a, the knockdown of RNF2 using down using shprohibitin-1 reduced the level of RNF2. RNA interference (RNAi) led to a reduction in the levels

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Relative Level of RNF2 protein (%) 0306090 Time(min after Addition of CHX) Figure 3 The stability of prohibitin and RNF2 is interdependently regulated at the post-translational level.( a) Total RNA was prepared from HEK293 cells transfected with empty or RNF2 shRNA expression vector.The relative amounts of endogenous prohibitin mRNA or RNF2 mRNA were analysed by quantitative-reverse transcription–PCR using prohibitin or RNF2 primers.Assays were performed in triplicate on a real-time PCR system.The results are presented as the means of three independent experiments ±s.d. (b) Total RNA was prepared from HEK293 cells transfected with an empty vector or prohibitin shRNA expression vector.( c) The effects of prohibitin on endogenous RNF2 protein stability in HEK293 cells.HEK293 cells were transfected with prohibitin or empty vectors, followed by exposure to the protein synthesis inhibitor cycloheximide for various times.Target proteins were detected by western blot analysis (top panel).The graph of the bottom panel shows a quantification using Multi Gauge program of the western blot results shown in the top panel.**P o0.005 versus the control (student’s t-test).RNF2, RING finger protein 2; shRNA, short interfering hairpin RNA.

Oncogene Interaction between prohibitin and RNF2 D Choi et al 1719 of RNF2 mRNA.However, RNF2 knockdown had no expression level of endogenous p16 is affected by the effect on the level of endogenous prohibitin mRNA.At overexpression or knockdown of RNF2 in HEK293 this point, we knocked down the expression of prohi- cells (Figure 4a).As expected, the downregulated bitin using shprohibitin-1, which did not reduce the level expression of RNF2 increased endogenous p16 protein of endogenous RNF2 mRNA (Figure 3b).These results levels.Interestingly, the overexpression of RNF2 did not show that the interdependent regulation of prohibitin increase or decrease the protein level of p16. and RNF2 stability is not carried out at the level of Since our study showed that RNF2 interacts with transcription. prohibitin, we investigated whether prohibitin also influ- We then investigated whether the interdependent ences the level of p16, as was shown with RNF2.We regulation of prohibitin and RNF2 stability occurs at investigated the endogenous p16 protein levels in HEK293 the post-translational level.HEK293 cells were trans- cells with overexpressed prohibitin or in HEK293 cells fected with the control vector or prohibitin (Figure 3c). with reduced prohibitin by RNAi (Figure 4b).The results At 48 h after transfection, the cells were treated with were the same as those observed with RNF2: prohibitin cycloheximide to block protein synthesis.As shown in knockdown resulted in a marked increase in p16 expres- Figure 3c, RNF2 showed a half-life of about 60 min. sion, but the level of p16 was not altered by prohibitin RNF2 decayed at a slower rate in the presence of overexpression.In addition, we tried to test other cell lines ectopically expressed prohibitin; the half-life of RNF2 and obtained similar results in MEF cells treated with was increased to more than 90 min.These observations prohibitin siRNA oligonucleotides (Figure 4b).These strongly suggest that RNF2 is more stabilized by its results demonstrate that the decrease in either RNF2 or interaction with prohibitin and that the interdependent prohibitin opens the door for p16 upregulation. regulation of prohibitin and RNF2 stability occurs at the post-translational level. The expression of p16is regulated by prohibitin through RNF2 Protein levels of p16are increased by prohibitin or RNF2 We defined the prohibitin region responsible for depletion regulating the stability of RNF2 and whose knockdown In the previous report, the expression of p16INK4a was increases the level of p16.We analysed the endogenous found to be upregulated in RNF2-knockout mice p16 protein levels in HEK293 cells transfected with (Voncken et al., 2003). We tested whether the protein deleted versions of prohibitin: prohibitin D185–214

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(HEK 293) (MEF) Figure 4 Protein levels of p16 are increased by a decrease of prohibitin or RNF2.( a) HEK293 cells were transfected with empty, RNF2 or RNF2 shRNA expression vectors.Western blot analysis with anti-RNF2 and anti-P16 showed that the protein level of p16 was not altered in RNF2-overexpressed cell lysates (left panel), but that it was increased in RNF2-knockdown cell lysates (right panel). (b) HEK293 cells were transfected with empty or prohibitin expression vectors or with control or shprohibitin-1 (left panel).MEF cells were transfected with siprohibitin-3 or siprohibitin-4 siRNA oligonucleotides (right panel).MEF, mouse embryonic fibroblast; RNF2, RING finger protein 2; shRNA, short interfering hairpin RNA; siRNA, short interfering RNA.

Oncogene Interaction between prohibitin and RNF2 D Choi et al 1720 (deletion of E2F1-binding site), prohibitin 1–185, prohibi- shRNA expression vector and RNF2.These results suggest tin D1–185 and prohibitin 1–214.As shown in Figure 5a, that absence of prohibitin might make RNF2 unstable, the protein levels of RNF2 were increased not only by full- which would promote the expression of p16. length prohibitin, but also by the deleted versions of prohibitin except for prohibitin D1–185, suggesting that the protein stability of RNF2 is not regulated by the Prohibitin is able to regulate E2F1 transcription activity E2F1-binding site of prohibitin, but rather by prohibitin via the p16–CDK4 pathway 1–185. Our results, presented in Figure 4, showed that the We analysed the p16 protein levels in HEK293 cells co- decrease in prohibitin expression induced the upregula- transfected with a prohibitin shRNA expression vector and tion of p16 expression, which gave us the idea that a deleted version of prohibitin (Figure 5b).The knock- prohibitin might indirectly regulate the transcriptional down of prohibitin expression using RNAi increased the activity of E2F1 through the p16–CDK4–Rb pathway. amount of p16 (Figure 5b, lane 2).However, the effect was To determine whether the level of phosphorylated Rb is rescued by the overexpression of either full-length prohi- decreased by prohibitin knockdown, we transfected bitin or the deleted versions.Moreover, similar results were prohibitin siRNA oligonucleotides into MEF cells and also obtained in cells co-transfected with a prohibitin observed the amount of phosphorylated Rb using western

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Figure 5 The protein level of p16 is regulated by deletion forms of prohibitin.( a) HEK293 cells were transfected with wild-type prohibitin or mutation constructs.( b) HEK293 cells were transfected with the plasmids indicated at the top of the figure.The p16 levels were increased by knockdown of either prohibitin or RNF2 expression (lanes 2 and 8), and this effect was rescued by the expression of RNF2 (lane 7), prohibitin (lane 3) or various mutation constructs of prohibitin (lanes 4–6).RNF2, RING finger protein 2.

Oncogene Interaction between prohibitin and RNF2 D Choi et al 1721 blotting and by probing with a specific antibody against a Gal4-E2F1 protein.However, as expected, co-transfec- short amino-acid sequence containing phosphorylated tion with Gal4-E2F1 and the E2F1-binding domain Ser 807 and Ser 811 of Rb.As shown in Figure 6a, the deletion mutant of prohibitin (prohibitin D185–214) had level of phosphorylated Rb was reduced in the cells no effect on the transcription induced by Gal4–E2F1. transfected with prohibitin siRNA oligonucleotides. To gain insight into the mechanism underlying the The region spanning amino-acid residues 185–214 of regulation of E2F1 transcriptional activity by prohibitin, prohibitin is the E2F1-binding site and is required for the we analysed the change in E2F1 transcriptional activity repression of E2F1 transcriptional activity.To confirm caused by the knockdown of prohibitin expression whether prohibitin residues 185–214 are required to (Figure 6c).Interestingly, the decreased level of prohibitin repress the transcriptional activity of E2F1, we con- did not drastically change the transcriptional activity of structed E2F1 fused to the Gal4-DNA-binding domain. E2F1.We also tested the transcriptional activity of E2F1 We then examined the effect of prohibitin on the activity in HEK293T cells co-transfected with prohibitin D185–214 of the Gal4-E2F1 protein by transient-transfection and shprohibitin-1.Although prohibitin D185–214, which experiments in HEK293T cells.As shown in Figure 6b, lacks the E2F1 binding domain, was not able to directly the Gal4-E2F1 construct could induce transcription from repress the transcriptional activity of E2F1, it reduced the apFRtrans-Reporter plasmid containing the luciferase p16 protein level that had been increased by prohibitin reporter , but co-transfection with Gal4-E2F1 and knockdown using the transfection of shprohibitin-1.It is prohibitin reduced the transcriptional activity of the important to note that shprohibitin-1 is not able to

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shprohibitin-1 -+++ prohibitin --+- prohibitin 185-214 ---+ Figure 6 Prohibitin regulates the transcriptional activity of E2F1 via the RNF2-p16–CDK4 pathway.( a) The amount of phosphorylated Rb was decreased in MEF cells transfected with siprohibitin-3 or siprohibitin-4.( b) HEK293T cells were co- transfected with Gal4-DBD-E2F1, pFR trans-Reporter plasmids and either an empty vector, wild-type prohibitin or prohibitin D185– 214.The transcriptional activity of E2F1 was decreased when prohibitin was overexpressed, but not when the mutant was expressed. (c) HEK293T cells were transfected with plasmids indicated in the figure.The results are presented as the means of three independent experiments±s.d. **Po0.01 versus the control (Student’s t-test).RNF2, RING finger protein 2.

Oncogene Interaction between prohibitin and RNF2 D Choi et al 1722 knockdown transfected prohibitins, including prohibitin luciferase gene and Gal4-E2F1 were co-transfected into D185–214, because it targets the 30UTR region of HEK293T cells.Unexpectedly, transfection with either prohibitin.The transcriptional activity of E2F1 was the RNF2 shRNA expression vector or RNF2 slightly remarkably increased in HEK293T cells co-transfected altered the transcriptional activity of E2F1 (Figures 7a with prohibitin D185–214 and shprohibitin-1.These and b).However, co-transfection with RNF2 and results suggest that prohibitin regulates the transcrip- prohibitin resulted in a synergistic repression of E2F1 tional activity of E2F1 not only by direct binding to transcriptional activity (Figure 7c), indicating that E2F1, but also through the p16–CDK4–Rb pathway. RNF2 plays roles not only in the regulation of the p16-Rb-E2F1 pathway, but also in the prohibitin- mediated repression of E2F1 activity. RNF2 is recruited to the E2F1-binding promoters and To confirm that RNF2 is involved in the prohibitin- plays a role in the prohibitin-mediated repression mediated repression of E2F1 activity, we investigated of E2F1 transcriptional activity whether RNF2 and prohibitin are co-recruited to the It was previously reported that the p16 expression level E2F1-binding promoters using chromatin immunopre- was increased in RNF2-knockout mice (Voncken et al., cipitations (ChIP).Using anti-prohibitin antibodies, we 2003).We also found that RNF2 interacted with initially immunoprecipitated chromatin from HEK293 prohibitin.We therefore used a luciferase reporter assay cells and performed ChIP PCR with E2F1, TK and p107 to assess whether RNF2 influenced the transcriptional primers, which were previously reported to be prohibi- activity of E2F1 through the p16–CDK4–Rb pathway. tin-recruiting promoters (Wang et al., 2002). As The pFR trans-Reporter plasmids containing the expected, prohibitin was recruited to the promoters

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Figure 7 The overexpression of RNF2 increases the prohibitin-mediated repression of E2F1 transcriptional activity.( a) HEK293T cells were co-transfected with Gal4-DBD-E2F1, pSV-b-Gal plasmid, pFR trans-luciferase Reporter plasmid and either shRNF2 or an empty vector.pSV- b-Gal plasmids were used to normalize the transfection efficiency.The relative luciferase activity was calculated by comparison to the empty vector, which was arbitrarily set to 100.( b) Increasing amounts of the RNF2 expression vector were co- transfected into the cells.( c) The cells were co-transfected with empty, prohibitin or both prohibitin and RNF2 vectors.The results are presented as the means of three independent experiments±s.d. **Po0.05 versus the prohibitin overexpressed samples (Student’s t-test).( d) ChIP experiments were performed with antibodies for prohibitin or RNF2, and the resulting immunoprecipitates were amplified with primer pairs corresponding to the genes shown.Both proteins were recruited at the indicated promoters.ChIP, chromatin immunoprecipitation; RNF2, RING finger protein 2.

Oncogene Interaction between prohibitin and RNF2 D Choi et al 1723 tested (Figure 7d, left panel).We also performed ChIP expression of E2F1 in quiescent cells results in S phase using anti-RNF2 antibody and ChIP PCR using the entry.This study showed that the transcriptional same primers.As shown in Figure 7d (right panel), activity of E2F1 was increased in cells co-transfected RNF2 also associated with the tested promoters, with prohibitin D185–214 and the shprohibitin-1 ex- suggesting that RNF2 and prohibitin are co-recruited pression vector and that it was decreased in cells co- to the E2F1-binding promoters. transfected with prohibitin and RNF2.To determine whether cell proliferation is affected by the interaction The change in cell proliferation by the interaction with between RNF2 and prohibitin, we investigated cell RNF2 and prohibitin proliferation using a colony formation assay after E2F1 regulates the expression of genes involved in the transfection with vectors encoding RNF2, prohibitin, cell cycle and proliferation, and thus, the exogenous prohibitin D185–214 and/or shprohibitin-1, as indicated

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Figure 8 The interaction between RNF2 and prohibitin regulates cell growth.HEK293 cells transiently transfected with the indicated vectors were seeded in a 100-mm dish and cultured in a G418-supplemented medium.After 15 days, G418-resistant colonies were stained with methylene blue dye, and colonies were counted.The results are expressed as the means of three independent experiments±s.d. (a) The cell growth rate was more increased in cells co-transfected with shprohibitin-1 and prohibitin D185–214 than in cells transfected with shprohibitin-1 alone.** Po0.05 versus the shprohibitin-1 alone (Student’s t-test).( b) Cell growth was dramatically repressed in the cells co-transfected with prohibitin and RNF2.** Po0.01 versus the prohibitin alone (Student’s t-test). RNF2, RING finger protein 2.

Oncogene Interaction between prohibitin and RNF2 D Choi et al 1724 in Figure 8.The proliferation rate was higher in cells co- transfected with prohibitin D185–214 and siprohibitin-1 than in cells transfected with shprohibitin-1 alone or with the control vector (Figure 8a).However, the rate of proliferation was much lower in cells co-transfected with prohibitin and RNF2 than in cells transfected with just the control vector, RNF2 or prohibitin (Figure 8b).In addition, Supplementary Figure S3 showed that the reduced number of colony formation was not caused by apoptosis.Taken together, these results demonstrate that the rate of cell proliferation is also influenced by the interaction between RNF2 and prohibitin.

Discussion

Prohibitin has been known to repress the transcriptional activity of E2F1 by physically interacting with E2F1. Our study shows that RNF2 is involved in the direct repression of the transcriptional activity of E2F1. Alternatively, Rb represses the transcriptional activity of E2Fs1–3 through the recruitment of histone deacetyl- ase 1, DNA methyltransferase and BrG1/Brm (Zhu, Figure 9 Mechanisms of E2F1 activity regulation.Prohibitin and 2005).At the beginning of the G1 phase of the cell cycle, RNF2 regulate the activity of E2F1 via dual pathways.( a) Direct Rb exists in a non-phosphorylated state, which allows pathway and (b) indirect pathway.RNF2, RING finger protein 2. the protein to bind E2F1, resulting in the inactivation of E2F1.The Cdk4/6–cyclin D complexes phosphorylate Rb, which dissociates Rb from the transcriptional We hypothesized that the E2F1-binding domain- activator E2F1, leading to the activation of E2F1.The mutated prohibitins might be tumorigenic because p16 protein binds to Cdk4/6 and blocks the interaction mutations in the E2F1-binding site would inhibit the between Cdk4/6 and cyclin D.Our results presented in interaction between prohibitin and E2F1, but the Figure 4 show that decreases in prohibitin result in the expression level of p16 would be maintained, as was upregulation of p16 expression, suggesting that prohi- shown in our study.In previous reports, we found cases bitin might indirectly regulate the transcriptional of sporadic breast cancer caused by mutations in the activity of E2F1 through the p16–CDK4–Rb pathway. E2F1-binding site (Sato et al., 1993). This case study Thus, we demonstrated that prohibitin and RNF2 also supports the finding that RNF2 and prohibitin regulate the function of E2F1 by dual pathways: the regulate E2F1 function by dual pathways. direct, prohibitin-mediated pathway and the indirect, p16-mediated E2F1 pathway of transcriptional regula- tion (Figure 9). Materials and methods Co-transfection with shprohibitin-1 and prohibitin D185–214, which lacks the E2F1-binding domain, would Plasmids, cell culture and transfection decrease the amount of prohibitin that directly represses Details are given in Supplementary Information. the transcriptional activity of E2F1, but would not change the levels of the p16 protein, resulting in an RNA interference of RNF2 and prohibitin increase in the transcriptional activity of E2F1.It is For vector-based RNF2 and prohibitin RNAi, 53 bp oligo- important to note that shprohibitin-1 is not able to nucleotide duplexes containing sequences specific to a portion knockdown transfected prohibitins, including prohibitin of the RNF2 or prohibitin coding sequences were cloned into D185–214, because it targets the 30UTR region of the pSilencer1.0-U6 shRNA expression vector according to prohibitin.However, transfected prohibitin DNA does the manufacturer’s instructions (Ambion, Austin, TX, USA). Details are submitted as Supplementary Information. not contain the 30UTR region.To prove this hypothesis, we tested the transcriptional activity of E2F1 in HEK293T cells co-transfected with prohibitin D185–214 Quantitative-reverse transcription–PCR Details on the method of quantitative-reverse transcription– and shprohibitin-1.Figures 5b and 6c show that the p16 PCR are given in Supplementary Information. protein levels were not changed and that the transcrip- tional activity of E2F1 was remarkably increased in Protein half-life experiments HEK293T cells co-transfected with prohibitin D185–214 We performed protein half-life experiments using a modifica- and shprohibitin-1.These results support our hypothesis tion of previously published methods (Kim et al., 2004). At that the decrease in prohibitin might downregulate 48 h after transfection, HEK293 cells were treated with the transcriptional activity of E2F1 through the p16– 40 mgmlÀ1 of cycloheximide (Sigma, St Louis, MO, USA) for CDK4–Rb pathway. the indicated durations.The cells were harvested and lysed.

Oncogene Interaction between prohibitin and RNF2 D Choi et al 1725 The extracts were analysed by western blot and probed with Colony formation assay the anti-RNF2 antibody. HEK293 cells (1 Â 105) transiently transfected with a control vector, prohibitin, RNF2, prohibitin D185–214 or Luciferase reporter assays an shprohibitin expression vector, were seeded in a 100-mm Details regarding luciferase reporter assays are given in dish and cultured in G418 (3 mg mlÀ1)-supplemented Supplementary Information. medium (DMEM plus 8% FBS).After 15 days, G418-resistant colonies were counted using methyl blue staining (Yoo Co-immunoprecipitation et al., 2005). Details are given in Supplementary Information. Acknowledgements Chromatin immunoprecipitations We performed ChIP using a modification of previously This work was supported by the Korea Research Foundation published methods (Shang et al., 2000). Details are submitted grant funded by the Korean Government (MOEHRD) (KRF- as Supplementary Information. 2005-C00350) and Korea University (K0401401).

References

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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