Degradation of Human RAP80 is Cell Cycle Regulated by Cdc20 and Cdh1 Ubiquitin Ligases

This article is featured in Highlights of This Issue, p. 571

Successful cell division occurs as a result of a well-ordered serial series of events called the cell cycle, which include DNA replication, spindle assembly, nuclear division, and cytokinesis. The eukaryotic cell cycle is tightly regulated by cyclin and cyclin-dependent kinase (Cdk) complexes, which are key regulatory complexes in cell-cycle progression. The Cdk4-cyclin D complex promotes initiation of the cell cycle, and Cdk2-cyclin E promotes cell-cycle progression from the G₁ to S phase (G₁–S transition). The Cdk1-cyclin B complex initiates the G₂–M transition (1). Another key regulatory mechanism of cell-cycle progression is ubiquitination/proteasome-mediated proteolysis of cell-cycle machinery. The Skp1/Cullin/F-box protein complex (SCF) and anaphase-promoting complex/cyclosome (APC/C) are 2 major E3 ligases involved in the regulation of cell-cycle progression. Although SCF mainly regulates the expression levels of many proteins during S phase, APC/C regulates the expression of proteins involved in the progression from mitosis to late G₁ (2, 3). Two different activators, Cdc20 and Cdh1, are required for the activation of APC/C and have specificity for substrates regulating mitotic cell-cycle progression. In the early mitotic phase, APC/C is activated by binding to Cdc20, and in the late mitotic and G₁ phase, Cdc20 is replaced by Cdh1 (4, 5).

Receptor-associated protein 80 (RAP80) plays an important role in signal transduction in the DNA damage response by recruiting the BRCA1-A complex to DNA damage sites in a lysine 63-mediated polyubiquitin-binding dependent manner (6–14). The BRCA1-A complex contains many subunits, including a coiled coil domain-containing protein (CCDC98/ABRAXAS), the BRCA1/BRCA2-containing complex subunit 45/brain and reproductive organ-expressed protein (BRCC45/BRE), the BRCA1/BRCA2-containing complex subunit 36 (BRCC36), mediator of RAP80 interactions and targeting subunit of 40/new component of the BRCA1-A complex (MERIT40/NBA1), and breast cancer 1 (BRCA1; refs. 6–14). This recruitment is required for the activity of BRCA1 at the DNA damage checkpoint and for DNA repair.

In this study, we show that the expression levels of RAP80 peak in the G₂ phase and early mitosis and decline during mitotic exit and progression into the G₁ phase. Fluctuating RAP80 protein during the cell cycle is dependent on the APC/C-ubiquitin-proteasome pathway. We also show that D box1, conserved in several mammalian species and located in the central region of RAP80, is involved in regulating RAP80 stability through ubiquitination during mitosis. Thus, we can conclude that the RAP80 protein is a target of the APC/C-ubiquitin-proteasome pathway and undergoes proteolysis during anaphase through the G₁ phase to allow for proper cell-cycle progression.

The SFB-RAP80 and Myc-ubiquitin expression vectors (Myc-Ubi) were previously described (6, 9). The small interfering RNA (siRNA) resistant RAP80 expression plasmid was generated by site-directed mutagenesis using the forward primer 5′-GGAACCTGGAGAAAAAGACGTTGAGACCACGAGTTCTGTCAGTG-3′ and reverse primer 5′-CACTGACAGAACTCGTGGTCTCAACGTCTTTTTCTCCAGGTTCC-3′. RAP80 Dbox1, 2, 3, or 4 deletion mutants were generated by site-directed mutagenesis and subcloned into a SFB-tagged mammalian expression vector. The HA-tagged Cdh1 expression plasmid was cloned into a HA-tagged mammalian expression vector. The Flag-Cdc20 expression vector was previously described (15).

HeLa and human embryonic kidney (HEK) 293T cell lines were purchased from American Type Culture Collection, and mouse embryonic fibroblasts (MEF) were previously described (9). The cell lines were maintained in Dulbecco modified Eagle's medium (Invitrogen) supplemented with 10% FBS (Invitrogen) and penicillin/streptomycin (100 IU/mL and 100 μg/mL, respectively) at 37°C in an atmosphere containing 5% CO₂ (v/v).

Control, RAP80 (6), Cdc20 (16), and Cdh1 siRNAs (17) were previously described. siRNAs were transfected into cells using Oligofectamine (Invitrogen) according to the manufacturer's instructions.

Anti-RAP80 antibody was previously described (9). Rabbit RAP80 polyclonal antibody was affinity purified using the Sulfolink Plus Immobilization and Purification Kit (Pierce). Anti-Flag, anti-HA, anti-Myc, and anti–β-actin antibodies were purchased from Sigma-Aldrich, and human cyclin E1, cyclin A2, cyclin B1, Cdc20, and Cdh1 antibodies were purchased from Cell Signaling Technology. Transient transfection was carried out using the Fugene 6 reagent (Roche Applied Science). For immunoprecipitation, cells were washed with ice-cold phosphate-buffered saline (PBS) and then lysed in NETN buffer [0.5% Nonidet P-40, 20 mmol/L Tris (pH 8.0), 50 mmol/L NaCl, 50 mmol/L NaF, 100 μmol/L Na₃VO₄, 1 mmol/L dithiothreitol, and 50 μg/mL phenylmethanesulfonylfluoride] at 4°C for 10 minutes. Crude lysates were cleared by centrifugation at 14,000 rpm at 4°C for 5 minutes, and supernatants were incubated with protein A agarose–conjugated primary antibodies. The immunocomplexes were washed 3 times with NETN buffer and subjected to PAGE. Western blotting was conducted using the antibodies indicated in the figure legend.

The establishment of stable cell lines was previously described (9). To establish cell lines stably expressing epitope-tagged proteins, HeLa cells were transfected with plasmids encoding RAP80 wild type (WT), or RAP80Dbox1 and puromycin-resistant protein. Forty-eight hours after transfection, the cells were split at a 10:1 ratio and cultured in medium containing puromycin (10 μg/mL) for 3 weeks. Individual puromycin-resistant colonies were isolated and screened by Western blotting for expression of the RAP80 protein.

Cells were synchronized at late G₁ phase with the thymidine double blocking method (18). Briefly, the cells were plated in 100 mm diameter Petri dishes and thymidine was added to a final concentration of 2 mmol/L after cell adherence. The cells were cultured for 16 hours. After removal of the thymidine and incubation for 10 hours in fresh medium, thymidine was added to a final concentration of 2 mmol/L for an additional 16 hours. After removal of thymidine, synchronized cells were cultured in fresh medium and collected at different times for cell-cycle analysis and Western blotting. Cells were synchronized in prometaphase with 17 hours of nocodazole treatment and then released into fresh medium for further incubation.

The double thymidine or nocodazole synchronized cells were collected at different times after release from a G₁–S boundary. After washing twice with PBS, cells were fixed with chilled 70% alcohol at 20°C for 24 hours. The fixed cells were collected by centrifugation (2,000 rpm; 5 minutes), washed twice with PBS, incubated with RNaseA (30 mg/mL) for 30 minutes at 37°C, stained with 50 μg/mL propidium iodide (Sigma-Aldrich) for 30 minutes at room temperature, and then analyzed by flow cytometry.

To investigate whether RAP80 expression level is cell cycle regulated, we synchronized human HeLa cells and MEFs at the G₁–S boundary with a double thymidine block; after release from this block, we harvested cells at the indicated time points (Fig. 1A and B). This analysis showed that the expression level of RAP80 was the highest in the G₂–M phase and declined during mitosis and progression into the G₁ phase. This may be consistent with a specific role of the RAP80 protein in the G₂–M phase and also may indicate that the RAP80 protein is degraded during mitosis and the G₁ phase. Cyclin B1 expression levels and flow cytometric analysis confirmed cell-cycle progression (Fig. 1A and B). We further confirmed the changes in RAP80 expression levels at different stages of the cell cycle. Low levels of RAP80 expression were observed when HeLa cells were in the G₁–S boundary and mitotic (M) phase (Fig. 1C). Elevated cyclin E1 expression, elevated cyclin A2 expression, or Ser10 phosphorylation of histone H3 (a marker of chromosome condensation) was observed for each respective stage and flow cytometric analysis confirmed cell-cycle progression. RAP80 expression levels during mitotic progression were also examined. HeLa cells arrested in prometaphase with nocodazole (Fig. 1D) or taxol (data not shown) were collected by shake-off and replated to allow progress through mitosis, and RAP80 protein levels were checked at different time points. RAP80 protein levels gradually decreased over time during mitosis and the G₁ phase.

Given the above findings, we were prompted to investigate whether RAP80 degradation could be regulated by the ubiquitin-proteasome pathway. Overexpressed RAP80 was polyubiquitinated in HEK 293T cells (Fig. 2A and B). We also investigated whether the ubiquitin-proteosome system was involved in regulating RAP80 levels in HeLa cell lines. HeLa cells incubated in the presence of the proteosome inhibitor MG132 were collected at indicated times and endogenous RAP80 expression levels were assessed by immunoblot analysis. RAP80 expression levels steadily increased in the presence of MG132 (Fig. 2C).

Many mitotic regulatory proteins and cyclins are degraded by the APC/cyclosome^Cdc20 (C^Cdc20) and/or the APC/C^Cdh1 complexes during mitosis and the G₁ phase. In addition, RAP80 downregulation in mitosis and the G₁ phase led us to test whether RAP80 degradation is dependent on Cdc20 or Cdh1. To show whether Cdh1 and Cdc20 are required for RAP80 degradation in vivo, we used siRNAs to reduce their expression and the transfected cells were arrested at a mitotic phase with nocodazole. Following the removal of nocodazole, control or Cdc20 siRNA-transfected cells were collected at the indicated times to detect RAP80 protein levels and cell-cycle progression was confirmed by flow cytometric analysis. In control siRNA-transfected cells, RAP80 levels decreased by 1 hour following nocodazole removal, which may correspond to anaphase entry, and further decreased by 3 hours following nocodazole removal, which may correspond to mixed late mitotic and G₁ phase (Fig. 3A). In contrast, RAP80 protein accumulated in mitotically synchronized HeLa cells transfected with Cdc20 siRNA up to 3 hours following nocodazole removal (Fig. 3A). Although this may seem to result in a delay into mitotic and G₁ phase, flow cytometry analysis shows that Cdc20 knockdown cells were in mixed mitotic and G₁ phase at 3 hours following nocodazole removal. Thus, our data suggest that Cdc20 is important for regulating the accumulation of RAP80 protein during mitotic progression. This accumulation in mitosis seems to be caused by RAP80 stabilization, as shown by measuring the RAP80 half-life (Fig. 3B). We also checked the effect of Cdh1 on RAP80 degradation. Control (Con) or Cdh1 siRNA-transfected HeLa cells were arrested in prometaphase with nocodazole, washed, and released. As Cdh1 is mainly activated in late mitosis and the G₁ phase, we checked RAP80 expression and turnover after 1 hour postrelease. As shown in Fig. 3C, RAP80 protein accumulated in mitotically synchronized HeLa cells transfected with Cdh1 siRNA compared with cells transfected with control siRNA. This accumulation in mitosis and the G₁ phase seems to be caused by RAP80 stabilization, as shown by measuring the RAP80 half-life (Fig. 3D). We also observed RAP80 degradation upon Cdc20 or Cdh1 overexpression in HEK 293T cells. In addition, treatment with MG132 inhibited Cdc20- or Cdh1-mediated RAP80 degradation (Fig. 3E). Furthermore, overexpressed Cdc20 or Cdh1 increased RAP80 polyubiquitination in HEK 293T cells (Fig. 3F and G). Thus, the data indicate that Cdc20 and Cdh1 stimulate APC/C-mediated ubiquitination and degradation of RAP80.

Many proteins containing a destruction box (D box) are degraded by the APC/C^Cdc20 and APC/C^Cdh1 complexes. The D box consensus sequence consists of RxxLxxxxN/E/D (x, any amino acid), although most substrates only contain the minimal RxxL motif. The RAP80 protein has 4 putative minimal RxxL motifs (D box1-4) that potentially serve as D boxes (Fig. 4A and B), but does not have any APC/C^Cdc20 and APC/C^Cdh1 complex recognition motifs, such as the KEN box or A box. As only the putative D box1 among these 4 putative D boxes is conserved in most mammalian species, including human, chimpanzee, monkey, dog, rat, and mouse (Fig. 4C and data not shown), we investigated the importance of the putative D box1 on the regulation of RAP80 ubiquitination and degradation. As shown in Fig. 4D and E, ubiquitination of the D box1 deletion mutant of RAP80 (SFP-RAP80Dbox1) was reduced compared with the wild type (SFP-RAP80). In addition, we further showed the dependence of Cdc20- or Cdh1-mediated RAP80 ubiquitination on the D box1 (Fig. 4F and G). Next, we checked whether the D box1 deletion mutant was resistant to degradation during mitosis and the G₁ phase. HeLa cells expressing wild-type SFP-RAP80, SFP-RAP80Dbox1, 2, 3, or 4 were arrested in prometaphase with nocodazole, collected by shake-off, and replated to allow progress through mitosis and G₁ phase. Subsequently, RAP80 protein levels were checked at different time points (Fig. 4H). Similar to endogenous RAP80, wild-type RAP80, SFP-RAP80Dbox2, 3, and 4 were degraded after nocodazole release. In contrast, the RAP80D box1 deletion mutant remained stable throughout anaphase and progression into the G₁ phase. These data indicate that D box1 is important for RAP80 degradation and ubiquitination by the APC/C^Cdc20 and APC/C^Cdh1 complexes.

The data in this article show that Cdc20 and Cdh1 are involved in mediating RAP80 ubiquitination and degradation. These data led us to check the possibility of an association between RAP80 and Cdc20 or Cdh1. Overexpressed RAP80 protein associated with endogenous Cdc20 or Cdh1 protein (Fig. 5A and B). To further show the role of the D box1 in binding between RAP80 and Cdc20 or Cdh1, we checked the ability of the RAP80Dbox1 deletion mutant to bind to Cdc20 or Cdh1. Compared with wild-type RAP80, the D box1 deletion mutant did not coimmunoprecipitate with Cdc20 or Cdh1 using overexpressed HEK 293T cells (Fig. 5C and D). These data indicate that RAP80 is a direct target of Cdc20 and Cdh1 and is degraded by the ubiquitination/proteosome pathway in mitosis.

We then investigated the biological significance of RAP80 protein during cell-cycle progression. The downregulation of RAP80 in mitosis and the G₁ phase of the cell cycle led us to examine the requirement of RAP80 for cell-cycle progression. We first generated a RAP80 wild-type expression plasmid resistant to the effects of RAP80 siRNA (R-RAP80) tagged with Flag and HA (Fig. 6A) to identify RAP80 functions in cell-cycle progression. Next, HeLa cell lines stably expressing control or R-RAP80 were generated (Fig. 6B). Using these stable cell lines, we examined whether RAP80 overexpression is important for mitotic cell-cycle progression. The established stable HeLa cell lines expressing control or R-RAP80 (Fig. 6B) were transfected with RAP80 siRNA to reduce the effects of endogenous RAP80 protein. When mitotic progression was examined using synchronized cells after release from a double thymidine block, mitotic delay was apparent in R-RAP80-overexpressing HeLa cells compared with control-expressing HeLa cells (Fig. 6C). To check whether the RAP80Dbox1 deletion mutant has any effect on mitotic progression, we established 2 different new R-RAP80- or R-RAP80Dbox1-expressing HeLa cells, which showed different expression levels (Fig. 6D). When mitotic progression was examined using synchronized cells after release from a double thymidine block, mitotic delay was apparent in R-RAP80Dbox1–expressing HeLa cells compared with R-RAP80–expressing HeLa cells in an expression-dependent manner (Fig. 6E). These data suggest that preventing the degradation of RAP80 is sufficient for mitotic progression. In addition, the RAP80Dbox1 deletion mutant remained stable throughout the cell cycle, indicating the importance of the putative D box1 motif for regulating degradation of RAP80 during the cell cycle (Fig. 6F). Next, we checked whether this mitotic function of RAP80 is distinctly different from its role in DNA damage response. The amount of γ-H2AX was determined throughout cell cycle. R-RAP80-2 or R-RAP80Dbox1-2 cells were synchronized at G₁–S by a double thymidine block and released into the cell cycle. At different times after the release from thymidine, the amount of γ-H2AX (an index of DNA DSBs) was determined with Western blots. As shown in Fig. 6G, no differences in the level of γ-H2AX were detected throughout the cell cycle. Because nocodazole has been reported to either cause DNA damage (19) or not to cause DNA damage (20), immunofluorescence microscopy staining for γH2AX foci (an index of DNA DSBs) was carried out. Nocodazole did not induce DNA damage at the dose used (Fig. 6H). This data indicated that overexpression of wild type or RAP80Dbox1 deletion mutant does not induce DNA damage. Therefore, RAP80-mediated delay of mitotic progression is induced in the absence of DNA damage. Initiation of anaphase and mitotic exit are dependent on active APC/C E3 ligase complex. To determine whether RAP80-mediated inhibition of mitotic progression was due to the inactivation of APC/C, we compared the protein levels of known APC/C substrates (cyclin A, cyclin B1, and securin) using control, wild type (R-RAP80-2), or RAP80Dbox1 deletion mutant (R-RAP80Dbox1-2)-stable cell lines synchronized with nocodazole. As expected, nocodazole-synchronized wild type or RAP80Dbox1 deletion mutant-stable cell lines expressed high levels of cyclin A, cyclin B1, and securin compared with control-stable cell lines (Fig. 6I).

In this study, we investigated the regulatory mechanism of RAP80 ubiquitination and degradation during cell-cycle progression and showed an unexpected role of RAP80 in the regulation of mitotic progression. Our results show that regulation of RAP80 expression is dependent on cell-cycle progression. RAP80 is a target molecule of APC/C^Cdc20 and APC/C^Cdh1 for degradation during mitosis and the G₁ phase.

A single conserved D box1 (RHCLTPLAD), in which deletion prevented RAP80 polyubiquitination and degradation, was identified in the middle region of RAP80 (254–262 amino acids). In addition, RAP80 bound to Cdc20 and Cdh1 through its D box1. Thus, this posttranslational modification of RAP80 seems to be a novel mechanism that regulates RAP80 stability and functions in mitotic cell-cycle progression. RAP80 D box1 is not required for translocation to DNA damage sites (data not shown) and no different level of phosphorylation of H2AX was detected throughout cell cycle using control, wild type, or RAP80Dbox1 deletion mutant-stable cell lines. These data indicate that overexpression of wild type or RAP80Dbox1 deletion mutant does not induce DNA damage and might regulate the mitotic progression independent of its involvement in the DNA damage response. Nocodazole-synchronized wild type or RAP80Dbox1 deletion mutant-stable cell lines expressed high levels of cyclin A, cyclin B1, and securin, which are the substrates of APC/C complexes, compared with control-stable cell lines. So, the RAP80-mediated inhibition of mitotic progression seems to be caused by the inactivation of APC/C. However, the inhibitory mechanism may not be the competition of RAP80 to bind to Cdc20 or Cdh1 with its substrates because cyclin A, cyclin B1, and securin also highly expressed levels in RAP80Dbox1 deletion mutant-stable cell lines. So, we cannot exclude the other possibility that RAP80 regulates the activity of upstream molecules of Cdc20 and Cdh1. Elevated Cdk1 activity triggered mitotic entry and Cdk1 inactivation (21), and the dephosphorylation of a subset of Cdk1 substrates (22) requires mitotic exit. Cdk1 participates in the activation of the APC/C, which is required for the metaphase–anaphase transition (21, 23). Recently, we obtained evidence that RAP80 binds to Cdk1 (unpublished data). It is possible that RAP80 may inhibit the Cdk1 activity to phosphorylate the substrate to be required for the metaphase–anaphase transition. However, the exact reason why RAP80 degradation is an essential step for mitotic cell-cycle progression is not clear. Presently, RAP80 protein levels fluctuated during the cell cycle. Its expression level peaked at the G₂ phase and declined during mitosis and progression into the G₁ phase. In addition, overexpressed and nondegradable RAP80 delays mitotic cell-cycle progression. These data may indicate that elevated RAP80 during the G₂ phase functions to inhibit the G₂–M transition and RAP80 inhibition of the G₂–M transition may be blocked to resume progression through the cell cycle. The degradation of RAP80 by APC/C^Cdc20 and APC/C^Cdh1 may be the major mechanism to progress mitotic cell cycle.

In summary, the decline in human RAP80 expression levels following entry into mitosis is regulated by the APC-ubiquitin-proteosome pathway during mitosis and the G₁ phase. Overexpressed RAP80 inhibits mitotic cell-cycle progression. It will be interesting to investigate whether other modifications of RAP80 are also important for regulating RAP80 stability throughout the cell cycle.

No potential conflicts of interest were disclosed.

Conception and design: J.H. Jeong, J. Kwon, H. Kim.

Writing, review, and/or revision of the manuscript: H.J. Cho, J.H. Jeong, J. Kwon, H. Kim.

Administrative, technical, or material support: H.J. Cho, E.H. Lee, S.H. Han, H.J. Chung.

The authors thank members of Dr. Kim's laboratory for helpful discussion and technical support.

This work was supported by a grant from the National R&D Program for Cancer Control, Ministry for Health, Welfare and Family Affairs, Republic of Korea (0920070), Korean Research Foundation grant funded by the Korean government (2009-0067028), and National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST; no. 20110030831).

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