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Previous Article | Next Article 
Molecular and Cellular Biology, January 2004, p. 514-526, Vol. 24, No. 2
0270-7306/04/$08.00+0 DOI: 10.1128/MCB.24.2.514-526.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.
Graduate Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei 100, Taiwan (Republic of China)
Received 10 July 2003/ Returned for modification 21 August 2003/ Accepted 21 October 2003
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ABSTRACT |
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INTRODUCTION |
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In this study, we investigated the molecular mechanism responsible for mitotic hTK1 proteolytsis. It is well established that the ubiquitin-proteasome pathway represents a fundamental mechanism for regulating protein level in the cell cycle (18). Ubiquitinylation of protein substrates is carried out by a series of enzymatic reactions catalyzed by E1, E2, and E3 enzymes (42). The SCF complex (Skp1-Cullin1/CDC53-F box) and the APC/C (anaphase-promoting complex/cyclosome) are two ubiquitin ligases (E3) that play a determining role in targeting proteins for cell cycle-dependent proteolysis (11, 34, 35). The SCF complex recognizes phosphorylated substrates, such as p27, cyclin D, cyclin E, E2F, I
B, and ß-catenin, to promote their degradation in mammalian cells via the so-called F box proteins (16, 22). The interaction between F box protein and Skp1, one subunit of the SCF complex, bridges phosphorylated substrate and F box protein to Cullin1, which organizes this E3 complex and helps recruit E2 ubiquitin-conjugating enzyme through interaction with the Roc1/UBC subunit (16). In contrast to the SCF complex, substrate phosphorylation is not a requisite recognized by the APC/C complex (17, 32, 35). APC/C-mediated ubiquitinylation and degradation require two different activators, Cdc20 and Cdh1 (21, 35, 49). Cdc20-APC/C is activated during mitosis and targets cyclin B (53, 55) and securin (54) for proteolysis through binding to the destruction box (D box; RXXLXXXXN) at their N termini. APC/C-Cdh1 recognizes either a D box or the KEN box (37, 38). To date, human Cdc6, Aurora-A kinase, Cdc20, Cdc25A, and mouse ribonucleotide reductase R2 protein have been found to be the substrates of APC/C-Cdh1 in late mitosis and G0/G1 transition (4, 12, 20, 28, 36).
The C-terminal region of mouse TK1 (mTK1) or hTK1 has been shown to play a crucial role in determining its protein stability during mitosis (25, 46). Other reports, however, have suggested that the capability of substrate binding (47) and the multimerization status of the mTK1 subunit might contribute to the stability of mTK1 protein in a proteasome-independent manner (40). On the other hand, with expression of hTK1 in the yeast systems, we found that serine 13 phosphorylation plays a role in hTK1 degradation, which requires the functional activity of SCF (26). Despite these experimental results, the molecular event that targets hTK1 for mitotic degradation in mammalian cells is still unknown. In this study, we provided the first evidence that in mammalian cells, the ubiquitin-proteasome pathway controls the expression level of hTK1 dependent on the APC/C-Cdh1 E3 ligase, which interacts with hTK1 by recognizing the KEN box located at its C-terminal region. Overall, the results obtained from this study concluded that hTK1 is another new target for APC/C-Cdh1-mediated proteolysis at mitosis in mammalian cells.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Construction of expression plasmids and site-directed mutagenesis. pCDNA3.1-hTK1 was generated by insertion of the EcoRI-XhoI fragment of the pBSK-hTK1 (6) into plasmid pCDNA3.1 (Invitrogen). cDNA encoding hTK1, devoid of the initiation ATG codon, was amplified by PCR with pBSK-hTK1 as the template and subcloned into the EcoRI site of plasmid pCVM2FLAG (Sigma), producing pFLAG-hTK1. The first methionine of hTK1 was deleted to eliminate alternative initiation of FLAG-hTK1 translation from the start codon of hTK1 (8), thus ensuring only the FLAG-hTK1 polypeptide derived from pFLAG-hTK1 is expressed in the cells. The glutathione S-transferase (GST)-fused hTK1 constructs cloned into pGEX-3X expression vectors were created as described previously (6). Mutations of hTK1 were generated by using the Quick-Change site-directed mutagenesis kit (Stratagene) with specific mutated primers. The following amino acid changes were made: the KEN box motif at positions 203 to 205 were mutated into AEN, KAA, and AAA, producing K mt, EN mt, KEN mt, respectively. For construction of retrovectors of hTK1, the SacI-XhoI restriction fragment of FLAG-fused wild-type or KEN box-mutated hTK1 in the corresponding pFLAG-hTK1 plasmid was individually inserted into the retroviral S2 vector (provided by Lih-Hwa Hwang, National Taiwan University, Taipei, Taiwan, Republic of China). pHA-Cdc20, pHA-Cdc6, and pHA-Cdh1 were kindly provided by Kristian Helin (European Institute of Oncology, Milan, Italy) (36). Mouse E1 and Xenopus UbcX expression plasmids were kindly provided by Tim Hunt (ICRF Clare Hall Laboratory, South Mimms, United Kingdom) (53). DNA fragments covering amino acids (aa) 1 to 120 of Cdc20, 1 to 125 of Cdh1, and 1 to 452 of Cullin1 were amplified by PCR with pHA-Cdc20, pHA-Cdh1, and pFLAG-Cullin1 as the templates and were cloned into pCMV2FLAG plasmid to generate pFLAG-Cdc20(1-120), pFLAG-Cdh1(1-125), and pFLAG-Cullin1(1-452). pFLAG-Cullin1(full length) was a gift from Zhen-Qiang Pan (The Mount Sinai School of Medicine, New York, N.Y.) (52). pCS2+ Myc-Cdc20 and pCS2+ Myc-Cdh1 were kindly provided by Marc W. Kirschner (Harvard Medical School, Boston, Mass.) (51). Human cyclin B1 was amplified by PCR from HeLa cDNA and inserted into the BamHI site of plasmid pCDNA3.1, yielding pCDNA3.1-hcyclinB1. pCDNA3.1-p27 was a gift provided by Joan Massague (Howard Hughes Medical Institute, Seattle, Wash.) (39). His-tagged Cdh1 of pET28a-Fzr/Cdh1 (provided by Jan-Michael Peters, Research Institute of Molecular Biology, Vienna, Austria) (27) was subcloned into the pCDNA3.1 vector, yielding pCDNA3.1-His-Cdh1. The DNA sequence of each plasmid used for this study was confirmed by dideoxynucleotide sequencing.
Cell culture, synchronization, FACS analysis, and transfection. LM-TK deficient (LM-TK-) and HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM; Life Technologies) supplemented with 10% fetal bovine serum plus 100 µg of streptomycin per ml and 100 U of penicillin per ml (Life Technologies) at 37°C under 5% CO2. For G2/M arrest, nocodazole (Sigma) was added to subconfluent HeLa and LM-TK- cells at a final concentration of 0.5 µg/ml for 20 h. To obtain early G1-phase cells, mitotic phase-arrested cells were shaken off, washed with phosphate-buffered saline (PBS), and incubated in fresh medium for an additional 3 to 5 h. The synchronized cells were fixed in 70% (vol/vol) ethanol, and the cell cycle profile was examined by fluorescence-activated cell sorter (FACS) analysis with a Becton Dickinson FACScan flow cytometer and CellQuest software. For ectopic expression experiments, cells plated on a 60-mm-diameter dish were transiently transfected with a mixture containing 3 µg of DNA with 18 µg of Lipofectamine (Invitrogene, Life Technologies) according to the manufacturer's instructions.
Protein extraction, Western blotting, and immunoprecipitation. Cell extracts were prepared and Western blotting was performed as described previously (6). Immunoprecipitations were performed as previous described (19). Equal amounts of protein in each cell lysate were incubated with anti-hTK1 antibodies at 4°C for 2 h, and the immunocomplexes were adsorbed onto protein A-Sepharose (Amersham Pharmacia), followed by being washed five times with lysis buffer and eluted with sample loading buffer at 95°C for 5 min.
RNA interference. RNA interference was performed essentially as described previously (12, 13). Duplex small interfering RNA (siRNA) against Cdh1, corresponding to nucleotide sequence 5'-AATGAGAAGTCTCCCAGTCAG-3', was kindly provided by Chi-Ying F. Huang (National Health Research Institute, Taipei, Taiwan, Republic of China). Transfection of siRNA was carried out with Oligofectamine (Invitrogene; Life Technologies) according to the manufacturer's instructions.
Expression and purification of recombinant proteins. His-tagged mouse E1and Xenopus UbcX were expressed in Escherichia coli BL21 (DE3) and were purified under native condition with Ni-nitrilotriacetic acid (NTA) beads (Qiagen). His-tagged Cdh1 protein was purified from a stable His-Cdh1-expressing LM-TK- cell line, which was established by transfection with pCDNA3.1-His-Cdh1, followed by G418 selection. The wild type and three KEN box mutants of GST-hTK1 were expressed in E. coli JM109 and purified by glutathione 4B beads (Amersham Pharmacia) as described previously (5).
Pulse-chase experiment. For metabolic labeling, cells were washed with PBS twice and incubated in 5 ml of methionine-free DMEM for 1 h of deprivation. Cells were then incubated in 2 ml of fresh methionine-free DMEM containing dialyzed 10% fetal bovine serum (FBS) and [35S]methionine (500 µCi; Amersham Pharmacia) for 30 min. To terminate labeling, complete DMEM was added for chasing prior to harvesting at the indicated time points. Immunoprecipitation of hTK1 was performed as described above, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and autoradiography.
Retrovirus-mediated expression of Flag-tagged hTK1 in HeLa cells. S2 retroviral vectors expressing Flag-tagged wild type or KEN box mutants of hTK1 were individually transfected to the packaging cell line PT67. Stable clones were selected in the medium supplemented with G418 antibiotic (400 µg/ml). Following propagation of each clone, viral supernatant from the individual culture was collected and stored at -80°C. To infect HeLa cells on a 6-cm-diameter dish, 3 ml of viral supernatant mixed with 5 µg of Polybrene per ml (Sigma) was incubated with cells for 48 h. Afterwards, HeLa cells were kept in the medium containing G418 for further selection. Each stable clone was propagated and confirmed by detection of FLAG-tagged hTK1 in the cell lysates by Western blotting with anti-hTK1 or anti-FLAG antibodies.
In vitro transcription and translation, in vitro binding assay, and GST pull-down assay. An in vitro transcription and translation coupling system (TNT; Promega) using plasmids was employed to prepare [35S]methionine-labeled in vitro-translated proteins. For in vitro binding assay, the cold in vitro-translated proteins were prepared with unlabeled methionine. The in vitro binding assay was performed as described in previous studies (37). Cold in vitro-translated Myc-tagged proteins were bound to anti-Myc antibody-adsorbed beads. After the adsorption, 35S-labeled substrates were added to the binding buffer (50 mM HEPES [pH 7.7], 50 mM NaCl, 1 mM MgCl2, 1 mM EDTA, 1-mg/ml bovine serum albumin, 0.2% Tween 20). Following 2 h of incubation on the rotator at 4°C, samples were washed four times with the binding buffer and eluted with an SDS-PAGE sample loading buffer at 95°C for 5 min. GST pull-down assays were performed as described previously (19).
Preparation of cell extract for in vitro ubiquitinylation. Early G1 cell extract was prepared as previously described (3). After washing twice with PBS and once with a hypotonic buffer (20 mM HEPES [pH 7.5], 5 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol [DTT]), cells were resuspended in a hypotonic buffer supplemented with a protease inhibitor cocktail (Sigma) and lysed by Dounce homogenization. After removal of cell debris by centrifugation at 2,000 x g, the supernatants were then recentrifuged at 14,000 x g at 4°C for 30 min to collect the clear cytoplasmic extracts, and aliquots of the extract were snap-frozen in liquid N2 and stored in 10% glycerol at -80°C.
Immunodepletion and purification of APC/C. Immunodepletion of the SCF complex and APC/C from cell extracts by using anti-Skp1 and anti-Cdc27 antibodies, respectively, was performed as described previously (38, 48). Ten micrograms of antibody was preadsorbed to 15 µl of protein A-Sepharose at 4°C for 2 h and then mixed with 40 µl of cell extracts for 1 h. Beads were removed by high-speed centrifugation, and supernatants were collected for in vitro assays.
For purification of HeLa cells mitotic APC/C, mitotic cell extracts were prepared and immunoprecipitated with Cdc27 antibody covalently linked to protein A-Sepharose by dimethylpimelimidate (Sigma) as described previously (15, 50). The Cdc27 antibody-linked beads were incubated with 5 volumes of extracts for 2 h at 4°C and then removed by centrifugation. The beads were further washed three times with XB buffer (10 mM HEPES [pH 7.7], 1 mM MgCl2, 100 mM KCl, 1 mM DTT, 0.1 mM CaCl2, 0.2 µM okadaic acid) supplemented with 400 mM KCl and 0.5% NP-40 and four times with XB buffer. After removal of the residual washing buffer, the APC/C beads were used for in vitro ubiquitinylation experiments.
In vitro ubiquitinylation. In vitro ubiquitinylation was carried out by incubating cell extracts with 1 µl of 35S-labeled hTK1 in a reaction mixture (20 µl) containing an energy regeneration system (2 mM ATP, 40 mM phosphocreatine, and 80-µg/ml creatine kinase), 1 µM ubiquitin aldehyde, and LLnL as described previously (30) at 30°C. The ubiquitinylated forms of hTK1 were analyzed by SDS-PAGE prior to autoradiography.
Experiments involving reconstitution of in vitro ubiquitinylation were performed as described previously (14). The APC/C beads were activated by incubation with in vitro-translated Cdc20 or Cdh1 for 1 h at room temperature. The activated APC/C beads were then washed four times with XB buffer. Each ubiquitinylation assay was performed in a total volume of 5 µl. The reaction mixture contained an energy regeneration system, 1 µM ubiquitin aldehyde, 1.25-mg/ml ubiquitin, 200-µg/ml mouse E1, 100-µg/ml Xenopus UbcX, 100 ng of substrate, and 3 µl of APC/C beads. All reaction mixtures were incubated at 37°C for 1 h, after which, the mixtures were analyzed by 5 to 15% polyacrylamide gradient SDS-PAGE and Western blotting.
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RESULTS |
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APC/C-Cdh1-dependent degradation of human TK1. Because SCF and APC/C complexes are two major E3 ligases targeting substrates for cell cycle-dependent proteolysis, we then determined which of these two E3 complexes is necessary for mitotic degradation of hTK1. It has been shown that mutant proteins containing an amino-terminal 125 aa of Cdh1 and 120 aa of Cdc20 are potential dominant-negative mutants for APC/C-mediated protein degradation (37), and these mutants indeed inhibit the proteolysis of their corresponding APC/C substrates in vivo (51). On this basis, we transfected HeLa cells with the expression vector of a dominant-negative mutant of Cdc20 or Cdh1. Following transfection, cells were synchronized by nocodazole treatment with the subsequent release from mitotic block. We found that overexpression of the dominant-negative Cdh1 mutant clearly blocked the degradation of endogenous hTK1 during the mitotic exit phase, whereas the Cdc20 mutant had little effect on hTK1 stabilization (Fig. 2A). All cells were cotransfected with pEGFP, whose expression levels under this experimental condition were used as an internal control to indicate similar transfection efficiency in different transfected cells. Since LLnL-sensitive degradation of hTK1 at mitosis was abrogated in those cells expressing the dominant-negative form of Cdh1, it is evident that the functional activity of APC/C-Cdh1 complex is necessary for proteasome-dependent hTK1 at mitosis.
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We then further tested the effect of depletion of endogenous Cdh1 on degradation of hTK1 in asynchronous and G1-phase cells by duplex siRNA. As shown in Fig. 2D, transfection with duplex siRNA against Cdh1 resulted in reduction of endogenous Cdh1 protein expression, which was accompanied with increasing accumulation of hTK1 after 24 to 48 h of transfection. To examine the effect of Cdh1 depletion on degradation of hTK1 in the G1 phase, HeLa cells were arrested in the G1/S phase by thymidine block and released for cell cycle progression. Four hours before the second round of thymidine block, cells were transfected with duplex siRNA against Cdh1. After double-thymidine blocks, cells were synchronized at M phase by nocodazole treatment and released to G1 progression as shown in Fig. 2E (upper panel). As expected, endogenous Cdh1 was significantly depleted in the cells with siRNA transfection at the M and G1 phases (Fig. 2E, lower panel). The expressed levels of hTK1 were increased at M phase regardless of siRNA transfection. In contrast, following G1 progression, depletion of endogenous Cdh1 clearly caused accumulation of hTK1 in the G1-phase cells (Fig. 2E). Thus, knockdown of Cdh1 prohibited hTK1 degradation in the G1 phase, resulting in accumulation of hTK1.
In the interest of determining whether the SCF complex is involved in hTK1 degradation in mammalian cells, we expressed Cul1(1-452), which lacks the Roc/UBC binding region while retaining its ability to bind to F box protein, as a dominant-negative mutant in cells to interfere with SCF-mediated degradation (12, 52). As shown in Fig. 3, endogenous hTK1 degradation in mitotic HeLa cells was unaffected by expressing dominant-negative mutant Cul1(1-452), while p27 is accumulated in response to this functional disruption (data not shown). Overexpression of full-length Cullin1 did not affect the expressed level of endogenous hTK1 either. Therefore, it is unlikely that the SCF complex plays a determining role in mitotic degradation of hTK1 in mammalian cells. Rather, the APC/C-Cdh1-mediated pathway is indeed necessary for controlling hTK1 degradation.
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DISCUSSION |
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It has been previously shown that deletion of the carboxyl-terminal 40 aa of hTK1 completely abolishes cell cycle regulation and stabilizes the protein throughout the cell cycle (25). The KEN box identified in this study is located in this C-terminal region, thus explaining how this region confers the degradation signal. By comparing the amino acid sequences of cytosolic TK1 among different eukaryotic cells, we found a KEN box or KEN-like box is present in C-terminal region of chicken, Chinese hamster, and mouse TK1. Notably, the vaccinia virus and related viral TK proteins are highly homologous to cytosolic hTK1, but lack this KEN box in their C-terminal regions. Therefore, it is logical to assume that absence of the KEN box signal in the C-terminal region of viral TK may provide a mechanism allowing its escape from the cell cycle-dependent degradation in the host.
Unlike mammalian cells, both budding and fission yeasts lack their cytosolic TK1. Our laboratory has previously used the inducible expression systems in yeast to study the degradation mechanism of hTK1 (26). Through this approach, we demonstrated that degradation of hTK1 is impaired in Saccharomyces cerevisiae carrying a temperature-sensitive mutation in proteasomal gene pre1-1 or the SCF complex gene cdc34 or cdc53, while mutation in cdc16, an essential component of APC/C in yeast, does not affect hTK1 proteolysis. In that study, we also found that mutation of serine 13 to alanine, which disrupts its phosphorylation, stabilizes the expression of hTK1 in budding and fission yeasts. These results suggested that the SCF-mediated pathway contributes to hTK1 degradation via the phosphorylation signal in the yeast system. However, in this study, we found that expression of a dominant-negative form of Cullin1 did not result in accumulation of hTK1 in the mitotic cells (Fig. 3), indicating that in mammalian cells, the SCF is not essential for targeting mitotic degradation of hTK1. This situation is similar to the case of Cdc6, in which degradation is via the SCF complex in yeast, yet requires the APC/C-Cdh1 complex in human cells (33, 36). In addition, we found that there is no difference in the stabilities of the wild-type and mutant form (S13A) of FLAG-tagged-TK1 at mitosis in the stable cell lines (Fig. 5), suggesting that serine 13 phosphorylation is probably not a necessary signal for its mitotic degradation. Previously, Littlepage and Ruderman (28) showed that mutation of serine 53 to aspartic acid, which mimics the phosphorylation state, indeed abrogates Aurora-A kinase degradation in the mitotic exit phase, indicating dephosphorylation could control timing of its destruction. In the case of hTK1, we found that conversion of serine 13 to either alanine or aspartic acid did not have any effect on the level of hTK1 expressed in response to Cdh1 overexpression (data not shown), suggesting that neither phosphorylation nor dephosphorylation of serine 13 is crucial in Cdh1-mediated degradation of hTK1. At this point, we have speculated that yeasts, lacking endogenous TK, apparently do not need TK to supply dTTP during DNA replication; therefore, it is possible that the ectopically expressed hTK1 is readily phosphorylated in yeast and is targeted for degradation by a not-yet-identified F box protein to avoid the unnecessary synthesis of dTTP prior to the onset of activation of APC/C-Cdh1 complex. After all, it remains to be seen whether this phosphorylation-dependent degradation pathway for hTK1 does exist in mammalian cells under other physiological conditions, such as inhibition of DNA synthesis in the S phase.
It has been shown that the dTTP pool in S-phase cells is 20 times larger than that in G0-phase cells, which coincides with the level of expression of TK1 (44). Presumably, this fluctuation is important for maintaining genome stability, since reports have shown that elevation of dTTP concentration increases in vitro and in vivo mutation rates (10, 29). Extending from these observations, it is noteworthy that dTTP has been identified as an allosteric effector for ribonucleotide reductase, directing its substrate specificity (7). An abnormal increase in the dTTP level has been shown to promote the catalytic efficiency of ribonucleotide reductase for converting rGDP to dGDP, which can result in increase of the dGTP pool (41). Given that a larger dGTP pool is mutagenic, it is proposed that a balance of deoxynucleoside triphosphate (dNTP) pools is important for the fidelity of DNA repair and replication. Apparently, restricting dTTP level in cells by TK1 degradation is a way of controlling the balance of dNTP pools in the G1 phase. While this article was in preparation, a report showed that mouse ribonucleotide reductase R2 (mR2) protein is targeted to the APC/C-Cdh1 complex through KEN box-mediated binding and gets degraded in late mitosis (4). Considering the coordination between the supply of dTTP and regulation of ribonucleotide reductase activity in providing the correct pool of dNTP, information gained from these two reports together suggests that during mitotic exit, a common proteolytic controller, APC/C-Cdh1, is utilized to degrade two dNTP pool regulators, mR2 and TK1, by which an unwanted supply of dNTP can be stopped concurrently at entry into the G1 phase.
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ACKNOWLEDGMENTS |
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This study was supported by grants NSC90-2320-B-002-065 and NSC 92-3112-B-002-016 from the National Science Council, Taiwan, Republic of China.
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FOOTNOTES |
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