Article |
Address correspondence to Tomoki Chiba, Dept. of Molecular Oncology, Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome, Bunkyo-Ku, Tokyo 113-8613, Japan. Tel. and fax: 81-3-3823-2237. E-mail: tchiba{at}rinshoken.or.jp
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Abstract |
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Key Words: NEDD8; ubiquitin; cullin; knock-out; cell cycle
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Introduction |
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The targets of NEDD8 are unknown apart from cullin (Cul) family proteins, which are also conserved across species (Liakopoulos et al., 1998; Osaka et al., 1998; Hori et al., 1999; Gray and Estelle, 2000). Cul-1 is a common subunit of a class of Skp1/Cul-1/F-box protein (SCF) complexes responsible for ubiquitylation of a multitude of proteins that regulate various biologically important processes such as cell cycle progression and signal transduction (Deshaies, 1999; Weissman, 2001; Pickart, 2001). Recently, other Cul family proteins have also been suggested to constitute a large family of distinct Ub protein ligase complexes (E3-enzymes), based on their interaction with a recently identified ROC1Rbx1Hrt1, a RING finger protein, which is essential for the catalytic activity of SCF (Deshaies, 1999; Kamura et al., 1999; Ohta et al., 1999). Indeed, Cul-2 and Cul-5, each complexed with elongin B and elongin C, are reported to have Ub protein ligase activities (Iwai et al., 1999; Lisztwan et al., 1999; Kamura et al., 2001) and Cul-3 as a component of Ub ligase to target cyclin E for ubiquitylation (Singer et al., 1999).
The modification of SCF by NEDD8 is essential in fission yeast. Pcu1K720R, a Pcu1 (Cul-1 homologue) mutant defective for NEDD8-ylation cannot complement the lethal phenotype of pcu1, and overexpression of Pcu1K720R results in accumulation of SCF complex substrates, Rum1, a cyclin-dependent kinase (CDK) inhibitor (CKI) (Osaka et al., 2000). In contrast, the budding yeast homologue Cdc53 does not require NEDD8 modification for its function (Lammer et al., 1998). In Arabidopsis thaliana, Axr1, an APP-BP1 homologue was identified from screening of mutants resistant to auxin response. However, Axr1-1 cells, which have no NEDD8-modifying activity remain viable (Gray and Estelle, 2000). Thus, the NEDD8 system plays a synergistic role with the SCF complex in various organisms, but its requirement may significantly differ among species.
Biochemical studies have shown that the NEDD8 modification of the SCF complex enhances the Ub ligase activity by recruiting E2 to the complex efficiently without affecting the stability, assembly, or substrate binding capacity of the SCF complex (Kawakami et al., 2001). However, the in vitro ubiquitylation activity of the SCF complex is still evident without its modification by NEDD8 (Podust et al., 2000; Read et al., 2000; Wu et al., 2000; Kawakami et al., 2001). Furthermore, it was reported recently that p9Suc1/Cks1 but not NEDD8 promotes the ubiquitylation activity of SCFSkp2 (Ganoth et al., 2001). Thus, although NEDD8 modification is essential for Cul function in fission yeast its precise molecular action remains to be unraveled.
To investigate the role of NEDD8 in mammalian organisms in vivo, we generated the Uba3-deficient mouse, which lacks the catalytic subunit of NEDD8 activating enzyme. Analysis of Uba3-deficient mice showed that the NEDD8 system is essential for cell cycle progression including the endoreduplication cycle. The endoreduplication cycle is an unusual mode of cell cycle that results in duplication of the chromosome without intervening mitosis (Varmuza et al., 1988; Edgar and Orr-Weaver, 2001). Although, this cycle is mutually exclusive with mitotic cell cycle, the two processes share common mechanisms such as fluctuation of CDKs activity (Traas et al., 1998). We found that Uba3-deficient trophoblastic cells could not enter S phase, and this cell cycle arrest was accompanied with the high expression of cyclin E and p57Kip2. Furthermore, ß-catenin, a mediator of the Wnt/wingless (Wg) signaling pathway, accumulated in the cytoplasm and nuclei of mutant cells, suggesting that the SCF complex and its modification by NEDD8 are essential for ß-catenin degradation. Since the Wnt/Wg signaling pathway regulates the orientation of cell polarity axis and tissue specific gene expression (Beddington and Robertson, 1999; Bellaiche et al., 2001), we propose that the regulation of the NEDD8 system may coordinate the cell cycle progression and cell polarity axis formation in the development of multicellular organism.
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Results |
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Discussion |
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We also showed aberrant expression of p57Kip2 in Uba3-/- trophoblasts. Consistent with this finding, it is reported that fluctuations of p57Kip2 level are essential for the entry to S phase of the endoreduplication cycle (Hattori et al., 2000). However, our results do not exclude the possibility that Uba3-/- trophoblasts arrest just before the degradation of p57Kip2 by other cell cycle defects. It is also possible that the presence of dysregulated Wnt/Wg signaling pathway might interfere with the endoreduplication cycle. It was reported recently that knirps and knirps-related transcription factors are regulated by the Wnt/Wg signaling pathway, and both inhibit endoreduplication in a spatio-temporal manner (Fuss et al., 2001). Furthermore, in Drosophila wing primordia Wnt/Wg induces arrests of the cells at G2 phase by downregulating mitosis-inducing phosphatase, Cdc25 (Johnston and Edgar, 1998).
Our results also showed that loss of the NEDD8 system led to selective apoptosis of ICM although the exact mechanism could not be identified. Since Uba3-/- ICM but not trophoblasts can enter the S phase, the apoptotic process may be linked to a DNA replication checkpoint. Based on previous findings and the results of our study, we postulate that loss of the NEDD8 system leads to impaired replication and/or repair of the DNA for the following reasons. First, subunit of global genomic repair protein xeroderma pigmentosa group E (XP-E) interacts with Cul4A (Shiyanov et al., 1999). XP-E is induced by UV irradiation and binds to UV-damaged DNA (Tang et al., 2000). Uba3-/- embryos were not exposed to UV; however, loss of the XP-E activity may fail to replicate DNA faithfully. Secondly, the presence of such DNA damage in Uba3-/- can be presumed from the report that p53 is highly expressed in Cul-1-/- embryos because p53 is known to be upregulated upon DNA damage (Dealy et al., 1999). Thus, the NEDD8 system may be essential for global genomic repair and prevention of high risk carcinogenesis in XP-E patients.
Upon UV irradiation, hamster cell line V79 fails to express UV-damaged DNA-binding activity, due to the low expression of XP-E (Tang et al., 2000). Ts41 cells, which harbor mutation in SMC gene, which is highly homologous to APP-BP1 (Chen et al., 2000), are derived from the cell line (Handeli and Weintraub, 1992). At nonpermissive temperature, ts41 cells undergo successive DNA replication without intervening mitosis nor apoptosis, suggesting that the XP-E activity may be relevant to the essential role of the NEDD8 system. Strikingly, XP-E gene homologue is present in fission yeast but not in budding yeast, which may also account for the indispensable role of the NEDD8 system in the former.
It has been reported that the SCF complex is exclusively modified by NEDD8 in the centrosomes (Freed et al., 1999). Furthermore, in the initiation of DNA replication NEDD8-ylated SCF complex is recruited to chromatin through the preinitiation complex (Laura et al., 2001). These results suggest that SCF complexes are active at those sites, and both centrosome duplication and DNA replication might be impaired in Uba3-/- mice.
Given the possible involvement of the NEDD8 system in DNA replication and repair and possible dissociation from the apoptotic pathway, it is worth noting that accumulation of cyclin E and ß-catenin, which was observed in Uba3 mutants, has been otherwise implicated in carcinogenesis (Nielsen et al., 1998; Spruck et al., 1999; Polakis, 2000). Moreover, one might expect that loss of the NEDD8 system should also lead to the dysfunction of tumor suppressor, pVHL (von Hippel-Lindau syndrome gene product), which acts as Ub ligase together with Cul-2. Thus, we propose that attenuation of the NEDD8 system may link to carcinogenesis or the unusual characters of cancer cells.
It is likely that NEDD8 modification is essential for all of the Cul family proteins because Uba3-deficient mice appear to have both phenotypes induced by loss of Cul-1 and Cul-3 and thus even severer than each Cul-1-/- and Cul-3-/- mouse. Why is NEDD8 modification essential for Cul function? NEDD8-ylation of Cul-1 recruits Ub-E2 to the SCF complex (Kawakami et al., 2001) and stimulates in vitro ubiquitylation of IB
and p27Kip1 by SCFßTrCP and SCFSkp2, respectively (Podust et al., 2000; Read et al., 2000; Wu et al., 2000). However, such NEDD8-ylation was not strictly required, at least for in vitro ubiquitylation activity (Ganoth et al., 2001). Thus, the NEDD8 pathway may have other roles in addition to recruit the E2 enzyme to the SCF complex. Since NEDD8-ylated SCF complexes are spatio temporally regulated during centrosome duplication and DNA replication, the NEDD8 modification may control the regulation in those processes. Alternatively, the NEDD8 system may be regulated in a spatio-temporal manner.
Finally, the NEDD8 system, which seemed to be diverged from the Ub system, is a regulator of the protein degradation machinery. However, unlike Ub NEDD8 targets a highly conserved family of Cul-based E3 enzymes for activation of their ultimate functions. This pathway may coordinate the regulation of cell cycle progression and morphogenetic pathway.
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Materials and methods |
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Histology and immunofluorescence
Embryos in utero were dissected at various stages of development, fixed in 4% paraformaldehyde (PFA), paraffin embedded, and sectioned sagittally. Sections were stained by Meyer's H&E. For immunofluorescence analysis, the following primary and secondary antibodies were used: anticytokeratin-endoA antibody (TROMA-1; Developmental Studies Hybridoma Bank), anticyclin E antibody (M-20; Santa Cruz Biotechnology, Inc.), anti-Uba3 antibody raised in rabbits in our laboratories, anti-p57Kip2 antibody (provided by Dr. H. Toyoshima, Tsukuba University, Ibaraki, Japan), anti-BrdU antibody (Bu20a, Dako), antiß-catenin antibody (clone14; Transduction Laboratories) and Alexa 488 nm antirabbit and Alexa 594 nm antimouse antibodies (Molecular Probes). Nuclei were counterstained with TOTO3 (Molecular Probes) or Hoechst 33342 (Sigma-Aldrich). Fluorescence images and differential interference contrast images were obtained using a confocal laser scanning microscope (LSM510; ZEISS) or a fluorescence microscope (AX70; Olympus) equipped with cooled charge-coupled device camera (Sensys/OL; Photometrics). Pictures were taken using IPlab image software (solution systems).
Blastocysts in vitro culture
Eight-cell embryos or blastocysts were flashed out and collected from pregnant female uteri at E2.5 or E3.5, then cultured in vitro in M16 medium (Sigma-Aldrich) at 37°C with 5% CO2. After hatching, individual blastocysts were transferred to ES cell medium and spread onto 0.1% gelatin-coated chambered coverglass (Lab-Tek; Nalgen).
BrdU incorporation
Expanded blastocysts were cultured for 48 h and labeled with 20 µM BrdU (Sigma Aldrich) for 30 h before fixation by 70% ethanol overnight at 4°C. Embryos were treated with 4N HCl and 0.5% Triton X-100 in PBS for 30 min at room temperature. After washes with PBS, embryos were subjected to immunocytochemistry as described above. In in vivo experiments, BrdU (100 µg/g of body weight) was injected i.p. to pregnant females at 5.75 d postcoitum. The females were killed 2 h after injection; the decidua were fixed in 4% PFA at 4°C overnight. Sagittal sections were treated with 2N HCl for 45 min, incubated in proteinase K (20 µg/ml) for 10 min at room temperature, and then processed for immunohistochemistry.
TUNEL assay
Apoptotic cells were detected by TdT-mediated dUTP-biotin nick end labeling (TUNEL) assay using Apoptag kit (Intergen). The assay was performed as per manufacture's instructions. In brief, the sections and PFA-fixed blastocysts were treated by proteinase K (20 µg/ml) for 15 min at room temperature. After equilibration, the specimens were incubated with digoxigenin-dNTP under the TdT enzyme drop for 1 h at 37°C. After stopping reaction, the tissues were incubated covered by antidigoxigenin conjugate for 30 min at room temperature. The digoxigenin conjugate was detected immunochemically by FITC-labeled antibody.
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Footnotes |
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Acknowledgments |
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This work was supported in part by grants-in-aid from the Ministry of Education, Science and Culture of Japan.
Submitted: 9 April 2001
Revised: 22 August 2001
Accepted: 2 October 2001
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References |
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