Multiple Functions of Jab1 Are Required for Early Embryonic Development and Growth Potential in Mice
2004; Elsevier BV; Volume: 279; Issue: 41 Linguagem: Inglês
10.1074/jbc.m406559200
ISSN1083-351X
AutoresKiichiro Tomoda, Noriko Yoneda‐Kato, Akihisa Fukumoto, Shinya Yamanaka, Jun‐ya Kato,
Tópico(s)14-3-3 protein interactions
ResumoJab1 interacts with a variety of signaling molecules and regulates their stability in mammalian cells. As the fifth component of the COP9 signalosome (CSN) complex, Jab1 (CSN5) plays a central role in the deneddylation of the cullin subunit of the Skp1-Cullin-F box protein ubiquitin ligase complex. In addition, a CSN-independent function of Jab1 is suggested but is less well characterized. To elucidate the function of Jab1, we targeted the Jab1 locus by homologous recombination in mouse embryonic stem cells. Jab1-null embryos died soon after implantation. Jab1-/- embryonic cells, which lacked other CSN components, expressed higher levels of p27, p53, and cyclin E, resulting in impaired proliferation and accelerated apoptosis. Jab1 heterozygous mice were healthy and fertile but smaller than their wild-type littermates. Jab1+/- mouse embryonic fibroblast cells, in which the amount of Jab1-containing small subcomplex, but not that of CSN, was selectively reduced, proliferated poorly, showed an inefficient down-regulation of p27 during G1, and was delayed in the progression from G0 to S phase by 3 h compared with the wild-type cells. Most interestingly, in Jab1+/- mouse embryonic fibroblasts, the levels of cyclin E and deneddylated Cul1 were unchanged, and p53 was not induced. Thus, Jab1 controls cell cycle progression and cell survival by regulating multiple cell cycle signaling pathways. Jab1 interacts with a variety of signaling molecules and regulates their stability in mammalian cells. As the fifth component of the COP9 signalosome (CSN) complex, Jab1 (CSN5) plays a central role in the deneddylation of the cullin subunit of the Skp1-Cullin-F box protein ubiquitin ligase complex. In addition, a CSN-independent function of Jab1 is suggested but is less well characterized. To elucidate the function of Jab1, we targeted the Jab1 locus by homologous recombination in mouse embryonic stem cells. Jab1-null embryos died soon after implantation. Jab1-/- embryonic cells, which lacked other CSN components, expressed higher levels of p27, p53, and cyclin E, resulting in impaired proliferation and accelerated apoptosis. Jab1 heterozygous mice were healthy and fertile but smaller than their wild-type littermates. Jab1+/- mouse embryonic fibroblast cells, in which the amount of Jab1-containing small subcomplex, but not that of CSN, was selectively reduced, proliferated poorly, showed an inefficient down-regulation of p27 during G1, and was delayed in the progression from G0 to S phase by 3 h compared with the wild-type cells. Most interestingly, in Jab1+/- mouse embryonic fibroblasts, the levels of cyclin E and deneddylated Cul1 were unchanged, and p53 was not induced. Thus, Jab1 controls cell cycle progression and cell survival by regulating multiple cell cycle signaling pathways. The successful identification and characterization of the COP9 signalosome (CSN) 1The abbreviations used are: CSN, COP9 signalosome; SCF, Skp1-Cullin-F box protein; MEF, mouse embryonic fibroblasts; FBS, fetal bovine serum; ES, embryonic stem; TUNEL, terminal dUTP nick-end labeling; ICM, inner cell mass; E, embryonic day; Cdk, cyclin-dependent kinase. 1The abbreviations used are: CSN, COP9 signalosome; SCF, Skp1-Cullin-F box protein; MEF, mouse embryonic fibroblasts; FBS, fetal bovine serum; ES, embryonic stem; TUNEL, terminal dUTP nick-end labeling; ICM, inner cell mass; E, embryonic day; Cdk, cyclin-dependent kinase. complex from yeast (1Mundt K.E. Porte J. Murray J.M. Brikos C. Christensen P.U. Caspari T. Hagan I.M. Millar J.B. Simanis V. Hofmann K. Carr A.M. Curr. Biol. 1999; 9: 1427-1430Abstract Full Text Full Text PDF PubMed Google Scholar, 2Maytal-Kivity V. 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Cell. 2002; 13: 646-655Crossref PubMed Scopus (57) Google Scholar, 28Mundt K.E. Liu C. Carr A.M. Mol. Biol. Cell. 2002; 13: 493-502Crossref PubMed Scopus (99) Google Scholar). Protein kinases capable of phosphorylating c-Jun, NF-κB, and p53 are associated with CSN (4Seeger M. Kraft R. Ferrell K. Bech-Otschir D. Dumdey R. Schade R. Gordon C. Naumann M. Dubiel W. FASEB J. 1998; 12: 469-478Crossref PubMed Scopus (308) Google Scholar, 29Uhle S. Medalia O. Waldron R. Dumdey R. Henklein P. Bech-Otschir D. Huang X. Berse M. Sperling J. Schade R. Dubiel W. EMBO J. 2003; 22: 1302-1312Crossref PubMed Scopus (168) Google Scholar, 30Sun Y. Wilson M.P. Majerus P.W. J. Biol. Chem. 2002; 277: 45759-45764Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). p53 is destabilized by CSN-mediated phosphorylation at Thr-155 in proliferating cells, and disruption of CSN leads to accumulation of p53 and eventual cell cycle arrest/apoptosis (31Bech-Otschir D. Kraft R. Huang X. Henklein P. Kapelari B. Pollmann C. Dubiel W. EMBO J. 2001; 20: 1630-1639Crossref PubMed Scopus (322) Google Scholar). CSN interacts with the Skp1-Cullin-F box protein (SCF) ubiquitin ligase and removes a ubiquitin-like polypeptide, Nedd8, from the Cul subunit (deneddylation) (32Cope G.A. Suh G.S. Aravind L. Schwarz S.E. Zipursky S.L. Koonin E.V. Deshaies R.J. Science. 2002; 298: 608-611Crossref PubMed Scopus (571) Google Scholar, 33Lyapina S. Cope G. Shevchenko A. Serino G. Tsuge T. Zhou C. Wolf D.A. Wei N. Deshaies R.J. Science. 2001; 292: 1382-1385Crossref PubMed Scopus (551) Google Scholar), thereby regulating the ligase activity (34Cope G.A. Deshaies R.J. Cell. 2003; 114: 663-671Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 35Wolf D.A. Zhou C. Wee S. Nat. Cell Biol. 2003; 5: 1029-1033Crossref PubMed Scopus (161) Google Scholar). The JAMM domain within the Jab1/CSN5 subunit plays an essential role in this reaction (32Cope G.A. Suh G.S. Aravind L. Schwarz S.E. Zipursky S.L. Koonin E.V. Deshaies R.J. Science. 2002; 298: 608-611Crossref PubMed Scopus (571) Google Scholar), but the monomeric form of the Jab1/CSN5 polypeptide alone failed to manifest the activity. Disruption of the CSN complex in Drosophila results in accumulation of the hyper-deneddylated Cul subunit and cyclin E polypeptide (one of the substrates of SCF) and failure of oogenesis (17Doronkin S. Djagaeva I. Beckendorf S.K. Dev. Cell. 2003; 4: 699-710Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). It is not clear whether all biological responses correlated with CSN are mediated either by phosphorylation or deneddylation (27Wang X. Kang D. Feng S. Serino G. Schwechheimer C. Wei N. Mol. Biol. Cell. 2002; 13: 646-655Crossref PubMed Scopus (57) Google Scholar). Other biochemical functions associated with CSN include regulation of the subcellular localization of the target protein (24Tomoda K. Kubota Y. Kato J. 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Although Jab1 was shown to play a critical role in other organisms such as Caenorhabditis elegans (14Smith P. Leung-Chiu W.M. Montgomery R. Orsborn A. Kuznicki K. Gressman-Coberly E. Mutapcic L. Bennett K. Dev. Biol. 2002; 251: 333-347Crossref PubMed Scopus (48) Google Scholar) and Drosophila (13Oron E. Mannervik M. Rencus S. Harari-Steinberg O. Neuman-Silberberg S. Segal D. Chamovitz D.A. Development (Camb.). 2002; 129: 4399-4409Crossref PubMed Google Scholar, 16Doronkin S. Djagaeva I. Beckendorf S.K. Development (Camb.). 2002; 129: 5053-5064PubMed Google Scholar), some Jab1 targets are unique to mammalian cells, and it is important to know how these targets are regulated in living organs. Jab1 was also found as a smaller form not part of the CSN complex in various species (12Freilich S. Oron E. Kapp Y. Nevo-Caspi Y. Orgad S. Segal D. Chamovitz D.A. Curr. Biol. 1999; 9: 1187-1190Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 13Oron E. Mannervik M. Rencus S. 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To understand better the function of Jab1 in development, cell proliferation, and oncogenesis, we targeted the Jab1 locus by homologous recombination in ES cells. Jab1-null embryos did not survive, whereas Jab1 heterozygous mice were viable and fertile but smaller than the wild-type littermates. Jab1-/- cells lacked other CSN components and expressed higher levels of p27, p53, and cyclin E, resulting in impaired proliferation and accelerated apoptosis. In contrast, Jab1+/- MEF cells, in which the amount of small Jab1 subcomplex but not that of CSN was selectively reduced, were delayed in the progression from G0 to S phase by 3 h due to an inefficient down-regulation of p27 during G1. Most interestingly, in Jab1+/- MEFs, the levels of cyclin E and neddylated Cul1 were unchanged, and p53 was not induced. Thus, Jab1 controls cell proliferation and survival in mice through multiple cell cycle regulatory pathways in both CSN-dependent and -independent ways. Targeted Disruption of the Mouse Jab1 Gene—The gene structure of mouse Jab1 was determined by PCR and DNA sequencing 2K. Tomoda, N. Yoneda-Kato, and J.-y. Kato, unpublished data. and subsequently confirmed by Blast (NCBI) analysis of the complete cDNA sequence (AF068223) and the genomic sequence (NT039169). The Jab1 targeting vector was constructed by subcloning a 1-kb genomic DNA fragment containing the sequence upstream from the initial methionine and a 5-kb genomic DNA fragment downstream of exon 6, both of which had been amplified by genomic PCR and confirmed by sequencing, into the ploxPNT vector at the EcoRI and XhoI sites, respectively. The targeting vector was linearized with XhoI and was electroporated into mouse RF8 ES cells (57Meiner V.L. Cases S. Myers H.M. Sande E.R. Bellosta S. Schambelan M. Pitas R.E. McGuire J. Herz J. Farese Jr., R.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14041-14046Crossref PubMed Scopus (237) Google Scholar). ES clones selected in 200 μg/ml G418 and 0.2 μm FIAU were subjected to Southern blot analysis by using probes external to both the 5′ and 3′ end of the targeting construct (Fig. 1, a and b). We did not detect truncated polypeptides from the putative open reading frame (corresponding to amino acids 286–334) in exons 7 and 8 by Western blotting by using antibody recognizing the C terminus of the Jab1 protein, indicating that the mutant allele is truly a null locus. Jab1+/- ES cells were microinjected into blastocyst stage C57BL/6 mouse embryos. Chimeric males were crossed to C57BL/6 females, and offspring were genotyped by genomic PCR using Jab1-specific primers as follows: a (5′-CTC TCT GTC CTG GGC TTT CAT TAC CAT TTC-3′), b (5′-GCT CTC CAC ACC CTT CAT CTC CCA CCC CTC-3′), and a neo gene-specific primer c (5′-CCT GCG TGC AAT CCA TCT TGT TCA CA-3′) (Fig. 1, a and c). p53+/- mice were purchased from Taconic Farms. p27+/- and p27-/- mice were generated basically according to the method described previously (58Kiyokawa H. Kineman R.D. Manova-Todorova K.O. Soares V.C. Hoffman E.S. Ono M. Khanam D. Hayday A.C. Frohman L.A. Koff A. Cell. 1996; 85: 721-732Abstract Full Text Full Text PDF PubMed Scopus (1132) Google Scholar). Histology and Immunohistochemistry—Uteri from pregnant females were dissected, fixed overnight in 4% paraformaldehyde, embedded in paraffin, cut into 4-μm sections, and stained with hematoxylin and eosin. For antibody staining, sections were deparaffinized, rehydrated, and placed in a 3% solution of hydrogen peroxide for 20 min. This was followed by blocking in 5% bovine serum albumin for 30 min. After incubation with primary antibodies overnight at 4 °C, peroxidase-conjugated secondary antibody was applied (Histofine Simple Stain MAX PO, Nichirei Co.). The staining was visualized with diaminobenzidine, and the sections were counterstained with hematoxylin. The antibodies used included rabbit polyclonal antibodies to Jab1/CSN5 (1:100) (24Tomoda K. Kubota Y. Kato J. Nature. 1999; 398: 160-165Crossref PubMed Scopus (549) Google Scholar), CSN1 (1:50) (39Tomoda K. Kubota Y. Arata Y. Mori S. Maeda M. Tanaka T. Yoshida M. Yoneda-Kato N. Kato J.Y. J. Biol. Chem. 2002; 277: 2302-2310Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar), cyclin E (M-20 Santa Cruz Biotechnology, 1:50), and Cul1 (Zymed Laboratories Inc., 1:50), and mouse monoclonal antibodies to p27 (Transduction Laboratories, 1:50) and p53 (Oncogene Science, 1:50 and Calbiochem, 1:250). We incubated sections with either no primary or no secondary antibodies to control for nonspecific staining (data not shown). For detection of apoptosis, TUNEL staining of the sections was carried out according to the manufacturer's instructions (ApopTag Red, Intergen). Blastocyst Outgrowth and Immunofluorescence Analysis—Blastocysts were isolated from the uterus at embryonic day 3.5 (E3.5), cultured in ES medium in 5% CO2 at 37 °C, and photographed. Cells cultured on a Lab-TekII Chamber (Nalge Nunc) were fixed in 3% paraformaldehyde, permeabilized in 0.5% Triton X-100, stained with primary antibodies, and incubated with fluorescein isothiocyanate-linked anti-mouse and Texas Red-linked anti-rabbit IgG (Amersham Biosciences). For the determination of BrdUrd incorporation, cells were incubated in 10 μm bromodeoxyuridine for 24 h, stained with anti-Jab1 rabbit polyclonal antibody followed by Texas Red-linked anti-rabbit IgG, treated with 1.5 m HCl, and stained with anti-BrdUrd mouse monoclonal antibody (Amersham Biosciences) and fluorescein isothiocyanate-linked anti-mouse IgG. The TUNEL assay was performed with Jab1-stained cells according to the manufacturer's instructions (see above). The cell samples were viewed by phase-contrast or fluorescence microscopy. The genotype of the cultured embryos was determined by anti-Jab1 immunofluorescence staining and by genomic PCR using primers a, b, and c (Fig. 1, a and c). MEF Assays—Primary MEFs were isolated from E13.5 embryos and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS). For growth curve assays, only early passage MEF cells (passage 2–4) were seeded at 105 cells on 6-cm plates and quantified at given time points. The 3T6 protocol was employed by plating 2 × 106 cells on 10-cm plates and replating at the same cell density every 3 days. To analyze S phase entry, 2 × 105 cells per 6-cm dish were starved in Dulbecco's modified Eagle's medium supplemented with 0.1% FBS for 48 h before being stimulated with 10% FBS for given periods. Collected cells were suspended in a 1-ml solution of 0.1% sodium citrate and 0.1% Triton X-100 containing 50 μg/ml of propidium iodide and treated with 1 μg/ml of RNase for 30 min at room temperature. Fluorescence from the propidium iodide-DNA complex was measured with a FACScan flow cytometer (BD Biosciences), and the percentages of cells in phases G1, S, and G2/M of the cell cycle were determined with Cell Fit cell cycle software. Protein Analyses—Cell lysis, gel electrophoresis, and immunoblotting were performed by using standard procedures (24Tomoda K. Kubota Y. Kato J. Nature. 1999; 398: 160-165Crossref PubMed Scopus (549) Google Scholar, 39Tomoda K. Kubota Y. Arata Y. Mori S. Maeda M. Tanaka T. Yoshida M. Yoneda-Kato N. Kato J.Y. J. Biol. Chem. 2002; 277: 2302-2310Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 59Kurokawa K. Tanaka T. Kato J. Oncogene. 1999; 18: 2718-2727Crossref PubMed Scopus (35) Google Scholar). Developed films were quantitatively analyzed with a densitograph (ATTO, Japan). Rabbit polyclonal antibodies against γ-tubulin, p27, Cul1, cyclin E, Cdk4, Skp2, p21, p53, MDM2, and p16 and mouse monoclonal antibody to cyclin D1 were purchased from Santa Cruz Biotechnology. Mouse monoclonal antibody to mouse p27 was obtained from Transduction Laboratories. For nondenaturing gel electrophoresis, cells were lysed in modified EBC buffer containing 0.1% digitonin as a detergent. Lysates were separated in a pre-made nondenaturing gel (Biocraft) without SDS and analyzed by immunoblotting. The fractions from the glycerol gradient centrifugation (39Tomoda K. Kubota Y. Arata Y. Mori S. Maeda M. Tanaka T. Yoshida M. Yoneda-Kato N. Kato J.Y. J. Biol. Chem. 2002; 277: 2302-2310Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar) containing only the large CSN complex or the small Jab1 complex were separated by nondenaturing gel electrophoresis, and the positions of each complex were determined. In this assay, the amount of the small Jab1 subcomplex was 10–20% of total Jab1 protein (equivalent to the results of the glycerol gradient centrifugation analysis), and the CSN complex migrated slower and appeared as a broad band, which contained all eight CSN subunits (CSN1–8), suggesting multiplicity of the modified CSN complex. 3K. Tomoda, A. Fukumoto, N. Yoneda-Kato, and J.-y. Kato, unpublished observations. The in vitro kinase assay for Cdk2 and Cdk4 was performed as described (59Kurokawa K. Tanaka T. Kato J. Oncogene. 1999; 18: 2718-2727Crossref PubMed Scopus (35) Google Scholar) using a recombinant retinoblastoma protein as a substrate. The phosphorylated retinoblastoma protein was separated by SDS-PAGE and quantified for 32P incorporation by a Fuji BAS-2500 analyzed image. To obtain the quantitative results, we routinely used several different MEF lines (usually five lines or more) prepared from different mice for each experiment. The averages and the standard deviations were calculated and are shown in the text. We show only the representative data in the figures. Targeted Disruption of the Jab1 Gene and Requirement of Jab1 in Early Embryogenesis—To examine the physiological requirement of Jab1 in development, cell proliferation, and cell survival, we attempted to disrupt the Jab1 gene in mice. The murine Jab1 gene spans about 13 kb, containing eight coding exons (Fig. 1a). Our strategy was to delete coding exons 1–6, including the start codon, and replace them with a neo marker gene through homologous recombination in ES cells. Correct targeting was confirmed in two independent ES clones by Southern blot hybridization analysis with 5′ and 3′ probes, respectively (Fig. 1b). One ES clone was injected into blastocysts from C57BL/6 mice to generate chimeras, and germ line transmission was established. Jab1+/- heterozygous mice were fertile and were intercrossed to produce Jab1-/- mice. No nullizygous mice were born live among 186 offspring of different heterozygous intercrosses, and the ratio of heterozygous mice to wild-type mice was 2.0 to 1 (Table I), indicating that loss of Jab1 was embryonic lethal. Embryos were isolated from timed heterozygous intercrosses from embryonic day 8.5 to as early as E3.5 (Table I) and were ge
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