NUB1, a NEDD8-interacting Protein, Is Induced by Interferon and Down-regulates the NEDD8 Expression

NEDD8, a ubiquitin-like protein, covalently conjugates to cullin family members. It appears to control vital biological events through its conjugation to cullins. To study how this conjugation pathway is regulated, we performed yeast two-hybrid screening by using NEDD8 as a bait and isolated a cDNA fragment encoding a potent down-regulator of the NEDD8 expression. Here, we report this novel regulator, NUB1 (NEDD8Ultimate Buster-1). NUB1 is composed of 601 residues with a calculated 69.1-kDa molecular mass. It is an interferon-inducible protein and predominantly localized in the nucleus. The NUB1 message is specifically expressed in adult human testis, ovary, heart, and skeletal muscle tissues and is developmentally down-regulated in mouse embryos. In biochemical analysis, we found that NUB1 overexpression leads to severe reduction of NEDD8 monomer and NEDD8 conjugates in cells. This reduction is not due to down-regulation of NEDD8 transcription, but due to post-transcriptional mechanism. As expected from this activity, overexpression of NUB1 had a profound growth-inhibitory effect on U2OS cells. Thus, NUB1 is a strong down-regulator of the NEDD8 expression and appears to play critical roles in regulating biological events, including cell growth.AF300717 NEDD8, a ubiquitin-like protein, covalently conjugates to cullin family members. It appears to control vital biological events through its conjugation to cullins. To study how this conjugation pathway is regulated, we performed yeast two-hybrid screening by using NEDD8 as a bait and isolated a cDNA fragment encoding a potent down-regulator of the NEDD8 expression. Here, we report this novel regulator, NUB1 (NEDD8Ultimate Buster-1). NUB1 is composed of 601 residues with a calculated 69.1-kDa molecular mass. It is an interferon-inducible protein and predominantly localized in the nucleus. The NUB1 message is specifically expressed in adult human testis, ovary, heart, and skeletal muscle tissues and is developmentally down-regulated in mouse embryos. In biochemical analysis, we found that NUB1 overexpression leads to severe reduction of NEDD8 monomer and NEDD8 conjugates in cells. This reduction is not due to down-regulation of NEDD8 transcription, but due to post-transcriptional mechanism. As expected from this activity, overexpression of NUB1 had a profound growth-inhibitory effect on U2OS cells. Thus, NUB1 is a strong down-regulator of the NEDD8 expression and appears to play critical roles in regulating biological events, including cell growth.AF300717 human cullin interferon hemagglutinin epitope RGS-poly(His) horseradish peroxidase glyceraldehyde-3-phosphate dehydrogenase ubiquitin C-terminal hydrolases ubiquitin-associated domain nuclear localization signal Skp1-Cullin-F-box kilobase(s) base pair(s) phosphate-buffered saline glutathione S-transferase activating enzyme conjugating enzyme ligating enzyme NEDD8 is a highly conserved 81-amino acid protein that shares 60“%” identity and 80“%” homology with ubiquitin. Expression of the NEDD8 message is highly restricted to the heart and skeletal muscle in adult human tissues (1Kamitani T. Kito K. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 28557-28562Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar) and is developmentally down-regulated in mouse embryos (1Kamitani T. Kito K. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 28557-28562Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar, 2Kumar S. Yoshida Y. Noda M. Biochem. Biophys. Res. Commun. 1993; 195: 393-399Crossref PubMed Scopus (117) Google Scholar). NEDD8 and its yeast homologue, Rub1 (3Lammer D. Mathias N. Laplaza J.M. Jiang W. Liu Y. Callis J. Goebl M. Estelle M. Genes Dev. 1998; 12: 914-926Crossref PubMed Scopus (279) Google Scholar, 4Liakopoulos D. Doenges G. Matuschewski K. Jentsch S. EMBO J. 1998; 17: 2208-2214Crossref PubMed Scopus (307) Google Scholar), belong to an expanding family of ubiquitin-like proteins that includes UCRP (5Haas A.L. Ahrens P. Bright P.M. Ankel H. J. Biol. Chem. 1987; 262: 11315-11323Abstract Full Text PDF PubMed Google Scholar), sentrin-1/SUMO1 (6Okura T. Gong L. Kamitani T. Wada T. Okura I. Wei C.-F. Chang H.-M. Yeh E.T.H. J. Immunol. 1996; 157: 4277-4281PubMed Google Scholar, 7Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), sentrin-2 (8Kamitani T. Kito K. Nguyen H.P. Fukuda-Kamitani T. Yeh E.T.H. J. Biol. Chem. 1998; 273: 11349-11353Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar), and sentrin-3 (9Kamitani T. Nguyen H.P. Kito K. Fukuda-Kamitani T. Yeh E.T.H. J. Biol. Chem. 1998; 273: 3117-3120Abstract Full Text Full Text PDF PubMed Scopus (177) Google Scholar). These proteins share a common distinction; the mature form is always translated in precursor form, with one or more amino acids following a Gly-Gly dipeptide that forms the C terminus of the mature protein (10Yeh E.T.H. Gong L. Kamitani T. Gene ( Amst. ). 2000; 248: 1-14Crossref PubMed Scopus (415) Google Scholar). In the NEDD8-conjugation process, the C-terminal tail of the precursor protein is cleaved off by a C-terminal hydrolase, such as UCH-L3 (11Wada H. Kito K. Caskey L.S. Yeh E.T.H. Kamitani T. Biochem. Biophys. Res. Commun. 1998; 251: 688-692Crossref PubMed Scopus (128) Google Scholar). The mature form has been shown to conjugate to a large number of nuclear proteins (1Kamitani T. Kito K. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 28557-28562Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). The pathway of NEDD8 conjugation is thought to be catalyzed by three enzymes, termed E1 (NEDD8-activating), E2 (NEDD8-conjugating), and E3 (NEDD8-ligating), in a manner analogous to ubiquitination and sentrinization (10Yeh E.T.H. Gong L. Kamitani T. Gene ( Amst. ). 2000; 248: 1-14Crossref PubMed Scopus (415) Google Scholar, 12Osaka F. Kawasaki H. Aida N. Saeki M. Chiba T. Kawashima S. Tanaka K. Kato S. Genes Dev. 1998; 12: 2263-2268Crossref PubMed Scopus (225) Google Scholar, 13Gong L. Yeh E.T.H. J. Biol. Chem. 1999; 274: 12036-12042Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). All known NEDD8 targets in mammalian cells are cullin (Cul)1 family members, including Cul-1, -2, -3, -4A, -4B, and -5 (14Kipreos E.T. Lander L.E. Wing J.P. He W.W. Hedgecock E.M. Cell. 1996; 85: 829-839Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar, 15Wada H. Yeh E.T.H. Kamitani T. Biochem. Biophys. Res. Commun. 1999; 257: 100-105Crossref PubMed Scopus (75) Google Scholar). Human Cul-1 is a major component of ubiquitin ligase, known as an SCF complex that catalyzes the ubiquitination of IκBα, “”β“”-catenin, and p27 (Kip1) (16Pagano M. FASEB J. 1997; 11: 1067-1075Crossref PubMed Scopus (196) Google Scholar, 17Lisztwan J. Marti A. Sutterluety H. Gstaiger M. Wirbelauer C. Krek W. EMBO J. 1998; 17: 368-383Crossref PubMed Scopus (178) Google Scholar, 18Laney J.D. Hochstrasser M. Cell. 1999; 97: 427-430Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). Interestingly, the NEDD8 conjugation to Cul-1 is required for the ubiquitin ligase activity of the SCF complex containing Cul-1 (19Read M.A. Brownell J.E. Gladysheva T.B. Hottelet M. Parent L.A. Coggins M.B. Pierce J.W. Podust V.N. Luo R.S. Chau V. Palombella V.J. Mol. Cell. Biol. 2000; 20: 2326-2333Crossref PubMed Scopus (329) Google Scholar,20Podust V.N. Brownell J.E. Gladysheva T.V. Lou R.S. Wang C. Coggins M.B. Pierce J.W. Lightcap E.S. Chau V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4579-4584Crossref PubMed Scopus (224) Google Scholar). Thus, NEDD8 conjugation seems to be involved in many important biological functions, including NFκB signaling and cell-cycle regulation by p27, and must be strictly regulated. However, the regulation system of NEDD8-conjugation is still unclear, with the exception of the recent discovery of USP21, a novel isopeptidase for NEDD8-conjugated proteins (21Gong L. Kamitani T. Millas S. Yeh E.T.H. J. Biol. Chem. 2000; 275: 14212-14216Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). To define the unknown regulators of NEDD8 conjugation, the yeast two-hybrid system was applied in this study. From library screening, we isolated a cDNA clone encoding a novel NEDD8-interacting protein, NUB1. Here, we report NUB1 as a strong down-regulator of the NEDD8 expression. We purchased the following human cell lines from American Type Culture Collection (Manassas, VA): rectal adenocarcinoma SW837, neuroblastoma SK-N-SH, malignant melanoma SK-MEL28, myeloid leukemia U937, Burkitt lymphoma Raji, T-cell leukemia Jurkat, chronic myelogenous leukemia K562, promyelocytic leukemia HL60, human embryonic kidney 293, osteosarcoma U2OS, renal cell carcinoma 786–0, and cervical adenocarcinoma HeLa. COS-M6 cells were a generous gift from Dr. Steve Goldring of Harvard Medical School. SW837, SK-N-SH, SK-MEL28, human embryonic kidney 293, U2OS, 786–0, HeLa, and COS-M6 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10“%” fetal calf serum and antibiotics. U937, Raji, Jurkat, K562, and HL60 cells were maintained in RPMI 1640 medium supplemented with 10“%” fetal calf serum and antibiotics. Human interferon-“”β“” (IFN-“”β“”) was purchased from Calbiochem (La Jolla, CA). Mouse monoclonal antibody 16B12 (Covance; Richmond, CA) is an antibody to the peptide sequence YPYDVPDYA of influenza hemagglutinin (HA). Rabbit anti-human NUB1 antiserum was generated by immunization with a GST fusion protein of NUB1 corresponding to amino acids 432–601 (NUB1432–601). Rabbit polyclonal anti-actin antibody (specific for the C-terminal actin fragment) was purchased from Sigma. To express proteins tagged with epitope at the N terminus in mammalian cells, plasmid vectors pcDNA3/HA-N (7Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar) and pcDNA3/RH-N (8Kamitani T. Kito K. Nguyen H.P. Fukuda-Kamitani T. Yeh E.T.H. J. Biol. Chem. 1998; 273: 11349-11353Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar) were used as described previously (1Kamitani T. Kito K. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 28557-28562Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). The human cDNAs used in this study have been described previously: ubiquitin (7Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), NEDD8 (1Kamitani T. Kito K. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 28557-28562Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar), sentrin-1 (7Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar), and Ubc12(C111S) (22Wada H. Yeh E.T.H. Kamitani T. J. Biol. Chem. 2000; 275: 17008-17015Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). These cDNAs were inserted into the aforementioned plasmid vectors. The sequence of each cDNA was confirmed by automated DNA sequencing. Plasmids were transfected into mammalian cells using FuGENE 6 (Roche Molecular Biochemicals). The transfected cells were processed for immunostaining, Western blotting, or Northern blotting 20 h after transfection. Yeast strain L40 was purchased from Invitrogen (Carlsbad, CA). Prey vector pGAD10 was purchased fromCLONTECH (Palo Alto, CA). The bait plasmid pHybLex/HA-NEDD8-GG (11Wada H. Kito K. Caskey L.S. Yeh E.T.H. Kamitani T. Biochem. Biophys. Res. Commun. 1998; 251: 688-692Crossref PubMed Scopus (128) Google Scholar) was transformed into L40 using the lithium acetate method (6Okura T. Gong L. Kamitani T. Wada T. Okura I. Wei C.-F. Chang H.-M. Yeh E.T.H. J. Immunol. 1996; 157: 4277-4281PubMed Google Scholar). The transformants were plated on YPD medium containing adenosine and Zeocin (YPAD/Zeo) and selected for 2 days at 30 °C. The L40 clone carrying pHybLex/HA-NEDD8-GG was cultured in YPAD/Zeo medium and sequentially transformed with 500 “μ”g of human heart cDNA (CLONTECH) fused to GAL4 DNA-activating domain vector, pGAD10. The transformed cells were incubated for 6 days at 30 °C on selection plates (Ura−, Lys−, His−, Leu−, and Zeocin+). The positive colonies were picked and replated on selection plates (Ura−, Lys−, His−, Leu−, and Zeocin+) and assayed for “”β“”-galactosidase activity on filter papers as described in the protocol ofCLONTECH. Domain search of NUB1 was performed by using several research tools as described below (all three programs are available via the World Wide Web). Coiled coil regions and ubiquitin-associated (UBA) domains were determined by the SMART program. Bipartite nuclear localization signal (NLS) was determined by the ProfileScan program. PEST sequence was determined by the PESTfind program. To demonstrate specificity of the immunoreactivity to NUB1, rabbit antiserum against GST-NUB1432–601 was preabsorbed with either GST or GST-NUB1432–601 and used for Western blot analysis as a primary antibody. For this preabsorption, of antiserum was incubated with GST or GST-NUB1432–601 fusion the the were by The was to with 20 containing and used for Western blot were at °C for h in of containing Western blotting was performed using the protocol with the system As horseradish peroxidase against mouse or rabbit CA) were was performed by the complex method T. H. S. Immunol. PubMed Scopus Google Scholar), using the system CA). cells on a were in for 20 and in for at with the cells were incubated with containing for to peroxidase activity and with The cells were incubated for with containing serum for by with antibody for 30 at °C. with the cells were incubated with for 30 at with and with the for 30 at °C. the was performed as previously T. H. S. Immunol. PubMed Scopus Google Scholar). To study the of NUB1 message in human tissues and mouse Northern blotting was of human NUB1 and NEDD8 cDNAs were from or (1Kamitani T. Kito K. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 28557-28562Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar), The were with by a The was with human Northern and a mouse Northern blot purchased To message by human Northern blotting was performed using cells were cultured with or human in and was using of were on a containing and to a by As of NUB1 and cDNAs were by and by a These were with by using the system for h and with at the were The signal was by the method using To the message of protein in Northern blot analysis was performed using a encoding epitope as a was using from COS-M6 cells transfected with the of was to on and a The for and were with using The for human was with using the DNA system The were to the in (CLONTECH) and in and was performed at °C. The was with after the was performed as described previously (22Wada H. Yeh E.T.H. Kamitani T. J. Biol. Chem. 2000; 275: 17008-17015Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, S. C. T. N. EMBO J. 1998; 17: PubMed Scopus Google Scholar). U2OS cells were plated in a and transfected by FuGENE 6 with “μ”g of control vector, or the cells were with and incubated with medium containing 10“%” fetal calf serum and The medium was 2 days after cells were and The were for by To NEDD8-interacting we primary library transformants as previously (11Wada H. Kito K. Caskey L.S. Yeh E.T.H. Kamitani T. Biochem. Biophys. Res. Commun. 1998; 251: 688-692Crossref PubMed Scopus (128) Google Scholar). of colonies on the selection of positive for “”β“”-galactosidase expression. analysis of DNA that of the human UCH-L3 (11Wada H. Kito K. Caskey L.S. Yeh E.T.H. Kamitani T. Biochem. Biophys. Res. Commun. 1998; 251: 688-692Crossref PubMed Scopus (128) Google Scholar). In of the to be a human of mouse yeast two-hybrid screening, we isolated of cDNA from a human heart cDNA library This cDNA a 69.1-kDa protein of 601 amino were found in the The was a is for M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). signal was found from the of the search of the base through the for that this protein was to mouse in amino acid that the protein is a human of mouse this protein the NEDD8 expression as described we this protein as NUB1 (NEDD8Ultimate and its cDNA sequence was to the cDNA sequence of mouse was in we not on its biological in the Thus, the of proteins NUB1 and was To the protein we or domains in NUB1 by using several research tools in As shown in were coil regions at the of NUB1. The coil was from to The coil was from to the C-terminal of NUB1, were a and a PEST The domain was from to The domain was from to The domain has been to in of the ubiquitin and K. P. Biochem. Sci. 1996; Full Text PDF PubMed Scopus Google Scholar). a was from to a PEST sequence S. M. PubMed Scopus Google Scholar) was from to on the domain search we that NUB1 was a nuclear protein and its was of the PEST To protein expression of NUB1 in human cell rabbit polyclonal antiserum for NUB1 was The expression of NUB1 was in human cell lines and cells by Western The antiserum was preabsorbed with either GST or not to demonstrate specificity of the immunoreactivity to NUB1. As shown in of a for NUB1 was in SK-N-SH, Raji, K562, and cells signal was in SW837, 293, HeLa, and cells. In SK-MEL28, Jurkat, and HL60 cells, the was the was in and U2OS cells, a to the In to this blotting, the antiserum preabsorbed with not not Thus, antiserum specifically NUB1, and its expression cell The sequence on a mouse of human NUB1, was to on and to number the of the base was an interferon-inducible with novel has not been in the To NUB1 is an protein, HeLa, 293, and U2OS cells were cultured with human and the of NUB1 was by Western As shown in the of with for h expression of the NUB1 protein in cells and cells but not in U2OS cells Thus, we found that NUB1 is an protein and that cells are to this we the of NUB1 protein by using cells for h with of As shown in of NUB1 protein was expressed in the cells and be with in a manner the of NUB1 was by using cells with for As shown in the of the expression of NUB1 protein was not by 2 h after with from h to h after that the NUB1 protein be by in a and In to was for its on of NUB1. In cells, NUB1 protein as as not To the expression of NUB1 message in human Northern blot were performed human NUB1 cDNA as As shown in of NUB1 message was in testis, ovary, heart, and skeletal In the message signal was or In a was in the but not in the As shown in the NEDD8 message was on the Interestingly, NEDD8 message was in the ovary, heart, and skeletal This specificity is to that of NUB1. NEDD8, with NUB1, was isolated as a developmentally down-regulated message in the mouse we the expression of NUB1 message in mouse embryos. As shown in of and NUB1 were The message was the message was and the message was These NUB1 were in the mouse and were in the and embryos In the of the NEDD8 message in the mouse and was in the and embryos Thus, of NUB1 and NEDD8 were developmentally down-regulated We the of NUB1. cells were for h with of and the of NUB1 message was by Northern blotting The NUB1 message not be in cells was by and in a manner Thus, the of NUB1. The localization of NUB1 was cells were transfected with a plasmid containing an of As we transfected an expression plasmid or a plasmid containing an of (21Gong L. Kamitani T. Millas S. Yeh E.T.H. J. Biol. Chem. 2000; 275: 14212-14216Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar) or The cells were and with antibody. As shown in be in the and the nucleus. and were restricted to the nucleus. This nuclear localization of NUB1 is with the that NUB1 has an in its sequence The yeast two-hybrid the NEDD8 and NUB1, that NUB1 the NEDD8-conjugation To this cell was NEDD8 was in cells with NUB1 or a control As ubiquitin or sentrin-1 was with vector or NUB1. As shown in was expressed with vector we a of and molecular of NEDD8-conjugated This NEDD8 expression was to (1Kamitani T. Kito K. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 28557-28562Abstract Full Text Full Text PDF PubMed Scopus (365) Google Scholar). was with we not the and forms of NEDD8 for a This that NUB1 the protein expression of NEDD8 monomer and its In was with or the overexpression of NUB1 not to reduction of the expression of ubiquitin or Thus, the down-regulation by NUB1 was to we the effect of NUB1 on expression of the NEDD8 protein, the molecular was NUB1 as an To this we the effect of NUB1 on expression of the NEDD8 As shown in overexpression of not the expression of message 2 This that NUB1 NEDD8 expression We previously of the NEDD8-conjugation The down-regulator is USP21, has isopeptidase activity with specificity for and NEDD8-conjugated The down-regulator is a that NEDD8 and NEDD8 These proteins U2OS cell (21Gong L. Kamitani T. Millas S. Yeh E.T.H. J. Biol. Chem. 2000; 275: 14212-14216Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, H. Yeh E.T.H. Kamitani T. J. Biol. Chem. 2000; 275: 17008-17015Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). We NUB1 has a biological As shown in overexpression of NUB1 cell to as with a cell to are a of with effect of is potent be on malignant and cells of many To this have been S. D. 2000; PubMed Google Scholar). In this we found three important of NUB1. NUB1. NUB1 the NEDD8 expression. NUB1 cell These that with NUB1 in of the NEDD8 expression and cell Thus, of cell be one of the of the effect of family are known as NEDD8 targets in mammalian cells H. Yeh E.T.H. Kamitani T. Biochem. Biophys. Res. Commun. 1999; 257: 100-105Crossref PubMed Scopus (75) Google Scholar). The cullin family is composed of Cul-1, -2, -3, -4A, -4B, and -5 (14Kipreos E.T. Lander L.E. Wing J.P. He W.W. Hedgecock E.M. Cell. 1996; 85: 829-839Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar). These SCF or have activity of E3 ubiquitin ligase (10Yeh E.T.H. Gong L. Kamitani T. Gene ( Amst. ). 2000; 248: 1-14Crossref PubMed Scopus (415) Google Scholar). Cul-1 an SCF complex to the ubiquitination of “”β“”-catenin, p27 and H. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar, M. S. M. M. N. K. I. A. K. K. EMBO J. 1999; PubMed Scopus Google Scholar, M. T. H. Biochem. Biophys. Res. Commun. 2000; PubMed Scopus Google Scholar, H. Chiba T. M. M. T. A. T. M. K. Tanaka K. Biochem. Biophys. Res. Commun. 1999; PubMed Scopus Google Scholar, M. F. A. P. H. J.P. F. F. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar, S. M. K. M. M. K. H. H. K. K. K. Proc. Natl. Acad. Sci. U. S. A. 1999; PubMed Scopus Google Scholar). an complex and is involved in the ubiquitination of T. S. K. M. J.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: PubMed Scopus Google Scholar). has been shown to for ubiquitination and control in mammalian cells J.D. M. J.M. Genes Dev. 1999; PubMed Scopus Google Scholar). Thus, cullin family play important roles in many biological several that NEDD8 conjugation to Cul-1 is required for the E3 ubiquitin ligase activity of the SCF complex (19Read M.A. Brownell J.E. Gladysheva T.B. Hottelet M. Parent L.A. Coggins M.B. Pierce J.W. Podust V.N. Luo R.S. Chau V. Palombella V.J. Mol. Cell. Biol. 2000; 20: 2326-2333Crossref PubMed Scopus (329) Google Scholar,20Podust V.N. Brownell J.E. Gladysheva T.V. Lou R.S. Wang C. Coggins M.B. Pierce J.W. Lightcap E.S. Chau V. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 4579-4584Crossref PubMed Scopus (224) Google Scholar, M. T. H. Biochem. Biophys. Res. Commun. 2000; PubMed Scopus Google Scholar). These that the ubiquitin ligase activity of SCF and is by conjugation of NEDD8 to cullins. the NEDD8 expression is down-regulated by NUB1, the expression of NUB1 control many biological events, including cell NFκB and biological to this we that overexpression of NUB1 leads to reduction of NEDD8 monomer and its This reduction from the down-regulation of NEDD8 expression by NUB1. NUB1 not NEDD8 expression at message the reduction appears to be by a post-transcriptional mechanism. are required to define the mechanism. We Nguyen and Dr. Wada for and