Characterization of NEDD8, a Developmentally Down-regulated Ubiquitin-like Protein

NEDD8 is a novel 81 amino acid polypeptide which is 60% identical and 80% homologous to ubiquitin. Northern blot analysis showed that the NEDD8 message was developmentally down-regulated. In adult tissues, NEDD8 expression was mostly restricted to the heart and skeletal muscle. Antiserum specific for NEDD8 detected a 6-kDa monomer in SK-N-SH, BJAB, and HL60 cell lysates. A 14-kDa band was also detected in BJAB, HL60, and SK-MEL28 but not in SK-N-SH and K562 cell lysates. An approximately 90-kDa band was detected in all cell lines tested. Thus, NEDD8 is likely to be conjugated to other proteins in a manner analogous to ubiquitination. However, the conjugation pattern of NEDD8 is entirely different from that of ubiquitin in all cell lines tested. To study NEDD8 conjugation in more detail, hemagglutinin-epitope-tagged NEDD8 was expressed in COS cells. Western blot analysis revealed an NEDD8 monomer and a series of higher molecular weight NEDD8-conjugated proteins or NEDD8 multimers. Immunocytochemical analysis showed that NEDD8 expression was highly enriched in the nucleus and was much weaker in the cytosol. In contrast, ubiquitin expression was detectable equally well in the nucleus and cytosol. Mutational analysis showed that the C terminus of NEDD8 was efficiently cleaved and that Gly-76 was required for conjugation of NEDD8 to other proteins. Taken together, NEDD8 provides another substrate for covalent protein modification and may play a unique role during development. NEDD8 is a novel 81 amino acid polypeptide which is 60% identical and 80% homologous to ubiquitin. Northern blot analysis showed that the NEDD8 message was developmentally down-regulated. In adult tissues, NEDD8 expression was mostly restricted to the heart and skeletal muscle. Antiserum specific for NEDD8 detected a 6-kDa monomer in SK-N-SH, BJAB, and HL60 cell lysates. A 14-kDa band was also detected in BJAB, HL60, and SK-MEL28 but not in SK-N-SH and K562 cell lysates. An approximately 90-kDa band was detected in all cell lines tested. Thus, NEDD8 is likely to be conjugated to other proteins in a manner analogous to ubiquitination. However, the conjugation pattern of NEDD8 is entirely different from that of ubiquitin in all cell lines tested. To study NEDD8 conjugation in more detail, hemagglutinin-epitope-tagged NEDD8 was expressed in COS cells. Western blot analysis revealed an NEDD8 monomer and a series of higher molecular weight NEDD8-conjugated proteins or NEDD8 multimers. Immunocytochemical analysis showed that NEDD8 expression was highly enriched in the nucleus and was much weaker in the cytosol. In contrast, ubiquitin expression was detectable equally well in the nucleus and cytosol. Mutational analysis showed that the C terminus of NEDD8 was efficiently cleaved and that Gly-76 was required for conjugation of NEDD8 to other proteins. Taken together, NEDD8 provides another substrate for covalent protein modification and may play a unique role during development. Ubiquitin is one of the most conserved eukaryotic proteins which can be conjugated to other proteins through a well defined enzymatic pathway (1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1992; 61: 761-807Crossref PubMed Scopus (1190) Google Scholar, 2Jentsch S. Annu. Rev. Genet. 1992; 26: 179-207Crossref PubMed Scopus (450) Google Scholar). The importance of ubiquitination is underscored by its involvement in antigen processing, in cell cycle regulation, in degradation of tumor suppressors, in receptor endocytosis, and in signal transduction (3Hochstrasser M. Cell. 1996; 84: 813-815Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar, 4Rock K.L. Gramm C. Rothstein L. Clark K. Stein R. Dick L. Hwang D. Goldberg A.L. Cell. 1994; 78: 761-771Abstract Full Text PDF PubMed Scopus (2172) Google Scholar, 5Murray A. 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Conjugation of ubiquitin to its target protein requires the initial activation of the conserved C-terminal Gly residue catalyzed by a specific ubiquitin-activating enzyme, E1 1The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase; HA, hemagglutinin; UCRP, ubiquitin cross-reactive protein; NEDD8, neural precursor cell-expressed developmentally down-regulated; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; HRP, horseradish peroxidase; GST, glutathione S-transferase; mAb, monoclonal antibody. 1The abbreviations used are: E1, ubiquitin-activating enzyme; E2, ubiquitin-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase; HA, hemagglutinin; UCRP, ubiquitin cross-reactive protein; NEDD8, neural precursor cell-expressed developmentally down-regulated; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; HRP, horseradish peroxidase; GST, glutathione S-transferase; mAb, monoclonal antibody. (1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1992; 61: 761-807Crossref PubMed Scopus (1190) Google Scholar, 2Jentsch S. Annu. Rev. Genet. 1992; 26: 179-207Crossref PubMed Scopus (450) Google Scholar, 11Hochstrasser M. Curr. Opin. Cell Biol. 1995; 7: 215-233Crossref PubMed Scopus (775) Google Scholar, 12Wilkinson K.D. Annu. Rev. Nutr. 1995; 15: 161-189Crossref PubMed Scopus (133) Google Scholar, 13Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2215) Google Scholar). Ubiquitin adenylate is formed by displacement of PPi from ATP and subsequently transferred to a thiol site in E1 with release of AMP. Next, ubiquitin is transferred to a ubiquitin-conjugating enzyme, E2, to form another thiol ester bond. Finally, ubiquitin is transferred from E2 to its target protein through an isopeptide linkage with the ε-amino group of the Lys residue of the target protein. The transfer of ubiquitin from E2 to the target protein requires the participation of a ligase, E3, in many instances. The biological specificity of the ubiquitination pathway appears to be regulated by a selective combination of E2 and E3 proteins (6Hopkin K. J. Natl. Inst. Health Res. 1997; 9: 36-42Google Scholar). Currently, more than 30 E2 and 10 E3 proteins have been identified. The complexity of the ubiquitination system is further compounded by the identification of other ubiquitin-like molecules, such as UCRP and sentrin. UCRP (ubiquitin cross-reactive protein) is a type 1 interferon-inducible protein which contains two ubiquitin domains (14Haas A.L. Ahrens P. Bright P.M. Ankel H. J. Biol. Chem. 1987; 262: 11315-11323Abstract Full Text PDF PubMed Google Scholar). UCRP has been shown to be conjugated to a large number of intracellular proteins (15Loeb K.R. Haas A.L. J. Biol. Chem. 1992; 267: 7806-7813Abstract Full Text PDF PubMed Google Scholar). The proteins which are modified by UCRP have not yet been identified. It is unknown whether UCRP and ubiquitin could share the same substrate. Recent results from Haas and co-workers (16Narasimhan J. Potter J.L. Haas A.L. J. Biol. Chem. 1996; 271: 324-330Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) suggest that UCRP conjugation proceeds through an enzyme pathway distinct from that of ubiquitin with respect to the activation step. Our laboratory has recently reported the cloning of a novel ubiquitin-like protein, sentrin, that protects cells against both anti-Fas and tumor necrosis factor-induced cell death (17Okura T. Gong L. Kamitani T. Wada T. Okura I. Wei C.K. Chang H.M. Yeh E.T.H. J. Immunol. 1996; 272: 4277-4281Google Scholar). We have further demonstrated that the C terminus of sentrin is efficiently processed, which allows sentrin to be transferred to a subset of nuclear proteins (18Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). It appears that sentrin targets a more limited substrate pool than ubiquitin (18Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Mahajan et al. (19Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (999) Google Scholar) and Matunis et al. (20Matunis M.J. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (948) Google Scholar) have independently identified a novel modification of RanGAP1 by a ubiquitin-like protein, respectively called SUMO-1 and GMP-1. Boddy et al. (21Boddy M.N. Howe K. Etkin L.D. Solomon E. Freemont P.S. Oncogene. 1996; 13: 971-982PubMed Google Scholar) and Shen et al. (22Shen Z. Pardington-Purtymun P.E. Comeaux J.C. Moyzis R.K. Chen D.J. Genomics. 1996; 36: 271-279Crossref PubMed Scopus (179) Google Scholar) have reported the interaction between PIC1 with PML or UBL1 with Rad51. Remarkably, PIC1, UBL1, SUMO-1, and GMP-1 are identical to sentrin. Kumar et al. (23Kumar S. Tomooka Y. Noda M. Biochem. Biophys. Res. Commun. 1992; 185: 1155-1161Crossref PubMed Scopus (439) Google Scholar) have reported another ubiquitin-like protein, NEDD8 (Neural precursor cell-Expressed Developmentally down-regulated), using a subtraction cloning approach. NEDD8 encodes a small novel protein of 81 amino acids, which is 60% identical and 80% homologous to ubiquitin (24Kumar S. Yoshida Y. Noda M. Biochem. Biophys. Res. Commun. 1993; 195: 393-399Crossref PubMed Scopus (113) Google Scholar). Although the authors speculated that NEDD8 could also be conjugated to other proteins, they did not provide any biochemical evidence for NEDD8 modification of other proteins. Here, we demonstrate that NEDD8 is indeed activated and transferred to other proteins in a process analogous to ubiquitination. Furthermore, NEDD8-conjugated proteins appear to reside predominately in the nucleus. The characterization of NEDD8 adds another level of complexity to the process of protein ubiquitination. The specific antiserum described in this manuscript will provide a powerful tool to study the biochemical and biological differences between NEDD8 modification and protein ubiquitination. SK-MEL28, SK-N-SH, HL60, and K562 were purchased from American Type Culture Collection (Rockville, MD). BJAB and COS-M6 cells were generous gifts from Dr. Fred Wang (Harvard University) and Dr. Steve Goldring (Harvard Medical School), respectively. These cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and antibiotics. 12CA5 (Boehringer Mannheim) and 16B12 (BAbCo, Richmond, CA) are mouse monoclonal antibodies to the peptide sequence YPYDVPDYA of influenza hemagglutinin (HA). Mouse anti-RH (specific for the amino acid sequence, RGSHHHH) monoclonal antibody was purchased from QIAGEN (Santa Clara, CA). Mouse anti-GST monoclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit polyclonal anti-human NEDD8 antiserum was generated by immunization with a peptide corresponding to amino acids 20–32 (TDKVERIKERVEE). The rabbit polyclonal anti-ubiquitin antibody was purchased from Sigma. To express HA-tagged proteins in COS-M6 cells, two vectors for N-terminal tagging (pcDNA3/HA-N) and C-terminal tagging (pcDNA3/HA-C) were constructed as described previously (18Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). HA adaptor duplexes were inserted into pcDNA3 (Invitrogen, San Diego, CA), and then the cDNAs of ubiquitin, sentrin, Rad51, and NEDD8 mutants were carried out by polymerase chain reaction using appropriate primers followed by ligation into the vector pcDNA3/HA-N or pcDNA3/HA-C. Plasmid encoding RGS(H)6-tagged RanGAP1 was constructed by inserting the human RanGAP1 cDNA into pcDNA3/RH-N vector. The sequence of each insert was confirmed by automated DNA sequencing. Plasmids were transfected into COS-M6 cells using LipofectAMINE (Life Technologies, Inc.). The transfected cells were harvested for Western blotting or immunostaining 16 h after transfection. -A full-length cDNA fragment of human NEDD8 from the plasmid pcDNA3/NEDD8-HA was labeled with α-32P-dCTP by a megaprime labeling kit (Amersham). The radioactive probe was hybridized with human multiple tissue Northern blots and mouse embryo multiple tissue Northern blot purchased from CLONTECH. 1 × 106 cells were harvested, washed twice with cold PBS, and centrifuged. To prevent protein degradation, the cell pellet was immediately transferred into liquid nitrogen, and then the frozen pellet was treated at 45 °C for 1 h in 300 μl of 2% SDS treating solution containing 5% β-mercaptoethanol. DNA in the sample was sheared with a 25 G needle. After SDS-PAGE, using 3 μl of the sample (equivalent to 1 × 104 cells) per each lane, Western blotting was performed by the protocol of ECL detection system (Amersham Corp.). As a secondary antibody, horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Santa Cruz Biotechnology) was used. RH-RanGAP1 was co-expressed with HA-Rad51, HA-ubiquitin, HA-NEDD8, or HA-sentrin in COS cells. Total cell lysate from the transfectants was prepared in the lysis buffer (pH 7.8) containing 6 m guanidine-HCl. The lysate was centrifuged at 100,000 × g at 15 °C for 30 min, and the supernatant was incubated with nickel-charged agarose resin beads (Invitrogen) for 1 h at room temperature. The beads were washed twice with the washing buffer (pH 7.8) containing 8m urea, followed by washing with a buffer (pH 6.0) containing 8 m urea. Finally, the beads were washed twice with PBS and treated in 2% SDS solution for SDS-PAGE. The solubilized proteins were analyzed by Western blotting using anti-HA antibody to detect RH-RanGAP1 conjugated with HA-tagged ubiquitin-like proteins and using anti-RH antibody to detect derivatives of RH-RanGAP1. Immunocytochemical staining was performed by the avidin-biotin-HRP complex (ABC-HRP) method (25Kamitani T. Suzuki H. Yano S. Clin. Immunol. Immunopathol. 1991; 58: 217-235Crossref PubMed Scopus (8) Google Scholar), using the VECTASTAIN ABC kit system (Vector, Burlingame, CA). Transfected COS-M6 cells on coverslip were fixed in 3.7% paraformaldehyde solution for 20 min and permeabilized in 0.1% Triton X-100 for 10 min at room temperature. After washing with PBS, the fixed cells were incubated with PBS containing 0.1% H2O2 for 10 min to quench endogenous peroxidase activity and then washed with PBS. The cells were incubated for 10 min with PBS containing 5% horse serum for blocking, followed by the further incubation with anti-HA antibody (16B12) for 30 min at 37 °C. After rinsing with PBS, the cells were incubated with biotinylated anti-mouse IgG for 30 min at 37 °C, washed with PBS, and then treated with the ABC reagent (avidin-biotin-HRP complex) for 30 min at 37 °C. Finally, the enzymatic disclosing procedure was performed as reported previously (25Kamitani T. Suzuki H. Yano S. Clin. Immunol. Immunopathol. 1991; 58: 217-235Crossref PubMed Scopus (8) Google Scholar). -The expression and purification were performed essentially as described previously (26Kaelin Jr., W.G. Pallas D.C. DeCaprio J.A. Kaye F.J. Livingston D.M. Cell. 1991; 64: 521-532Abstract Full Text PDF PubMed Scopus (433) Google Scholar). Logarithmically growing cultures (500 ml,A 600 = 0.8) of Escherichia coli BL 21 (Stratagene, La Jolla, CA) transformed with the pGEX-2TK (Pharmacia Biotech Inc.) recombinants were incubated with 0.1 mmisopropyl-β-d-thiogalactopyranoside (Stratagene) at room temperature for 2.5 h. The cells were then pelleted, resuspended in 50 ml of ice-cold NETN buffer (100 mm NaCl, 1 mm EDTA, 20 mm Tris (pH 8.0), 0.5% Nonidet P-40) containing 100 μg/ml egg white lysozyme (Sigma). The bacteria were then lysed on ice by and centrifuged at × g for 30 min at °C. were for 1 h at °C with μl of beads which been previously washed and resuspended in The beads were then washed with analysis of proteins, the beads were incubated in the sample buffer containing 2% SDS and at 45 °C for 1 h and then 1 ml of antiserum was incubated with or The beads were by and the supernatant was used for Western blotting NEDD8 was from a subtraction prepared by a cDNA of mouse neural precursor cells with adult (23Kumar S. Tomooka Y. Noda M. Biochem. Biophys. Res. Commun. 1992; 185: 1155-1161Crossref PubMed Scopus (439) Google Scholar). It encodes an acid polypeptide with 60% to human ubiquitin. 1 the amino acid of and NEDD8 with ubiquitin. As NEDD8 is highly conserved in is a amino acid between the human and mouse Furthermore, its C terminus contains the which are for conjugation of ubiquitin to other proteins. The which an role in the of is also To the expression of NEDD8 in human tissues, Northern blot were performed human NEDD8 cDNA as As shown in NEDD8 message is highly enriched in the heart and skeletal but much in all other NEDD8 was as a developmentally message in the mouse we also its expression in the mouse As shown in NEDD8 message was in the mouse embryo and was in 15 and blot analysis of NEDD8 expression in mouse of different A cDNA fragment of human NEDD8 from pcDNA3/NEDD8-HA was used as a probe was used as a To further NEDD8 protein expression in or cell polyclonal antibody specific for NEDD8 was As shown in antiserum with protein but not with In contrast, anti-GST antibody was to detect both and and The expression of NEDD8 was in different cell lines using antiserum as described As shown in antiserum specific for NEDD8 identified a 6-kDa monomer in SK-N-SH, BJAB, and HL60 A 14-kDa band was also in BJAB, HL60, and Furthermore, a 90-kDa band was in all cell lines tested. The of the and 90-kDa is In a analysis with anti-ubiquitin antiserum a 6-kDa band was in SK-N-SH, BJAB, HL60, and K562 Furthermore, a of molecular weight proteins was in Western blot and is that the expression pattern of NEDD8 is distinct from that of blot analysis of NEDD8 expression in human cell Total cell were analyzed by Western blotting using rabbit antiserum with or as a blot analysis of ubiquitin expression in human cell Total cell were analyzed by Western blotting using rabbit anti-ubiquitin antiserum with or as a To study the of NEDD8 modification in more detail, NEDD8 was transfected into COS cells as described previously (18Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). Transfected cell were analyzed by Western blot analysis with anti-HA antibody. ubiquitin as a As shown in NEDD8 expression and conjugation are distinct from that of ubiquitin anti-HA monoclonal antibodies were used in Western blot 12CA5 is more than However, and were in the 12CA5 blot as previously reported (18Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). that NEDD8 modification is much than ubiquitination. The 90-kDa band in the COS cell lysate is also of However, is that this band is RanGAP1 The using a more anti-HA antibody revealed a series of in a pattern to in the These could conjugation of proteins by NEDD8 the residue is conserved in However, this to be The 90-kDa band shown in is of RanGAP1 modified by sentrin. To this a COS cell was RanGAP1 was in the terminus with the amino acid sequence of which for purification of the RanGAP1 protein with resin Plasmids encoding for HA-tagged sentrin, HA-tagged ubiquitin, HA-tagged NEDD8, and HA-tagged were with plasmid encoding RH-RanGAP1 into COS cells as described The prepared from the transfectants were with resin beads The were with Western blot analysis using anti-HA antibody. As shown in RanGAP1 is in However, HA-tagged ubiquitin HA-tagged NEDD8 were to form a with The HA-tagged Rad51, also could not to The of the protocol was confirmed in a Western blot analysis anti-RH monoclonal antibody. As shown in both RanGAP1 and RanGAP1 could be equally well in all It be that was from RanGAP1 modified by sentrin in COS cells. is further by the of a in the sample The band is most likely and the band is most likely RanGAP1 Taken together, NEDD8 could not form a with The of in to be Transfected COS cells were also with anti-HA antibody (16B12) as described previously (18Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). As shown in NEDD8 is expressed predominately in the nucleus with In contrast, ubiquitin is in both nucleus and cytosol. The pattern of NEDD8 is of sentrin, which is more restricted to the nuclear (18Kamitani T. Nguyen H.P. Yeh E.T.H. J. Biol. Chem. 1997; 272: 14001-14004Abstract Full Text Full Text PDF PubMed Scopus (145) Google Scholar). The C terminus of human NEDD8 contains to the conserved and Gly-76 In for NEDD8 conjugation to this C-terminal be As shown in the was at the C terminus of NEDD8, is efficiently cleaved in transfected COS cells. Thus, anti-HA antibody could not detect any NEDD8 monomer or proteins. is further confirmed by the As antiserum could detect a 6-kDa band in COS cells transfected with vector and a 6-kDa or and The band is in cell prepared from COS cells transfected with NEDD8 with HA at the C of for Gly at or did not of the C terminus of the C terminus of NEDD8 contains Gly is to which Gly residue is in the of NEDD8 As shown in Gly-76 is required for the of NEDD8 Thus, in the the NEDD8 monomer is Taken together, the C-terminal and conjugation is identical to that of ubiquitin and sentrin. The of proteins modified by NEDD8 is proteins for NEDD8 modification are nuclear proteins expressed in the heart and skeletal or in and Furthermore, is unknown whether a protein can be modified by ubiquitin or NEDD8 or both ubiquitin and NEDD8 the conserved is also of to whether of NEDD8 and ubiquitin The antiserum described in this will provide an tool to in the We L. Gong and L. for of this