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

Abstract

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 (1, 2). 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 (3-10). 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 1 (1, 2, 11-13). 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 ⑀-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 (6). 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 (14). UCRP has been shown to be ...

Building and remodelling Cullin–RING E3 ubiquitin ligases

“…Early studies demonstrated that cullin neddylation is required for efficient ubiquitylation and/or turnover of CRL substrates in vivo, which has been borne out through the development of MLN4924, a small-molecule inhibitor of NAE [14,[48][49][50]. Inhibition of CRL activity by MLN4924 results in accumulation of a host of CRL targets [14,43,51,52].…”

Targeting inhibitors of apoptosis proteins suppresses medulloblastoma cell proliferation via G2/M phase arrest and attenuated neddylation of p21

“…The RING domain of IAPs acts as an E3 ligase, leading to ubiquitination of IAPs themselves and their client proteins, such as caspases. Recent studies discovered that neuronal precursor cell‐expressed developmentally downregulated protein 8 (NEDD8), a ubiquitin‐like protein,17 is activated by IAPs in the regulation of apoptosis through neddylation of caspases 18, 19, 20. IAPs function in ways beyond inhibition of apoptotic proteins.…”

Covalent modification of all members of human cullin family proteins by NEDD8

“…The elevation of NEDD8 expression was observed in various human tumor cell lines, such as leukemia, HeLa, K562, and MOLT-4 cells but was low in Raji, SW480, and G361 cells. No appreciable change in the NEDD8 expression was observed during embryonic development of mice, although it was reported to be down-regulated developmentally (Kumar et al, 1993;Kamitani et al, 1997).…”