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 ...

Acute promyelocytic leukemia arises following a reciprocal chromosome translocation t(15;17), which generates PML-retinoic acid receptor ␣ fusion proteins (PML-RAR␣). We have shown previously that wild type PML, but not PML-RAR␣, is covalently modified by the sentrin family of ubiquitin-like proteins (Kamitani, T., Nguyen, H. P., Kito, K., Fukuda-Kamitani, T., and Yeh, E. T. H. (1998) J. Biol. Chem. 273, 3117-3120). To understand the mechanisms underlying the differential sentrinization of PML versus PML-RAR␣, extensive mutational analysis was carried out to determine which Lys residues are sentrinized. We show that Lys 65 in the RING finger domain, Lys 160 in the B1 Box, and Lys 490 in the nuclear localization signal contributes three major sentrinization sites. The PML mutant with Lys to Arg substitutions in all three sites is expressed normally, but cannot be sentrinized. Furthermore, the triple substitution mutant is localized predominantly to the nucleoplasm, in contrast to wild type PML, which is localized to the nuclear bodies. Thus, sentrinization of PML, in the context of the RING finger and the B1 box, regulates nuclear body formation. Furthermore, we showed that sentrinization of PML-RAR␣ could be restored by overexpression of sentrin, but not by retinoic acid treatment. These studies provide novel insight into the pathobiochemistry of acute promyelocytic leukemia and the sentrinization pathway.

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PML, a RING finger protein with tumor suppressor activity, has been implicated in the pathogenesis of acute promyelocytic leukemia that arises following a reciprocal chromosomal translocation that fuses the PML gene with the retinoic acid receptor ␣ (RAR␣) gene. Immunocytochemical analysis has demonstrated that PML is co-localized with a novel ubiquitin-like protein in the nuclear bodies, which could be disrupted by the PML-RAR␣ fusion protein. The physical nature of this co-localization is unknown. Using a COS cell expression system, we show that PML is covalently modified by all three members of the sentrin family of ubiquitin-like proteins. Covalent modification of PML requires the conserved Gly residue near the C termini of sentrin proteins. Sentrinization of PML is highly specific because neither NEDD8 nor ubiquitin could modify PML. Similar specificity is also observed for the covalent modification of RanGAP1 by the sentrin member of ubiquitin-like proteins. These observations highlight the fine substrate specificity of the sentrinization pathway. In acute promyelocytic leukemia, two forms of PML-RAR␣ fusion proteins have been reported. Remarkably, both forms of PML-RAR␣ fusion proteins could not be sentrinized. Thus differential sentrinization of PML and PML-RAR␣ could play an important role in regulating the biological function of PML and in the pathogenesis of acute promyelocytic leukemia.Chromosomal translocation t(15;17), detected in the majority of patients with acute promyelocytic leukemia, generates a fusion protein composed of portions of the retinoic acid receptor ␣ (RAR␣) 1 and RING finger protein called PML (1-4). In normal myeloid cells, PML is localized to a nuclear multiprotein complex called nuclear bodies or PML oncogenic domains (5, 6). In cell lines derived from patients with acute promyelocytic leukemia, the nuclear bodies are disrupted into a microparticulate pattern, which is reversible by treatment with retinoic acid (5, 6). PML has also been shown to suppress the transformation of NIH3T3 cells by the activated neu oncogene (7). Using fulllength PML as bait in a yeast two-hybrid interaction screening, Boddy et al. have isolated a novel ubiquitin-like protein called PIC1 that interacted specifically with PML in the yeast interaction assay (8). They have further shown that PIC1 was colocalized with PML to the nuclear bodies. In NB4 cells, which are derived from a patient with acute promyelocytic leukemia, there was no significant co-localization of PIC1 with PML. However, following retinoic acid treatment, a significant relocalization of PIC1 with PML was observed. These observations suggest that the association of PIC1 with PML may play an important role in the pathogenesis of acute promyelocytic leukemia.Our laboratory has recently reported the cloning of a novel ubiquitin-like molecule, called sentrin, which interacts specifically with the death domains of both Fas and tumor necrosis factor receptor 1 (9). Overexpression of sentrin protects cells against anti-Fas or tumor necrosis f...

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NEDD8 is a ubiquitin-like protein that controls vital biological events through its conjugation to cullin family members. Recently, we identified a negative regulator of the NEDD8 conjugation system, NUB1, which interacts with NEDD8 and down-regulates NEDD8 ex- These results suggest that NUB1 recruits NEDD8 and its conjugates to the proteasome for degradation, providing a direct functional link between the NEDD8 conjugation system and the proteasomal degradation pathway.

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 (NEDD8 Ultimate Buster-1). NUB1 is composed of 601 residues with a calculated 69.1-kDa molecular mass. It is an interferoninducible 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.