Nuclear domain 10 (ND10), also referred to as nuclear bodies, are discrete interchromosomal accumulations of several proteins including promyelocytic leukemia protein (PML) and Sp100. In this study, we investigated the mechanism of ND10 assembly by identifying proteins that are essential for this process using cells lines that lack individual ND10-associated proteins. We identified the adapter protein Daxx and BML, the RecQ helicase missing in Bloom syndrome, as new ND10-associated proteins. PML, but not BLM or Sp100, was found to be responsible for the proper localization of all other ND10-associated proteins since they are dispersed in PML−/− cells. Introducing PML into this cell line by transient expression or fusion with PML-producing cells recruited ND10-associated proteins into de novo formed ND10 attesting to PMLs essential nature in ND10 formation. In the absence of PML, Daxx is highly enriched in condensed chromatin. Its recruitment to ND10 from condensed chromatin requires a small ubiquitin-related modifier (SUMO-1) modification of PML and reflects the interaction between the COOH-terminal domain of Daxx and PML. The segregation of Daxx from condensed chromatin in the absence of PML to ND10 by increased accumulation of SUMO-1–modified PML suggests the presence of a variable equilibrium between these two nuclear sites. Our findings identify the basic requirements for ND10 formation and suggest a dynamic mechanism for protein recruitment to these nuclear domains controlled by the SUMO-1 modification state of PML.

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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.

Sentrin is a novel ubiquitin-like protein that protects cells against both anti-Fas and tumor necrosis factorinduced cell death. Antiserum recognizing the N terminus of sentrin revealed the presence of a 18-kDa sentrin monomer, a 90-kDa band (p90), and multiple high molecular mass bands. Because sentrin possesses the conserved Gly-Gly residues near the C terminus, it is likely that these additional bands represent conjugation of sentrin to other proteins in a manner that is similar to the ubiquitination pathway. Transient expression of hemagglutinin epitope-tagged sentrin mutants in COS cells demonstrated that the sentrin C terminus is cleaved, which allows it to be conjugated to other proteins via the conserved C-terminal Gly residue. Immunocytochemical staining and cell fractionation analysis demonstrated that sentrin monomer is localized predominantly to the cytosol. However, p90 and the majority of sentrinized proteins appeared to be localized to the nucleus. When the conserved Gly-Gly residues of sentrin were changed to Gly-Ala, only sentrin monomer and p90 but not the high molecular mass bands were observed. Thus, p90 generation appears to be required for the formation of high molecular mass bands in the nucleus. Taken together, sentrinization represents a novel pathway for nuclear protein modification, which is distinct from ubiquitination.Sentrin was originally isolated in a yeast two hybrid screen using the death domain of Fas as bait (1). It also interacts with tumor necrosis factor (TNF) 1 receptor 1 death domain but not with the death domain of FADD/MORT1 or CD40. When overexpressed in mammalian cells, sentrin protects cells against both anti-Fas and TNF-induced cell death. The mechanism of action of sentrin has not been clearly elucidated. Sentrin could block cell death signaling by blocking the assembly of the death-inducing signal complex. Alternatively, due to its homology to ubiquitin (18% identical and 48% similar), sentrin could exert its anti-death effect through modification of other proteins in a process similar to ubiquitination.Protein modification by ubiquitin is critical for targeting proteins to be degraded by proteasomes (2-4). Conjugation of ubiquitin to other proteins requires initial activation of the conserved C-terminal Gly residue catalyzed by a specific ubiquitin-activating enzyme, E1. An intermediate, ubiquitin adenylate, is formed by displacement of PPi from ATP. Ubiquitin adenylate is then transferred to a thiol site in E1 with release of AMP. Next, ubiquitin is transferred to a family of ubiquitin carrier proteins, E2, through transacylation. 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 may require the participation of a ligase, E3. The internal Lys of ubiquitin, in particular Lys 48 , can also be modified by another ubiquitin to form multiubiquitin chains that may be crucial for proteosome recognition (5)....