Ubiquitin-like protein activation Danny T Huang 1,2 , Helen Walden 1,2 , David Duda 1,2 and Brenda A Schulman* ,1,2 1 Department of Structural Biology, St Jude Children’s Research Hospital, Memphis, TN 38105, USA; 2 Department of Genetics, Tumor Cell Biology, St Jude Children’s Research Hospital, Memphis, TN 38105, USA Post-translational covalent attachment of ubiquitin and ubiquitin-like proteins (ubls) has emerged as a predomi- nant cellular regulatory mechanism, with important roles in controlling cell division, signal transduction, embryonic development, endocytic trafficking and the immune response. Ubls function by remodeling the surface of their target proteins, changing their target’s half-life, enzy- matic activity, protein–protein interactions, subcellular localization or other properties. At least 10 different ubiquitin-like modifications exist in mammals, and attachment of different ubls to a target leads to different biological consequences. Ubl-conjugation cascades are initiated by activating enzymes, which also coordinate the ubls with their downstream pathways. A number of biochemical and structural studies have provided insights into the mechanism of ubl-activating enzymes and their roles in ubl conjugation cascades. Oncogene (2004) 23, 1958–1971. doi:10.1038/sj.onc.1207393 Keywords: ubiquitin; E1; ubiquitin activating enzyme; SUMO; NEDD8; MoeB Introduction Ubiquitin and its relatives are small proteins that are covalently attached to other proteins and macromole- cules via their C-termini. Post-translational modification by ubiquitin and ubiquitin-like proteins (ubls) has emerged as a predominant cellular regulatory mechanism (reviewed in Hochstrasser, 2000b; Pickart, 2001), with important roles in cell division, the immune response, development, endocytic trafficking, biosynthetic path- ways, cancer and many other biological processes (for examples and reviews, see Scheffner et al., 1990; Glotzer et al., 1991; Rock et al., 1994; Pagano et al., 1995; Aberle et al., 1997; Honda et al., 1997; Kishino et al., 1997; Matsuura et al., 1997; Salceda and Caro, 1997; Ghosh et al., 1998; Hicke, 1999, 2001a; Joazeiro et al., 1999; Kamura et al., 1999; Koepp et al., 1999; Lorick et al., 1999; Sidow et al., 1999; York et al., 1999; Yewdell and Bennink, 2001; Kwon et al., 2002). Modification by ubls rapidly and reversibly changes the function of the target. The different ubl modifications can affect different properties of the target (Hoege et al., 2002), including the protein’s half-life, enzymatic activity, subcellular localization and protein–protein interactions (reviewed in Hochstrasser, 1996, 2000a,b; Jentsch and Pyrowolakis, 2000; Yeh et al., 2000; Pickart, 2001). The best under- stood consequence of these modifications is ubiquitin- mediated proteolysis, in which polymeric chains contain- ing four or more ubiquitins, isopeptide-linked between Lys48 on the surface of one ubiquitin and the C-terminus of the next in the chain, direct proteins for degradation by the proteasome (reviewed in Rechsteiner, 1998). Many other types of ubiquitin modification also play funda- mental cellular regulatory roles. For example, multi- ubiquitin chains with linkages via Lys63, rather than Lys48, activate the IkB kinase (Deng et al., 2000), and monoubiquitylation plays a role in processes ranging from endocytosis to transcriptional activation (reviewed in Hicke, 2001b). Other ubls that structurally resemble ubiquitin and are conjugated to macromolecules in vivo are being discovered at a rapid rate (reviewed in Hochstrasser, 1998, 2000a,b; Jentsch and Pyrowolakis, 2000; Melchior, 2000; Yeh et al., 2000; Hay, 2001; Muller et al., 2001; Ohsumi, 2001; Schwartz and Hochstrasser, 2003). For example, the ubl NEDD8 (Rub1p in Saccharomyces cerevisiae) activates SCF ubiquitin ligases and is involved in cell cycle control, signaling and embryogenesis (Lammer et al., 1998; Liakopoulos et al., 1998; Pozo et al., 1998; Jones and Candido, 2000; Tateishi et al., 2001; Kurz et al., 2002). ISG15 is involved in the antiviral interferon response (Haas et al., 1987; Loeb and Haas, 1992; Narasimhan et al., 1996; Nicholl et al., 2000; Yuan and Krug, 2001; MacQuillan et al., 2003). Apg8p (also called Aut7p and Cvt5p) modifies the lipid phosphatidylethanolamine to modulate membrane dynamics (Mizushima et al., 1998a; Ichimura et al., 2000). Hub1p modifies cell polarity factors and plays a role in cell polarization (Dittmar et al., 2002). SUMO family members, including Smt3p in S. cerevisiae, modify a number of different proteins involved in cell division, nuclear transport, the stress response and signal trans- duction (reviewed in Hay, 2001; Johnson and Gupta, 2001; Muller et al., 2001; Takahashi et al., 2001; Tatham et al., 2001; Pichler et al., 2002; Schmidt and Muller, 2002). There are many other ubls, including FAT10, Urm1p and Apg12p whose important biological func- tions are just beginning to be discovered (Liu et al., 1999; Furukawa et al., 2000; Raasi et al., 2001). The best understood conjugation pathway is that for ubiquitin. Following proteolytic cleavage at the C- terminus to end with the sequence Gly–Gly (reviewed *Correspondence: BA Schulman, Department of Structural Biology, MS #311, St Jude Children’s Research Hospital, Memphis, TN 38105, USA; E-mail: Brenda.schulman@stjude.org Oncogene (2004) 23, 1958–1971 & 2004 Nature Publishing Group All rights reserved 0950-9232/04 $25.00 www.nature.com/onc