Activation of NF-kappaB is achieved by ubiquitination and proteasome-mediated degradation of IkappaBalpha. We have detected modified IkappaBalpha, conjugated to the small ubiquitin-like protein SUMO-1, which is resistant to signal-induced degradation. In the presence of an E1 SUMO-1-activating enzyme, Ubch9 conjugated SUMO-1 to IkappaBalpha primarily on K21, which is also utilized for ubiquitin modification. Thus, SUMO-1-modified IkappaBalpha cannot be ubiquitinated and is resistant to proteasome-mediated degradation. As a result, overexpression of SUMO-1 inhibits signal-induced activation of NF-kappaB-dependent transcription. Unlike ubiquitin modification, which requires phosphorylation of S32 and S36, SUMO-1 modification of IkappaBalpha is inhibited by phosphorylation. Thus, while ubiquitination targets proteins for rapid degradation, SUMO-1 modification acts antagonistically to generate proteins resistant to degradation.

Conjugation of the small ubiquitin-like modifier SUMO-1/SMT3C/Sentrin-1 to proteins in vitro is dependent on a heterodimeric E1 (SAE1/SAE2) and an E2 (Ubc9). Although SUMO-2/SMT3A/Sentrin-3 and SUMO-3/SMT3B/Sentrin-2 share 50% sequence identity with SUMO-1, they are functionally distinct. Inspection of the SUMO-2 and SUMO-3 sequences indicates that they both contain the sequence KXE, which represents the consensus SUMO modification site. As a consequence SAE1/ SAE2 and Ubc9 catalyze the formation of polymeric chains of SUMO-2 and SUMO-3 on protein substrates in vitro, and SUMO-2 chains are detected in vivo. The ability to form polymeric chains is not shared by SUMO-1, and although all SUMO species use the same conjugation machinery, modification by SUMO-1 and SUMO-2/-3 may have distinct functional consequences.The small ubiquitin-like modifier SUMO-1 1 (also known as SMT3C, Sentrin, GMP1, UBL1, and PIC1) is a member of the ubiquitin-like protein family (1). SUMO-1 is known to be covalently conjugated to a variety of cellular substrates via a three-step enzymatic pathway analogous to that of ubiquitin conjugation. The E1-like enzymes for both SUMO-1 and the yeast homologue Smt3p exist as heterodimers known as SAE1/ SAE2 and Uba2p/Aos1p, respectively (2-5). In the first step the SAE1/SAE2 heterodimer utilizes ATP to adenylate the C-terminal glycine of SUMO-1. Formation of a thioester bond between the C-terminal glycine of SUMO-1 and a cysteine residue in SAE2 is accompanied by the release of AMP. The second step is a transesterification reaction, which transfers SUMO-1 from the E1 to a cysteine residue within the SUMO-specific E2-conjugating enzyme (Cys 93 in Ubc9). In the third step, Ubc9 catalyzes the formation of an isopeptide bond between the C terminus of SUMO-1 and the ⑀-amino group of lysine in the target protein. In contrast to the ubiquitin conjugation pathway no activity equivalent to an E3 ligase is required for SUMO-1 conjugation in vitro (2, 4), suggesting that the specificity for target proteins is conferred by Ubc9 itself or the Ubc9⅐SUMO-1 thioester complex. This is supported by the observations that almost all SUMO-1-conjugated proteins bind Ubc9 in two-hybrid assays, and the acceptor lysine residues on target proteins appear to exist within the consensus motif KXE (where represents a large hydrophobic amino acid, and X represents any amino acid) (6 -8). Furthermore, SUMO-1 is thought not to form SUMO-1-SUMO-1 polymers, which are characteristic of ubiquitination.Unlike the majority of ubiquitinated proteins, acceptors of SUMO-1 modifications are not targeted for degradation. In fact, in the case of the transcriptional inhibitor IB␣ the target lysine for SUMO-1 modification is the same as that of ubiquitin conjugation, thus blocking ubiquitination at that residue and stabilizing the protein (8). Transcriptional activity of specific proteins appears to be affected by SUMO-1 modification. For example, conjugation at a single site in the C terminus of p53 activates its transcriptional response (9, 10)...

The p53 tumour suppressor protein is regulated by ubiquitin-mediated proteasomal degradation. In normal cells p53 is constitutively ubiquitylated by the Mdm2 ubiquitin ligase. When the p53 response is activated by stress signals p53 levels rise due to inhibition of this degradative pathway. Here we show that p53 is modified by the small ubiquitin-like protein SUMO-1 at a single site, K386, in the C-terminus of the protein. Modification in vitro requires only SUMO-1, the SUMO-1 activating enzyme and ubc9. SUMO-1 and ubiquitin modification do not compete for the same lysine acceptor sites in p53. Overexpression of SUMO-1 activates the transcriptional activity of wildtype p53, but not K386R p53 where the SUMO-1 acceptor site has been mutated. The SUMO-1 modification pathway therefore acts as a potential regulator of the p53 response and may represent a novel target for the development of therapeutically useful modulators of the p53 response.

In normal cells, p53 is maintained at a low level by ubiquitin-mediated proteolysis, but after genotoxic insult this process is inhibited and p53 levels rise dramatically. Ubiquitination of p53 requires the ubiquitinactivating enzyme Ubc5 as a ubiquitin conjugation enzyme and Mdm2, which acts as a ubiquitin protein ligase. In addition to the N-terminal region, which is required for interaction with Mdm2, the C-terminal domain of p53 modulates the susceptibility of p53 to Mdm2-mediated degradation. To analyze the role of the C-terminal domain in p53 ubiquitination, we have generated p53 molecules containing single and multiple lysine-toarginine changes between residues 370 and 386. Although wild-type (WT) and mutant molecules show similar subcellular distributions, the mutants display a higher transcriptional activity than WT p53. Simultaneous mutation of lysine residues 370, 372, 373, 381, 382, and 386 to arginine residues (6KR p53 mutant) generates a p53 molecule with potent transcriptional activity that is resistant to Mdm2-induced degradation and is refractory to Mdm2-mediated ubiquitination. In contrast to WT p53, transcriptional activity directed by the 6KR p53 mutant fails to be negatively regulated by Mdm2. Those differences are also manifest in HeLa cells which express the human papillomavirus E6 protein, suggesting that p53 C-terminal lysine residues are also implicated in E6-AP-mediated ubiquitination. These data suggest that p53 C-terminal lysine residues are the main sites of ubiquitin ligation, which target p53 for proteasome-mediated degradation.

Ubiquitin conjugating enzymes participate in the thioester cascade that leads to protein ubiquitination. Although Ubc9 is homologous to E2 ubiquitin conjugating enzymes we have shown that it is unable to form a thioester with ubiquitin, but can form a thioester with the small ubiquitin-like protein SUMO. Thus Ubc9 is a SUMO conjugating enzyme rather than a ubiquitin conjugating enzyme. Transacetylation of Ubc9 by SUMO is not mediated by the El ubiquitin activating enzyme, but by a distinct enzymatic activity. SUMO conjugation to target proteins is mediated by a different, but parallel pathway to ubiquitination.

ADAR1 and ADAR2 are editing enzymes that deaminate adenosine to inosine in long double stranded RNA duplexes and specific pre-mRNA transcripts. Here, we show that full-length and N-terminally truncated forms of ADAR1 are simultaneously expressed in HeLa and COS7 cells owing to the usage of alternative starting methionines. Because the N-terminus of ADAR1 contains a nuclear export signal, the full-length protein localizes predominantly in the cytoplasm, whereas the N-terminally truncated forms are exclusively nuclear and accumulate in the nucleolus. ADAR2, which lacks a region homologous to the N-terminal domain of ADAR1, localizes exclusively to the nucleus and similarly accumulates in the nucleolus.Within the nucleolus, ADAR1 and ADAR2 co-localize in a novel compartment. Photobleaching experiments demonstrate that, in live cells, ADAR1 and ADAR2 are in constant flux in and out of the nucleolus. When cells express the editing-competent glutamate receptor GluR-B RNA, endogenous ADAR1 and ADAR2 de-localize from the nucleolus and accumulate at sites where the substrate transcripts accumulate. This suggests that ADAR1 and ADAR2 are constantly moving through the nucleolus and might be recruited onto specific editing substrates present elsewhere in the cell.

The ubiquitin-like protein SUMO-1 is conjugated to a variety of proteins including Ran GTPase-activating protein 1 (RanGAP1), IB␣, and PML. SUMO-1-modified proteins display altered subcellular targeting and/or stability. We have purified the SUMO-1-activating enzyme from human cells and shown that it contains two subunits of 38 and 72 kDa. Isolation of cDNAs for each subunit indicates that they are homologous to ubiquitinactivating enzymes and to the Saccharomyces cerevisiae enzymes responsible for conjugation of Smt3p and Rub1p. In vitro, recombinant SAE1/SAE2 (SUMO-1-activating enzyme) was capable of catalyzing the ATP-dependent formation of a thioester linkage between SUMO-1 and SAE2. The addition of the SUMO-1-conjugating enzyme Ubch9 resulted in efficient transfer of the thioester-linked SUMO-1 from SAE2 to Ubch9. In the presence of SAE1/SAE2, Ubch9, and ATP, SUMO-1 was efficiently conjugated to the protein substrate IB␣. As SAE1/SAE2, Ubch9, SUMO-1, and IB␣ are all homogeneous, recombinant proteins, it appears that SUMO-1 conjugation of IB␣ in vitro does not require the equivalent of an E3 ubiquitin protein ligase activity.

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