In acute promyelocytic leukaemia (APL), the promyelocytic leukaemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). This disease can be treated effectively with arsenic, which induces PML modification by small ubiquitin-like modifiers (SUMO) and proteasomal degradation. Here we demonstrate that the RING-domain-containing ubiquitin E3 ligase, RNF4 (also known as SNURF), targets poly-SUMO-modified proteins for degradation mediated by ubiquitin. RNF4 depletion or proteasome inhibition led to accumulation of mixed, polyubiquitinated, poly-SUMO chains. PML protein accumulated in RNF4-depleted cells and was ubiquitinated by RNF4 in a SUMO-dependent fashion in vitro. In the absence of RNF4, arsenic failed to induce degradation of PML and SUMO-modified PML accumulated in the nucleus. These results demonstrate that poly-SUMO chains can act as discrete signals from mono-SUMOylation, in this case targeting a poly-SUMOylated substrate for ubiquitin-mediated proteolysis.

SUMMARYUbiquitin modification is mediated by a large family of specificity determining ubiquitin E3 ligases. To facilitate ubiquitin transfer, RING E3 ligases bind both substrate and a ubiquitin E2 conjugating enzyme linked to ubiquitin via a thioester bond, but the mechanism of transfer has remained elusive. Here we report the crystal structure of the dimeric RING of RNF4 in complex with E2 (UbcH5a) linked by an isopeptide bond to ubiquitin. While the E2 contacts a single protomer of the RING, ubiquitin is folded back onto the E2 by contacts from both RING protomers. The C-terminal tail of ubiquitin is locked into an active site groove on the E2 by an intricate network of interactions, resulting in changes at the E2 active site. This arrangement is primed for catalysis as it can deprotonate the incoming substrate lysine residue and stabilise the consequent tetrahedral transition state intermediate.

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 ETS domain transcription factor Elk-1 is a direct target of the MAP kinase pathways. Phosphorylation of the Elk-1 transcriptional activation domain by MAP kinases triggers its activation. However, Elk-1 also contains two domains with repressive activities. One of these, the R motif, appears to function by suppressing the activity of the activation domain. Here, we demonstrate that SUMO modification of the R motif is required for this repressive activity. A dynamic interplay exists between the activating ERK MAP kinase pathway and the repressive SUMO pathway. ERK pathway activation leads to both phosphorylation of Elk-1 and loss of SUMO conjugation and, hence, to the loss of the repressive activity of the R motif. Thus, the reciprocal regulation of the activation and repressive activities are coupled by MAP kinase modification of Elk-1.

Posttranslational modification with small ubiquitin-like modifiers (SUMOs) alters the function of proteins involved in diverse cellular processes. SUMO-specific enzymes conjugate SUMOs to lysine residues in target proteins. Although proteomic studies have identified hundreds of sumoylated substrates, methods to identify the modified lysines on a proteomic scale are lacking. We developed a method that enabled proteome-wide identification of sumoylated lysines that involves the expression of polyhistidine (6His)-tagged SUMO2 with Thr(90) mutated to Lys. Endoproteinase cleavage with Lys-C of 6His-SUMO2(T90K)-modified proteins from human cell lysates produced a diGly remnant on SUMO2(T90K)-conjugated lysines, enabling immunoprecipitation of SUMO2(T90K)-modified peptides and producing a unique mass-to-charge signature. Mass spectrometry analysis of SUMO-enriched peptides revealed more than 1000 sumoylated lysines in 539 proteins, including many functionally related proteins involved in cell cycle, transcription, and DNA repair. Not only can this strategy be used to study the dynamics of sumoylation and other potentially similar posttranslational modifications, but also, these data provide an unprecedented resource for future research on the role of sumoylation in cellular physiology and disease.

Mammalian RNF4 is a dimeric RING ubiquitin E3 ligase that ubiquitylates poly-SUMOylated proteins. We found that RNF4 bound ubiquitin-charged UbcH5a tightly but free UbcH5a weakly. To provide insight into the mechanism of RING-mediated ubiquitylation we docked the UbcH5~ubiquitin thioester onto the RNF4 RING structure. This revealed that with E2 bound to one monomer of RNF4, the thioester-linked ubiquitin could reach across the dimer to engage the other monomer. In this model the "Ile44 hydrophobic patch" of ubiquitin is predicted to engage a conserved tyrosine located at the dimer interface of the RING and mutation of these residues blocked ubiquitylation activity. Thus, dimeric RING ligases are not simply inert scaffolds that bring substrate and E2-loaded ubiquitin into close proximity. Instead, they facilitate ubiquitin transfer by preferentially binding the E2~ubiquitin thioester across the dimer and activating the thioester bond for catalysis.

The androgen receptor (AR) is a nuclear hormone receptor superfamily member that conveys both trans repression and ligand-dependent trans-activation function. Activation of the AR by dihydrotestosterone (DHT) regulates diverse physiological functions including secondary sexual differentiation in the male and the induction of apoptosis by the JNK kinase, MEKK1. The AR is posttranslationally modified on lysine residues by acetylation and sumoylation. The histone acetylases p300 and P/CAF directly acetylate the AR in vitro at a conserved KLKK motif. To determine the functional properties governed by AR acetylation, point mutations of the KLKK motif that abrogated acetylation were engineered and examined in vitro and in vivo. Steroid receptors, including the androgen receptor (AR), are members of the nuclear receptor (NR) superfamily which generally function as ligand-dependent transcriptional regulators (9, 31, 84). The AR is expressed in a variety of cell types and plays an important role in development, male sexual differentiation, and prostate cellular proliferation. The functional domains of the AR (termed A to F) are conserved with other members of the classical receptor subclass. The C-terminal region of the AR, including the hinge region and ligand-binding domain (LBD), is responsible for ligand binding and dimerization. The well-conserved DNA binding domain consists of 68 amino acids with two zinc finger structures. The N-terminal region contributes to transcriptional activation through its activation function 1 (AF-1) (5). In contrast to several other hormone-regulated NRs, the AR lacks an intrinsic AF-2 function in the LBD. The LBD, which consists of 12 ␣ helices projecting away from the hormone-binding pocket in the absence of ligand, undergoes substantial conformational changes in the presence of ligand. The folding of the most carboxyl-terminal helix 12 over the ligand-binding pocket in turn creates new structural surfaces that bind coactivators required for efficient transactivation.Several AR coactivators have been identified, including the p160 proteins, the p300/CREB-binding protein (CBP) family, Ubc9, ARA70, ARA55, and TIP60 (1,5,15,68,92). The efficient recruitment of coactivators to the AR involves an association between both the AR amino terminus and the LBD (5, 34). The coactivator proteins regulate gene expression through several distinct mechanisms. CBP and the related functional homologue p300 (CBP/p300) convey a bridging function between the DNA-bound transcription factor and the basal apparatus and provide a scaffold to assemble high-molecular-weight enhanceosomes (reviewed in reference 26). In addition, the cointegrator proteins p300/CBP share the capacity to acetylate histones, which correlates, under certain circumstances, with their transcriptional coactivator function (54,87). Acetylation facilitates binding of transcription factors to * Corresponding author. Mailing address: The Albert Einstein Can-

The SUMO family in vertebrates includes at least three distinct proteins (SUMO-1, -2, and -3) that are added as post-translational modifications to target proteins. A considerable number of SUMO-1 target proteins have been identified, but little is known about SUMO-2. A stable HeLa cell line expressing His 6 -tagged SUMO-2 was established and used to label and purify novel endogenous SUMO-2 target proteins. Tagged forms of SUMO-2 were functional and localized predominantly in the nucleus. His 6 -tagged SUMO-2 conjugates were affinity-purified from nuclear fractions and identified by mass spectrometry. Eight novel potential SUMO-2 target proteins were identified by at least two peptides. Three of these proteins, SART1, heterogeneous nuclear ribonucleoprotein (RNP) M, and the U5 small nuclear RNP 200-kDa helicase, play a role in RNA metabolism. SART1 and heterogeneous nuclear RNP M were both shown to be genuine SUMO targets, confirming the validity of the approach.The small ubiquitin-like modifier (SUMO) 1 is linked to target proteins by post-translational conjugation and may thereby influence protein function and/or localization (1-3). The name derives from the relationship of SUMO to the better characterized post-translational modifier ubiquitin (4). They are 18% identical at the amino acid level and show a clear similarity in their respective three-dimensional structures (5). Whereas yeast and nematodes have a single SUMO gene, in humans and mice, the SUMO family consists of three members, SUMO-1, -2, and -3, which are encoded by separate genes. The mature forms of SUMO-2 and SUMO-3 are similar (ϳ95% identical), but less closely related to SUMO-1 (ϳ50% identical) (6 -8).Despite their close similarity, there is evidence that SUMO-1 and SUMO-2/3 are preferentially conjugated to distinct sets of target proteins. Whereas RanGAP1 is modified by SUMO-1, many as yet unidentified proteins are modified by SUMO-2/3 after exposure of cells to various stress stimuli (7).Several groups independently identified SUMO-1, which is also known as PIC1, GMP1, SMT3C, UBL1, and sentrin (9 -13). In contrast to most polyubiquitinylated proteins, sumoylated proteins are not thereby targeted for degradation. SUMO-1 can even compete with ubiquitin for the same acceptor lysine in the target protein. In the case of IB␣, SUMO modification serves to protect the protein from ubiquitin-mediated degradation (14), whereas in yeast, DNA repair can proceed only after proliferating cell nuclear antigen is desumoylated to allow ubiquitination. Here, ubiquitination does not target the protein for degradation, but functions as a switch to activate the DNA repair function of proliferating cell nuclear antigen (15). Therefore, despite their structural relationship, it appears that SUMO and ubiquitin conjugation can play distinct roles in modulating target proteins. Sumoylation can also regulate the subcellular localization of target proteins. For example, SUMO conjugation is required for both the nuclear pore and kinetochore localization of RanGAP1 (1...