The DNA repair function of the breast cancer susceptibility protein BRCA1 depends in part on its interaction with RAP80, which targets BRCA1 to DNA double strand breaks (DSBs) through recognition of K63-linked polyubiquitin chains. The localization of BRCA1 to DSBs also requires sumoylation. Here, we demonstrated that, in addition to having ubiquitin-interacting motifs, RAP80 also contains a SUMO-interacting motif (SIM) that is critical for recruitment to DSBs. In combination with the ubiquitin-binding activity of RAP80, this SIM enabled RAP80 to bind with nanomolar affinity to hybrid chains consisting of ubiquitin conjugated to SUMO. Furthermore, RNF4, a SUMO-targeted ubiquitin E3 ligase that synthesizes hybrid SUMO-ubiquitin chains, localized to DSBs and was critical for the recruitment of RAP80 and BRCA1 to sites of DNA damage. Our findings, therefore, connect ubiquitin-dependent and SUMO-dependent DSB recognition, revealing that RNF4 synthesized hybrid SUMO-ubiquitin chains are recognized by RAP80 to promote BRCA1 recruitment and DNA repair.

OTUB1 is a Lys48-specific deubiquitinating enzyme that forms a complex in vivo with E2 ubiquitin conjugating enzymes including UBC13 and UBCH5. OTUB1 binds to E2~Ub thioester intermediates and prevent ubiquitin transfer, thereby non-catalytically inhibiting accumulation of polyubiquitin. We report here that a second role of OTUB1-E2 interactions is to stimulate OTUB1 cleavage of Lys48 polyubiquitin, and that this stimulation is regulated by the ratio of charged to uncharged E2 and by the concentration of Lys48-linked polyubiquitin and free ubiquitin. Structural and biochemical studies of human and worm OTUB1 and UBCH5B show that the E2 stimulates binding of the Lys48 polyubiquitin substrate by stabilizing folding of the OTUB1 N-terminal ubiquitin-binding helix. Our results suggest that OTUB1-E2 complexes in the cell are poised to regulate polyubiquitin chain elongation or degradation in response to changing levels of E2 charging and available free ubiquitin.

Small ubiquitin-related modifiers (SUMOs) regulate diverse cellular processes through their covalent attachment to target proteins. Vertebrates express three SUMO paralogs: SUMO-1, SUMO-2, and SUMO-3 (SUMO-2 and SUMO-3 are ϳ96% identical and referred to as SUMO-2/3). SUMO-1 and SUMO-2/3 are conjugated, at least in part, to unique subsets of proteins and thus regulate distinct cellular pathways. However, how different proteins are selectively modified by SUMO-1 and SUMO-2/3 is unknown. We demonstrate that BLM, the RecQ DNA helicase mutated in Bloom syndrome, is preferentially modified by SUMO-2/3 both in vitro and in vivo. Our findings indicate that non-covalent interactions between SUMO and BLM are required for modification at non-consensus sites and that preferential SUMO-2/3 modification is determined by preferential SUMO-2/3 binding. We also present evidence that sumoylation of a C-terminal fragment of HIPK2 is dependent on SUMO binding, indicating that non-covalent interactions between SUMO and target proteins provide a general mechanism for SUMO substrate selection and possible paralogselective modification.Post-translational protein modifications play essential roles in regulating all aspects of cell function. Small ubiquitin-related modifiers (SUMOs) 2 are unusual post-translational modifications, because they themselves are proteins of ϳ100 amino acids (1, 2). Through covalent attachment to lysine residues in target proteins, sumoylation regulates a wide range of processes, including transcription activation, DNA synthesis and repair, nucleocytoplasmic transport, and chromosome segregation. The molecular mechanisms by which sumoylation affects individual proteins, and thus this diversity of processes, are in many cases target protein-specific. An emerging theme, however, is that sumoylation often promotes interactions between modified proteins and downstream factors containing SUMO-interacting motifs (SIMs) (1). To date, a single conserved SIM has been identified that consists of a hydrophobic core ((V/I)X(V/I)(V/I)) followed or preceded by a negatively charged cluster of amino acids (3-6). Although only a limited number of SIM-containing proteins has been functionally characterized to date, a large number of proteins contains this motif and is thus predicted to interact non-covalently with SUMO.Invertebrate organisms express only a single SUMO, whereas vertebrates express three paralogs capable of covalent conjugation to other proteins: SUMO-1, SUMO-2, and SUMO-3. SUMO-2 and SUMO-3 are ϳ96% identical to each other (and thus referred to collectively as SUMO-2/3); however, they are only ϳ45% identical to SUMO-1. Increasingly, evidence suggests that SUMO-1 and SUMO-2/3 have distinct cellular functions. Proteomic studies have, for example, shown that SUMO-1 and SUMO-2/3 are conjugated to only partially overlapping subsets of proteins (7,8). In addition, localization studies indicate that SUMO-1 and SUMO-2/3 are conjugated to unique subsets of proteins that localize to different subcellular domains (9, 10)...

E pstein-Barr virus (EBV), first discovered in association withBurkitt's lymphoma (27), is linked to a variety of human diseases, including infectious mononucleosis, nasopharyngeal carcinoma, gastric carcinoma, and posttransplant lymphoproliferative disease (105). EBV infection results in either lytic replication or the establishment of viral latency. Both latent and lytic EBV gene products have been implicated in the development of cancer (28,51,72,105). EBV can be reactivated from latency by various reagents, such as 5-bromodeoxyuridine (39, 46), phorbol esters (110), anti-Ig antibodies (23,92,97), sodium butyrate (71), methotrexate (28), bortezomib (33, 34), thapsigargin (88, 95), and arsenic trioxide (89). The transition from latency to lytic replication is mediated by two EBV immediate-early genes, BZLF1 and BRLF1. The encoded proteins, ZTA and RTA, function as transcriptional activators that regulate the expression of EBV lytic cycle genes and lytic viral DNA replication (16,21,22,31,36,69,70,83,86,96). The lytic induction of EBV has been postulated as a therapeutic strategy for the treatment of virus-associated tumors (29,30,33,77).The small ubiquitin-related modifier (SUMO) was first identified as a posttranslational modifier of RanGAP1 (73,75). Similarly to the ubiquitination pathway, SUMOylation involves a series of sequential enzymatic reactions. The SUMO precursor protein is first cleaved by sentrin-specific proteases (SENPs) to generate a C-terminal diglycine motif. This then forms an E1ϳSUMO thioester, which is transferred to the E2-conjugating enzyme UBC9. E2ϳSUMO directly transfers SUMO to the substrate at lysine residues to form an isopeptide bond. E3 SUMO protein ligases facilitate this process by recruiting E2ϳSUMO to specific substrates and by enhancing the transfer process. SUMOylated targets can be de-SUMOylated by the SENP removal of SUMO (37). SUMOylation has been implicated in a variety of cellular processes, including transcriptional regulation, cell cycle regulation, signal transduction, the DNA damage response (DDR), and the regulation of protein-protein interactions (38). Both latent and lytic EBV proteins interact with the SUMO system. EBNA3C is SUMOylated (84), while LMP1 modulates the SUMOylation processes by interaction with UBC9 (6). SUMOylation regulates the transcriptional activity of ZTA and RTA (10,13,14,45,47,80). Noncovalent SUMO-protein interactions can also occur through a SUMO interaction motif (SIM) in the target proteins (3,57,90,91,93). EBNA3C contains a SIM motif and upregulates EBNA2-mediated gene activation by binding to a SUMOylated protein (84).In this study, we used an EBV protein microarray to identify additional EBV proteins that bind to SUMO. One of the identified proteins was the conserved protein kinase BGLF4. BGLF4 is present in the virion and expressed at an early stage of the lytic cycle (40,41,99). BGLF4 phosphorylates multiple EBV proteins, including BMRF1 (18, 42), EBNA2 (106), EBNA-LP (55), ZTA (4), EBNA1, and virion proteins (108). BGLF4 also phosp...

Proteomics studies have revealed that SUMOylation is a widely used post-translational modification (PTM) in eukaryotes. However, how SUMO E1/2/3 complexes use different SUMO isoforms and recognize substrates remains largely unknown. Using a human proteome microarray-based activity screen, we identified over 2500 proteins that undergo SUMO E3-dependent SUMOylation. We next constructed a SUMO isoform- and E3 ligase-dependent enzyme-substrate relationship network. Protein kinases were significantly enriched among SUMOylation substrates, suggesting crosstalk between phosphorylation and SUMOylation. Cell-based analyses of tyrosine kinase, PYK2, revealed that SUMOylation at four lysine residues promoted PYK2 autophosphorylation at tyrosine 402, which in turn enhanced its interaction with SRC and full activation of the SRC-PYK2 complex. SUMOylation on WT but not the 4KR mutant of PYK2 further elevated phosphorylation of the downstream components in the focal adhesion pathway, such as paxillin and Erk1/2, leading to significantly enhanced cell migration during wound healing. These studies illustrate how our SUMO E3 ligase-substrate network can be used to explore crosstalk between SUMOylation and other PTMs in many biological processes.

SUMO-binding proteins interact with SUMO modified proteins to mediate a wide range of functional consequences. Here, we report the identification of a new SUMO-binding protein, ZNF261. Four human proteins including ZNF261, ZNF198, ZNF262, and ZNF258 contain a stretch of tandem zinc fingers called myeloproliferative and mental retardation (MYM)-type zinc fingers. We demonstrated that MYM-type zinc fingers from ZNF261 and ZNF198 are necessary and sufficient for SUMO-binding and that individual MYM-type zinc fingers function as SUMO-interacting motifs (SIMs). Our binding studies revealed that the MYM-type zinc fingers from ZNF261 and ZNF198 interact with the same surface on SUMO-2 recognized by the archetypal consensus SIM. We also present evidence that MYM-type zinc fingers in ZNF261 contain zinc, but that zinc is not required for SUMO-binding. Immunofluorescence microscopy studies using truncated fragments of ZNF198 revealed that MYM-type zinc fingers of ZNF198 are necessary for localization to PML-nuclear bodies (PML-NBs). In summary, our studies have identified and characterized the SUMO-binding activity of the MYM-type zinc fingers in ZNF261 and ZNF198.

Background: Post-translational SUMO modification of TDG weakens its DNA binding and was proposed to regulate dissociation of a tight enzyme-product complex. Results: In vitro sumoylation of TDG by SUMO-1 and SUMO-2 is efficient for free and DNA-bound TDG. Conclusion: E2-mediated sumoylation is not selective for product-bound TDG but could potentially stimulate product release. Significance: Our findings inform the mechanism and role of TDG sumoylation.

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