Unique binding interactions among Ubc9, SUMO and RanBP2 reveal a mechanism for SUMO paralog selection

Tatham, Michael H.; Kim, Suhkmann; Jaffray, Ellis; Song, Jing; Chen, Yuan; Hay, Ronald T.

doi:10.1038/nsmb878

Cited by 133 publications

(146 citation statements)

References 30 publications

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“…The SIM in the TAD is involved in fine-tuning the sumoylation of c-Myb in the EVES domain, such that, mutating the SIM reduces the sumoylation of c-Myb slightly (Figure 4a). Such interdependency has been shown for other SUMO targets, and reflects the fact that noncovalent binding of SUMO is an important mechanistic step in the conjugation reaction, orientating the SUMO moiety for optimal transfer (Reverter and Lima, 2005;Tatham et al, 2005).…”

Section: Discussionmentioning

confidence: 80%

“…Thus, we expressed increasing amounts of nonconjugatable SUMO1 and -2 (mono-Gly in C terminus) in the presence of our c-Myb mutants. We expected the nonconjugatable SUMO to interfere mainly with SUMO binding, but also indirectly with conjugation, as the conjugation process is dependent on SUMO interactions (Reverter and Lima, 2005;Tatham et al, 2005). As can be seen in Figure 6a, co-expression of SUMO1-1G in the presence of wild-type c-Myb led to an increase in transactivation, whereas no significant change in activity was observed for c-Myb ANAA 2KR.…”

Section: Sumo Binding and The Transforming Ability Of C-mybmentioning

confidence: 93%

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A functional SUMO-interacting motif in the transactivation domain of c-Myb regulates its myeloid transforming ability

et al. 2010

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Section: Discussionmentioning

confidence: 80%

Section: Sumo Binding and The Transforming Ability Of C-mybmentioning

confidence: 93%

A functional SUMO-interacting motif in the transactivation domain of c-Myb regulates its myeloid transforming ability

et al. 2010

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Section: Introductionmentioning

confidence: 99%

“…Three SUMO E3s have been identified so far: the mammalian protein inhibitors of activated STAT (PIAS; Sachdev et al, 2001), the nucleoporin RanBP2 (Azuma and Dasso, 2002;Pichler et al, 2002), and the polycomb group protein PC2 (Kagey et al, 2003). Recent structural data provide novel insights into the mechanism used by E3s to enhance SUMO conjugation (Duda and Schulman, 2005;Reverter and Lima, 2005;Tatham et al, 2005).Removal of SUMO from proteins is carried out by specific cysteine proteases that have both hydrolase and isopeptidase activity Hochstrasser, 1999, 2000). Most enzymes involved in the SUMO pathway are localized in the nucleus, and it is therefore believed that sumoylation is predominantly a nuclear process (Rodriguez et al, 2001;Zhang et al, 2002;Seeler and Dejean, 2003).…”

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confidence: 99%

SUMO-1 Modification Alters ADAR1 Editing Activity

Desterro

Keegan

Jaffray

et al. 2005

MBoC

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106

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We identify ADAR1, an RNA-editing enzyme with transient nucleolar localization, as a novel substrate for sumoylation. We show that ADAR1 colocalizes with SUMO-1 in a subnucleolar region that is distinct from the fibrillar center, the dense fibrillar component, and the granular component. Our results further show that human ADAR1 is modified by SUMO-1 on lysine residue 418. An arginine substitution of K418 abolishes SUMO-1 conjugation and although it does not interfere with ADAR1 proper localization, it stimulates the ability of the enzyme to edit RNA both in vivo and in vitro. Moreover, modification of wild-type recombinant ADAR1 by SUMO-1 reduces the editing activity of the enzyme in vitro. Taken together these data suggest a novel role for sumoylation in regulating RNA-editing activity. INTRODUCTIONA defining feature of eukaryotic cells is the generation of protein diversity either posttranscriptionally by alternative splicing and RNA editing or posttranslationally by modification of amino acids in proteins. One of the most recently discovered posttranslational modification mechanism in eukaryotes involves the covalent attachment of the small ubiquitinlike modifier, SUMO, to target proteins. Modification of proteins by SUMO, or sumoylation, plays crucial regulatory roles in eukaryotes. Proteins known to be modified by SUMO include, among others, RanGAP1, PCNA, I B␣, p53, c-jun, topoisomerases, promyelocytic leukemia protein (PML), Sp100, and the mitogen-activated protein kinase kinase 1 (MEKK1). Many SUMO substrates are transcription factors and cofactors, or proteins implicated in DNA repair and replication (reviewed by Hay, 2001;Melchior et al., 2003;Seeler and Dejean, 2003;Hay, 2005). Although it is well established that SUMO can affect target protein function by altering its subcellular localization, activity, or stability, for many substrates the biological functions of sumoylation remain unknown.Sumoylation is a reversible and highly dynamic process that involves formation of an isopeptide bond between the C-terminus of SUMO and the ⑀-amino group of a lysine residue of the target protein. The most intensely studied human form of SUMO is the SUMO-1 protein, which is 48% identical to yeast Smt3 (Bayer et al., 1998;Mossessova and Lima, 2000). In vertebrates there are at least three additional proteins. SUMO-2 and SUMO-3 are ϳ45% identical to SUMO-1 (Saitoh and Hinchey, 2000), and SUMO-4 shows an 86% amino acid homology to SUMO-2 (Bohren et al., 2004). SUMO is conjugated to protein substrates via an ATP-dependent enzymatic pathway that is mechanistically similar to ubiquitination. The reaction requires a SUMO protease that removes four amino acids from the C-terminus of the 101-amino acid SUMO-1 precursor to generate the mature form; an heterodimeric SUMO-activating enzyme, SAE1/2; Ubc9, a SUMO-conjugating enzyme that ligates directly to its protein target; and an E3-like SUMO ligase (reviewed Melchior et al., 2003). Three SUMO E3s have been identified so far: the mammalian protein inhibitors of activated S...

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“…The stable interactions observed between RanGAP1 and E2; E3, SUMO and E2; and the covalent interaction between RanGAP1-SUMO suggested that our structure represented a trapped product complex. To test this, conjugation assays using RanGAP1 under multiple turnover conditions with and without Nup358/RanBP2 IR1* showed, in contrast to other substrates 12,18,19 , that IR1* inhibited SUMO conjugation. Importantly, SUMO-RanGAP1 accumulated to near stochiometric ratios of product, E2, and E3 ( Figure 4a).…”

mentioning

confidence: 99%

Insights into E3 ligase activity revealed by a SUMO–RanGAP1–Ubc9–Nup358 complex

Reverter

Lima

2005

Nature

442

560

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The Small Ubiquitin-related Modifier SUMO-1 belongs to the ubiquitin (Ub) and ubiquitin-like (Ubl) protein family. SUMO conjugation occurs on specific lysine residues within protein targets, regulating pathways involved in differentiation, apoptosis, the cell cycle, and responses to stress by altering protein function through changes in activity, cellular localization, or by protecting substrates from ubiquitination 1,2 . Ub/Ubl conjugation occurs in sequential steps and requires the concerted action of E2 conjugating proteins and E3 ligases 1,2 . In addition to being a SUMO E3, the nucleoporin Nup358/RanBP2 localizes SUMO conjugated RanGAP1 to the cytoplasmic face of the nuclear pore complex (NPC) via interactions in a complex that also includes Ubc9, the SUMO E2 conjugating protein 3-6 . We present the 3.0 Å crystal structure of a four protein complex between Ubc9, a Nup358/ RanBP2 E3 ligase domain (IR1-M), and SUMO-1 conjugated to the C-terminal domain of RanGAP1. Structural insights, combined with biochemical and kinetic data using additional substrates, support a model whereby Nup358/RanBP2 acts as an E3 by binding both SUMO and Ubc9 to position the SUMO-E2-thioester in an optimal orientation to enhance conjugation.Ub/Ubls are activated by E1 and transferred to E2 to form E2-Ub/Ubl-thioesters. While competent for Ub/Ubl ligation to lysine ɛ-amino groups, E2s generally require E3 ligases to specifically recognize substrate lysine residues. Most E3s belong to either RING or HECT families and while RING E3s recruit substrate and bind the E2-Ub/Ubl via a zinc domain to promote conjugation to lysine residues 7 , HECT E3s recruit E2-Ub/Ubl to generate E3-Ub/Ublthioesters for conjugation 8 . The SUMO E2 can directly bind the consensus sequence Ψ-K-x-D/E where Ψ is hydrophobic, K is the substrate lysine, x is any amino acid, and D or E is acidic 9 , although several SUMO E3s facilitate conjugation in vivo and in vitro and include RING-type E3s 10,11 , Nup358/RanBP2 12 , and Pc2 13 . Nup358/RanBP2 and Pc2 appear unrelated to either RING or HECT E3 ligases.One of the first discovered functions for SUMO-1 was its role in nucleocytoplasmic trafficking 3-5 . SUMO conjugation localizes RanGAP1 to the NPC in a complex that includes the SUMO E2 Ubc9 and Nup358/RanBP2, a multi-domain 3,224 amino acid (aa) nucleoporin that also interacts with Ran and other nuclear transport factors 3-6,14,15 . The SUMORanGAP1-Ubc9-Nup358/RanBP2 complex does not dissociate upon entry into mitosis and NPC disassembly, but redistributes to kinetochores and contributes to the stability of kinetochore-microtubule interactions 16 . A ~30kDa Nup358/RanBP2 fragment named IR1-M-IR2 binds Ubc9 and promotes SUMO E3 activity in vitro and in vivo 12,17-19 , although domains flanking IR1-M-IR2 also contribute to SUMO-RanGAP1 interactions 6 . IR1-M-IR2 was parsed into three elements: IR1 (aa To determine the molecular details of this system, SUMO-1 was conjugated to RanGAP1, combined with Ubc9 and Nup358/RanBP2, purified by gel filtration, and cry...

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Unique binding interactions among Ubc9, SUMO and RanBP2 reveal a mechanism for SUMO paralog selection

Cited by 133 publications

References 30 publications

A functional SUMO-interacting motif in the transactivation domain of c-Myb regulates its myeloid transforming ability

A functional SUMO-interacting motif in the transactivation domain of c-Myb regulates its myeloid transforming ability

SUMO-1 Modification Alters ADAR1 Editing Activity

Insights into E3 ligase activity revealed by a SUMO–RanGAP1–Ubc9–Nup358 complex

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