Crystal structures of the human SUMO‐2 protein at 1.6 Å and 1.2 Å resolution

Huang, Wen‐Chen; Ko, Tzu‐Ping; Li, Steven S.‐L.; Wang, Andrew H.-J.

doi:10.1111/j.1432-1033.2004.04349.x

Cited by 63 publications

(36 citation statements)

References 29 publications

(41 reference statements)

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“…Similarly, expression of Gal4-S2-Sp3KR and Gal4-S1-Sp3KR repressed 98% and 96% of Gal4-Sp3K539R activity, respectively (Fig. 2), whereas Gal4-S2-Sp3KR and Gal4-S1-Sp3KR had no effect on control pRL-tk reporter activity, which lacks upstream Gal4-binding sites (10).…”

Section: Identification Of Sumo-2 Residues Required For Transcriptionmentioning

confidence: 79%

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NXP-2 association with SUMO-2 depends on lysines required for transcriptional repression

Rosendorff

Sakakibara

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

104

100

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Small ubiquitin-like modifier (SUMO) modification of transcription factors is generally associated with repression. Reverse genetic analysis of SUMO-1, and -2 conserved residues emphasized the importance of dual charge reversals in abrogating the critical role of SUMO-2 K33, K35, and K42 in repression. GST-SUMO-2-affinity chromatography followed by liquid chromatography (LC)-MS analysis identified proteins that appeared to bind preferentially to WT SUMO-2 versus SUMO-2 K33E and K35E. LSD1, NXP-2, KIAA0809 (ARIP4), SAE2, RanGAP1, PELP1, and SETDB1 bound to SUMO-2 and not to SUMO-2 K33E, K42E, or K35E and K42E. Although LSD1 is a histone lysine demethylase, and histone H3K4 was demethylated at a SUMO-2-repressed promoter, neither overexpression of a dominant-negative LSD1 nor LSD1 depletion with RNA interference affected SUMO-2-mediated repression, indicating that LSD1 is not essential for repression, in this context. When tethered to a promoter by fusion to Gal4, NXP-2 repressed transcription, consistent with a role for NXP-2 in SUMO-mediated repression. SUMO-2-associated proteins identified in this study may contribute to SUMO-dependent regulation of transcription or other processes.regulation ͉ repressor ͉ ubiquitin-like S mall ubiquitin-like modifier (SUMO) is a ubiquitin-like protein that is reversibly covalently attached to lysines in target proteins, resulting in altered protein function, localization, or stability. SUMO modification of corepressors, coactivators, histones, histone-modifying enzymes, or sequence-specific transcription factors usually down-regulates transcription. For example, mutation so as to prevent SUMO modification increases transcription-factor activity associated with Elk-1, Sp3, c-myb, c-jun, AP2, or the p300 N terminus (1-5). In contrast to SUMO, ubiquitin modification is more frequently associated with proteasome-dependent degradation or activation of transcription. In fact, activation of transcription by the VP16 activation domain, in yeast, requires an E3 ubiquitin ligase (6). Addition of a SUMO consensus acceptor site to the Gal4-VP16 activation domain reduces transcription 10-fold (7). Thus, ubiquitin and SUMO modifications can have opposite effects on transcription.Despite these differences, SUMO-1, -2, and -3 are ubiquitinlike proteins and have ubiquitin-like structures. SUMO-2 and -3 are 95% identical and are 46% and 48%, respectively, identical to SUMO-1. In contrast, SUMO-1, -2, and -3 share only 18% identity with ubiquitin (Fig. 1). The differences in primary sequence and their role in the opposing transcriptional properties of SUMO and ubiquitin are being elucidated. SUMO interaction with histone deacetylases has been proposed as one mechanism of repression (1, 8). However, an unbiased directed screen to identify candidate SUMO-associated repressors has not been undertaken.The objective of our experiments was to identify proteins whose association with SUMO-2 depends on SUMO-2 residues required for repression. Such proteins would be candidate SUMO corepressors. We have u...

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Section: Identification Of Sumo-2 Residues Required For Transcriptionmentioning

confidence: 79%

“…2) by using the G5Luc reporter (10). In these assays, Gal4-SUMO-2 expression repressed 90-95% of the of the luciferase reporter activity observed after expression of the Gal4 DNA-binding domain alone (data not shown).…”

Section: Identification Of Sumo-2 Residues Required For Transcriptionmentioning

confidence: 89%

NXP-2 association with SUMO-2 depends on lysines required for transcriptional repression

Rosendorff

Sakakibara

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

104

100

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“…The structures of all three SUMO paralogues resemble the globular and compact Ub-like fold (6,7). The differences of SUMO1 and SUMO2 are mostly found in the second ␤-strand and the ␣-helix of both proteins (7).…”

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confidence: 96%

“…The differences of SUMO1 and SUMO2 are mostly found in the second ␤-strand and the ␣-helix of both proteins (7). In cells, different SUMO paralogues appear to share common properties but also have some distinct functions.…”

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confidence: 99%

Specification of SUMO1- and SUMO2-interacting Motifs

Hecker

Rabiller

Haglund

et al. 2006

Journal of Biological Chemistry

523

499

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SUMO proteins are ubiquitin-related modifiers implicated in the regulation of gene transcription, cell cycle, DNA repair, and protein localization. The molecular mechanisms by which the sumoylation of target proteins regulates diverse cellular functions remain poorly understood. Here we report isolation and characterization of SUMO1-and SUMO2-binding motifs. Using yeast two-hybrid system, bioinformatics, and NMR spectroscopy we define a common SUMO-interacting motif (SIM) and map its binding surfaces on SUMO1 and SUMO2. This motif forms a ␤-strand that could bind in parallel or antiparallel orientation to the ␤ 2 -strand of SUMO due to the environment of the hydrophobic core. A negative charge imposed by a stretch of neighboring acidic amino acids and/or phosphorylated serine residues determines its specificity in binding to distinct SUMO paralogues and can modulate the spatial orientation of SUMO-SIM interactions. SUMO proteins are small ubiquitin (Ub)5 -related modifiers that become conjugated to cellular substrates and regulate diverse cellular processes including cell cycle progression, intracellular trafficking, transcription, and DNA repair (1-3). Like Ub, a SUMO protein is covalently attached to target proteins through an isopeptide bond by a mechanism similar to that of ubiquitination, which involves E1, E2, and E3 enzymes (4). In mammals, three SUMO paralogues are commonly expressed: SUMO1 shares about 45% identity to SUMO2 and SUMO3, while SUMO2 and SUMO3 are 96% identical to each other (2, 5).The structures of all three SUMO paralogues resemble the globular and compact Ub-like fold (6, 7). The differences of SUMO1 and SUMO2 are mostly found in the second ␤-strand and the ␣-helix of both proteins (7). In cells, different SUMO paralogues appear to share common properties but also have some distinct functions. For example, the promyelocytic leukemia protein is conjugated to all three SUMO paralogs (8, 9), whereas RanGAP1 is preferentially modified with SUMO1 (10) and topoisomerase II with SUMO2/3 during mitosis (11). Furthermore, the distribution of the SUMO paralogues within cells seems to be different. SUMO1 is uniquely found within the nucleoli, the nuclear envelope, and cytoplasmic foci, whereas SUMO2/3 are accrued on chromosomes at an earlier point in the nuclear reformation process (12). Interestingly, there is a larger pool of free, non-conjugated SUMO2/3 than of SUMO1 (10).In addition to targeting different substrate proteins, the functional properties of SUMO isoforms in vivo might also reflect their ability to mediate distinct protein-protein interactions. Indeed, recent studies have shown that SUMO paralogues can promote non-covalent binding to other proteins containing specific motifs that recognize SUMO paralogues. Minty and coworkers defined a Ser-Xaa-Ser motif surrounded by hydrophobic and acidic amino acids as a SUMO-interacting motif (SIM) (13). Biophysical studies of the SIM in PIAS revealed that the small hydrophobic region is an essential determinant of SUMO recognition (14). Moreover...

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“…SUMO proteins, SUMO-1 through -4, form a Ub-like fold in the central region, containing an ␣-helix packed on a ␤-sheet (8,9). However, its N-terminal extension, which is not found in ubiquitin, is highly flexible and does not adopt any defined three-dimensional structure in solution.…”

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confidence: 99%

Doxorubicin Down-regulates Krüppel-associated Box Domain-associated Protein 1 Sumoylation That Relieves Its Transcription Repression on p21WAF1/CIP1 in Breast Cancer MCF-7 Cells

Lee

Thomas

Yang

et al. 2007

Journal of Biological Chemistry

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The role of post-translational modification, such as sumoylation, in modulating the efficacy of doxorubicin (Dox) treatment remains unclear. Transcriptional cofactor KRAB domain-associated protein 1 (KAP1) has been shown to complex with the KRAB zinc finger protein, ZBRK1, to repress the transcription of target genes. Through a combination of proteomic screening and sitedirected mutagenesis approaches, we have identified lysines 554, 779, and 804 as the major sumoylation sites in KAP1. We then present evidence that Dox-mediated induction of cell cycle regulator p21 expression is differentially regulated by KAP1 sumoylation status. Moreover, the KAP1 sumoylation level was transiently decreased upon Dox exposure, and transfection with the KAP1 sumoylation mimetic, SUMO-1-KAP1, desensitizes breast cancer MCF-7 cells to Dox-elicited cell death. The sumoylation-dependent stimulation of KAP1 function is achieved by enhancing the methylation of H3-K9 and attenuating the acetylation of H3-K9 and H3-K14 at the p21 core promoter. We also show that occupancy of ZBRK1 response elements located at the p21 promoter by ZBRK1⅐KAP1 is independent of KAP1 sumoylation. Hence, sumoylation of KAP1 represses p21 transcription via a chromatinsilencing process without affecting interaction between KAP1⅐ZBRK1 and DNA, thus providing a novel mechanistic basis for the understanding of Dox-induced de-repression of p21 transcription. Taken together, our results suggest that Dox-induced decrease in KAP1 sumoylation is essential for Dox to induce p21 expression and subsequent cell growth inhibition in MCF-7 cells. Covalent attachment of ubiquitin (Ub)3 and small ubiquitinlike modifier (SUMO) to proteins plays a major role in regulating cellular functions (reviewed in Ref. 1). Although ubiquitination generally promotes protein degradation, sumoylation regulates a variety of cellular processes, including nuclear transport, genome integrity, signal transduction, and transcriptional regulation (1-3). Briefly, SUMO is activated in an ATPdependent manner by an E1 activating enzyme, transferred to the E2 conjugating enzyme, and subsequently attached covalently to the lysine acceptor site, within a particular sequence of ⌿KX(D/E) (where ⌿ represents hydrophobic amino acid) of SUMO-target proteins by distinct E3 ligase. Similar to phosphorylation, sumoylation is a reversible process catalyzed by de-sumoylation enzymes. The list of known SUMO targets has grown substantially in recent years, and, remarkably, over half of the presently identified SUMO targets are transcriptional factors or co-regulators. In many cases, sumoylation leads to a modified protein-protein interaction of target protein(s), thus altering the biological consequences (4, 5). Recently, a SUMO-binding motif that interacts non-covalently with sumoylated proteins has been described (6), and this SUMO-binding motif is found to exist in nearly all proteins known to be involved in SUMO-dependent processes (7).SUMO proteins, SUMO-1 through -4, form a Ub-like fold in the central region, co...

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Crystal structures of the human SUMO‐2 protein at 1.6 Å and 1.2 Å resolution

Cited by 63 publications

References 29 publications

NXP-2 association with SUMO-2 depends on lysines required for transcriptional repression

NXP-2 association with SUMO-2 depends on lysines required for transcriptional repression

Specification of SUMO1- and SUMO2-interacting Motifs

Doxorubicin Down-regulates Krüppel-associated Box Domain-associated Protein 1 Sumoylation That Relieves Its Transcription Repression on p21WAF1/CIP1 in Breast Cancer MCF-7 Cells

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