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1.
Human Topors, which was originally identified as cellular binding partner of DNA topoisomerase I and of p53, has recently been shown to function as an ubiquitin E3 ligase for p53 in a manner dependent on its N'-terminally located RING finger. Here, we demonstrate that Topors also enhances the conjugation of the small ubiquitin-like modifier 1 (SUMO-1) to p53 in vivo and in a reconstituted in vitro system. The Topors SUMO-1 E3 ligase activity does not depend upon its RING finger motif. In HeLa cells, Topors induced p53 sumoylation was accompanied by an increase in endogenous p53 protein levels. Furthermore, Topors enhances the sumoylation of a variety of other, yet unidentified, cellular proteins. 相似文献
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Over the past years, modification by covalent attachment of SUMO (small ubiquitin-like modifier) has been demonstrated for of a number of cellular and viral proteins. While increasing evidence suggests a role for SUMO modification in the regulation of protein-protein interactions and/or subcellular localization, most SUMO targets are still at large. In this report we show that Topors, a Topoisomerase I and p53 interacting protein of hitherto unknown function, presents a novel cellular target for SUMO-1 modification. In a yeast two-hybrid system, Topors interacted with both SUMO-1 and the SUMO-1 conjugating enzyme UBC9. Multiple SUMO-1 modified forms of Topors could be detected after cotransfection of exogenous SUMO-1 and Topors induced the colocalization of a YFP tagged SUMO-1 protein in a speckled pattern in the nucleus. A subset of these Topors' nuclear speckles were closely associated with the PML nuclear bodies (POD, ND10). A central domain comprising Topors residues 437 to 574 was sufficient for both sumolation and localization to nuclear speckles. One SUMO-1 acceptor site at lysine residue 560 could be identified within this region. However, sumolation-deficient Topors mutants showed that sumolation obviously is not required for localization to nuclear speckles. 相似文献
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Human Topors has originally been identified as binding partner of p53 and DNA topoisomerase I (TOP1). It can function as both an ubiquitin and SUMO-1 E3 ligase for p53. Here we demonstrate that Topors enhances the formation of high-molecular weight SUMO-1 conjugates of TOP1 in a reconstituted in vitro system and also in human osteosarcoma cells, similar to treatment with CPT. In contrast to the situation observed with p53, overall sumoylation levels were rather unaffected. Experiments with TOP1 point mutants strongly suggest that the high-molecular weight conjugates represent SUMO-1 chains formed on a limited number of SUMO-1 acceptor sites. 相似文献
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Xiaoming Yang Hongchang Li Zinan Zhou Wen-Horng Wang Anping Deng Ourania Andrisani Xiaoqi Liu 《The Journal of biological chemistry》2009,284(28):18588-18592
Polo-like kinase 1 (Plk1) overexpression is associated with tumorigenesis by an unknown mechanism. Likewise, Plk1 was suggested to act as a negative regulator of tumor suppressor p53, but the mechanism remains to be determined. Herein, we have identified topoisomerase I-binding protein (Topors), a p53-binding protein, as a Plk1 target. We show that Plk1 phosphorylates Topors on Ser718 in vivo. Significantly, expression of a Plk1-unphosphorylatable Topors mutant (S718A) leads to a dramatic accumulation of p53 through inhibition of p53 degradation. Topors is an ubiquitin and small ubiquitin-like modifier ubiquitin-protein isopeptide ligase (SUMO E3) ligase. Plk1-mediated phosphorylation of Topors inhibits Topors-mediated sumoylation of p53, whereas p53 ubiquitination is enhanced, leading to p53 degradation. These results demonstrate that Plk1 modulates Topors activity in suppressing p53 function and identify a likely mechanism for the tumorigenic potential of Plk1.Polo-like kinase-1 (Plk1)3 has multiple functions required for cell cycle progression, and overexpression of Plk1 is observed in various types of human tumors (1, 2). Thus, Plk1 has been proposed as a novel diagnostic marker for cancers. Accumulating evidence suggests that Plk1 negatively regulates the function of the tumor suppressor p53, whose loss-of-function mutations have been observed in nearly 50% of human tumors (1). In our earlier studies, we were the first to demonstrate that Plk1 depletion results in increased p53 level in HeLa cells (3) and that human cells with different levels of p53 respond to Plk1 depletion differently (4). Subsequently, it was shown that Plk1 directly binds to the DNA-binding domain of p53 through its N-terminal kinase domain and inhibits the transactivation as well as the proapoptotic function of p53 (5). Although it has been suggested that Plk1 might regulate p53 through direct phosphorylation (5), our repeated efforts to prove p53 as a direct target of Plk1 have been unsuccessful.Topors was discovered in a screen searching for proteins that bind to DNA topoisomerase I (6) and was also identified as a p53-binding protein (7). Although Topors is widely expressed in normal human tissues, its expression is decreased or undetectable in colon, lung, and brain adenocarcinomas, indicating that it might function as a tumor suppressor (8). Topors contains an N-terminal C3HC4-type RING domain that is closely related in sequence to the RING domains of known E3 ligases (see Fig. 1A) and is the first example of a protein that has both ubiquitin and SUMO-1 E3 ligase activity. Topors functions as an E3 ubiquitin ligase for p53 and NKX3.1, and Topors-mediated ubiquitination leads to the degradation of these proteins (9, 10). Substrates of the SUMO-1 E3 ligase activity of Topors include DNA topoisomerase I and p53 (11, 12). In contrast to ubiquitination-induced protein degradation, Topors-induced p53 sumoylation is accompanied by an increase in the level of p53 protein (11). Taken together, these studies indicate that Topors functions both as an ubiquitin and as a SUMO-1 E3 ligase for p53. Therefore, it is likely that the effects of Topors on p53 depend on cellular context (10).Open in a separate windowFIGURE 1.Plk1 phosphorylates Topors at Ser718in vitro and in vivo. A, schematic representation of the domain structure of Topors. Two separate regions encoding putative p53-binding domains are aa 456–731 and 854–916. Amino acid residues in the putative Ring finger motif are shown in a black box. PEST, sequences rich in Pro, Glu, Ser, and Thr; RS domain, Arg- and Ser-rich domain; NLS, nuclear localization sequence; NB, nuclear bodies. B, purified Plk1 was incubated with purified GST-Topors (aa 1–510) or GST-Topors (aa 511–1045) for 30 min at 30 °C in the presence of [γ-32P]ATP (32P). Reaction mixtures were resolved by SDS-PAGE followed by autoradiography. Coom., Coomassie Blue. C and D, Plk1 phosphorylates Topors (aa 679–760). Purified Plk1 was incubated with purified GST-Topors fragments (aa 1–250, 251–510, 511–760, 756–1045, 511–596, 597–678, and 679–760). Kinase assays were performed as described in B. E, Ser718 of Topors is a Plk1 phosphorylation site in vitro. Purified Plk1 was incubated with the indicated serine to alanine Topors (aa 679–760) mutants and analyzed as in B. F, Topors is phosphorylated in vivo at Ser718 by Plk1. HEK293T cells were transfected with WT-Topors-Myc (lanes 1 and 3) or S718A-Topors-Myc (lane 2) and depleted of Plk1 by using double-stranded RNA targeting Plk1 (lane 3). After overnight incubation, cells were treated with nocodazole for 10 h and metabolically labeled with [32P]orthophosphate. Phosphoproteins were immunoprecipitated with anti-Myc antibodies, resolved by SDS-PAGE, and subjected to autoradiography. Relative 32P (Rel. 32P) incorporations of Topors are indicated on the bottom.In this study, we provide evidence that Plk1 phosphorylates Topors on Ser718. Significantly, we demonstrate that the Plk1-mediated phosphorylation of Topors results in reduced sumoylation of p53, whereas the ubiquitination activity toward p53 is increased, thereby facilitating p53 degradation. 相似文献
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Kawabe Y Seki M Seki T Wang WS Imamura O Furuichi Y Saitoh H Enomoto T 《The Journal of biological chemistry》2000,275(28):20963-20966
Werner's syndrome is a potential model of accelerated human aging. The gene responsible for Werner's syndrome encodes a protein that has a helicase domain homologous to Escherichia coli RecQ. To identify binding partners that regulate the function in concert with Wrn, we screened for proteins using the yeast two-hybrid system with mouse Wrn as bait and found three. One was a novel protein, and the other two were mouse Ubc9 and SUMO-1. Ubc9 also interacted with the mouse homologue of the Bloom's syndrome gene product, another eukaryotic RecQ-type helicase, but not mouse DNA helicase Q1/RecQL (RecQL1). Deletion experiments indicated that both proteins interacted with the N-terminal segment of Wrn (amino acid 272-514). The interaction between Wrn and SUMO-1 was weaker than that between Wrn and Ubc9. Positive interaction was observed in the heterogeneous combination of Wrn and yeast Ubc9 (yUbc9), as well as yUbc9 and SUMO-1, in the two-hybrid system. The interaction between yUbc9 and SUMO-1 was abolished by deleting the C-terminal Gly residue of SUMO-1, which is reportedly required for the formation of Ubc9-SUMO-1 thioester linkage. The interaction of Wrn and SUMO-1 was also abolished by deleting the Gly residue, indicating that the interaction of Wrn and SUMO-1 is mediated by yUbc9 in the two-hybrid system. Finally, we confirmed by immunoblotting with an anti-SUMO-1 antibody that Wrn was covalently attached with SUMO-1. 相似文献
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SUMO (small ubiquitin-like modifier)/Smt3 (suppressor of mif two) is a member of the ubiquitin-related protein family and is known to conjugate with many proteins. In the sumoylation pathway, SUMO/Smt3 is transferred to substrate lysine residues through the thioester cascade of E1 (activating enzyme) and E2 (conjugating enzyme), and E3 (SUMO ligase) functions as an adaptor between E2 and each substrate. Yeast Ull1 (ubiquitin-like protein ligase 1)/Siz1, a PIAS (protein inhibitor of activated STAT)-type SUMO ligase, modifies both cytoplasmic and nuclear proteins. In this paper, we performed a domain analysis of Ull1/Siz1 by constructing various deletion mutants. A novel conserved N-terminal domain, called PINIT, as well as the RING-like domain (SP-RING) were required for the SUMO ligase activity in the in vitro conjugation system and for interaction with Smt3 in an in vitro binding assay. The most distal N-terminal region, which contains a putative DNA-binding SAF-A/B, Acinus, and PIAS (SAP) motif, was not required for the ligase activity but was involved in nuclear localization. A strong SUMO-binding motif was identified, which interacted with Smt3 in the two-hybrid system but was not necessary for the ligase activity. The most distal C-terminal domain was important for stable localization at the bud neck region and thereby for the substrate recognition of septins. Furthermore, the C-terminal half conferred protein instability on Ull1/Siz1. Taken together, we conclude that the SP-RING and PINIT of Ull1/Siz1 are core domains of the SUMO ligase, and the other domains are regulatory for protein stability and subcellular localization. 相似文献
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Tallec LP Kirsh O Lecomte MC Viengchareun S Zennaro MC Dejean A Lombès M 《Molecular endocrinology (Baltimore, Md.)》2003,17(12):2529-2542
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Nakaishi A Hirose M Yoshimura M Oneyama C Saito K Kuki N Matsuda M Honma N Ohnishi H Matozaki T Okada M Nakagawa A 《Journal of molecular biology》2008,375(3):650-660
SRC homology 2 domain-containing protein tyrosine phosphatase substrate 1 (SHPS-1 or SIRPα/BIT) is an immunoglobulin (Ig) superfamily transmembrane receptor and a member of the signal regulatory protein (SIRP) family involved in cell-cell interaction. SHPS-1 binds to its ligand CD47 to relay an inhibitory signal for cellular responses, whereas SIRPβ, an activating member of the same family, does not bind to CD47 despite sharing a highly homologous ligand-binding domain with SHPS-1. To address the molecular basis for specific CD47 recognition by SHPS-1, we present the crystal structure of the ligand-binding domain of murine SHPS-1 (mSHPS-1). Folding topology revealed that mSHPS-1 adopts an I2-set Ig fold, but its overall structure resembles IgV domains of antigen receptors, although it has an extended loop structure (C′E loop), which forms a dimer interface in the crystal. Site-directed mutagenesis studies of mSHPS-1 identified critical residues for CD47 binding including sites in the C′E loop and regions corresponding to complementarity-determining regions of antigen receptors. The structural and functional features of mSHPS-1 are consistent with the human SHPS-1 structure except that human SHPS-1 has an additional β-strand D. These results suggest that the variable complementarity-determining region-like loop structures in the binding surface of SHPS-1 are generally required for ligand recognition in a manner similar to that of antigen receptors, which may explain the diverse ligand-binding specificities of SIRP family receptors. 相似文献
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The negative effect of human immunodeficiency virus type 1 Nef on cell surface CD4 expression is not species specific and requires the cytoplasmic domain of CD4. 总被引:25,自引:15,他引:10
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The nef gene product of human immunodeficiency virus type 1 has been shown to induce CD4 downregulation from the surface of human cells. To determine if this effect is species specific, we used a retroviral vector to transduce the human immunodeficiency virus type 1 nef gene into murine cells expressing human, chimpanzee, or murine CD4. Our results indicate that Nef induces cell surface downregulation of all three molecules. We also determined that Nef is functional in murine T cells and induces downregulation of both murine CD4 and CD8 (Ly-2) from the cell surface. In contrast, Nef does not downregulate cell surface expression of human CD8 in either murine or human cells. By using a mutant of human CD4 lacking its cytoplasmic domain and a human CD4/CD8 chimera, we determined that the cytoplasmic domain of CD4 is required for its downregulation by Nef. Transduction with a control vector had no effect on CD4 cell surface levels, indicating that retroviral transduction by itself has no significant effect on the cell surface levels of CD4. These results show that the observed downregulation of CD4 by Nef is independent of human-specific factors, is not species specific, and requires the cytoplasmic domain of CD4. 相似文献
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TRIM family of E3 ubiquitin ligases have an amino-terminal conserved tripartite motif consisting of RING, B-Box, coiled-coil domain and different C-terminal domain leading it to classification into 11 subclasses. TRIM72 is an E3 ligase of class IV and subclass 1 with its role in a multitude of cellular processes. Despite being crucial in multiple cellular processes, TRIM72 still hasn't been biochemically characterized. In the present study, we have characterized the oligomeric status of TRIM72 and found that it forms both monomers, dimers, and tetramers. We have screened a set of 12 E2s and identified two novel E2 enzymes (Ubch5c and Ubch10) that work in cooperation with TRIM72. Nevertheless, E3 ligase activity is minimal and we propose that additional regulation is required to enhance its E3 ligase activity. We have also used surface plasmon resonance to study interaction with one of its substrate proteins, IRS1, and identified the PH domain of IRS1 is mediating interaction with the TRIM72 E3 ligase while the PTB domain of IRS1, does not show any interaction. 相似文献
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Zhao H Li CC Pardo J Chu PC Liao CX Huang J Dong JG Zhou X Huang Q Huang B Bennett MK Molineaux SM Lu H Daniel-Issakani S Payan DG Masuda ES 《Journal of immunology (Baltimore, Md. : 1950)》2005,174(9):5288-5297
TRAC-1 (T cell RING (really interesting new gene) protein identified in activation screen) is a novel E3 ubiquitin ligase identified from a retroviral vector-based T cell surface activation marker screen. The C-terminal truncated TRAC-1 specifically inhibited anti-TCR-mediated CD69 up-regulation in Jurkat cells, a human T leukemic cell line. In this study, we show that TRAC-1 is a RING finger ubiquitin E3 ligase with highest expression in lymphoid tissues. Point mutations that disrupt the Zn(2+)-chelating ability of its amino-terminal RING finger domain abolished TRAC-1's ligase activity and the dominant inhibitory effect of C-terminal truncated TRAC-1 on TCR stimulation. The results of in vitro biochemical studies indicate that TRAC-1 can stimulate the formation of both K48- and K63-linked polyubiquitin chains and therefore could potentially activate both degradative and regulatory ubiquitin-dependent pathways. Antisense oligonucleotides to TRAC-1 specifically reduced TRAC-1 mRNA levels in Jurkat and primary T cells and inhibited their activation in response to TCR cross-linking. Collectively, these results indicate that the E3 ubiquitin ligase TRAC-1 functions as a positive regulator of T cell activation. 相似文献
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CD84 functions as a homophilic adhesion molecule and enhances IFN-gamma secretion: adhesion is mediated by Ig-like domain 1 总被引:6,自引:0,他引:6
Martin M Romero X de la Fuente MA Tovar V Zapater N Esplugues E Pizcueta P Bosch J Engel P 《Journal of immunology (Baltimore, Md. : 1950)》2001,167(7):3668-3676
CD84 is a member of the CD2 subset of the Ig superfamily of cell surface molecules. Its cytoplasmic tail binds to Src homology 2 domain-containing protein 1A (signaling lymphocytic activation molecule-associated protein), a protein encoded by the X-linked lymphoproliferative disease gene. It is preferentially expressed on B lymphocytes, monocytes, and platelets. We show that it is also expressed on thymocytes and T cells. CD84 was positive on CD4-CD8- thymocytes, and its expression decreased with cell maturation. It is expressed on mature T cells preferentially on CD45RO+. To identify the CD84 ligand, we generated a soluble Ig fusion protein containing the human CD84 extracellular domains (CD84-Ig). Because receptor-ligand interactions occur between several members of this subfamily, we assayed CD84-Ig binding with all members of the CD2 family. CD84-Ig bound to CD84-transfected cells, whereas no binding was detected with cells expressing other CD2 subfamily receptors, showing that CD84 binds to itself. Anti-CD84 mAbs recognizing epitopes wholly within domain 1 of CD84 blocked the binding of the CD84-Ig fusion protein to CD84-transfected cells and platelets. Data from CD84 domain human/mouse chimeras further revealed that only the first extracellular domain of the molecule is involved in the ligand receptor recognition. The CD84-CD84 interaction was independent of its cytoplasmic tail. Finally, concurrent ligation of human CD84 with mAbs or CD84-Ig and CD3 enhanced IFN-gamma secretion in human lymphocytes. Thus, CD84 is its own ligand and acts as a costimulatory molecule. 相似文献
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Mdm2 is an E3 ubiquitin ligase for the p53 tumor suppressor protein. We demonstrate that Mdm2 is conjugated with SUMO-1 (sumoylated) at Lys-446, which is located within the RING finger domain and plays a critical role in Mdm2 self-ubiquitination. Whereas mutant Mdm2(K446R) is stabilized, it elicits increased degradation of p53 and concomitant inhibition of p53-mediated apoptosis. In vitro sumoylation of Mdm2 abrogates its self-ubiquitination and increases its ubiquitin ligase activity toward p53. Radiation caused a dose- and time-dependent decrease in the degree of Mdm2 SUMO-1 modification, which is inversely correlated with the levels of p53. Our results suggest that the maintenance of the intrinsic activity of a RING finger E3 ubiquitin ligase is sumoylation dependent and that reduced Mdm2 sumoylation in response to DNA damage contributes to p53 stability. 相似文献
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SUMO-1 Modification and Its Role in Targeting the Ran GTPase-activating Protein, RanGAP1, to the Nuclear Pore Complex 总被引:24,自引:2,他引:22
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RanGAP1 is the GTPase-activating protein for Ran, a small ras-like GTPase involved in regulating nucleocytoplasmic transport. In vertebrates, RanGAP1 is present in two forms: one that is cytoplasmic, and another that is concentrated at the cytoplasmic fibers of nuclear pore complexes (NPCs). The NPC-associated form of RanGAP1 is covalently modified by the small ubiquitin-like protein, SUMO-1, and we have recently proposed that SUMO-1 modification functions to target RanGAP1 to the NPC. Here, we identify the domain of RanGAP1 that specifies SUMO-1 modification and demonstrate that mutations in this domain that inhibit modification also inhibit targeting to the NPC. Targeting of a heterologous protein to the NPC depended on determinants specifying SUMO-1 modification and also on additional determinants in the COOH-terminal domain of RanGAP1. SUMO-1 modification and these additional determinants were found to specify interaction between the COOH-terminal domain of RanGAP1 and a region of the nucleoporin, Nup358, between Ran-binding domains three and four. Together, these findings indicate that SUMO-1 modification targets RanGAP1 to the NPC by exposing, or creating, a Nup358 binding site in the COOH-terminal domain of RanGAP1. Surprisingly, the COOH-terminal domain of RanGAP1 was also found to harbor a nuclear localization signal. This nuclear localization signal, and the presence of nine leucine-rich nuclear export signal motifs, suggests that RanGAP1 may shuttle between the nucleus and the cytoplasm. 相似文献