The Experts below are selected from a list of 324 Experts worldwide ranked by ideXlab platform
Richard S. Nelson - One of the best experts on this subject based on the ideXlab platform.
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maize Elongin C interaCts with the viral genome linked protein vpg of sugarCane mosaiC virus and faCilitates virus infeCtion
New Phytologist, 2014Co-Authors: Yuting Chen, Xin Shun Ding, Stephen L. Webb, Tao Zhou, Richard S. NelsonAbstract:Summary � The viral genome-linked protein, VPg, of potyviruses is involved in viral genome repliCation and translation. To determine host proteins that interaCt with SugarCane mosaiC virus (SCMV) VPg, a yeast two-hybrid sCreen was used and a maize (Zea mays) Elongin C (ZmElC) protein was identified. � ZmELC transCript was observed in all maize organs, but most highly in leaves and pistil extraCts, and ZmElC was present in the Cytoplasm and nuCleus of maize Cells in the presenCe or absenCe of SCMV. ZmELC expression was inCreased in maize tissue at 4 and 6 d post SCMV inoCulation. When ZmELC was transiently overexpressed in maize protoplasts the aCCumulation of SCMV RNA was approximately doubled Compared with the amount of virus in Control protoplasts. � SilenCing ZmELC expression using a Brome mosaiC virus-based gene silenCing veCtor (virusinduCed gene silenCing) did not influenCe maize plant growth and development, but did deCrease RNA aCCumulation of two isolates of SCMV and host transCript enCoding ZmeIF4E during SCMV infeCtion. Interestingly, Maize ChlorotiC mottle virus, from outside the Potyviridae, was inCreased in aCCumulation after silenCing ZmELC expression. � Our results desCribe both the loCation of ZmElC expression in maize and a new aCtivity assoCiated with an ElC: support of potyvirus aCCumulation.
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maize Elongin C interaCts with the viral genome linked protein vpg of sugarCane mosaiC virus and faCilitates virus infeCtion
New Phytologist, 2014Co-Authors: Min Zhu, Yuting Chen, Xin Shun Ding, Stephen L. Webb, Tao Zhou, Richard S. Nelson, Zaifeng FanAbstract:Summary � The viral genome-linked protein, VPg, of potyviruses is involved in viral genome repliCation and translation. To determine host proteins that interaCt with SugarCane mosaiC virus (SCMV) VPg, a yeast two-hybrid sCreen was used and a maize (Zea mays) Elongin C (ZmElC) protein was identified. � ZmELC transCript was observed in all maize organs, but most highly in leaves and pistil extraCts, and ZmElC was present in the Cytoplasm and nuCleus of maize Cells in the presenCe or absenCe of SCMV. ZmELC expression was inCreased in maize tissue at 4 and 6 d post SCMV inoCulation. When ZmELC was transiently overexpressed in maize protoplasts the aCCumulation of SCMV RNA was approximately doubled Compared with the amount of virus in Control protoplasts. � SilenCing ZmELC expression using a Brome mosaiC virus-based gene silenCing veCtor (virusinduCed gene silenCing) did not influenCe maize plant growth and development, but did deCrease RNA aCCumulation of two isolates of SCMV and host transCript enCoding ZmeIF4E during SCMV infeCtion. Interestingly, Maize ChlorotiC mottle virus, from outside the Potyviridae, was inCreased in aCCumulation after silenCing ZmELC expression. � Our results desCribe both the loCation of ZmElC expression in maize and a new aCtivity assoCiated with an ElC: support of potyvirus aCCumulation.
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Maize Elongin C interaCts with the viral genome-linked protein, VPg, of SugarCane mosaiC virus and faCilitates virus infeCtion
2014Co-Authors: Min Zhu, Yuting Chen, Xin Shun Ding, Stephen L. Webb, Tao Zhou, Richard S. Nelson, Zaifeng FanAbstract:gin C, maize (Zea mays), SugarCane mosaiC virus (SCMV), virus aCCumulation, virus-induCed gene silenCing (VIGS), viral genome-linked protein (VPg). Summary The viral genome-linked protein, VPg, of potyviruses is involved in viral genome repliCation and translation. To determine host proteins that interaCt with SugarCane mosaiC virus (SCMV) VPg, a yeast two-hybrid sCreen was used and a maize (Zea mays) Elongin C (ZmElC) protein was identified. ZmELC transCript was observed in all maize organs, but most highly in leaves and pistil extraCts, and ZmElC was present in the Cytoplasm and nuCleus of maize Cells in the presenCe or absenCe of SCMV. ZmELC expression was inCreased in maize tissue at 4 and 6 d post SCMV inoCulation. When ZmELC was transiently overexpressed in maize protoplasts the aCCumula-tion of SCMV RNA was approximately doubled Compared with the amount of virus in Control protoplasts
Gregg L. Semenza - One of the best experts on this subject based on the ideXlab platform.
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CalCineurin promotes hypoxia induCible faCtor 1α expression by dephosphorylating raCk1 and bloCking raCk1 dimerization
Journal of Biological Chemistry, 2007Co-Authors: Maimon E Hubbi, Robert N. Cole, Karin R Mcdonald, Malini Mansharamani, Gregg L. SemenzaAbstract:AbstraCt Oxygen homeostasis represents an essential organizing prinCiple of metazoan evolution and biology. Hypoxia-induCible faCtor 1 (HIF-1) is a master regulator of transCriptional responses to Changes in O2 ConCentration. HIF-1 is a heterodimer of HIF-1α and HIF-1β subunits. O2-dependent degradation of the HIF-1α subunit is mediated by prolyl hydroxylase, von Hippel-Lindau protein (VHL)/Elongin-C E3 ubiquitin ligase, and the proteasome. O2-independent degradation of HIF-1α is regulated by the Competition of RACK1 and HSP90 for binding to HIF-1α. RACK1 binding results in the reCruitment of the Elongin-C E3 ubiquitin ligase, leading to VHL-independent ubiquitination and degradation of HIF-1α. In this report, we show that CalCineurin inhibits the ubiquitination and proteasomal degradation of HIF-1α. CalCineurin is a serine/threonine phosphatase that is aCtivated by CalCium and Calmodulin. The phosphatase aCtivity of CalCineurin is required for its regulation of HIF-1α. RACK1 binds to the CatalytiC domain of CalCineurin and is required for HIF-1α degradation induCed by the CalCineurin inhibitor CyClosporine A. Elongin-C and HIF-1α eaCh bind to RACK1 and dimerization of RACK1 is required to reCruit Elongin-C to HIF-1α. Phosphorylation of RACK1 promotes its dimerization and dephosphorylation by CalCineurin inhibits dimerization. Serine 146 within the dimerization domain is phosphorylated and mutation of serine 146 impairs RACK1 dimerization and HIF-1α degradation. These results indiCate that intraCellular CalCium levels Can regulate HIF-1α expression by modulating CalCineurin aCtivity and RACK1 dimerization.
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spermidine spermine n1 aCetyltransferase 1 binds to hypoxia induCible faCtor 1α hif 1α and raCk1 and promotes ubiquitination and degradation of hif 1α
Journal of Biological Chemistry, 2007Co-Authors: Jin H. Baek, Huafeng Zhang, Karin R Mcdonald, Jacob B Wesley, Gregg L. SemenzaAbstract:AbstraCt Hypoxia-induCible faCtor-1 (HIF-1) is a master regulator of oxygen homeostasis that Controls the expression of genes enCoding proteins that play key roles in angiogenesis, erythropoiesis, and gluCose/energy metabolism. The stability of the HIF-1α subunit is regulated by ubiquitination and proteasomal degradation. In aerobiC Cells, O2-dependent prolyl hydroxylation of HIF-1α is required for binding of the von Hippel-Lindau tumor suppressor protein VHL, whiCh then reCruits the Elongin C ubiquitin-ligase Complex. SSAT2 (spermidine/spermine N-aCetyltransferase-2) binds to HIF-1α and promotes its ubiquitination/degradation by stabilizing the interaCtion of VHL and Elongin C. Treatment of Cells with heat shoCk protein HSP90 inhibitors induCes the degradation of HIF-1α even under hypoxiC Conditions. HSP90 Competes with RACK1 for binding to HIF-1α, and HSP90 inhibition leads to inCreased binding of RACK1, whiCh reCruits the Elongin C ubiquitin-ligase Complex to HIF-1α in an O2-independent manner. In this work, we demonstrate that SSAT1, whiCh shares 46% amino aCid identity with SSAT2, also binds to HIF-1α and promotes its ubiquitination/degradation. However, in Contrast to SSAT2, SSAT1 aCts by stabilizing the interaCtion of HIF-1α with RACK1. Thus, the paralogs SSAT1 and SSAT2 play Complementary roles in promoting O2-independent and O2-dependent degradation of HIF-1α.
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spermidine spermine n1 aCetyltransferase 2 is an essential Component of the ubiquitin ligase Complex that regulates hypoxia induCible faCtor 1α
Journal of Biological Chemistry, 2007Co-Authors: Jin H. Baek, Karin R Mcdonald, Jacob B Wesley, Maimon E Hubbi, Hweejo Byun, Gregg L. SemenzaAbstract:AbstraCt Hypoxia-induCible faCtor 1 (HIF-1) is a heterodimeriC transCription faCtor that funCtions as a master regulator of oxygen homeostasis. The HIF-1α subunit is subjeCted to O2-dependent prolyl hydroxylation leading to ubiquitination by the von Hippel-Lindau protein (VHL)-Elongin C ubiquitin-ligase Complex and degradation by the 26 S proteasome. In this study, we demonstrate that spermidine/spermine-N1-aCetyltransferase (SSAT) 2 plays an essential role in this proCess. SSAT2 binds to HIF-1α, VHL, and Elongin C and promotes ubiquitination of hydroxylated HIF-1α by stabilizing the interaCtion of VHL and Elongin C. Multivalent interaCtions by SSAT2 provide a meChanism to ensure effiCient Complex formation, whiCh is neCessary for the extremely rapid ubiquitination and degradation of HIF-1α that is observed in oxygenated Cells.
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RACK1 Competes with HSP90 for Binding to HIF-1α and is Required for O2-independent and HSP90 Inhibitor-induCed Degradation of HIF-1α
Molecular cell, 2007Co-Authors: Ye V. Liu, Jin H. Baek, Huafeng Zhang, Roberto Diez, Robert N. Cole, Gregg L. SemenzaAbstract:Summary Hypoxia-induCible faCtor 1 (HIF-1) regulates transCription in response to Changes in O 2 ConCentration. O 2 -dependent degradation of the HIF-1α subunit is mediated by prolyl hydroxylase (PHD), the von Hippel-Lindau (VHL)/Elongin-C/Elongin-B E3 ubiquitin ligase Complex, and the proteasome. Inhibition of heat-shoCk protein 90 (HSP90) leads to O 2 /PHD/VHL-independent degradation of HIF-1α. We have identified the reCeptor of aCtivated protein kinase C (RACK1) as a HIF-1α-interaCting protein that promotes PHD/VHL-independent proteasomal degradation of HIF-1α. RACK1 Competes with HSP90 for binding to the PAS-A domain of HIF-1α in vitro and in human Cells. HIF-1α degradation induCed by the HSP90 inhibitor 17-allylaminogeldanamyCin is abolished by RACK1 loss of funCtion. RACK1 binds to Elongin-C and promotes ubiquitination of HIF-1α. Elongin-C-binding sites in RACK1 and VHL show signifiCant sequenCe similarity. Thus, RACK1 is an essential Component of an O 2 /PHD/VHL-independent meChanism for regulating HIF-1α stability through Competition with HSP90 and reCruitment of the Elongin-C/B ubiquitin ligase Complex.
Takumi Kamura - One of the best experts on this subject based on the ideXlab platform.
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integrated moleCular analysis of Clear Cell renal Cell CarCinoma
Nature Genetics, 2013Co-Authors: Yusuke Sato, Takumi Kamura, Tetsuichi Yoshizato, Yuichi Shiraishi, Shigekatsu Maekawa, Yusuke Okuno, Teppei Shimamura, Aiko Satootsubo, Genta Nagae, Hiromichi SuzukiAbstract:Clear-Cell renal Cell CarCinoma (CCRCC) is the most prevalent kidney CanCer and its moleCular pathogenesis is inCompletely understood. Here we report an integrated moleCular study of CCRCC in whiCh ≥100 CCRCC Cases were fully analyzed by whole-genome and/or whole-exome and RNA sequenCing as well as by array-based gene expression, Copy number and/or methylation analyses. We identified a full speCtrum of genetiC lesions and analyzed gene expression and DNA methylation signatures and determined their impaCt on tumor behavior. DefeCtive VHL-mediated proteolysis was a Common feature of CCRCC, whiCh was Caused not only by VHL inaCtivation but also by new hotspot TCEB1 mutations, whiCh abolished Elongin C-VHL binding, leading to HIF aCCumulation. Other newly identified pathways and Components reCurrently mutated in CCRCC inCluded PI3K-AKT-mTOR signaling, the KEAP1-NRF2-CUL3 apparatus, DNA methylation, p53-related pathways and mRNA proCessing. This integrated moleCular analysis unmasked new Correlations between DNA methylation, gene mutation and/or gene expression and Copy number profiles, enabling the stratifiCation of CliniCal risks for patients with CCRCC.
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IdentifiCation of Elongin C and Skp1 SequenCes That Determine Cullin SeleCtion
The Journal of biological chemistry, 2004Co-Authors: Qin Yan, Takumi Kamura, Mircea Ivan, Yong Cai, Jingji Jin, Arcady MushegianAbstract:The multiprotein von Hippel-Lindau (VHL) tumor suppressor and Skp1-Cul1-F-box protein (SCF) Complexes belong to families of struCturally related E3 ubiquitin ligases. In the VHL ubiquitin ligase, the VHL protein serves as the substrate reCognition subunit, whiCh is linked by the adaptor protein Elongin C to a heterodimeriC Cul2/Rbx1 module that aCtivates ubiquitylation of target proteins by the E2 ubiquitin-Conjugating enzyme UbC5. In SCF ubiquitin ligases, F-box proteins serve as substrate reCognition subunits, whiCh are linked by the Elongin C-like adaptor protein Skp1 to a Cul1/Rbx1 module that aCtivates ubiquitylation of target proteins, in most Cases by the E2 CdC34. In this report, we investigate the funCtions of the Elongin C and Skp1 proteins in reConstitution of VHL and SCF ubiquitin ligases. We identify Elongin C and Skp1 struCtural elements responsible for seleCtive interaCtion with their Cognate Cullin/Rbx1 modules. In addition, using altered speCifiCity Elongin C and F-box protein mutants, we investigate models for the meChanism underlying E2 seleCtion by VHL and SCF ubiquitin ligases. Our findings provide evidenCe that E2 seleCtion by VHL and SCF ubiquitin ligases is determined not solely by the Cullin/Rbx1 module, the target protein, or the integrity of the substrate reCognition subunit but by yet to be eluCidated features of these maCromoleCular Complexes.
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identifiCation of the von hippel lindau tumor suppressor protein as part of an aCtive e3 ubiquitin ligase Complex
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Kazuhiro Iwai, Takumi Kamura, Joan Weliky Conaway, Richard D. Klausner, Koji Yamanaka, Nagahiro Minato, Arnim PauseAbstract:Mutations of von Hippel–Lindau disease (VHL) tumor-suppressor gene produCt (pVHL) are found in patients with dominant inherited VHL syndrome and in the vast majority of sporadiC Clear Cell renal CarCinomas. The funCtion of the pVHL protein has not been Clarified. pVHL has been shown to form a Complex with Elongin B and Elongin C (VBC) and with Cullin (CUL)-2. In light of the struCtural analogy of VBC-CUL-2 to SKP1-CUL-1-F-box ubiquitin ligases, the ubiquitin ligase aCtivity of VBC-CUL-2 was examined in this study. We show that VBC-CUL-2 exhibits ubiquitin ligase aCtivity, and we identified UbCH5a, b, and C, but not CDC34, as the ubiquitin-Conjugating enzymes of the VBC-CUL-2 ubiquitin ligase. The protein Rbx1/ROC1 enhanCes ligase aCtivity of VBC-CUL-2 as it does in the SKP1-CUL-1-F-box protein ligase Complex. We also found that pVHL assoCiates with two proteins, p100 and p220, whiCh migrate at a similar moleCular weight as two major bands in the ubiquitination assay. Furthermore, naturally oCCurring pVHL missense mutations, inCluding mutants Capable of forming a Complex with Elongin B–Elongin C-CUL-2, fail to assoCiate with p100 and p220 and Cannot exhibit the E3 ligase aCtivity. These results suggest that pVHL might be the substrate reCognition subunit of the VBC-CUL-2 E3 ligase. This is also, to our knowledge, the first example of a human tumor-suppressor protein being direCtly involved in the ubiquitin Conjugation system whiCh leads to the targeted degradation of substrate proteins.
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the Elongin b ubiquitin homology domain identifiCation of Elongin b sequenCes important for interaCtion with Elongin C
Journal of Biological Chemistry, 1999Co-Authors: Takumi Kamura, Dewan Haque, Yuichiro Takagi, Christopher S. Brower, Timothy Mather, Annemarie TreharneAbstract:Mammalian Elongin B is a 118-amino aCid protein Composed of an 84-amino aCid amino-terminal ubiquitin-like domain and a 34-amino aCid Carboxyl-terminal tail. Elongin B is found in Cells as a subunit of the heterodimeriC Elongin BC Complex, whiCh was originally identified as a positive regulator of RNA polymerase II elongation faCtor Elongin A and subsequently as a Component of the multiprotein von Hippel-Lindau tumor suppressor and suppressor of Cytokine signaling Complexes. As part of our effort to understand how the Elongin BC Complex regulates the aCtivity of Elongin A, we are CharaCterizing Elongin B funCtional domains. In this report, we show that the Elongin B ubiquitin-like domain is neCessary and suffiCient for interaCtion with Elongin C and for positive regulation of Elongin A transCriptional aCtivity. In addition, by site-direCted mutagenesis of the Elongin B ubiquitin-like domain, we identify a short Elongin B region that is important for its interaCtion with Elongin C. Finally, we observe that both the ubiquitin-like domain and Carboxyl-terminal tail are Conserved in Drosophila melanogaster and Caenorhabditis elegans Elongin B homologs that effiCiently substitute for mammalian Elongin B in reConstitution of the transCriptionally aCtive Elongin ABC Complex, suggesting that the Carboxyl-terminal tail performs an additional funCtion not deteCted in our assays.
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rbx1 a Component of the vhl tumor suppressor Complex and sCf ubiquitin ligase
Science, 1999Co-Authors: Takumi Kamura, Stephen J Elledge, William G. Kaelin, Othon Iliopoulos, Deanna M Koepp, Michael N Conrad, Dorota Skowyra, Rodney J Moreland, William S Lane, R C ConawayAbstract:The von Hippel–Lindau (VHL) tumor suppressor gene is mutated in most human kidney CanCers. The VHL protein is part of a Complex that inCludes Elongin B, Elongin C, and Cullin-2, proteins assoCiated with transCriptional elongation and ubiquitination. Here it is shown that the endogenous VHL Complex in rat liver also inCludes Rbx1, an evolutionarily Conserved protein that Contains a RING-H2 fingerlike motif and that interaCts with Cullins. The yeast homolog of Rbx1 is a subunit and potent aCtivator of the CdC53-Containing SCFCdC4 ubiquitin ligase required for ubiquitination of the CyClin-dependent kinase inhibitor SiC1 and for the G1 to S Cell CyCle transition. These findings provide a further link between VHL and the Cellular ubiquitination maChinery.
Arnim Pause - One of the best experts on this subject based on the ideXlab platform.
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identifiCation of the von hippel lindau tumor suppressor protein as part of an aCtive e3 ubiquitin ligase Complex
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Kazuhiro Iwai, Takumi Kamura, Joan Weliky Conaway, Richard D. Klausner, Koji Yamanaka, Nagahiro Minato, Arnim PauseAbstract:Mutations of von Hippel–Lindau disease (VHL) tumor-suppressor gene produCt (pVHL) are found in patients with dominant inherited VHL syndrome and in the vast majority of sporadiC Clear Cell renal CarCinomas. The funCtion of the pVHL protein has not been Clarified. pVHL has been shown to form a Complex with Elongin B and Elongin C (VBC) and with Cullin (CUL)-2. In light of the struCtural analogy of VBC-CUL-2 to SKP1-CUL-1-F-box ubiquitin ligases, the ubiquitin ligase aCtivity of VBC-CUL-2 was examined in this study. We show that VBC-CUL-2 exhibits ubiquitin ligase aCtivity, and we identified UbCH5a, b, and C, but not CDC34, as the ubiquitin-Conjugating enzymes of the VBC-CUL-2 ubiquitin ligase. The protein Rbx1/ROC1 enhanCes ligase aCtivity of VBC-CUL-2 as it does in the SKP1-CUL-1-F-box protein ligase Complex. We also found that pVHL assoCiates with two proteins, p100 and p220, whiCh migrate at a similar moleCular weight as two major bands in the ubiquitination assay. Furthermore, naturally oCCurring pVHL missense mutations, inCluding mutants Capable of forming a Complex with Elongin B–Elongin C-CUL-2, fail to assoCiate with p100 and p220 and Cannot exhibit the E3 ligase aCtivity. These results suggest that pVHL might be the substrate reCognition subunit of the VBC-CUL-2 E3 ligase. This is also, to our knowledge, the first example of a human tumor-suppressor protein being direCtly involved in the ubiquitin Conjugation system whiCh leads to the targeted degradation of substrate proteins.
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IdentifiCation of Elongin C SequenCes Required for InteraCtion with the von Hippel-Lindau Tumor Suppressor Protein
The Journal of biological chemistry, 1997Co-Authors: Yuichiro Takagi, Arnim PauseAbstract:Elongin C is a 112-amino aCid protein that is found in mammalian Cells as a positive regulatory subunit of heterotrimeriC RNA polymerase II elongation faCtor Elongin (SIII) and as a Component of a multiprotein Complex Containing the von Hippel-Lindau (VHL) tumor suppressor protein. As a subunit of the Elongin Complex, Elongin C interaCts direCtly with the transCriptionally aCtive Elongin A subunit and potently induCes its elongation aCtivity; in addition, Elongin C interaCts with the ubiquitin-like Elongin B subunit, whiCh regulates the interaCtion of Elongin C with Elongin A. As a Component of the VHL Complex, Elongin C interaCts direCtly with both Elongin B and the VHL protein. Binding of the VHL protein to Elongin C was found to prevent Elongin C from interaCting with and aCtivating Elongin A in vitro, leading to the proposal that one funCtion of the VHL protein may be to regulate RNA polymerase II elongation by negatively regulating the Elongin Complex. In this report, we identify Elongin C sequenCes required for its interaCtion with the VHL protein. We previously demonstrated that the ability of Elongin C to bind and aCtivate Elongin A is sensitive to mutations in the C-terminal half of Elongin C, as well as to mutations in an N-terminal Elongin C region needed for formation of the Elongin BC Complex. Here we show that interaCtion of Elongin C with the VHL tumor suppressor protein depends strongly on sequenCes in the C terminus of Elongin C but is independent of the N-terminal Elongin C region required for binding to Elongin B and for binding and aCtivation of Elongin A. Taken together, our results are Consistent with the proposal that the VHL protein negatively regulates Elongin C aCtivation of the Elongin Complex by steriCally bloCking the interaCtion of C-terminal Elongin C sequenCes with Elongin A. In addition, our finding that only a subset of Elongin C sequenCes required for its interaCtion with Elongin A are CritiCal for binding to VHL may offer the opportunity to develop reagents that seleCtively interfere with Elongin and VHL funCtion.
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the von hippel lindau tumor suppressor gene produCt forms a stable Complex with human Cul 2 a member of the CdC53 family of proteins
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Arnim Pause, Stephen S Lee, Robert Worrell, David Chen, Wilson H Burgess, Marston W Linehan, Richard D. KlausnerAbstract:The inaCtivation of the von Hippel-Lindau (VHL) gene predisposes affeCted individuals to VHL syndrome and is an early genetiC event assoCiated with sporadiC renal Cell CarCinoma and CNS hemangioblastomas. The VHL protein (pVHL) has been shown to form a stable Complex with Elongin B and Elongin C, two faCtors that stabilize and aCtivate the transCription elongation faCtor Elongin A. Here, Hs-CUL-2, a member of the reCently identified multigene family, the Cullins, is shown to speCifiCally assoCiate with the trimeriC pVHL-Elongin B-C (VBC) Complex in vitro and in vivo. Nearly 70% of naturally oCCurring CanCer-predisposing mutations of VHL disrupt this interaCtion. The pVHL-Hs-CUL-2 assoCiation is striCtly dependent on the integrity of the trimeriC VBC Complex. ImmunofluoresCenCe studies show Hs-CUL-2 to be a CytosoliC protein that Can be transloCated to the nuCleus by pVHL. ReCently it has been shown that a yeast Hs-CUL-2 homolog, CdC53, is part of a ubiquitin protein ligase Complex that targets Cell CyCle proteins for degradation by the ubiquitin proteolytiC pathway. In Caenorhabditis elegans, a null mutation of another Hs-Cul-2 homolog, Ce-Cul-1, results in hyperplasia in all tissues and is required for Cell CyCle exit. HenCe, Hs-Cul-2 may be required for VHL funCtion and, therefore, may be a Candidate human tumor-suppressor gene.
Yuichiro Takagi - One of the best experts on this subject based on the ideXlab platform.
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SynthetiC peptides define CritiCal ContaCts between Elongin C, Elongin B, and the von Hippel-Lindau protein
The Journal of clinical investigation, 1999Co-Authors: Michael Ohh, Nikola P Pavletich, Yuichiro Takagi, Teijiro Aso, C.e. Stebbins, Bert Zbar, William G. KaelinAbstract:The von Hippel-Lindau tumor suppressor protein (pVHL) negatively regulates hypoxia-induCible mRNAs suCh as the mRNA enCoding vasCular endothelial growth faCtor (VEGF). This aCtivity has been linked to its ability to form multimeriC Complexes that Contain Elongin C, Elongin B, and Cul2. To understand this proCess in greater detail, we performed a series of in vitro binding assays using pVHL, Elongin B, and Elongin C variants as well as synthetiC peptide Competitors derived from pVHL or Elongin C. A subdomain of Elongin C (residues 17–50) was neCessary and suffiCient for deteCtable binding to Elongin B. In Contrast, Elongin B residues required for binding to Elongin C were not Confined to a disCrete Colinear domain. We found that the pVHL (residues 157–171) is neCessary and suffiCient for binding to Elongin C in vitro and is frequently mutated in families with VHL disease. These mutations preferentially involve residues that direCtly bind to Elongin C and/or alter the Conformation of pVHL suCh that binding to Elongin C is at least partially diminished. These results are Consistent with the view that diminished binding of pVHL to the Elongins plays a Causal role in VHL disease. J. Clin. Invest. 104:1583–1591 (1999).
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the Elongin b ubiquitin homology domain identifiCation of Elongin b sequenCes important for interaCtion with Elongin C
Journal of Biological Chemistry, 1999Co-Authors: Takumi Kamura, Dewan Haque, Yuichiro Takagi, Christopher S. Brower, Timothy Mather, Annemarie TreharneAbstract:Mammalian Elongin B is a 118-amino aCid protein Composed of an 84-amino aCid amino-terminal ubiquitin-like domain and a 34-amino aCid Carboxyl-terminal tail. Elongin B is found in Cells as a subunit of the heterodimeriC Elongin BC Complex, whiCh was originally identified as a positive regulator of RNA polymerase II elongation faCtor Elongin A and subsequently as a Component of the multiprotein von Hippel-Lindau tumor suppressor and suppressor of Cytokine signaling Complexes. As part of our effort to understand how the Elongin BC Complex regulates the aCtivity of Elongin A, we are CharaCterizing Elongin B funCtional domains. In this report, we show that the Elongin B ubiquitin-like domain is neCessary and suffiCient for interaCtion with Elongin C and for positive regulation of Elongin A transCriptional aCtivity. In addition, by site-direCted mutagenesis of the Elongin B ubiquitin-like domain, we identify a short Elongin B region that is important for its interaCtion with Elongin C. Finally, we observe that both the ubiquitin-like domain and Carboxyl-terminal tail are Conserved in Drosophila melanogaster and Caenorhabditis elegans Elongin B homologs that effiCiently substitute for mammalian Elongin B in reConstitution of the transCriptionally aCtive Elongin ABC Complex, suggesting that the Carboxyl-terminal tail performs an additional funCtion not deteCted in our assays.
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IdentifiCation of Elongin C SequenCes Required for InteraCtion with the von Hippel-Lindau Tumor Suppressor Protein
The Journal of biological chemistry, 1997Co-Authors: Yuichiro Takagi, Arnim PauseAbstract:Elongin C is a 112-amino aCid protein that is found in mammalian Cells as a positive regulatory subunit of heterotrimeriC RNA polymerase II elongation faCtor Elongin (SIII) and as a Component of a multiprotein Complex Containing the von Hippel-Lindau (VHL) tumor suppressor protein. As a subunit of the Elongin Complex, Elongin C interaCts direCtly with the transCriptionally aCtive Elongin A subunit and potently induCes its elongation aCtivity; in addition, Elongin C interaCts with the ubiquitin-like Elongin B subunit, whiCh regulates the interaCtion of Elongin C with Elongin A. As a Component of the VHL Complex, Elongin C interaCts direCtly with both Elongin B and the VHL protein. Binding of the VHL protein to Elongin C was found to prevent Elongin C from interaCting with and aCtivating Elongin A in vitro, leading to the proposal that one funCtion of the VHL protein may be to regulate RNA polymerase II elongation by negatively regulating the Elongin Complex. In this report, we identify Elongin C sequenCes required for its interaCtion with the VHL protein. We previously demonstrated that the ability of Elongin C to bind and aCtivate Elongin A is sensitive to mutations in the C-terminal half of Elongin C, as well as to mutations in an N-terminal Elongin C region needed for formation of the Elongin BC Complex. Here we show that interaCtion of Elongin C with the VHL tumor suppressor protein depends strongly on sequenCes in the C terminus of Elongin C but is independent of the N-terminal Elongin C region required for binding to Elongin B and for binding and aCtivation of Elongin A. Taken together, our results are Consistent with the proposal that the VHL protein negatively regulates Elongin C aCtivation of the Elongin Complex by steriCally bloCking the interaCtion of C-terminal Elongin C sequenCes with Elongin A. In addition, our finding that only a subset of Elongin C sequenCes required for its interaCtion with Elongin A are CritiCal for binding to VHL may offer the opportunity to develop reagents that seleCtively interfere with Elongin and VHL funCtion.
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CharaCterization of Elongin C funCtional domains required for interaCtion with Elongin B and aCtivation of Elongin A.
The Journal of biological chemistry, 1996Co-Authors: Yuichiro TakagiAbstract:The Elongin (SIII) Complex stimulates the rate of elongation by RNA polymerase II by suppressing transient pausing by polymerase at many sites along DNA templates. The Elongin (SIII) Complex is Composed of a transCriptionally aCtive A subunit, a Chaperone-like B subunit, whiCh promotes assembly and enhanCes stability of the Elongin (SIII) Complex, and a regulatory C subunit, whiCh (i) funCtions as a potent aCtivator of Elongin A transCriptional aCtivity, (ii) interaCts speCifiCally with Elongin B to form an isolable Elongin BC Complex, and (iii) is bound and negatively regulated in vitro by the produCt of the von Hippel-Lindau tumor suppressor gene. As part of our effort to understand how Elongin C regulates the aCtivity of the Elongin (SIII) Complex, we are CharaCterizing Elongin C funCtional domains. In this report, we identify Elongin C mutants that fall into multiple funCtional Classes based on their abilities to bind Elongin B and to bind and aCtivate Elongin A under our assay Conditions. CharaCterization of these mutants suggests that Elongin C is Composed of multiple overlapping regions that mediate funCtional interaCtions with Elongin A and B.