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Ronald C Conaway

Publications and source records attributed to Ronald C Conaway.

At least 37 records · Page 2Linked to original sources
PubMed2004

Both BC-box motifs of adenovirus protein E4orf6 are required to efficiently assemble an E3 ligase complex that degrades p53.

Small DNA tumor viruses typically encode proteins that either inactivate or degrade p53. Human adenoviruses encode products, including E4orf6 and E1B55K, that do both. Each independently binds to p53 and inhibits its ability to activate gene expression; however, in combination they induce p53 degradation by the ubiquitin pathway. We have shown previously that p53 degradation relies on interactions of E4orf6 with the cellular proteins Cul5, Rbx1, and elongins B and C to form an E3 ligase similar to the SCF and VBC complexes. Here we show that, like other elongin BC-interacting proteins, including elongin A, von Hippel-Lindau protein, and Muf1, the interaction of E4orf6 is mediated by the BC-box motif; however, E4orf6 uniquely utilizes two BC-box motifs for degradation of p53 and another target, Mre11. In addition, our data suggest that the interaction of E1B55K with E4orf6 depends on the ability of E4orf6 to form the E3 ligase complex and that such complex formation may be required for all E4orf6-E1B55K functions.

Adenovirus E1B Proteins↗
PubMed2004

In vivo requirement of the RNA polymerase II elongation factor elongin A for proper gene expression and development.

A number of transcription factors that increase the catalytic rate of mRNA synthesis by RNA polymerase II (Pol II) have been purified from higher eukaryotes. Among these are the ELL family, DSIF, and the heterotrimeric elongin complex. Elongin A, the largest subunit of the elongin complex, is the transcriptionally active subunit, while the smaller elongin B and C subunits appear to act as regulatory subunits. While much is known about the in vitro properties of elongin A and other members of this class of elongation factors, the physiological role(s) of these proteins remain largely unclear. To elucidate in vivo functions of elongin A, we have characterized its Drosophila homologue (dEloA). dEloA associates with transcriptionally active puff sites within Drosophila polytene chromosomes and exhibits many of the expected biochemical and cytological properties consistent with a Pol II-associated elongation factor. RNA interference-mediated depletion of dEloA demonstrated that elongin A is an essential factor that is required for proper metamorphosis. Consistent with this observation, dEloA expression peaks during the larval stages of development, suggesting that this factor may be important for proper regulation of developmental events during these stages. The discovery of the role of elongin A in an in vivo model system defines the novel contribution played by RNA polymerase II elongation machinery in regulation of gene expression that is required for proper development.

Amino Acid Sequence↗
PubMed2003

A mammalian mediator subunit that shares properties with Saccharomyces cerevisiae mediator subunit Cse2.

The multiprotein Mediator complex is a coactivator required for activation of RNA polymerase II transcription by DNA bound transcription factors. We previously identified and partially purified a mammalian Mediator complex from rat liver nuclei (Brower, C.S., Sato, S., Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner, R.D., Malik, S., Lane, W.S., Sorokina, I., Roeder, R.G., Conaway, J.W., and Conaway, R.C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10353-10358). Analysis by tandem mass spectrometry of proteins present in the most highly purified rat Mediator fractions led to the identification of a collection of new mammalian Mediator subunits, as well as several potential Mediator subunits including a previously uncharacterized protein encoded by the FLJ10193 open reading frame. In this study, we present direct biochemical evidence that the FLJ10193 protein, which we designate Med25, is a bona fide subunit of the mammalian Mediator complex. In addition, we present evidence that Med25 shares structural and functional properties with Saccharomyces cerevisiae Mediator subunit Cse2 and may be a mammalian Cse2 ortholog. Taken together, our findings identify a novel mammalian Mediator subunit and shed new light on the architecture of the mammalian Mediator complex.

Amino Acid Sequence↗
PubMed2003

A mammalian homolog of Drosophila melanogaster transcriptional coactivator intersex is a subunit of the mammalian Mediator complex.

The multiprotein Mediator complex is a coactivator required for transcriptional activation of RNA polymerase II transcribed genes by DNA binding transcription factors. We previously partially purified a Med8-containing Mediator complex from rat liver nuclei (Brower, C. S., Sato, S., Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner, R. D., Malik, S., Lane, W. S., Sorokina, I., Roeder, R. G., Conaway, J. W., and Conaway, R. C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10353-10358). Analysis of proteins present in the most highly enriched Mediator fractions by tandem mass spectrometry led to the identification of several new mammalian Mediator subunits, as well as several potential Mediator subunits. Here we identify one of these proteins, encoded by the previously uncharacterized AK000411 open reading frame, as a new subunit of the mammalian Mediator complex. The AK000411 protein, which we designate hIntersex (human Intersex), shares significant sequence similarity with the Drosophila melanogaster intersex protein, which has functional properties expected of a transcriptional coactivator specific for the Drosophila doublesex transactivator. In addition, we show that hIntersex assembles into a subcomplex with Mediator subunits p28b and TRFP. Taken together, our findings identify a new subunit of the mammalian Mediator and shed new light on the architecture of the mammalian Mediator complex.

Amino Acid Sequence↗
PubMed2003

Identification of new subunits of the multiprotein mammalian TRRAP/TIP60-containing histone acetyltransferase complex.

The mammalian ATM/PI 3-kinase-related TRRAP protein was previously found to be a component of a multi-protein histone acetyltransferase (HAT) complex containing the HAT TIP60. In this report, we identify a previously uncharacterized protein encoded by the FLJ10914 ORF, which we designate MRGBP, as a new component of the TRRAP/TIP60 HAT complex. In addition, through purification of MRGBP and its associated proteins from HeLa cell nuclear extracts, we identify the thyroid receptor coactivating protein (TRCp120), DMAP1, and the related MRG15 and MRGX proteins as MRGBP-associating proteins, and we present biochemical evidence that they are previously unrecognized components of the TRRAP/TIP60 HAT complex. Taken together, our findings shed new light on the structure and function of the mammalian TRRAP/TIP60 histone acetyltransferase complex.

Acetyltransferases↗
PubMed2003

The RNA polymerase II elongation complex.

Synthesis of eukaryotic mRNA by RNA polymerase II is an elaborate biochemical process that requires the concerted action of a large set of transcription factors. RNA polymerase II transcription proceeds through multiple stages designated preinitiation, initiation, and elongation. Historically, studies of the elongation stage of eukaryotic mRNA synthesis have lagged behind studies of the preinitiation and initiation stages; however, in recent years, efforts to elucidate the mechanisms governing elongation have led to the discovery of a diverse collection of transcription factors that directly regulate the activity of elongating RNA polymerase II. Moreover, these studies have revealed unanticipated roles for the RNA polymerase II elongation complex in such processes as DNA repair and recombination and the proper processing and nucleocytoplasmic transport of mRNA. Below we describe these recent advances, which highlight the important role of the RNA polymerase II elongation complex in regulation of eukaryotic gene expression.

Chromatin↗
PubMed2003

von Hippel-Lindau protein binds hyperphosphorylated large subunit of RNA polymerase II through a proline hydroxylation motif and targets it for ubiquitination.

The transition from transcription initiation to elongation involves phosphorylation of the large subunit (Rpb1) of RNA polymerase II on the repetitive carboxyl-terminal domain. The elongating hyperphosphorylated Rpb1 is subject to ubiquitination, particularly in response to UV radiation and DNA-damaging agents. By using computer modeling, we identified regions of Rpb1 and the adjacent subunit 6 of RNA polymerase II (Rpb6) that share sequence and structural similarity with the domain of hypoxia-inducible transcription factor 1 alpha (HIF-1 alpha) that binds von Hippel-Lindau tumor suppressor protein (pVHL). pVHL confers substrate specificity to the E3 ligase complex, which ubiquitinates HIF-alpha and targets it for proteasomal degradation. In agreement with the computational model, we show biochemical evidence that pVHL specifically binds the hyperphosphorylated Rpb1 in a proline-hydroxylation-dependent manner, targeting it for ubiquitination. This interaction is regulated by UV radiation.

Amino Acid Motifs↗
PubMed2003

Identification of mammalian Mediator subunits with similarities to yeast Mediator subunits Srb5, Srb6, Med11, and Rox3.

The Mediator is a multiprotein coactivator required for activation of RNA polymerase II transcription by DNA binding transactivators. We recently identified a mammalian homologue of yeast Mediator subunit Med8 and partially purified a Med8-containing Mediator complex from rat liver nuclei (Brower, C. S., Sato, S., Tomomori-Sato, C., Kamura, T., Pause, A., Stearman, R., Klausner, R. D., Malik, S., Lane, W. S., Sorokina, I., Roeder, R. G., Conaway, J. W., and Conaway, R. C. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 10353-10358). Analysis of proteins present in the most highly purified Med8-containing fractions by tandem mass spectrometry led to the identification of many known mammalian Mediator subunits, as well as four potential Mediator subunits exhibiting sequence similarity to yeast Mediator subunits Srb5, Srb6, Med11, and Rox3. Here we present direct biochemical evidence that these four proteins are bona fide mammalian Mediator subunits. In addition, we identify direct pairwise binding partners of these proteins among the known mammalian Mediator subunits. Taken together, our findings identify a collection of novel mammalian Mediator subunits and shed new light on the underlying architecture of the mammalian Mediator complex.

Animals↗
PubMed2003

Multiple splice variants of the human HIF-3 alpha locus are targets of the von Hippel-Lindau E3 ubiquitin ligase complex.

Functional inactivation of the von Hippel-Lindau (VHL) tumor suppressor protein is the cause of familial VHL disease and sporadic kidney cancer. The VHL gene product (pVHL) is a component of an E3 ubiquitin ligase complex that targets the hypoxia-inducible factor (HIF) 1 and 2 alpha subunits for polyubiquitylation. This process is dependent on the hydroxylation of conserved proline residues on the alpha subunits of HIF-1/2 in the presence of oxygen. In our effort to identify orphan HIF-like proteins in the data base that are potential targets of the pVHL complex, we report multiple splice variants of the human HIF-3 alpha locus as follows: hHIF-3 alpha 1, hHIF-3 alpha 2 (also referred to as hIPAS; human inhibitory PAS domain protein), hHIF-3 alpha 3, hHIF-3 alpha 4, hHIF-3 alpha 5, and hHIF-3 alpha 6. We demonstrate that the common oxygen-dependent degradation domain of hHIF-3 alpha 1-3 splice variants is targeted for ubiquitylation by the pVHL complex in vitro and in vivo. This activity is enhanced in the presence of prolyl hydroxylase and is dependent on a proline residue at position 490. Furthermore, the ubiquitin conjugation occurs on lysine residues at position 465 and 568 within the oxygen-dependent degradation domain. These results demonstrate additional targets of the pVHL complex and suggest a growing complexity in the regulation of hypoxia-inducible genes by the HIF family of transcription factors.

Alternative Splicing↗
PubMed2002

Biochemical purification and pharmacological inhibition of a mammalian prolyl hydroxylase acting on hypoxia-inducible factor.

The product of the von Hippel-Lindau gene, pVHL, targets the alpha subunits of the heterodimeric transcription factor hypoxia-inducible factor (HIF) for polyubiquitination in the presence of oxygen. The binding of pVHL to HIF is governed by the enzymatic hydroxylation of conserved prolyl residues within peptidic motifs present in the HIFalpha family members. By using a biochemical purification strategy, we have identified a human homolog of Caenorhabditis elegans Egl9 as a HIF prolyl hydroxylase. In addition, we studied the activity of a structurally diverse collection of low molecular weight inhibitors of procollagen prolyl 4-hydroxylase as potential inhibitors of the HIF hydroxylase. A model compound of this series stabilized HIF in a variety of cells, leading to the increased production of its downstream target, vascular endothelial growth factor.

Amino Acid Sequence↗
PubMed2002

Mammalian mediator subunit mMED8 is an Elongin BC-interacting protein that can assemble with Cul2 and Rbx1 to reconstitute a ubiquitin ligase.

The heterodimeric Elongin BC complex has been shown to interact in vitro and in cells with a conserved BC-box motif found in an increasing number of proteins including RNA polymerase II elongation factor Elongin A, suppressor of cytokine signaling (SOCS)-box proteins, and the von Hippel-Lindau tumor suppressor protein. Recently, the Elongin BC complex was found to function as an adaptor that links these BC-box proteins to a module composed of Cullin family members Cul2 or Cul5 and RING-H2 finger protein Rbx1 to reconstitute a family of E3 ubiquitin ligases that activate ubiquitylation by the E2 ubiquitin-conjugating enzyme Ubc5. As part of our effort to understand the functions of Elongin BC-based ubiquitin ligases, we exploited a modified yeast two-hybrid screen to identify a mammalian BC-box protein similar in sequence to Saccharomyces cerevisiae Mediator subunit Med8p. In this report we demonstrate (i) that mammalian MED8 is a subunit of the mammalian Mediator complex and (ii) that MED8 can assemble with Elongins B and C, Cul2, and Rbx1 to reconstitute a ubiquitin ligase. Taken together, our findings are consistent with the model that MED8 could function to recruit ubiquitin ligase activity directly to the RNA polymerase II transcriptional machinery.

Amino Acid Sequence↗
PubMed2002

A molecular basis for stabilization of the von Hippel-Lindau (VHL) tumor suppressor protein by components of the VHL ubiquitin ligase.

The multiprotein von Hippel-Lindau (VHL) tumor suppressor (CBC(VHL), Cul2-Elongin BC-VHL) and SCF (Skp1-Cul1/Cdc53-F-box protein) complexes are members of structurally related families of E3 ubiquitin ligases that use a heterodimeric module composed of a member of the Cullin protein family and the RING finger protein Rbx1 (ROC1/Hrt1) to activate ubiquitylation of target proteins by the E2 ubiquitin-conjugating enzymes Ubc5 and Cdc34. VHL and F-box proteins function as the substrate recruitment subunits of CBC(VHL) and SCF complexes, respectively. In cells, many F-box proteins are short lived and are proposed to be ubiquitylated by an autocatalytic mechanism and destroyed by the proteasome following assembly into SCF complexes. In contrast, the VHL protein is stabilized by interaction with the Elongin B and C subunits of CBC(VHL) in cells. In this report, we have presented direct biochemical evidence that unlike the F-box protein Cdc4, which is ubiquitylated in vitro by Cdc34 in the context of the SCF, the VHL protein is protected from Ubc5-catalyzed ubiquitylation following assembly into the CBC(VHL) complex. CBC(VHL) is continuously required for negative regulation of hypoxia-inducible transcription factors in normoxic cells and of SCF complexes, many of which function only transiently during the cell cycle or in response to cellular signals. Our findings provide a molecular basis for the different modes of cellular regulation of VHL and F-box proteins and are consistent with the known roles of CBC(VHL).

Animals↗
PubMed2002

Emerging roles of ubiquitin in transcription regulation.

Ubiquitin is a small protein that was initially found to function as a tag that can be covalently attached to proteins to mark them for destruction by a multisubunit, adenosine 5'-triphosphate-dependent protease called the proteasome. Ubiquitin is now emerging as a key regulator of eukaryotic messenger RNA synthesis, a process that depends on the RNA synthetic enzyme RNA polymerase II and the transcription factors that control its activity. Ubiquitin controls messenger RNA synthesis not only by mechanisms involving ubiquitin-dependent destruction of transcription factors by the proteasome, but also by an intriguing collection of previously unknown and unanticipated mechanisms that appear to be independent of the proteasome.

Animals↗
PubMed2002

Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex.

SCF complexes are the largest family of E3 ubiquitin-protein ligases and mediate the ubiquitination of diverse regulatory and signalling proteins. Here we present the crystal structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF complex, which shows that Cul1 is an elongated protein that consists of a long stalk and a globular domain. The globular domain binds the RING finger protein Rbx1 through an intermolecular beta-sheet, forming a two-subunit catalytic core that recruits the ubiquitin-conjugating enzyme. The long stalk, which consists of three repeats of a novel five-helix motif, binds the Skp1-F boxSkp2 protein substrate-recognition complex at its tip. Cul1 serves as a rigid scaffold that organizes the Skp1-F boxSkp2 and Rbx1 subunits, holding them over 100 A apart. The structure suggests that Cul1 may contribute to catalysis through the positioning of the substrate and the ubiquitin-conjugating enzyme, and this model is supported by Cul1 mutations designed to eliminate the rigidity of the scaffold.

Amino Acid Motifs↗