The Experts below are selected from a list of 1992 Experts worldwide ranked by ideXlab platform
Joan W. Conaway - One of the best experts on this subject based on the ideXlab platform.
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Imaging-based assays for investigating functions of the RNA polymerase II elongation factor Elongin and the Elongin ubiquitin ligase.
Methods (San Diego Calif.), 2019Co-Authors: Juston C. Weems, Ronald C. Conaway, Jay R. Unruh, Brian D. Slaughter, Joan W. ConawayAbstract:Elongin A binds to Elongins B and C to form the RNA polymerase II transcription elongation factor Elongin. It also functions as the substrate recognition subunit of a ubiquitin ligase that is formed by binding of Elongin to Cullin protein CUL5 and RING finger protein RBX2 and that targets RNA polymerase II for ubiquitination. In this article, we describe use of acceptor photobleaching fluorescence resonance energy transfer (AP-FRET) and laser microirradiation-based assays to study regulated assembly of the Elongin ubiquitin ligase and its recruitment to regions of localized DNA damage.
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cockayne syndrome b protein regulates recruitment of the Elongin a ubiquitin ligase to sites of dna damage
Journal of Biological Chemistry, 2017Co-Authors: Juston C. Weems, Jay R. Unruh, Brian D. Slaughter, Teijiro Aso, Takashi Yasukawa, Shawn M. Hall, Merry B. Mclaird, Stefan Boeing, Jesper Q Svejstrup, Joan W. ConawayAbstract:Elongin A performs dual functions as the transcriptionally active subunit of RNA polymerase II (Pol II) elongation factor Elongin and as the substrate recognition subunit of a Cullin-RING E3 ubiquitin ligase that ubiquitylates Pol II in response to DNA damage. Assembly of the Elongin A ubiquitin ligase and its recruitment to sites of DNA damage is a tightly regulated process induced by DNA-damaging agents and α-amanitin, a drug that induces Pol II stalling. In this study, we demonstrate (i) that Elongin A and the ubiquitin ligase subunit CUL5 associate in cells with the Cockayne syndrome B (CSB) protein and (ii) that this interaction is also induced by DNA-damaging agents and α-amanitin. In addition, we present evidence that the CSB protein promotes stable recruitment of the Elongin A ubiquitin ligase to sites of DNA damage. Our findings are consistent with the model that the Elongin A ubiquitin ligase and the CSB protein function together in a common pathway in response to Pol II stalling and DNA damage.
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Transcriptional properties of mammalian Elongin A and its role in stress response
The Journal of biological chemistry, 2013Co-Authors: Junya Kawauchi, Ronald C. Conaway, Joan W. Conaway, Teijiro Aso, Takashi Yasukawa, Mizue Fukuda, Makoto Inoue, Yohei Uchida, Shigetaka KitajimaAbstract:Elongin A was shown previously to be capable of potently activating the rate of RNA polymerase II (RNAPII) transcription elongation in vitro by suppressing transient pausing by the enzyme at many sites along DNA templates. The role of Elongin A in RNAPII transcription in mammalian cells, however, has not been clearly established. In this report, we investigate the function of Elongin A in RNAPII transcription. We present evidence that Elongin A associates with the IIO form of RNAPII at sites of newly transcribed RNA and is relocated to dotlike domains distinct from those containing RNAPII when cells are treated with the kinase inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole. Significantly, Elongin A is required for maximal induction of transcription of the stress response genes ATF3 and p21 in response to several stimuli. Evidence from structure-function studies argues that Elongin A transcription elongation activity, but not its ubiquitination activity, is most important for its function in induction of transcription of ATF3 and p21. Taken together, our data provide new insights into the function of Elongin A in RNAPII transcription and bring to light a previously unrecognized role for Elongin A in the regulation of stress response genes.
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mammalian Elongin a complex mediates dna damage induced ubiquitylation and degradation of rpb1
The EMBO Journal, 2008Co-Authors: Takashi Yasukawa, Takumi Kamura, Ronald C. Conaway, Joan W. Conaway, Shigetaka Kitajima, Teijiro AsoAbstract:The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II (pol II) by suppressing transient pausing of the pol II at many sites along the DNA. Elongin is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, which can form an isolable Elongin BC subcomplex. Here, we have shown that both the ubiquitylation and proteasomal degradation of the largest subunit of pol II (Rpb1) following UV-irradiation are significantly suppressed in Elongin A-deficient cells; however, in both cases suppression is rescued by transfection of wild-type Elongin A. Moreover, we have demonstrated that the Elongin A–Elongin BC complex is capable of assembling with the Cul5/Rbx2 module, and that this hetero-pentamer complex efficiently ubiquitylates Rpb1 in vitro. Mechanistic studies indicate that colocalization of Elongin A and Cul5 in cells and the interaction of Elongin A with the Ser5-phosphorylated form of Rpb1 are strongly enhanced following UV-irradiation. Taken together, our results suggest that mammalian Elongin A is directly involved in ubiquitylation and degradation of Rpb1 following DNA damage.
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Mammalian Elongin A complex mediates DNA‐damage‐induced ubiquitylation and degradation of Rpb1
The EMBO journal, 2008Co-Authors: Takashi Yasukawa, Takumi Kamura, Ronald C. Conaway, Joan W. Conaway, Shigetaka Kitajima, Teijiro AsoAbstract:The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II (pol II) by suppressing transient pausing of the pol II at many sites along the DNA. Elongin is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, which can form an isolable Elongin BC subcomplex. Here, we have shown that both the ubiquitylation and proteasomal degradation of the largest subunit of pol II (Rpb1) following UV-irradiation are significantly suppressed in Elongin A-deficient cells; however, in both cases suppression is rescued by transfection of wild-type Elongin A. Moreover, we have demonstrated that the Elongin A–Elongin BC complex is capable of assembling with the Cul5/Rbx2 module, and that this hetero-pentamer complex efficiently ubiquitylates Rpb1 in vitro. Mechanistic studies indicate that colocalization of Elongin A and Cul5 in cells and the interaction of Elongin A with the Ser5-phosphorylated form of Rpb1 are strongly enhanced following UV-irradiation. Taken together, our results suggest that mammalian Elongin A is directly involved in ubiquitylation and degradation of Rpb1 following DNA damage.
Ronald C. Conaway - One of the best experts on this subject based on the ideXlab platform.
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Imaging-based assays for investigating functions of the RNA polymerase II elongation factor Elongin and the Elongin ubiquitin ligase.
Methods (San Diego Calif.), 2019Co-Authors: Juston C. Weems, Ronald C. Conaway, Jay R. Unruh, Brian D. Slaughter, Joan W. ConawayAbstract:Elongin A binds to Elongins B and C to form the RNA polymerase II transcription elongation factor Elongin. It also functions as the substrate recognition subunit of a ubiquitin ligase that is formed by binding of Elongin to Cullin protein CUL5 and RING finger protein RBX2 and that targets RNA polymerase II for ubiquitination. In this article, we describe use of acceptor photobleaching fluorescence resonance energy transfer (AP-FRET) and laser microirradiation-based assays to study regulated assembly of the Elongin ubiquitin ligase and its recruitment to regions of localized DNA damage.
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Transcriptional properties of mammalian Elongin A and its role in stress response
The Journal of biological chemistry, 2013Co-Authors: Junya Kawauchi, Ronald C. Conaway, Joan W. Conaway, Teijiro Aso, Takashi Yasukawa, Mizue Fukuda, Makoto Inoue, Yohei Uchida, Shigetaka KitajimaAbstract:Elongin A was shown previously to be capable of potently activating the rate of RNA polymerase II (RNAPII) transcription elongation in vitro by suppressing transient pausing by the enzyme at many sites along DNA templates. The role of Elongin A in RNAPII transcription in mammalian cells, however, has not been clearly established. In this report, we investigate the function of Elongin A in RNAPII transcription. We present evidence that Elongin A associates with the IIO form of RNAPII at sites of newly transcribed RNA and is relocated to dotlike domains distinct from those containing RNAPII when cells are treated with the kinase inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole. Significantly, Elongin A is required for maximal induction of transcription of the stress response genes ATF3 and p21 in response to several stimuli. Evidence from structure-function studies argues that Elongin A transcription elongation activity, but not its ubiquitination activity, is most important for its function in induction of transcription of ATF3 and p21. Taken together, our data provide new insights into the function of Elongin A in RNAPII transcription and bring to light a previously unrecognized role for Elongin A in the regulation of stress response genes.
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mammalian Elongin a complex mediates dna damage induced ubiquitylation and degradation of rpb1
The EMBO Journal, 2008Co-Authors: Takashi Yasukawa, Takumi Kamura, Ronald C. Conaway, Joan W. Conaway, Shigetaka Kitajima, Teijiro AsoAbstract:The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II (pol II) by suppressing transient pausing of the pol II at many sites along the DNA. Elongin is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, which can form an isolable Elongin BC subcomplex. Here, we have shown that both the ubiquitylation and proteasomal degradation of the largest subunit of pol II (Rpb1) following UV-irradiation are significantly suppressed in Elongin A-deficient cells; however, in both cases suppression is rescued by transfection of wild-type Elongin A. Moreover, we have demonstrated that the Elongin A–Elongin BC complex is capable of assembling with the Cul5/Rbx2 module, and that this hetero-pentamer complex efficiently ubiquitylates Rpb1 in vitro. Mechanistic studies indicate that colocalization of Elongin A and Cul5 in cells and the interaction of Elongin A with the Ser5-phosphorylated form of Rpb1 are strongly enhanced following UV-irradiation. Taken together, our results suggest that mammalian Elongin A is directly involved in ubiquitylation and degradation of Rpb1 following DNA damage.
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Mammalian Elongin A complex mediates DNA‐damage‐induced ubiquitylation and degradation of Rpb1
The EMBO journal, 2008Co-Authors: Takashi Yasukawa, Takumi Kamura, Ronald C. Conaway, Joan W. Conaway, Shigetaka Kitajima, Teijiro AsoAbstract:The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II (pol II) by suppressing transient pausing of the pol II at many sites along the DNA. Elongin is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, which can form an isolable Elongin BC subcomplex. Here, we have shown that both the ubiquitylation and proteasomal degradation of the largest subunit of pol II (Rpb1) following UV-irradiation are significantly suppressed in Elongin A-deficient cells; however, in both cases suppression is rescued by transfection of wild-type Elongin A. Moreover, we have demonstrated that the Elongin A–Elongin BC complex is capable of assembling with the Cul5/Rbx2 module, and that this hetero-pentamer complex efficiently ubiquitylates Rpb1 in vitro. Mechanistic studies indicate that colocalization of Elongin A and Cul5 in cells and the interaction of Elongin A with the Ser5-phosphorylated form of Rpb1 are strongly enhanced following UV-irradiation. Taken together, our results suggest that mammalian Elongin A is directly involved in ubiquitylation and degradation of Rpb1 following DNA damage.
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Characterization of Cullin-box sequences that direct recruitment of Cul2-Rbx1 and Cul5-Rbx2 modules to Elongin BC-based ubiquitin ligases.
The Journal of biological chemistry, 2008Co-Authors: Nawel Mahrour, Ronald C. Conaway, Michael P. Washburn, Laurence Florens, William B. Redwine, Selene K. Swanson, Skylar Martin-brown, William D. Bradford, Karen Staehling-hampton, Joan W. ConawayAbstract:The Elongin BC-box protein family includes the von Hippel-Lindau tumor suppressor and suppressor of cytokine signaling proteins, which are substrate recognition subunits of structurally related classes of E3 ubiquitin ligases composed of Elongin C-Elongin B-Cullin 2-Rbx1 (Cul2 ubiquitin ligases) or of Elongin C-Elongin B-Cullin 5-Rbx2 (Cul5 ubiquitin ligases). The Elongin BC complex acts as an adaptor that links a substrate recognition subunit to heterodimers of either Cullin 2 (Cul2) and RING finger protein Rbx1 or Cullin 5 (Cul5) and Rbx2. It has been shown ( Kamura, T., Maenaka, K., Kotoshiba, S., Matsumoto, M., Kohda, D., Conaway, R. C., Conaway, J. W., and Nakayama, K. I. (2004) Genes Dev. 18, 3055-3065 ) that interaction of BC-box proteins with their cognate Cul-Rbx module is determined by specific regions, called Cul2- or Cul5-boxes, located immediately downstream of their BC-boxes. Here, we investigate further the mechanisms governing assembly of BC-box proteins with their specific Cul-Rbx modules. Through purification and characterization of a larger collection of BC-box proteins that serve as substrate recognition subunits of Cul2 and Cul5 ubiquitin ligases and through structure-function studies, we define Cul2- and Cul5-boxes in greater detail. Although it previously appeared that there was little sequence similarity between Cul5- and Cul2-box motifs, analyses of newly identified BC-box proteins reveal that residues conserved in the Cul2-box represent a subset of those conserved in the Cul5-box. The sequence motif LPPhiP, which is conserved in most Cul5-boxes and has been suggested to specify assembly of Cul5 ligases, is compatible with Cul2 interaction. Finally, the spacing between BC- and Cullin-boxes is much more flexible than has been appreciated and can vary from as few as 3 and as many as approximately 80 amino acids. Taken together, our findings shed new light on the mechanisms by which BC-box proteins direct recruitment of Cullin-Rbx modules during reconstitution of ubiquitin ligases.
Teijiro Aso - One of the best experts on this subject based on the ideXlab platform.
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cockayne syndrome b protein regulates recruitment of the Elongin a ubiquitin ligase to sites of dna damage
Journal of Biological Chemistry, 2017Co-Authors: Juston C. Weems, Jay R. Unruh, Brian D. Slaughter, Teijiro Aso, Takashi Yasukawa, Shawn M. Hall, Merry B. Mclaird, Stefan Boeing, Jesper Q Svejstrup, Joan W. ConawayAbstract:Elongin A performs dual functions as the transcriptionally active subunit of RNA polymerase II (Pol II) elongation factor Elongin and as the substrate recognition subunit of a Cullin-RING E3 ubiquitin ligase that ubiquitylates Pol II in response to DNA damage. Assembly of the Elongin A ubiquitin ligase and its recruitment to sites of DNA damage is a tightly regulated process induced by DNA-damaging agents and α-amanitin, a drug that induces Pol II stalling. In this study, we demonstrate (i) that Elongin A and the ubiquitin ligase subunit CUL5 associate in cells with the Cockayne syndrome B (CSB) protein and (ii) that this interaction is also induced by DNA-damaging agents and α-amanitin. In addition, we present evidence that the CSB protein promotes stable recruitment of the Elongin A ubiquitin ligase to sites of DNA damage. Our findings are consistent with the model that the Elongin A ubiquitin ligase and the CSB protein function together in a common pathway in response to Pol II stalling and DNA damage.
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6 21. Mammalian Elongin A complex mediates DNA-damage-induced ubiquitylation and degradation of Rpb1
2015Co-Authors: Takashi Yasukawa, Takumi Kamura, Shigetaka Kitajima, Aya Tsutsui, Yasufumi Kondo, Teijiro AsoAbstract:The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II (pol II) by suppressing transient pausing of the pol II at many sites along the DNA. Elongin is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, which bind stably to each other to form a binary complex that interacts with Elongin A and strongly induces its transcriptional activity. We have recently shown that Elongin A homozygous mutant (Elongin A-/-) mice are embryonic lethal, and primary fibroblasts derived from Elongin A-/- embryos display not only increased apoptosis but also senescence-like phenotypes accompanied by the activation of p38 mitogen-activated protein kinase and p53. In this report, we have shown that both the ubiquitylation and proteasomal degradation of the largest subunit of pol II (Rpb1) following UV-irradiation are significantly suppressed in Elongin A-deficient cells, however those suppressions are rescued by transfection of wild-type Elongin A. Moreover, using recombinant proteins expressed in insect cells, we have demonstrated that the Elongin A-Elongin BC complex is capable of assembling with the Cul5/Rbx2 module, and this hetero-pentamer complex efficiently ubiquitylates Rpb1 in vitro. Mechanistic studies indicate that colocalization of Elongin A and Cul5 in cells and the interaction of Elongin A with the Ser5-phosphorylated form of Rpb1 are strongly enhanced followin
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Assembly of the Elongin A Ubiquitin Ligase Is Regulated by Genotoxic and Other Stresses
The Journal of biological chemistry, 2015Co-Authors: Juston C. Weems, Jay R. Unruh, Brian D. Slaughter, Takashi Yasukawa, Shawn M. Hall, Merry B. Mclaird, Joshua M. Gilmore, Michael P. Washburn, Laurence Florens, Teijiro AsoAbstract:Elongin A performs dual functions in cells as a component of RNA polymerase II (Pol II) transcription elongation factor Elongin and as the substrate recognition subunit of a Cullin-RING E3 ubiquitin ligase that has been shown to target Pol II stalled at sites of DNA damage. Here we investigate the mechanism(s) governing conversion of the Elongin complex from its elongation factor to its ubiquitin ligase form. We report the discovery that assembly of the Elongin A ubiquitin ligase is a tightly regulated process. In unstressed cells, Elongin A is predominately present as part of Pol II elongation factor Elongin. Assembly of Elongin A into the ubiquitin ligase is strongly induced by genotoxic stress; by transcriptional stresses that lead to accumulation of stalled Pol II; and by other stimuli, including endoplasmic reticulum and nutrient stress and retinoic acid signaling, that activate Elongin A-dependent transcription. Taken together, our findings shed new light on mechanisms that control the Elongin A ubiquitin ligase and suggest that it may play a role in Elongin A-dependent transcription.
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Transcriptional properties of mammalian Elongin A and its role in stress response
The Journal of biological chemistry, 2013Co-Authors: Junya Kawauchi, Ronald C. Conaway, Joan W. Conaway, Teijiro Aso, Takashi Yasukawa, Mizue Fukuda, Makoto Inoue, Yohei Uchida, Shigetaka KitajimaAbstract:Elongin A was shown previously to be capable of potently activating the rate of RNA polymerase II (RNAPII) transcription elongation in vitro by suppressing transient pausing by the enzyme at many sites along DNA templates. The role of Elongin A in RNAPII transcription in mammalian cells, however, has not been clearly established. In this report, we investigate the function of Elongin A in RNAPII transcription. We present evidence that Elongin A associates with the IIO form of RNAPII at sites of newly transcribed RNA and is relocated to dotlike domains distinct from those containing RNAPII when cells are treated with the kinase inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole. Significantly, Elongin A is required for maximal induction of transcription of the stress response genes ATF3 and p21 in response to several stimuli. Evidence from structure-function studies argues that Elongin A transcription elongation activity, but not its ubiquitination activity, is most important for its function in induction of transcription of ATF3 and p21. Taken together, our data provide new insights into the function of Elongin A in RNAPII transcription and bring to light a previously unrecognized role for Elongin A in the regulation of stress response genes.
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mammalian Elongin a complex mediates dna damage induced ubiquitylation and degradation of rpb1
The EMBO Journal, 2008Co-Authors: Takashi Yasukawa, Takumi Kamura, Ronald C. Conaway, Joan W. Conaway, Shigetaka Kitajima, Teijiro AsoAbstract:The Elongin complex stimulates the rate of transcription elongation by RNA polymerase II (pol II) by suppressing transient pausing of the pol II at many sites along the DNA. Elongin is composed of a transcriptionally active A subunit and two small regulatory B and C subunits, which can form an isolable Elongin BC subcomplex. Here, we have shown that both the ubiquitylation and proteasomal degradation of the largest subunit of pol II (Rpb1) following UV-irradiation are significantly suppressed in Elongin A-deficient cells; however, in both cases suppression is rescued by transfection of wild-type Elongin A. Moreover, we have demonstrated that the Elongin A–Elongin BC complex is capable of assembling with the Cul5/Rbx2 module, and that this hetero-pentamer complex efficiently ubiquitylates Rpb1 in vitro. Mechanistic studies indicate that colocalization of Elongin A and Cul5 in cells and the interaction of Elongin A with the Ser5-phosphorylated form of Rpb1 are strongly enhanced following UV-irradiation. Taken together, our results suggest that mammalian Elongin A is directly involved in ubiquitylation and degradation of Rpb1 following DNA damage.
Arnim Pause - One of the best experts on this subject based on the ideXlab platform.
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Mammalian mediator subunit mMED8 is an Elongin BC-interacting protein that can assemble with Cul2 and Rbx1 to reconstitute a ubiquitin ligase
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Christopher S. Brower, Takumi Kamura, Shigeo Sato, Arnim Pause, Robert Stearman, Richard D. Klausner, Chieri Tomomori-sato, Sohail Malik, William S. Lane, Irina SorokinaAbstract: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.
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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, Ronald C. Conaway, 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 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, Ronald C. Conaway, Joan W. 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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Studying interactions of four proteins in the yeast two-hybrid system: Structural resemblance of the pVHL/Elongin BC/hCUL-2 complex with the ubiquitin ligase complex SKP1/cullin/F-box protein
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Arnim Pause, Brett Peterson, Gregor Schaffar, Robert Stearman, Richard D. KlausnerAbstract:The yeast two-hybrid system is a powerful technique that detects interactions between two proteins and has been useful in identifying new binding partners. However, the system fails to detect protein–protein interactions that require the presence of additional components of a multisubunit complex. Here we demonstrate that the vector YIpDCE1 can be used to express Elongins B and C in yeast, and that these proteins form a stable complex that interacts with the von Hippel–Lindau tumor-suppressor gene product (pVHL). Only when pVHL and Elongins B and C (VBC) are present does an interaction with the cullin family member, hCUL-2, occur, forming the heterotetrameric pVHL/Elongin BC/hCUL-2 complex. This system was then used to map the binding region of hCUL-2 for the VBC complex. The first amino-terminal 108 aa of hCUL-2 are necessary for interaction with the VBC complex. The Elongin BC dimer acts as a bridge between pVHL and hCUL-2 because pVHL and hCUL-2 can form distinct complexes with Elongins B and C. These results reveal a striking structural resemblance of pVHL/Elongin BC/hCUL-2 complex with the E3-like ubiquitin ligase complex SKP1/Cullin/F-box protein with respect to protein composition and sites of interactions. Thus, it seems possible that pVHL/Elongin BC/hCUL-2 complex will possess ubiquitin ligase activity targeting specific proteins for degradation by the proteasome.
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studying interactions of four proteins in the yeast two hybrid system structural resemblance of the pvhl Elongin bc hcul 2 complex with the ubiquitin ligase complex skp1 cullin f box protein
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Arnim Pause, Brett Peterson, Gregor Schaffar, Robert Stearman, Richard D. KlausnerAbstract:The yeast two-hybrid system is a powerful technique that detects interactions between two proteins and has been useful in identifying new binding partners. However, the system fails to detect protein–protein interactions that require the presence of additional components of a multisubunit complex. Here we demonstrate that the vector YIpDCE1 can be used to express Elongins B and C in yeast, and that these proteins form a stable complex that interacts with the von Hippel–Lindau tumor-suppressor gene product (pVHL). Only when pVHL and Elongins B and C (VBC) are present does an interaction with the cullin family member, hCUL-2, occur, forming the heterotetrameric pVHL/Elongin BC/hCUL-2 complex. This system was then used to map the binding region of hCUL-2 for the VBC complex. The first amino-terminal 108 aa of hCUL-2 are necessary for interaction with the VBC complex. The Elongin BC dimer acts as a bridge between pVHL and hCUL-2 because pVHL and hCUL-2 can form distinct complexes with Elongins B and C. These results reveal a striking structural resemblance of pVHL/Elongin BC/hCUL-2 complex with the E3-like ubiquitin ligase complex SKP1/Cullin/F-box protein with respect to protein composition and sites of interactions. Thus, it seems possible that pVHL/Elongin BC/hCUL-2 complex will possess ubiquitin ligase activity targeting specific proteins for degradation by the proteasome.
Richard D. Klausner - One of the best experts on this subject based on the ideXlab platform.
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Mammalian mediator subunit mMED8 is an Elongin BC-interacting protein that can assemble with Cul2 and Rbx1 to reconstitute a ubiquitin ligase
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Christopher S. Brower, Takumi Kamura, Shigeo Sato, Arnim Pause, Robert Stearman, Richard D. Klausner, Chieri Tomomori-sato, Sohail Malik, William S. Lane, Irina SorokinaAbstract: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.
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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, Ronald C. Conaway, 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 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, Ronald C. Conaway, Joan W. 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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Studying interactions of four proteins in the yeast two-hybrid system: Structural resemblance of the pVHL/Elongin BC/hCUL-2 complex with the ubiquitin ligase complex SKP1/cullin/F-box protein
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Arnim Pause, Brett Peterson, Gregor Schaffar, Robert Stearman, Richard D. KlausnerAbstract:The yeast two-hybrid system is a powerful technique that detects interactions between two proteins and has been useful in identifying new binding partners. However, the system fails to detect protein–protein interactions that require the presence of additional components of a multisubunit complex. Here we demonstrate that the vector YIpDCE1 can be used to express Elongins B and C in yeast, and that these proteins form a stable complex that interacts with the von Hippel–Lindau tumor-suppressor gene product (pVHL). Only when pVHL and Elongins B and C (VBC) are present does an interaction with the cullin family member, hCUL-2, occur, forming the heterotetrameric pVHL/Elongin BC/hCUL-2 complex. This system was then used to map the binding region of hCUL-2 for the VBC complex. The first amino-terminal 108 aa of hCUL-2 are necessary for interaction with the VBC complex. The Elongin BC dimer acts as a bridge between pVHL and hCUL-2 because pVHL and hCUL-2 can form distinct complexes with Elongins B and C. These results reveal a striking structural resemblance of pVHL/Elongin BC/hCUL-2 complex with the E3-like ubiquitin ligase complex SKP1/Cullin/F-box protein with respect to protein composition and sites of interactions. Thus, it seems possible that pVHL/Elongin BC/hCUL-2 complex will possess ubiquitin ligase activity targeting specific proteins for degradation by the proteasome.
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studying interactions of four proteins in the yeast two hybrid system structural resemblance of the pvhl Elongin bc hcul 2 complex with the ubiquitin ligase complex skp1 cullin f box protein
Proceedings of the National Academy of Sciences of the United States of America, 1999Co-Authors: Arnim Pause, Brett Peterson, Gregor Schaffar, Robert Stearman, Richard D. KlausnerAbstract:The yeast two-hybrid system is a powerful technique that detects interactions between two proteins and has been useful in identifying new binding partners. However, the system fails to detect protein–protein interactions that require the presence of additional components of a multisubunit complex. Here we demonstrate that the vector YIpDCE1 can be used to express Elongins B and C in yeast, and that these proteins form a stable complex that interacts with the von Hippel–Lindau tumor-suppressor gene product (pVHL). Only when pVHL and Elongins B and C (VBC) are present does an interaction with the cullin family member, hCUL-2, occur, forming the heterotetrameric pVHL/Elongin BC/hCUL-2 complex. This system was then used to map the binding region of hCUL-2 for the VBC complex. The first amino-terminal 108 aa of hCUL-2 are necessary for interaction with the VBC complex. The Elongin BC dimer acts as a bridge between pVHL and hCUL-2 because pVHL and hCUL-2 can form distinct complexes with Elongins B and C. These results reveal a striking structural resemblance of pVHL/Elongin BC/hCUL-2 complex with the E3-like ubiquitin ligase complex SKP1/Cullin/F-box protein with respect to protein composition and sites of interactions. Thus, it seems possible that pVHL/Elongin BC/hCUL-2 complex will possess ubiquitin ligase activity targeting specific proteins for degradation by the proteasome.