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Steven F Dowdy - One of the best experts on this subject based on the ideXlab platform.
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differential regulation of retinoblastoma Tumor Suppressor Protein by g1 cyclin dependent kinase complexes in vivo
Molecular and Cellular Biology, 2001Co-Authors: Sergei A Ezhevsky, Alan L Ho, Michelle Beckerhapak, Penny K Davis, Steven F DowdyAbstract:The retinoblastoma Tumor Suppressor Protein (pRB) negatively regulates early-G1 cell cycle progression, in part, by sequestering E2F transcription factors and repressing E2F-responsive genes. Although pRB is phosphorylated on up to 16 cyclin-dependent kinase (Cdk) sites by multiple G1 cyclin-Cdk complexes, the active form(s) of pRB in vivo remains unknown. pRB is present as an unphosphorylated Protein in G0 quiescent cells and becomes hypophosphorylated (;2 mol of PO4 to 1 mol of pRB) in early G1 and hyperphosphorylated (;10 mol of PO4 to 1 mol of pRB) in late G1 phase. Here, we report that hypophosphorylated pRB, present in early G1, represents the biologically active form of pRB in vivo that is assembled with E2Fs and E1A but that both unphosphorylated pRB in G0 and hyperphosphorylated pRB in late G1 fail to become assembled with E2Fs and E1A. Furthermore, using transducible dominant-negative TAT fusion Proteins that differentially target cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes, namely, TAT-p16 and TAT‐ dominant-negative Cdk2, respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G1 and that cyclin E-Cdk2 complexes inactivate pRB by hyperphosphorylation in late G1. Moreover, we found that cycling human Tumor cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion of the p16 INK4a gene, contained hypophosphorylated pRB that was bound to E2Fs in early G1 and that E2F-responsive genes, including those for dihydrofolate reductase and cyclin E, were transcriptionally repressed. Thus, we conclude that, physiologically, pRB is differentially regulated by G1 cyclin-Cdk complexes.
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Differential Regulation of Retinoblastoma Tumor Suppressor Protein by G1 Cyclin-Dependent Kinase Complexes In Vivo
Molecular and cellular biology, 2001Co-Authors: Sergei A Ezhevsky, Penny K Davis, Michelle Becker-hapak, Steven F DowdyAbstract:The retinoblastoma Tumor Suppressor Protein (pRB) negatively regulates early-G1 cell cycle progression, in part, by sequestering E2F transcription factors and repressing E2F-responsive genes. Although pRB is phosphorylated on up to 16 cyclin-dependent kinase (Cdk) sites by multiple G1 cyclin-Cdk complexes, the active form(s) of pRB in vivo remains unknown. pRB is present as an unphosphorylated Protein in G0 quiescent cells and becomes hypophosphorylated (;2 mol of PO4 to 1 mol of pRB) in early G1 and hyperphosphorylated (;10 mol of PO4 to 1 mol of pRB) in late G1 phase. Here, we report that hypophosphorylated pRB, present in early G1, represents the biologically active form of pRB in vivo that is assembled with E2Fs and E1A but that both unphosphorylated pRB in G0 and hyperphosphorylated pRB in late G1 fail to become assembled with E2Fs and E1A. Furthermore, using transducible dominant-negative TAT fusion Proteins that differentially target cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes, namely, TAT-p16 and TAT‐ dominant-negative Cdk2, respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G1 and that cyclin E-Cdk2 complexes inactivate pRB by hyperphosphorylation in late G1. Moreover, we found that cycling human Tumor cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion of the p16 INK4a gene, contained hypophosphorylated pRB that was bound to E2Fs in early G1 and that E2F-responsive genes, including those for dihydrofolate reductase and cyclin E, were transcriptionally repressed. Thus, we conclude that, physiologically, pRB is differentially regulated by G1 cyclin-Cdk complexes.
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differential regulation of retinoblastoma Tumor Suppressor Protein by g 1 cyclin dependent kinase complexes in vivo
Molecular and Cellular Biology, 2001Co-Authors: Sergei A Ezhevsky, Michelle Beckerhapak, Penny K Davis, Steven F DowdyAbstract:The retinoblastoma Tumor Suppressor Protein (pRB) negatively regulates early-G1 cell cycle progression, in part, by sequestering E2F transcription factors and repressing E2F-responsive genes. Although pRB is phosphorylated on up to 16 cyclin-dependent kinase (Cdk) sites by multiple G1 cyclin-Cdk complexes, the active form(s) of pRB in vivo remains unknown. pRB is present as an unphosphorylated Protein in G0 quiescent cells and becomes hypophosphorylated (;2 mol of PO4 to 1 mol of pRB) in early G1 and hyperphosphorylated (;10 mol of PO4 to 1 mol of pRB) in late G1 phase. Here, we report that hypophosphorylated pRB, present in early G1, represents the biologically active form of pRB in vivo that is assembled with E2Fs and E1A but that both unphosphorylated pRB in G0 and hyperphosphorylated pRB in late G1 fail to become assembled with E2Fs and E1A. Furthermore, using transducible dominant-negative TAT fusion Proteins that differentially target cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes, namely, TAT-p16 and TAT‐ dominant-negative Cdk2, respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G1 and that cyclin E-Cdk2 complexes inactivate pRB by hyperphosphorylation in late G1. Moreover, we found that cycling human Tumor cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion of the p16 INK4a gene, contained hypophosphorylated pRB that was bound to E2Fs in early G1 and that E2F-responsive genes, including those for dihydrofolate reductase and cyclin E, were transcriptionally repressed. Thus, we conclude that, physiologically, pRB is differentially regulated by G1 cyclin-Cdk complexes.
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, 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, 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.
Sergei A Ezhevsky - One of the best experts on this subject based on the ideXlab platform.
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differential regulation of retinoblastoma Tumor Suppressor Protein by g1 cyclin dependent kinase complexes in vivo
Molecular and Cellular Biology, 2001Co-Authors: Sergei A Ezhevsky, Alan L Ho, Michelle Beckerhapak, Penny K Davis, Steven F DowdyAbstract:The retinoblastoma Tumor Suppressor Protein (pRB) negatively regulates early-G1 cell cycle progression, in part, by sequestering E2F transcription factors and repressing E2F-responsive genes. Although pRB is phosphorylated on up to 16 cyclin-dependent kinase (Cdk) sites by multiple G1 cyclin-Cdk complexes, the active form(s) of pRB in vivo remains unknown. pRB is present as an unphosphorylated Protein in G0 quiescent cells and becomes hypophosphorylated (;2 mol of PO4 to 1 mol of pRB) in early G1 and hyperphosphorylated (;10 mol of PO4 to 1 mol of pRB) in late G1 phase. Here, we report that hypophosphorylated pRB, present in early G1, represents the biologically active form of pRB in vivo that is assembled with E2Fs and E1A but that both unphosphorylated pRB in G0 and hyperphosphorylated pRB in late G1 fail to become assembled with E2Fs and E1A. Furthermore, using transducible dominant-negative TAT fusion Proteins that differentially target cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes, namely, TAT-p16 and TAT‐ dominant-negative Cdk2, respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G1 and that cyclin E-Cdk2 complexes inactivate pRB by hyperphosphorylation in late G1. Moreover, we found that cycling human Tumor cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion of the p16 INK4a gene, contained hypophosphorylated pRB that was bound to E2Fs in early G1 and that E2F-responsive genes, including those for dihydrofolate reductase and cyclin E, were transcriptionally repressed. Thus, we conclude that, physiologically, pRB is differentially regulated by G1 cyclin-Cdk complexes.
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Differential Regulation of Retinoblastoma Tumor Suppressor Protein by G1 Cyclin-Dependent Kinase Complexes In Vivo
Molecular and cellular biology, 2001Co-Authors: Sergei A Ezhevsky, Penny K Davis, Michelle Becker-hapak, Steven F DowdyAbstract:The retinoblastoma Tumor Suppressor Protein (pRB) negatively regulates early-G1 cell cycle progression, in part, by sequestering E2F transcription factors and repressing E2F-responsive genes. Although pRB is phosphorylated on up to 16 cyclin-dependent kinase (Cdk) sites by multiple G1 cyclin-Cdk complexes, the active form(s) of pRB in vivo remains unknown. pRB is present as an unphosphorylated Protein in G0 quiescent cells and becomes hypophosphorylated (;2 mol of PO4 to 1 mol of pRB) in early G1 and hyperphosphorylated (;10 mol of PO4 to 1 mol of pRB) in late G1 phase. Here, we report that hypophosphorylated pRB, present in early G1, represents the biologically active form of pRB in vivo that is assembled with E2Fs and E1A but that both unphosphorylated pRB in G0 and hyperphosphorylated pRB in late G1 fail to become assembled with E2Fs and E1A. Furthermore, using transducible dominant-negative TAT fusion Proteins that differentially target cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes, namely, TAT-p16 and TAT‐ dominant-negative Cdk2, respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G1 and that cyclin E-Cdk2 complexes inactivate pRB by hyperphosphorylation in late G1. Moreover, we found that cycling human Tumor cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion of the p16 INK4a gene, contained hypophosphorylated pRB that was bound to E2Fs in early G1 and that E2F-responsive genes, including those for dihydrofolate reductase and cyclin E, were transcriptionally repressed. Thus, we conclude that, physiologically, pRB is differentially regulated by G1 cyclin-Cdk complexes.
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differential regulation of retinoblastoma Tumor Suppressor Protein by g 1 cyclin dependent kinase complexes in vivo
Molecular and Cellular Biology, 2001Co-Authors: Sergei A Ezhevsky, Michelle Beckerhapak, Penny K Davis, Steven F DowdyAbstract:The retinoblastoma Tumor Suppressor Protein (pRB) negatively regulates early-G1 cell cycle progression, in part, by sequestering E2F transcription factors and repressing E2F-responsive genes. Although pRB is phosphorylated on up to 16 cyclin-dependent kinase (Cdk) sites by multiple G1 cyclin-Cdk complexes, the active form(s) of pRB in vivo remains unknown. pRB is present as an unphosphorylated Protein in G0 quiescent cells and becomes hypophosphorylated (;2 mol of PO4 to 1 mol of pRB) in early G1 and hyperphosphorylated (;10 mol of PO4 to 1 mol of pRB) in late G1 phase. Here, we report that hypophosphorylated pRB, present in early G1, represents the biologically active form of pRB in vivo that is assembled with E2Fs and E1A but that both unphosphorylated pRB in G0 and hyperphosphorylated pRB in late G1 fail to become assembled with E2Fs and E1A. Furthermore, using transducible dominant-negative TAT fusion Proteins that differentially target cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes, namely, TAT-p16 and TAT‐ dominant-negative Cdk2, respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G1 and that cyclin E-Cdk2 complexes inactivate pRB by hyperphosphorylation in late G1. Moreover, we found that cycling human Tumor cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion of the p16 INK4a gene, contained hypophosphorylated pRB that was bound to E2Fs in early G1 and that E2F-responsive genes, including those for dihydrofolate reductase and cyclin E, were transcriptionally repressed. Thus, we conclude that, physiologically, pRB is differentially regulated by G1 cyclin-Cdk complexes.
Joan Weliky Conaway - 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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regulation of hypoxia inducible mrnas by the von hippel lindau Tumor Suppressor Protein requires binding to complexes containing elongins b c and cul2
Molecular and Cellular Biology, 1998Co-Authors: Takumi Kamura, Joan Weliky Conaway, Kim M Lonergan, Othon Iliopoulos, Michael OhhAbstract:The von Hippel-Lindau Tumor Suppressor Protein (pVHL) binds to elongins B and C and posttranscriptionally regulates the accumulation of hypoxia-inducible mRNAs under normoxic (21% O2) conditions. Here we report that pVHL binds, via elongin C, to the human homolog of the Caenorhabditis elegans Cul2 Protein. Coimmunoprecipitation and chromatographic copurification data suggest that pVHL-Cul2 complexes exist in native cells. pVHL mutants that were unable to bind to complexes containing elongin C and Cul2 were likewise unable to inhibit the accumulation of hypoxia-inducible mRNAs. A model for the regulation of hypoxia-inducible mRNAs by pVHL is presented based on the apparent similarity of elongin C and Cul2 to Skp1 and Cdc53, respectively. These latter Proteins form complexes that target specific Proteins for ubiquitin-dependent proteolysis.
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Regulation of Hypoxia-Inducible mRNAs by the von Hippel-Lindau Tumor Suppressor Protein Requires Binding to Complexes
1997Co-Authors: Containing Elongins B/c, Joan Weliky Conaway, Ronald C. Conaway, William G. KaelinAbstract:The von Hippel-Lindau Tumor Suppressor Protein (pVHL) binds to elongins B and C and posttranscription-ally regulates the accumulation of hypoxia-inducible mRNAs under normoxic (21 % O2) conditions. Here we report that pVHL binds, via elongin C, to the human homolog of the Caenorhabditis elegans Cul2 Protein. Coimmunoprecipitation and chromatographic copurification data suggest that pVHL-Cul2 complexes exist in native cells. pVHL mutants that were unable to bind to complexes containing elongin C and Cul2 were likewise unable to inhibit the accumulation of hypoxia-inducible mRNAs. A model for the regulation of hypoxia-inducible mRNAs by pVHL is presented based on the apparent similarity of elongin C and Cul2 to Skp1 and Cdc53, respectively. These latter Proteins form complexes that target specific Proteins for ubiquitin-dependent proteolysis. von Hippel-Lindau (VHL) disease, a hereditary cancer syn-drome characterized by the development of renal carcinomas, pheochromocytomas, and vascular Tumors of the central ner-vous system and retina, is caused by germ line mutations in the VHL Tumor Suppressor gene (9, 17, 27, 35). Tumor develop-ment in this setting is due to somatic loss or mutation of th
Kazuhiro Iwai - 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 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, 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.
