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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 <B>ElonginB> B and <B>ElonginB> 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 <B>ElonginB> B–<B>ElonginB> 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 <B>ElonginB> B and <B>ElonginB> 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 <B>ElonginB> B–<B>ElonginB> 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 <B>ElonginB> C Sequences Required for Interaction with the von Hippel-Lindau Tumor Suppressor Protein
The Journal of biological chemistry, 1997Co-Authors: Yuichiro Takagi, Arnim PauseAbstract:<B>ElonginB> 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 <B>ElonginB> (SIII) and as a component of a multiprotein complex containing the von Hippel-Lindau (VHL) tumor suppressor protein. As a suBunit of the <B>ElonginB> complex, <B>ElonginB> C interacts directly with the transcriptionally active <B>ElonginB> A suBunit and potently induces its elongation activity; in addition, <B>ElonginB> C interacts with the uBiquitin-like <B>ElonginB> B suBunit, which regulates the interaction of <B>ElonginB> C with <B>ElonginB> A. As a component of the VHL complex, <B>ElonginB> C interacts directly with Both <B>ElonginB> B and the VHL protein. Binding of the VHL protein to <B>ElonginB> C was found to prevent <B>ElonginB> C from interacting with and activating <B>ElonginB> 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 <B>ElonginB> complex. In this report, we identify <B>ElonginB> C sequences required for its interaction with the VHL protein. We previously demonstrated that the aBility of <B>ElonginB> C to Bind and activate <B>ElonginB> A is sensitive to mutations in the C-terminal half of <B>ElonginB> C, as well as to mutations in an N-terminal <B>ElonginB> C region needed for formation of the <B>ElonginB> BC complex. Here we show that interaction of <B>ElonginB> C with the VHL tumor suppressor protein depends strongly on sequences in the C terminus of <B>ElonginB> C But is independent of the N-terminal <B>ElonginB> C region required for Binding to <B>ElonginB> B and for Binding and activation of <B>ElonginB> A. Taken together, our results are consistent with the proposal that the VHL protein negatively regulates <B>ElonginB> C activation of the <B>ElonginB> complex By sterically Blocking the interaction of C-terminal <B>ElonginB> C sequences with <B>ElonginB> A. In addition, our finding that only a suBset of <B>ElonginB> C sequences required for its interaction with <B>ElonginB> A are critical for Binding to VHL may offer the opportunity to develop reagents that selectively interfere with <B>ElonginB> 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 <B>ElonginB> B and <B>ElonginB> C, two factors that staBilize and activate the transcription elongation factor <B>ElonginB> A. Here, Hs-CUL-2, a memBer of the recently identified multigene family, the cullins, is shown to specifically associate with the trimeric pVHL-<B>ElonginB> 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.
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inhiBition of transcription elongation By the vhl tumor suppressor protein
Science, 1995Co-Authors: D R Duan, Joan Weliky Conaway, R C Conaway, Arnim Pause, W H Burgess, T Aso, D Y T Chen, K P Garrett, W M Linehan, Richard D. KlausnerAbstract:Germline mutations in the von Hippel-Lindau tumor suppressor gene (VHL) predispose individuals to a variety of tumors, including renal carcinoma, hemangioBlastoma of the central nervous system, and pheochromocytoma. Here, a cellular transcription factor, <B>ElonginB> (SIII), is identified as a functional target of the VHL protein. <B>ElonginB> (SIII) is a heterotrimer consisting of a transcriptionally active suBunit (A) and two regulatory suBunits (B and C) that activate transcription elongation By RNA polymerase II. The VHL protein was shown to Bind tightly and specifically to the <B>ElonginB> B and C suBunits and to inhiBit <B>ElonginB> (SIII) transcriptional activity in vitro. These findings reveal a potentially important transcriptional regulatory network in which the VHL protein may play a key role.
Richard D. Klausner - 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 <B>ElonginB> B and <B>ElonginB> 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 <B>ElonginB> B–<B>ElonginB> 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 <B>ElonginB> B and <B>ElonginB> 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 <B>ElonginB> B–<B>ElonginB> 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 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 <B>ElonginB> B and <B>ElonginB> C, two factors that staBilize and activate the transcription elongation factor <B>ElonginB> A. Here, Hs-CUL-2, a memBer of the recently identified multigene family, the cullins, is shown to specifically associate with the trimeric pVHL-<B>ElonginB> 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.
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inhiBition of transcription elongation By the vhl tumor suppressor protein
Science, 1995Co-Authors: D R Duan, Joan Weliky Conaway, R C Conaway, Arnim Pause, W H Burgess, T Aso, D Y T Chen, K P Garrett, W M Linehan, Richard D. KlausnerAbstract:Germline mutations in the von Hippel-Lindau tumor suppressor gene (VHL) predispose individuals to a variety of tumors, including renal carcinoma, hemangioBlastoma of the central nervous system, and pheochromocytoma. Here, a cellular transcription factor, <B>ElonginB> (SIII), is identified as a functional target of the VHL protein. <B>ElonginB> (SIII) is a heterotrimer consisting of a transcriptionally active suBunit (A) and two regulatory suBunits (B and C) that activate transcription elongation By RNA polymerase II. The VHL protein was shown to Bind tightly and specifically to the <B>ElonginB> B and C suBunits and to inhiBit <B>ElonginB> (SIII) transcriptional activity in vitro. These findings reveal a potentially important transcriptional regulatory network in which the VHL protein may play a key role.
Takumi Kamura - One of the best experts on this subject based on the ideXlab platform.
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2009a) Degradation of phosphorylated p53 By viral proteinECS E3 ligase complex
2016Co-Authors: Yoshitaka Sato, Takumi Kamura, Noriko Shirata, Takayuki Murata, Ayumi Kudoh, Satoko IwahoriAbstract:p53-signaling is modulated By viruses to estaBlish a host cellular environment advantageous for their propagation. The Epstein-Barr virus (EBV) lytic program induces phosphorylation of p53, which prevents interaction with MDM2. Here, we show that induction of EBV lytic program leads to degradation of p53 via an uBiquitin-proteasome pathway independent of MDM2. The BZLF1 protein directly functions as an adaptor component of the ECS (<B>ElonginB> B/C-Cul2/5-SOCS-Box protein) uBiquitin ligase complex targeting p53 for degradation. Intringuingly, C-terminal phosphorylation of p53 resulting from activated DNA damage response By viral lytic replication enhances its Binding to BZLF1 protein. Purified BZLF1 protein-associated ECS could Be shown to catalyze uBiquitination of phospho-mimetic p53 more efficiently than the wild-type in vitro. The compensation of p53 at middle and late stages of the lytic infection inhiBits viral DNA replication and production during lytic infection, suggesting that the degradation of p53 is required for efficient viral propagation. Taken together, these findings demonstrate a role for the BZLF1 protein-associated ECS ligase complex in regulation of p53 phosphorylated By activated DNA damage signaling during viral lytic infection
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Degradation of phosphorylated p53 By viral protein-ECS E3 ligase complex.
Public Library of Science (PLoS), 2009Co-Authors: Yoshitaka Sato, Takumi Kamura, Noriko Shirata, Takayuki Murata, Ayumi Kudoh, Satoko Iwahori, Sanae Nakayama, Hiroki Isomura, Yukihiro Nishiyama, Tatsuya TsurumiAbstract:p53-signaling is modulated By viruses to estaBlish a host cellular environment advantageous for their propagation. The Epstein-Barr virus (EBV) lytic program induces phosphorylation of p53, which prevents interaction with MDM2. Here, we show that induction of EBV lytic program leads to degradation of p53 via an uBiquitin-proteasome pathway independent of MDM2. The BZLF1 protein directly functions as an adaptor component of the ECS (<B>ElonginB> B/C-Cul2/5-SOCS-Box protein) uBiquitin ligase complex targeting p53 for degradation. Intringuingly, C-terminal phosphorylation of p53 resulting from activated DNA damage response By viral lytic replication enhances its Binding to BZLF1 protein. Purified BZLF1 protein-associated ECS could Be shown to catalyze uBiquitination of phospho-mimetic p53 more efficiently than the wild-type in vitro. The compensation of p53 at middle and late stages of the lytic infection inhiBits viral DNA replication and production during lytic infection, suggesting that the degradation of p53 is required for efficient viral propagation. Taken together, these findings demonstrate a role for the BZLF1 protein-associated ECS ligase complex in regulation of p53 phosphorylated By activated DNA damage signaling during viral lytic infection
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vhl Box and socs Box domains determine Binding specificity for cul2 rBx1 and cul5 rBx2 modules of uBiquitin ligases
Genes & Development, 2004Co-Authors: Takumi Kamura, Joan Weliky Conaway, Katsumi Maenaka, Shuhei Kotoshiba, Masaki Matsumoto, Daisuke Kohda, Ronald C Conaway, Keiichi I NakayamaAbstract:The ECS (<B>ElonginB> B/C-Cul2/Cul5-SOCS-Box protein) complex is a memBer of a family of uBiquitin ligases that share a Cullin-RBx module. SOCS-Box proteins recruit suBstrates to the ECS complex and are linked to Cullin-RBx via <B>ElonginB> B/C. VHL has Been implicated as a SOCS-Box protein, But lacks a C-terminal sequence (downstream of the BC Box) of the SOCS Box. We now show that VHL specifically interacts with endogenous Cul2-RBx1 in mammalian cells, whereas SOCS-Box proteins associate with Cul5-RBx2. We also identify LRR-1 and FEM1B as proteins that share a region of homology with VHL (the VHL Box, including the BC Box and downstream residues) and associate with Cul2-RBx1. ECS complexes can thus Be classified into two distinct protein assemBlies, that is, those that contain a suBunit with a VHL Box (composed of the BC Box and a downstream Cul2 Box) that interacts with Cul2-RBx1, and those that contain a suBunit with a SOCS Box (BC Box and downstream Cul5 Box) that interacts with Cul5-RBx2. Domain-swapping analyses showed that the specificity of interaction of VHL-Box and SOCS-Box proteins with Cullin-RBx modules is determined By the Cul2 and Cul5 Boxes, respectively. Finally, RNAi-mediated knockdown of the Cul2-RBx1 inhiBited the VHL-mediated degradation of HIF-2alpha, whereas knockdown of Cul5-RBx2 did not affect it. These data suggest that the functions of the Cul2-RBx1 and Cul5-RBx2 modules are distinct.
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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 <B>ElonginB> B and <B>ElonginB> 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 <B>ElonginB> B–<B>ElonginB> 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 <B>ElonginB> B and <B>ElonginB> 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 <B>ElonginB> B–<B>ElonginB> 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.
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 <B>ElonginB> C, <B>ElonginB> 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 <B>ElonginB> C, <B>ElonginB> B, and Cul2. To understand this process in greater detail, we performed a series of in vitro Binding assays using pVHL, <B>ElonginB> B, and <B>ElonginB> C variants as well as synthetic peptide competitors derived from pVHL or <B>ElonginB> C. A suBdomain of <B>ElonginB> C (residues 17–50) was necessary and sufficient for detectaBle Binding to <B>ElonginB> B. In contrast, <B>ElonginB> B residues required for Binding to <B>ElonginB> C were not confined to a discrete colinear domain. We found that the pVHL (residues 157–171) is necessary and sufficient for Binding to <B>ElonginB> C in vitro and is frequently mutated in families with VHL disease. These mutations preferentially involve residues that directly Bind to <B>ElonginB> C and/or alter the conformation of pVHL such that Binding to <B>ElonginB> C is at least partially diminished. These results are consistent with the view that diminished Binding of pVHL to the <B>ElonginB>s plays a causal role in VHL disease. J. Clin. Invest. 104:1583–1591 (1999).
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the <B>ElonginB> B uBiquitin homology domain identification of <B>ElonginB> B sequences important for interaction with <B>ElonginB> c
Journal of Biological Chemistry, 1999Co-Authors: Takumi Kamura, Dewan Haque, Yuichiro Takagi, Christopher S. Brower, Timothy Mather, Annemarie TreharneAbstract:Mammalian <B>ElonginB> 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. <B>ElonginB> B is found in cells as a suBunit of the heterodimeric <B>ElonginB> BC complex, which was originally identified as a positive regulator of RNA polymerase II elongation factor <B>ElonginB> 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 <B>ElonginB> BC complex regulates the activity of <B>ElonginB> A, we are characterizing <B>ElonginB> B functional domains. In this report, we show that the <B>ElonginB> B uBiquitin-like domain is necessary and sufficient for interaction with <B>ElonginB> C and for positive regulation of <B>ElonginB> A transcriptional activity. In addition, By site-directed mutagenesis of the <B>ElonginB> B uBiquitin-like domain, we identify a short <B>ElonginB> B region that is important for its interaction with <B>ElonginB> C. Finally, we oBserve that Both the uBiquitin-like domain and carBoxyl-terminal tail are conserved in Drosophila melanogaster and CaenorhaBditis elegans <B>ElonginB> B homologs that efficiently suBstitute for mammalian <B>ElonginB> B in reconstitution of the transcriptionally active <B>ElonginB> ABC complex, suggesting that the carBoxyl-terminal tail performs an additional function not detected in our assays.
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Identification of <B>ElonginB> C Sequences Required for Interaction with the von Hippel-Lindau Tumor Suppressor Protein
The Journal of biological chemistry, 1997Co-Authors: Yuichiro Takagi, Arnim PauseAbstract:<B>ElonginB> 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 <B>ElonginB> (SIII) and as a component of a multiprotein complex containing the von Hippel-Lindau (VHL) tumor suppressor protein. As a suBunit of the <B>ElonginB> complex, <B>ElonginB> C interacts directly with the transcriptionally active <B>ElonginB> A suBunit and potently induces its elongation activity; in addition, <B>ElonginB> C interacts with the uBiquitin-like <B>ElonginB> B suBunit, which regulates the interaction of <B>ElonginB> C with <B>ElonginB> A. As a component of the VHL complex, <B>ElonginB> C interacts directly with Both <B>ElonginB> B and the VHL protein. Binding of the VHL protein to <B>ElonginB> C was found to prevent <B>ElonginB> C from interacting with and activating <B>ElonginB> 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 <B>ElonginB> complex. In this report, we identify <B>ElonginB> C sequences required for its interaction with the VHL protein. We previously demonstrated that the aBility of <B>ElonginB> C to Bind and activate <B>ElonginB> A is sensitive to mutations in the C-terminal half of <B>ElonginB> C, as well as to mutations in an N-terminal <B>ElonginB> C region needed for formation of the <B>ElonginB> BC complex. Here we show that interaction of <B>ElonginB> C with the VHL tumor suppressor protein depends strongly on sequences in the C terminus of <B>ElonginB> C But is independent of the N-terminal <B>ElonginB> C region required for Binding to <B>ElonginB> B and for Binding and activation of <B>ElonginB> A. Taken together, our results are consistent with the proposal that the VHL protein negatively regulates <B>ElonginB> C activation of the <B>ElonginB> complex By sterically Blocking the interaction of C-terminal <B>ElonginB> C sequences with <B>ElonginB> A. In addition, our finding that only a suBset of <B>ElonginB> C sequences required for its interaction with <B>ElonginB> A are critical for Binding to VHL may offer the opportunity to develop reagents that selectively interfere with <B>ElonginB> and VHL function.
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Characterization of <B>ElonginB> C functional domains required for interaction with <B>ElonginB> B and activation of <B>ElonginB> A.
The Journal of biological chemistry, 1996Co-Authors: Yuichiro TakagiAbstract:The <B>ElonginB> (SIII) complex stimulates the rate of elongation By RNA polymerase II By suppressing transient pausing By polymerase at many sites along DNA templates. The <B>ElonginB> (SIII) complex is composed of a transcriptionally active A suBunit, a chaperone-like B suBunit, which promotes assemBly and enhances staBility of the <B>ElonginB> (SIII) complex, and a regulatory C suBunit, which (i) functions as a potent activator of <B>ElonginB> A transcriptional activity, (ii) interacts specifically with <B>ElonginB> B to form an isolaBle <B>ElonginB> 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 <B>ElonginB> C regulates the activity of the <B>ElonginB> (SIII) complex, we are characterizing <B>ElonginB> C functional domains. In this report, we identify <B>ElonginB> C mutants that fall into multiple functional classes Based on their aBilities to Bind <B>ElonginB> B and to Bind and activate <B>ElonginB> A under our assay conditions. Characterization of these mutants suggests that <B>ElonginB> C is composed of multiple overlapping regions that mediate functional interactions with <B>ElonginB> A and B.
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The pVHL-associated SCF uBiquitin ligase complex: Molecular genetic analysis of <B>ElonginB> B and C, RBx1 and HIF-1α in renal cell carcinoma
Oncogene, 2001Co-Authors: Steven C Clifford, Dewi Astuti, Laura Hooper, Patrick H Maxwell, Peter J Ratcliffe, Eamonn R. MaherAbstract:The VHL gene product (pVHL) forms a multimeric complex with the <B>ElonginB> B and C, Cul2 and RBx1 proteins (VCBCR complex), which is homologous to the SCF family of uBiquitin ligase complexes. The VCBCR complex Binds HIF-1α and HIF-2α, transcription factors critically involved in cellular responses to hypoxia, and targets them for uBiquitin-mediated proteolysis. Germline mutations in the VHL gene cause susceptiBility to haemangioBlastomas, renal cell carcinoma (RCC), phaeochromocytoma and other tumours. In addition somatic inactivation of the VHL gene occurs in most sporadic clear cell RCC (CC-RCC). However, the aBsence of somatic VHL inactivation in 30–40% of CC-RCC implies the involvement of other gatekeeper genes in CC-RCC development. We reasoned that in CC-RCC without VHL inactivation, other pVHL-interacting proteins might Be defective. To assess the role of <B>ElonginB> B/C, RBx1 and HIF-1α in RCC tumorigenesis we (a) mapped the genes to chromosomes 8q(cen) (<B>ElonginB> C), 16p13.3 (<B>ElonginB> B) and 22q11.2 (RBx1) By FISH, monochromosomal somatic cell hyBrid panel screening and in silico GenBank homology searching; (B) determined the genomic organisation of <B>ElonginB> C (By direct sequencing of PAC clones), RBx1 and <B>ElonginB> B (By GenBank homology searching); and (c) performed mutation analysis of exons comprising the coding regions of <B>ElonginB>s B, C and RBx1 and the oxygen-dependent degradation domain of HIF-1α By SSCP screening and direct sequencing in 35 sporadic clear cell RCC samples without VHL gene inactivation and in 13 individuals with familial non-VHL clear cell RCC. No coding region sequence variations were detected for the <B>ElonginB> B, <B>ElonginB> C or RBx1 genes. Two amino acid suBstitutions (Pro582Ser and Ala588Thr) were identified in the oxygen-dependent degradation/pVHL Binding domain of HIF-1α, however neither suBstitution was oBserved exclusively in tumour samples. Association analysis in panels of CC-RCC and non-neoplastic samples using the RFLPs generated By each variant did not reveal allelic frequency differences Between RCC patients and controls ( P >0.32 By chi-squared analysis). Nevertheless, the significance of these variations and their potential for modulation of HIF-1α function merits further investigation in Both other tumour types and in non-neoplastic disease. Taken together with our previous Cul2 mutation analysis these data suggest that development of sporadic and familial RCC is not commonly contriButed to By genetic events altering the destruction domain of HIF-1α, or components of the HIF-α destruction complex other than VHL itself. Although (a) activation of HIF could occur through mutation of another region of HIF-a, and (B) epigenetic silencing of <B>ElonginB> B/C, Cul2 or RBx1 cannot Be excluded, these findings suggest that pVHL may represent the sole mutational target through which the VCBR complex is disrupted in CC-RCC. HIF response is activated in CC-RCC tumorigenesis.
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the pvhl associated scf uBiquitin ligase complex molecular genetic analysis of <B>ElonginB> B and c rBx1 and hif 1alpha in renal cell carcinoma
Oncogene, 2001Co-Authors: Steven C Clifford, Dewi Astuti, Laura Hooper, Patrick H Maxwell, Peter J Ratcliffe, Eamonn R. MaherAbstract:The pVHL-associated SCF uBiquitin ligase complex: Molecular genetic analysis of <B>ElonginB> B and C, RBx1 and HIF-1α in renal cell carcinoma