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Yue Xiong - One of the best experts on this subject based on the ideXlab platform.
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BTB protein Keap1 targets antioxidant transcription factor Nrf2 for ubiquitination by the Cullin 3-Roc1 ligase
2016Co-Authors: Manabu Furukawa, Yue XiongAbstract:The concentrations and functions of many eukaryotic proteins are regulated by the ubiquitin pathway, which consists of ubiquitin activation (E1), conjugation (E2), and ligation (E3). Cullins are a family of evolutionarily conserved proteins that assemble by far the largest family of E3 ligase complexes. Cullins, via a conserved C-terminal domain, bind with the RING finger protein Roc1 to recruit the catalytic function of E2. Via a distinct N-terminal domain, individual cullins bind to a protein motif present in multiple proteins to recruit specific substrates. Cullin 3 (CUL3), but not other cullins, binds directly with BTB domains to constitute a potentially large number of BTB-CUL3-ROC1 E3 ubiquitin ligases. Here we report that the human BTB-Kelch protein Keap1, a negative regulator of the antioxidative transcription factor Nrf2, binds to CUL3 and Nrf2 via its BTB and Kelch domains, respectively. The KEAP1-CUL3-ROC1 complex promoted NRF2 ubiquitination in vitro and knocking down Keap1 or CUL3 by short interfering RNA resulted in NRF2 protein accumulation in vivo. We suggest that Keap1 negatively regulates Nrf2 function in part by targeting Nrf2 for ubiquitination by the CUL3-ROC1 ligase and subsequent degradation by the proteasome. Blocking NRF2 degradation in cells expressing both KEAP1 and NRF2 by either inhibiting the proteasome activity or knocking down CUL3, resulted in NRF2 accumulation in the cytoplasm. These results may reconcile previously observed cytoplasmic seques-tration of NRF2 by KEAP1 and suggest a possible regulatory step between KEAP1-NRF2 binding and NRF
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deubiquitination of ci gli by usp7 hausp regulates hedgehog signaling
Developmental Cell, 2015Co-Authors: Zizhang Zhou, Yue Xiong, Shuang Li, Xiaohua Dong, Yun Zhao, Jin Jiang, Qing ZhangAbstract:Summary Hedgehog (Hh) signaling plays essential roles in animal development and tissue homeostasis, and its misregulation causes congenital diseases and cancers. Regulation of the ubiquitin/proteasome-mediated proteolysis of Ci/Gli transcription factors is central to Hh signaling, but whether deubiquitinase is involved in this process remains unknown. Here, we show that Hh stimulates the binding of a ubiquitin-specific protease Usp7 to Ci, which positively regulates Hh signaling activity through inhibiting Ci ubiquitination and degradation mediated by both Slimb-Cul1 and Hib-CUL3 E3 ligases. Furthermore, we find that Usp7 forms a complex with GMP-synthetase (GMPS) to promote Hh pathway activity. Finally, we show that the mammalian counterpart of Usp7, HAUSP, positively regulates Hh signaling by modulating Gli ubiquitination and stability. Our findings reveal a conserved mechanism by which Ci/Gli is stabilized by a deubiquitination enzyme and identify Usp7/HUASP as a critical regulator of Hh signaling and potential therapeutic target for Hh-related cancers.
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Deubiquitination of Ci/Gli by Usp7/HAUSP Regulates Hedgehog Signaling
Developmental Cell, 2015Co-Authors: Zizhang Zhou, Yue Xiong, Shuang Li, Xiaohua Dong, Yun Zhao, Jin Jiang, Qing ZhangAbstract:Summary Hedgehog (Hh) signaling plays essential roles in animal development and tissue homeostasis, and its misregulation causes congenital diseases and cancers. Regulation of the ubiquitin/proteasome-mediated proteolysis of Ci/Gli transcription factors is central to Hh signaling, but whether deubiquitinase is involved in this process remains unknown. Here, we show that Hh stimulates the binding of a ubiquitin-specific protease Usp7 to Ci, which positively regulates Hh signaling activity through inhibiting Ci ubiquitination and degradation mediated by both Slimb-Cul1 and Hib-CUL3 E3 ligases. Furthermore, we find that Usp7 forms a complex with GMP-synthetase (GMPS) to promote Hh pathway activity. Finally, we show that the mammalian counterpart of Usp7, HAUSP, positively regulates Hh signaling by modulating Gli ubiquitination and stability. Our findings reveal a conserved mechanism by which Ci/Gli is stabilized by a deubiquitination enzyme and identify Usp7/HUASP as a critical regulator of Hh signaling and potential therapeutic target for Hh-related cancers.
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human immunodeficiency virus type 1 vpr binding protein vprbp a wd40 protein associated with the ddb1 cul4 e3 ubiquitin ligase is essential for dna replication and embryonic development
Molecular and Cellular Biology, 2008Co-Authors: Chad M. Mccall, Paula Miliani L De Marval, Paul D Chastain, Sarah Jackson, Yojiro Kotake, Jeanette Gowen Cook, Yue XiongAbstract:Ubiquitin ligases play a critical role in cellular function by recruiting various protein substrates for covalent modification by the small protein ubiquitin (15, 31). Ubiquitin modification, either monomeric or in polyubiquitin chains, leads to various changes in cellular protein function, most prominently the targeting of polyubiquitin-conjugated proteins to the 26S proteasome for proteolytic degradation. The cullin family of ubiquitin ligases performs remarkably broad functions due to their ability to assemble a large number of distinct cullin-RING E3 ligase complexes through modular interaction with substrate receptors containing specific protein-protein interaction motifs (29). Cullins interact with their substrate receptors either directly, as in the case of CUL3, which interacts with one of more than 200 BTB domain-containing receptors (10, 11, 32, 43), or indirectly through a conserved linker protein, such as the SKP1 protein that bridges one of more than 70 F-box-containing receptors to CUL1 (2, 9, 36) and the heterodimer of elongins B and C that links one of the more than 30 VHL-box or SOCS-box receptors with CUL2 or CUL5 (21, 22, 38, 44). Through interaction with these common motifs, the cullin-RING E3 ligase complexes may potentially ubiquitinate a large number of substrates. We along with other groups recently discovered that damaged DNA binding protein 1, DDB1, acts a linker protein for CUL4 and recruits substrates through interaction with a subset of WD40 proteins (1, 14, 16, 19). Mammalian cells contain at least 90 DDB1-binding WD40 (DWD) proteins (also known as DCAF for Ddb1- and Cul4-associated factors and CDW for CUL4 and DDB1-associated WD40 repeats), suggesting that CUL4-ROC1 ligases may also promote ubiquitination of a large number of substrates. One of the more than 30 mammalian DWD proteins that have been experimentally demonstrated to bind DDB1-CUL4 is VprBP/DCAF1 (accession number NM014703), a 170-kDa protein that was initially identified through coimmunoprecipitation and peptide sequencing of HIV-1 Vpr-binding proteins (VprBPs) (45). Very recently, it has been shown that VprBP is required for the G2 cell cycle arrest caused by Vpr expression (3, 8, 17, 24, 35, 39, 42). The physiological significance underlying the Vpr-VprBP interaction for human immunodeficiency virus (HIV) viral propagation remains unclear. VprBP orthologs are found in Drosophila melanogaster (36% identical to human VprBP), Caenorhabditis elegans (31%), and Arabidopsis thaliana (27%), but no obvious ortholog is recognizable in yeast cells. VprBP is broadly expressed in most, if not all, human and mouse tissues that have been examined (45). These features suggest an unknown but conserved and possibly critical function of VprBP in multicellular organisms. This study is directed toward elucidating this issue.
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btb protein keap1 targets antioxidant transcription factor nrf2 for ubiquitination by the cullin 3 roc1 ligase
Molecular and Cellular Biology, 2005Co-Authors: Manabu Furukawa, Yue XiongAbstract:The concentrations and functions of many eukaryotic proteins are regulated by the ubiquitin pathway, which consists of ubiquitin activation (E1), conjugation (E2), and ligation (E3). Cullins are a family of evolutionarily conserved proteins that assemble by far the largest family of E3 ligase complexes. Cullins, via a conserved C-terminal domain, bind with the RING finger protein Roc1 to recruit the catalytic function of E2. Via a distinct N-terminal domain, individual cullins bind to a protein motif present in multiple proteins to recruit specific substrates. Cullin 3 (CUL3), but not other cullins, binds directly with BTB domains to constitute a potentially large number of BTB-CUL3-ROC1 E3 ubiquitin ligases. Here we report that the human BTB-Kelch protein Keap1, a negative regulator of the antioxidative transcription factor Nrf2, binds to CUL3 and Nrf2 via its BTB and Kelch domains, respectively. The KEAP1-CUL3-ROC1 complex promoted NRF2 ubiquitination in vitro and knocking down Keap1 or CUL3 by short interfering RNA resulted in NRF2 protein accumulation in vivo. We suggest that Keap1 negatively regulates Nrf2 function in part by targeting Nrf2 for ubiquitination by the CUL3-ROC1 ligase and subsequent degradation by the proteasome. Blocking NRF2 degradation in cells expressing both KEAP1 and NRF2 by either inhibiting the proteasome activity or knocking down CUL3, resulted in NRF2 accumulation in the cytoplasm. These results may reconcile previously observed cytoplasmic sequestration of NRF2 by KEAP1 and suggest a possible regulatory step between KEAP1-NRF2 binding and NRF2 degradation.
Anil K. Jaiswal - One of the best experts on this subject based on the ideXlab platform.
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retraction prothymosin α mediates nuclear import of the inrf2 CUL3 rbx1 complex to degrade nuclear nrf2
Journal of Biological Chemistry, 2017Co-Authors: Suryakant K Niture, Anil K. JaiswalAbstract:Nrf2-mediated coordinated induction of a battery of defensive genes is a critical mechanism in cellular protection and survival. INrf2 (Keap1), an inhibitor of Nrf2, functions as an adaptor for CUL3·Rbx1-mediated degradation of Nrf2. A majority of the INrf2/CUL3·Rbx1 complex is localized in the cytosol that degrades cytosolic Nrf2. However, 10-15% of INrf2 is also localized inside the nucleus. INrf2 does not contain a defined nuclear import signal, and the mechanism of nuclear import and its function inside the nucleus remain obscure. Present studies demonstrate that the DGR region of INrf2 is required for nuclear import of INrf2. Studies also demonstrate that CUL3 and Rbx1 are also imported inside the nucleus in complex with INrf2. Interestingly, Nrf2 and prothymosin-α both bind to the DGR region of INrf2. However, it is prothymosin-α and not Nrf2 that mediates nuclear import of INrf2/CUL3·Rbx1 complex. Antioxidant treatment increases nuclear import of INrf2/CUL3·Rbx1 complex. The INrf2/CUL3·Rbx1 complex inside the nucleus exchanges prothymosin-α with Nrf2, resulting in degradation of Nrf2. These results led to the conclusion that prothymosin-α-mediated nuclear import of INrf2/CUL3·Rbx1 complex leads to ubiquitination and degradation of Nrf2 inside the nucleus presumably to regulate nuclear level of Nrf2 and rapidly switch off the activation of Nrf2 downstream gene expression.
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antioxidant induced inrf2 keap1 tyrosine 85 phosphorylation controls the nuclear export and degradation of the inrf2 CUL3 rbx1 complex to allow normal nrf2 activation and repression
Journal of Cell Science, 2012Co-Authors: James W Kaspar, Suryakant K Niture, Anil K. JaiswalAbstract:INrf2 (Keap1) serves as a negative regulator of the cytoprotective transcription factor Nrf2. At basal levels, INrf2 functions as a substrate adaptor to sequester Nrf2 into the CUL3–Rbx1 E3 ligase complex for ubiquitylation and proteasomal degradation. In response to antioxidants, Nrf2 is released from the INrf2–CUL3–Rbx1 complex and translocates into the nucleus, where it activates ARE-mediated cytoprotective gene expression. The present studies demonstrate that INrf2, CUL3 and Rbx1 export out of the nucleus and are degraded during the early or pre-induction response to antioxidants. Mutation of Tyr85 in INrf2 stymied the nuclear export of INrf2, suggesting that tyrosine phosphorylation controls the pre-induction nuclear export and degradation in response to antioxidants. The nuclear export of CUL3–Rbx1 were also blocked when INrf2Tyr85 was mutated, suggesting that INrf2–CUL3–Rbx1 undergo nuclear export as a complex. INrf2 siRNA also inhibited the nuclear export of CUL3–Rbx1, confirming that CUL3–Rbx1 requires INrf2 for nuclear export. Newly synthesized INrf2–CUL3–Rbx1 is imported back into the nucleus during the post-induction period to ubiquitylate and degrade Nrf2. Mutation of INrf2Tyr85 had no effect on activation of Nrf2 but led to nuclear accumulation of Nrf2 during the post-induction period owing to reduced export and degradation of Nrf2. Our results also showed that nuclear export and degradation followed by the new synthesis of INrf2–CUL3–Rbx1 controls the cellular abundance of the proteins during different phases of antioxidant responses. In conclusion, the early or pre-induction nuclear export of INrf2 in response to antioxidants is controlled by tyrosine phosphorylation, whereas the nuclear export of CUL3 and Rbx1 is controlled by INrf2, allowing normal activation or repression of Nrf2.
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an autoregulatory loop between nrf2 and CUL3 rbx1 controls their cellular abundance
Journal of Biological Chemistry, 2010Co-Authors: James W Kaspar, Anil K. JaiswalAbstract:The INrf2 (Keap1)/CUL3-Rbx1 complex constantly degrades Nrf2 under normal conditions. When a cell encounters oxidative or electrophilic stress, Nrf2 dissociates from the INrf2/CUL3-Rbx1 complex and translocates into the nucleus. In the nucleus, Nrf2 activates a myriad of antioxidant and defensive genes that protect cells. Nrf2 is then exported out of the nucleus and degraded. INrf2 serves as a substrate adaptor to link Nrf2 to CUL3 and Rbx1. CUL3 and Rbx1 make up the ubiquitin ligase complex that is responsible for the ubiquitination and degradation of Nrf2. Previously we have shown a feedback autoregulatory loop between Nrf2 and INrf2 indicating that Nrf2 regulates INrf2 by controlling its transcription. Here we are extending this research by demonstrating the presence of another feedback autoregulatory loop between CUL3-Rbx1 and Nrf2. Experiments using Hepa-1 and HepG2 cells indicate that Nrf2 controls its own degradation by regulating expression and induction of CUL3-Rbx1 genes. Treatment with the antioxidant tert-Butylhydroquinone (t-BHQ) leads to induction of CUL3-Rbx1 genes. Mutagenesis and transfection experiments identified an antioxidant response element in the forward and reverse strands of the proximal CUL3 and Rbx1 promoters, respectively, that Nrf2 binds and regulates expression and antioxidant induction of the CUL3-Rbx1 genes. In addition, short interfering RNA inhibition and overexpression of Nrf2 led to a respective decrease and increase in CUL3-Rbx1 gene expression. The increase in CUL3-Rbx1 leads to ubiquitination and degradation of Nrf2. These data suggest that Nrf2 regulates CUL3-Rbx1 by controlling regulation of expression and induction of CUL3-Rbx1. The induction of CUL3-Rbx1 control Nrf2 by increasing degradation.
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prothymosin alpha mediates nuclear import of the inrf2 CUL3 rbx1 complex to degrade nuclear nrf2
Journal of Biological Chemistry, 2009Co-Authors: Suryakant K Niture, Anil K. JaiswalAbstract:Abstract Nrf2-mediated coordinated induction of a battery of defensive genes is a critical mechanism in cellular protection and survival. INrf2 (Keap1), an inhibitor of Nrf2, functions as an adaptor for CUL3·Rbx1-mediated degradation of Nrf2. A majority of the INrf2/CUL3·Rbx1 complex is localized in the cytosol that degrades cytosolic Nrf2. However, 10-15% of INrf2 is also localized inside the nucleus. INrf2 does not contain a defined nuclear import signal, and the mechanism of nuclear import and its function inside the nucleus remain obscure. Present studies demonstrate that the DGR region of INrf2 is required for nuclear import of INrf2. Studies also demonstrate that CUL3 and Rbx1 are also imported inside the nucleus in complex with INrf2. Interestingly, Nrf2 and prothymosin-α both bind to the DGR region of INrf2. However, it is prothymosin-α and not Nrf2 that mediates nuclear import of INrf2/CUL3·Rbx1 complex. Antioxidant treatment increases nuclear import of INrf2/CUL3·Rbx1 complex. The INrf2/CUL3·Rbx1 complex inside the nucleus exchanges prothymosin-α with Nrf2, resulting in degradation of Nrf2. These results led to the conclusion that prothymosin-α-mediated nuclear import of INrf2/CUL3·Rbx1 complex leads to ubiquitination and degradation of Nrf2 inside the nucleus presumably to regulate nuclear level of Nrf2 and rapidly switch off the activation of Nrf2 downstream gene expression.
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prothymosin α mediates nuclear import of the inrf2 CUL3 rbx1 complex to degrade nuclear nrf2
Journal of Biological Chemistry, 2009Co-Authors: Suryakant K Niture, Anil K. JaiswalAbstract:Nrf2-mediated coordinated induction of a battery of defensive genes is a critical mechanism in cellular protection and survival. INrf2 (Keap1), an inhibitor of Nrf2, functions as an adaptor for CUL3·Rbx1-mediated degradation of Nrf2. A majority of the INrf2/CUL3·Rbx1 complex is localized in the cytosol that degrades cytosolic Nrf2. However, 10-15% of INrf2 is also localized inside the nucleus. INrf2 does not contain a defined nuclear import signal, and the mechanism of nuclear import and its function inside the nucleus remain obscure. Present studies demonstrate that the DGR region of INrf2 is required for nuclear import of INrf2. Studies also demonstrate that CUL3 and Rbx1 are also imported inside the nucleus in complex with INrf2. Interestingly, Nrf2 and prothymosin-α both bind to the DGR region of INrf2. However, it is prothymosin-α and not Nrf2 that mediates nuclear import of INrf2/CUL3·Rbx1 complex. Antioxidant treatment increases nuclear import of INrf2/CUL3·Rbx1 complex. The INrf2/CUL3·Rbx1 complex inside the nucleus exchanges prothymosin-α with Nrf2, resulting in degradation of Nrf2. These results led to the conclusion that prothymosin-α-mediated nuclear import of INrf2/CUL3·Rbx1 complex leads to ubiquitination and degradation of Nrf2 inside the nucleus presumably to regulate nuclear level of Nrf2 and rapidly switch off the activation of Nrf2 downstream gene expression.
Manabu Furukawa - One of the best experts on this subject based on the ideXlab platform.
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BTB protein Keap1 targets antioxidant transcription factor Nrf2 for ubiquitination by the Cullin 3-Roc1 ligase
2016Co-Authors: Manabu Furukawa, Yue XiongAbstract:The concentrations and functions of many eukaryotic proteins are regulated by the ubiquitin pathway, which consists of ubiquitin activation (E1), conjugation (E2), and ligation (E3). Cullins are a family of evolutionarily conserved proteins that assemble by far the largest family of E3 ligase complexes. Cullins, via a conserved C-terminal domain, bind with the RING finger protein Roc1 to recruit the catalytic function of E2. Via a distinct N-terminal domain, individual cullins bind to a protein motif present in multiple proteins to recruit specific substrates. Cullin 3 (CUL3), but not other cullins, binds directly with BTB domains to constitute a potentially large number of BTB-CUL3-ROC1 E3 ubiquitin ligases. Here we report that the human BTB-Kelch protein Keap1, a negative regulator of the antioxidative transcription factor Nrf2, binds to CUL3 and Nrf2 via its BTB and Kelch domains, respectively. The KEAP1-CUL3-ROC1 complex promoted NRF2 ubiquitination in vitro and knocking down Keap1 or CUL3 by short interfering RNA resulted in NRF2 protein accumulation in vivo. We suggest that Keap1 negatively regulates Nrf2 function in part by targeting Nrf2 for ubiquitination by the CUL3-ROC1 ligase and subsequent degradation by the proteasome. Blocking NRF2 degradation in cells expressing both KEAP1 and NRF2 by either inhibiting the proteasome activity or knocking down CUL3, resulted in NRF2 accumulation in the cytoplasm. These results may reconcile previously observed cytoplasmic seques-tration of NRF2 by KEAP1 and suggest a possible regulatory step between KEAP1-NRF2 binding and NRF
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the CUL3 klhl18 ligase regulates mitotic entry and ubiquitylates aurora a
Biology Open, 2012Co-Authors: Saili Moghe, Fei Jiang, Yoshie Miura, Ronald L Cerny, Ming Ying Tsai, Manabu FurukawaAbstract:The cullin-RING family of ubiquitin ligases regulates diverse cellular functions, such as cell cycle control, via ubiquitylation of specific substrates. CUL3 targets its substrates through BTB proteins. Here we show that depletion of CUL3 and the BTB protein KLHL18 causes a delay in mitotic entry. Centrosomal activation of Aurora-A, a kinase whose activity is required for entry into mitosis, is also delayed in depleted cells. Moreover, we identify Aurora-A as a KLHL18-interacting partner. Overexpression of KLHL18 and CUL3 promotes Aurora-A ubiquitylation in vivo, and the CUL3-KLHL18-ROC1 ligase ubiquitylates Aurora-A in vitro. Our study reveals that the CUL3-KLHL18 ligase is required for timely entry into mitosis, as well as for the activation of Aurora-A at centrosomes. We propose that the CUL3-KLHL18 ligase regulates mitotic entry through an Aurora-A-dependent pathway.
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characterization of rhobtb dependent CUL3 ubiquitin ligase complexes evidence for an autoregulatory mechanism
Experimental Cell Research, 2008Co-Authors: Jessica Berthold, Manabu Furukawa, Yoshie Miura, Kristina Schenkova, Sonia Ramos, Pontus Aspenstrom, Francisco RiveroAbstract:RhoBTB proteins are atypical members of the Rho family of small GTPases. Two of the three RhoBTB proteins, RhoBTB1 and RhoBTB2, have been proposed as tumor suppressors and might function as adaptors of CUL3-dependent ubiquitin ligase complexes. Using yeast two-hybrid analysis and co-immunoprecipitation we show that all three RhoBTB proteins interact with CUL3. The interaction requires the N-terminal region of CUL3 and the first BTB domain of RhoBTB. RhoBTB3, the only RhoBTB with a prenylation motif, associates with vesicles that are frequently found in the vicinity of microtubules, suggesting a participation in some aspects of vesicle trafficking. We also show that RhoBTB2 and RhoBTB3 are capable of homo and heterodimerizing through the BTB domain region. The GTPase domain, which does not bind GTP, is able to interact with the BTB domain region, thus preventing proteasomal degradation of RhoBTB. This fits into a model in which an intramolecular interaction maintains RhoBTB in an inactive state, preventing the formation or the functionality of CUL3-dependent complexes. We also report a significantly decreased expression of RHOBTB and CUL3 genes in kidney and breast tumor samples and a very good correlation in the expression changes between RHOBTB and CUL3 that suggests that these genes are subject to a common inactivation mechanism in tumors.
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btb protein keap1 targets antioxidant transcription factor nrf2 for ubiquitination by the cullin 3 roc1 ligase
Molecular and Cellular Biology, 2005Co-Authors: Manabu Furukawa, Yue XiongAbstract:The concentrations and functions of many eukaryotic proteins are regulated by the ubiquitin pathway, which consists of ubiquitin activation (E1), conjugation (E2), and ligation (E3). Cullins are a family of evolutionarily conserved proteins that assemble by far the largest family of E3 ligase complexes. Cullins, via a conserved C-terminal domain, bind with the RING finger protein Roc1 to recruit the catalytic function of E2. Via a distinct N-terminal domain, individual cullins bind to a protein motif present in multiple proteins to recruit specific substrates. Cullin 3 (CUL3), but not other cullins, binds directly with BTB domains to constitute a potentially large number of BTB-CUL3-ROC1 E3 ubiquitin ligases. Here we report that the human BTB-Kelch protein Keap1, a negative regulator of the antioxidative transcription factor Nrf2, binds to CUL3 and Nrf2 via its BTB and Kelch domains, respectively. The KEAP1-CUL3-ROC1 complex promoted NRF2 ubiquitination in vitro and knocking down Keap1 or CUL3 by short interfering RNA resulted in NRF2 protein accumulation in vivo. We suggest that Keap1 negatively regulates Nrf2 function in part by targeting Nrf2 for ubiquitination by the CUL3-ROC1 ligase and subsequent degradation by the proteasome. Blocking NRF2 degradation in cells expressing both KEAP1 and NRF2 by either inhibiting the proteasome activity or knocking down CUL3, resulted in NRF2 accumulation in the cytoplasm. These results may reconcile previously observed cytoplasmic sequestration of NRF2 by KEAP1 and suggest a possible regulatory step between KEAP1-NRF2 binding and NRF2 degradation.
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targeting of protein ubiquitination by btb cullin 3 roc1 ubiquitin ligases
Nature Cell Biology, 2003Co-Authors: Manabu Furukawa, Christoph H Borchers, Yue XiongAbstract:The concentrations and functions of many cellular proteins are regulated by the ubiquitin pathway. Cullin family proteins bind with the RING-finger protein Roc1 to recruit the ubiquitin-conjugating enzyme (E2) to the ubiquitin ligase complex (E3). Cul1 and Cul7, but not other cullins, bind to an adaptor protein, Skp1. Cul1 associates with one of many F-box proteins through Skp1 to assemble various SCF–Roc1 E3 ligases that each selectively ubiquitinate one or more specific substrates. Here, we show that CUL3, but not other cullins, binds directly to multiple BTB domains through a conserved amino-terminal domain. In vitro, CUL3 promoted ubiquitination of Caenorhabditis elegans MEI-1, a katanin-like protein whose degradation requires the function of both CUL3 and BTB protein MEL-26. We suggest that in vivo there exists a potentially large number of BCR3 (BTB–CUL3–Roc1) E3 ubiquitin ligases.
Jeffrey D. Singer - One of the best experts on this subject based on the ideXlab platform.
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CUL3 is required for normal development of the mammary gland
Cell and Tissue Research, 2021Co-Authors: Cristina M. Cummings, Jeffrey D. SingerAbstract:Cullin 3 (CUL3) has recently been implicated in a multitude of different processes, including the oxidative stress response, autophagy, tumorigenesis, and differentiation. To investigate the role of CUL3 in mammary gland development, we created a mouse model system using Cre-lox targeting where CUL3 is specifically deleted from the mammary gland. Such MMTV-Cre CUL3^Flx/Flx mice examined at 2 and 3 months of age show delays and defects in mammary gland development. Mammary ductal trees from CUL3-deficient mammary glands exhibit delayed forward growth through the mammary fat pad, dilation of the ducts, and abnormal morphology of some of the epithelial structures within the gland. Additionally, terminal end buds are larger and less plentiful in MMTV-Cre CUL3^Flx/Flx mammary glands, and there is significantly less primary and secondary branching compared to control animals. In contrast, by 6 months of age, the mammary ductal tree has grown to fill the entire mammary fat pad in glands lacking CUL3. However, distorted epithelial structures and dilated ducts persist. MMTV-Cre CUL3^Flx/Flx mothers are able to nourish their litters, but the process of involution is slightly delayed in mammary glands lacking CUL3. Therefore, we conclude that while CUL3 is not essential for mammary gland function, CUL3 is required for the mammary gland to proceed normally through development.
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CUL3 regulates cyclin E1 protein abundance via a degron located within the N-terminal region of cyclin E.
Journal of cell science, 2019Co-Authors: Brittney Davidge, Katia Graziella De Oliveira Rebola, Larry N. Agbor, Curt D. Sigmund, Jeffrey D. SingerAbstract:Cyclin E and its binding partner Cdk2 control the G1/S transition in mammalian cells. Increased levels of cyclin E are found in some cancers. Additionally, proteolytic removal of the cyclin E N-terminus occurs in some cancers and is associated with increased cyclin E-Cdk2 activity and poor clinical prognosis. Cyclin E levels are tightly regulated and controlled in part through ubiquitin-mediated degradation initiated by one of two E3 ligases, Cul1 and CUL3. Cul1 ubiquitylates phosphorylated cyclin E, but the mechanism through which CUL3 ubiquitylates cyclin E is poorly understood. In experiments to ascertain how CUL3 mediates cyclin E destruction, we identified a degron on cyclin E that CUL3 targets for ubiquitylation. Recognition of the degron and binding of CUL3 does not require a BTB domain-containing adaptor protein. Additionally, this degron is lacking in N-terminally truncated cyclin E. Our results describe a mechanism whereby N-terminally truncated cyclin E can avoid the CUL3-mediated degradation pathway. This mechanism helps to explain the increased activity that is associated with the truncated cyclin E variants that occurs in some cancers.
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disruption of CUL3 mediated ubiquitination causes proximal tubule injury and kidney fibrosis
Scientific Reports, 2019Co-Authors: Rafael Kramann, Turgay Saritas, Catherina A Cuevas, Mohammed Z Ferdaus, Christoph Kuppe, Marcus J Moeller, Jurgen Floege, Jeffrey D. SingerAbstract:Cullin 3 (CUL3) is part of the ubiquitin proteasomal system and controls several cellular processes critical for normal organ function including the cell cycle, and Keap1/Nrf2 signaling. Kidney tubule-specific CUL3 disruption causes tubulointerstitial fibrosis, but little is known about the mechanisms. Therefore, we tested the hypothesis that dysregulation of the cell cycle and Keap1/Nrf2 pathway play a role in initiating the kidney injury upon CUL3 disruption. CUL3 deletion increased expression of cyclin E and p21, associated with uncontrolled proliferation, DNA damage, and apoptosis, all of which preceded proximal tubule injury. The cdk2-cyclin E inhibitor roscovitine did not prevent the effects of CUL3 deletion, but instead exacerbated the kidney injury. Injury occurred despite accumulation and activation of CUL3 substrate Keap1/Nrf2, proposed to be protective in kidney injury. CUL3 disruption led to progressive interstitial inflammation, functionally relevant renal fibrosis and death. Finally, we observed reduced CUL3 expression in several AKI and CKD mouse models and in fibrotic human kidney tissue. These data establish CUL3 knockout mice as a novel genetic CKD model in which dysregulation of the cell cycle may play a primary role in initiating tubule injury, and that CUL3 dysregulation could contribute to acute and fibrotic kidney disease.
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btb zf factors recruit the e3 ligase cullin 3 to regulate lymphoid effector programs
Nature, 2012Co-Authors: Rebecca Mathew, Jeffrey D. Singer, Michael P Seiler, Seth T Scanlon, Aiping Mao, Michael G Constantinides, Clara Bertozzivilla, Albert BendelacAbstract:The differentiation of several T- and B-cell effector programs in the immune system is directed by signature transcription factors that induce rapid epigenetic remodelling. Here we report that promyelocytic leukaemia zinc finger (PLZF), the BTB-zinc finger (BTB-ZF) transcription factor directing the innate-like effector program of natural killer T-cell thymocytes, is prominently associated with cullin 3 (CUL3), an E3 ubiquitin ligase previously shown to use BTB domain-containing proteins as adaptors for substrate binding. PLZF transports CUL3 to the nucleus, where the two proteins are associated within a chromatin-modifying complex. Furthermore, PLZF expression results in selective ubiquitination changes of several components of this complex. CUL3 was also found associated with the BTB-ZF transcription factor BCL6, which directs the germinal-centre B cell and follicular T-helper cell programs. Conditional CUL3 deletion in mice demonstrated an essential role for CUL3 in the development of PLZF- and BCL6-dependent lineages. We conclude that distinct lineage-specific BTB-ZF transcription factors recruit CUL3 to alter the ubiquitination pattern of their associated chromatin-modifying complex. We propose that this new function is essential to direct the differentiation of several T- and B-cell effector programs, and may also be involved in the oncogenic role of PLZF and BCL6 in leukaemias and lymphomas.
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the CUL3 klhdc5 e3 ligase regulates p60 katanin and is required for normal mitosis in mammalian cells
Journal of Biological Chemistry, 2009Co-Authors: Cristina M. Cummings, Peter W. Baas, Cornelia A. Bentley, Sarah A. Perdue, Jeffrey D. SingerAbstract:The proper regulation of factors involved in mitosis is crucial to ensure normal cell division. Levels and activities of proteins are regulated in many ways, one of which is ubiquitin-mediated protein degradation. E3 ubiquitin ligases are involved in targeting specific substrates for degradation by facilitating their ubiquitination. In seeking to elucidate additional biological roles for CUL3 we performed a two-hybrid screen and identified Ctb9/KLHDC5 as a CUL3-interacting protein. Overexpression of Ctb9/KLHDC5 resulted in an increase in microtubule density as well as persistent microtubule bridges between post-mitotic cells. Conversely, down-regulation of Ctb9/KLHDC5 showed a pronounced reduction in microtubule density. Based on these observations, we examined the interactions between CUL3, Ctb9/KLHDC5, and the microtubule-severing protein, p60/katanin. Here we show that p60/katanin interacts with a complex consisting of CUL3 and Ctb9/KLHDC5, which results in ubiquitin laddering of p60/katanin. Also, CUL3-deficient cells or Ctb9/KLHDC5-deficient cells show an increase in p60/katanin levels, indicating that CUL3/Ctb9/KLHDC5 is required for efficient p60/katanin removal. We demonstrate a novel regulatory mechanism for p60/katanin that occurs at the level of targeted proteolysis to allow normal mitotic progression in mammalian cells.
Suryakant K Niture - One of the best experts on this subject based on the ideXlab platform.
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retraction prothymosin α mediates nuclear import of the inrf2 CUL3 rbx1 complex to degrade nuclear nrf2
Journal of Biological Chemistry, 2017Co-Authors: Suryakant K Niture, Anil K. JaiswalAbstract:Nrf2-mediated coordinated induction of a battery of defensive genes is a critical mechanism in cellular protection and survival. INrf2 (Keap1), an inhibitor of Nrf2, functions as an adaptor for CUL3·Rbx1-mediated degradation of Nrf2. A majority of the INrf2/CUL3·Rbx1 complex is localized in the cytosol that degrades cytosolic Nrf2. However, 10-15% of INrf2 is also localized inside the nucleus. INrf2 does not contain a defined nuclear import signal, and the mechanism of nuclear import and its function inside the nucleus remain obscure. Present studies demonstrate that the DGR region of INrf2 is required for nuclear import of INrf2. Studies also demonstrate that CUL3 and Rbx1 are also imported inside the nucleus in complex with INrf2. Interestingly, Nrf2 and prothymosin-α both bind to the DGR region of INrf2. However, it is prothymosin-α and not Nrf2 that mediates nuclear import of INrf2/CUL3·Rbx1 complex. Antioxidant treatment increases nuclear import of INrf2/CUL3·Rbx1 complex. The INrf2/CUL3·Rbx1 complex inside the nucleus exchanges prothymosin-α with Nrf2, resulting in degradation of Nrf2. These results led to the conclusion that prothymosin-α-mediated nuclear import of INrf2/CUL3·Rbx1 complex leads to ubiquitination and degradation of Nrf2 inside the nucleus presumably to regulate nuclear level of Nrf2 and rapidly switch off the activation of Nrf2 downstream gene expression.
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antioxidant induced inrf2 keap1 tyrosine 85 phosphorylation controls the nuclear export and degradation of the inrf2 CUL3 rbx1 complex to allow normal nrf2 activation and repression
Journal of Cell Science, 2012Co-Authors: James W Kaspar, Suryakant K Niture, Anil K. JaiswalAbstract:INrf2 (Keap1) serves as a negative regulator of the cytoprotective transcription factor Nrf2. At basal levels, INrf2 functions as a substrate adaptor to sequester Nrf2 into the CUL3–Rbx1 E3 ligase complex for ubiquitylation and proteasomal degradation. In response to antioxidants, Nrf2 is released from the INrf2–CUL3–Rbx1 complex and translocates into the nucleus, where it activates ARE-mediated cytoprotective gene expression. The present studies demonstrate that INrf2, CUL3 and Rbx1 export out of the nucleus and are degraded during the early or pre-induction response to antioxidants. Mutation of Tyr85 in INrf2 stymied the nuclear export of INrf2, suggesting that tyrosine phosphorylation controls the pre-induction nuclear export and degradation in response to antioxidants. The nuclear export of CUL3–Rbx1 were also blocked when INrf2Tyr85 was mutated, suggesting that INrf2–CUL3–Rbx1 undergo nuclear export as a complex. INrf2 siRNA also inhibited the nuclear export of CUL3–Rbx1, confirming that CUL3–Rbx1 requires INrf2 for nuclear export. Newly synthesized INrf2–CUL3–Rbx1 is imported back into the nucleus during the post-induction period to ubiquitylate and degrade Nrf2. Mutation of INrf2Tyr85 had no effect on activation of Nrf2 but led to nuclear accumulation of Nrf2 during the post-induction period owing to reduced export and degradation of Nrf2. Our results also showed that nuclear export and degradation followed by the new synthesis of INrf2–CUL3–Rbx1 controls the cellular abundance of the proteins during different phases of antioxidant responses. In conclusion, the early or pre-induction nuclear export of INrf2 in response to antioxidants is controlled by tyrosine phosphorylation, whereas the nuclear export of CUL3 and Rbx1 is controlled by INrf2, allowing normal activation or repression of Nrf2.
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prothymosin alpha mediates nuclear import of the inrf2 CUL3 rbx1 complex to degrade nuclear nrf2
Journal of Biological Chemistry, 2009Co-Authors: Suryakant K Niture, Anil K. JaiswalAbstract:Abstract Nrf2-mediated coordinated induction of a battery of defensive genes is a critical mechanism in cellular protection and survival. INrf2 (Keap1), an inhibitor of Nrf2, functions as an adaptor for CUL3·Rbx1-mediated degradation of Nrf2. A majority of the INrf2/CUL3·Rbx1 complex is localized in the cytosol that degrades cytosolic Nrf2. However, 10-15% of INrf2 is also localized inside the nucleus. INrf2 does not contain a defined nuclear import signal, and the mechanism of nuclear import and its function inside the nucleus remain obscure. Present studies demonstrate that the DGR region of INrf2 is required for nuclear import of INrf2. Studies also demonstrate that CUL3 and Rbx1 are also imported inside the nucleus in complex with INrf2. Interestingly, Nrf2 and prothymosin-α both bind to the DGR region of INrf2. However, it is prothymosin-α and not Nrf2 that mediates nuclear import of INrf2/CUL3·Rbx1 complex. Antioxidant treatment increases nuclear import of INrf2/CUL3·Rbx1 complex. The INrf2/CUL3·Rbx1 complex inside the nucleus exchanges prothymosin-α with Nrf2, resulting in degradation of Nrf2. These results led to the conclusion that prothymosin-α-mediated nuclear import of INrf2/CUL3·Rbx1 complex leads to ubiquitination and degradation of Nrf2 inside the nucleus presumably to regulate nuclear level of Nrf2 and rapidly switch off the activation of Nrf2 downstream gene expression.
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prothymosin α mediates nuclear import of the inrf2 CUL3 rbx1 complex to degrade nuclear nrf2
Journal of Biological Chemistry, 2009Co-Authors: Suryakant K Niture, Anil K. JaiswalAbstract:Nrf2-mediated coordinated induction of a battery of defensive genes is a critical mechanism in cellular protection and survival. INrf2 (Keap1), an inhibitor of Nrf2, functions as an adaptor for CUL3·Rbx1-mediated degradation of Nrf2. A majority of the INrf2/CUL3·Rbx1 complex is localized in the cytosol that degrades cytosolic Nrf2. However, 10-15% of INrf2 is also localized inside the nucleus. INrf2 does not contain a defined nuclear import signal, and the mechanism of nuclear import and its function inside the nucleus remain obscure. Present studies demonstrate that the DGR region of INrf2 is required for nuclear import of INrf2. Studies also demonstrate that CUL3 and Rbx1 are also imported inside the nucleus in complex with INrf2. Interestingly, Nrf2 and prothymosin-α both bind to the DGR region of INrf2. However, it is prothymosin-α and not Nrf2 that mediates nuclear import of INrf2/CUL3·Rbx1 complex. Antioxidant treatment increases nuclear import of INrf2/CUL3·Rbx1 complex. The INrf2/CUL3·Rbx1 complex inside the nucleus exchanges prothymosin-α with Nrf2, resulting in degradation of Nrf2. These results led to the conclusion that prothymosin-α-mediated nuclear import of INrf2/CUL3·Rbx1 complex leads to ubiquitination and degradation of Nrf2 inside the nucleus presumably to regulate nuclear level of Nrf2 and rapidly switch off the activation of Nrf2 downstream gene expression.