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ABSTRACT: Hyper-activation of mechanistic target of rapamycin complex 1 (mTORC1) and its effector kinase s6 kinase 1 (S6K1) is known to trigger multisite seryl phosphorylation of insulin receptor substrate 1 (IRS1), leading to its ubiquitination and degradation. This negative feedback inhibition functions to restrain phospho inositide 3-kinase (PI3-K) activity, and plays critical roles in the pathogenesis of cancer and type II diabetes. Recent work has implicated a role for cullin-RING E3 ubiquitin ligase 7 (CRL7) in targeting IRS1 for mTORC1/S6K1-dependent degradation. In the present study we have employed both cell-based degradation and reconstituted ubiquitination approaches to define molecular features associated with IRS1 critical for CRL7-mediated ubiquitination and degradation. We have mapped IRS1 degradation signal sequence to its N-terminal 574 amino acid residues, of which the integrity of Ser307/Ser312 and Ser527, each constituting a S6K1 phosphorylation consensus site, was indispensible for supporting CRL7-forced degradation. In vitro, S6K1 was able to support the ubiquitination of bacterially expressed IRS1 N-terminal fragment by CRL7 but at low levels. In contrast, CRL7 supported efficient ubiquitination of IRS1 N-terminal fragment in hyper-phosphorylated form, which was isolated from infected insect cells, suggesting requirement of additional phosphorylation by kinases yet to be identified. Finally, removal of IRS1 amino acids 1-260 led to substantial reduction of ubiquitination efficiency suggesting a role for this region in mediating productive interactions with CRL7. The requirement of multisite phosphorylation and IRS1 N-terminus for its turnover may ensure that complete IRS1 degradation occurs only when mTORC1 and S6K1 reach exceedingly high levels.
Journal of Biological Chemistry 10/2012; · 4.77 Impact Factor
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ABSTRACT: RING E3 ligases are proteins that must selectively recruit an E2-conjugating enzyme and facilitate ubiquitin transfer to a substrate. It is not clear how a RING E3 ligase differentiates a naked E2 enzyme from the E2∼ubiquitin-conjugated form or how this is altered upon ubiquitin transfer. RING-box protein 1 (Rbx1/ROC1) is a key protein found in the Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligase complex that functions with the E2 ubiquitin conjugating enzyme CDC34. The solution structure of Rbx1/ROC1 revealed a globular RING domain (residues 40-108) stabilized by three structural zinc ions (root mean square deviation 0.30 ± 0.04 Å) along with a disordered N terminus (residues 12-39). Titration data showed that Rbx1/ROC1 preferentially recruits CDC34 in its ubiquitin-conjugated form and favors this interaction by 50-fold compared with unconjugated CDC34. Furthermore, NMR and biochemical assays identified residues in helix α2 of Rbx1/ROC1 that are essential for binding and activating CDC34∼ubiquitin for ubiquitylation. Taken together, this work provides the first direct structural and biochemical evidence showing that polyubiquitylation by the RING E3 ligase Rbx1/ROC1 requires the preferential recruitment of an E2∼ubiquitin complex and subsequent release of the unconjugated E2 protein upon ubiquitin transfer to a substrate or ubiquitin chain.
Journal of Biological Chemistry 03/2012; 287(21):17374-85. · 4.77 Impact Factor
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ABSTRACT: Conjugation of Nedd8 to a cullin protein, termed neddylation, is an evolutionarily conserved process that functions to activate the cullin-RING family E3 ubiquitin ligases, leading to increased proteasomal degradation of a wide range of substrate proteins. Recent emerging evidence demonstrates that cellular neddylation requires the action of Dcn1, which, in humans, consists of five homologues designated as hDCNL1-5. Here we revealed a previously unknown mechanism that regulates hDCNL1. In cultured mammalian cells ectopically expressed hDCNL1 was mono-ubiquitinated predominantly at K143, K149, and K171. Using a classical chromatographic purification strategy, we identified Nedd4-1 as an E3 ligase that can catalyze mono-ubiquitination of hDCNL1 in a reconstituted ubiquitination system. In addition, the hDCNL1 N-terminal ubiquitin-binding domain is necessary and sufficient to mediate mono-ubiquitination. Finally, fluorescence microscopic and subcellular fractionation analyses revealed a role for mono-ubiquitination in driving nuclear export of hDCNL1. Taken together, these results suggest a mono-ubiquitination-mediated mechanism that governs nuclear-cytoplasmic trafficking of hDCNL1, thereby regulating hDCNL1-dependent activation of the cullin-RING E3 ubiquitin ligases in selected cellular compartments.
Journal of Biological Chemistry 08/2011; 286(39):34060-70. · 4.77 Impact Factor
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ABSTRACT: Cullin proteins are molecular scaffolds that have crucial roles in the post-translational modification of cellular proteins involving ubiquitin. The mammalian cullin protein family comprises eight members (CUL1 to CUL7 and PARC), which are characterized by a cullin homology domain. CUL1 to CUL7 assemble multi-subunit Cullin-RING E3 ubiquitin ligase (CRL) complexes, the largest family of E3 ligases with more than 200 members. Although CUL7 and PARC are present only in chordates, other members of the cullin protein family are found in Drosophila melanogaster, Caenorhabditis elegans, Arabidopsis thaliana and yeast. A cullin protein tethers both a substrate-targeting unit, often through an adaptor protein, and the RING finger component in a CRL. The cullin-organized CRL thus positions a substrate close to the RING-bound E2 ubiquitin-conjugating enzyme, which catalyzes the transfer of ubiquitin to the substrate. In addition, conjugation of cullins with the ubiquitin-like molecule Nedd8 modulates activation of the corresponding CRL complex, probably through conformational regulation of the interactions between cullin's carboxy-terminal tail and CRL's RING subunit. Genetic studies in several model organisms have helped to unravel a multitude of physiological functions associated with cullin proteins and their respective CRLs. CRLs target numerous substrates and thus have an impact on a range of biological processes, including cell growth, development, signal transduction, transcriptional control, genomic integrity and tumor suppression. Moreover, mutations in CUL7 and CUL4B genes have been linked to hereditary human diseases.
Genome biology 04/2011; 12(4):220. · 6.63 Impact Factor
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ABSTRACT: SAG (sensitive to apoptosis gene; also known as RBX2 or ROC2) is a dual-function protein with antioxidant activity when acting alone or E3 ligase activity when complexed with other components of SCF (Skp1, cullins, F-box proteins) E3 ubiquitin ligases. SAG acts as a survival protein to inhibit apoptosis induced by a variety of stresses. Our recent work showed that SAG siRNA silencing sensitized cancer cells to radiation but the mechanism responsible remains elusive. Here we report that complete elimination of Sag expression via a gene-trapping strategy significantly sensitized mouse embryonic stem (ES) cells to radiation, with a sensitizing enhancement rate of 1.5-1.6. Radiosensitization was associated with increased steady-state levels of intracellular ROS (including superoxide) 24h after irradiation as well as enhancement of radiation-induced apoptosis. Furthermore, Sag elimination abrogated IkappaBalpha degradation leading to inhibition of NF-kappaB activation. Further detailed analysis revealed that IkappaBalpha is a direct substrate of SAG-SCF(beta-TrCP) E3 ubiquitin ligase. Taken together, these results support the hypothesis that Sag elimination via gene disruption sensitizes ES cells to radiation-induced cell killing by mechanisms that involve increased steady-state levels of ROS and decreased activation of NF-kappaB.
Free radical biology & medicine 09/2010; 49(6):976-83. · 5.42 Impact Factor
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ABSTRACT: We describe a mechanistic model of polyubiquitination by the SCF(beta TrCP2) E3 ubiquitin (Ub) ligase using human I kappaB alpha as a substrate. Biochemical reconstitution experiments revealed that the polyubiquitination of I kappaB alpha began with the action of the UbcH5 E2 Ub-conjugating enzyme, transferring a single Ub to I kappaB alpha K21/K22 rapidly and efficiently. Subsequently, the Cdc34 E2 functioned in the formation of polyubiquitin chains. It was determined that a Ub fused at I kappaB alpha K21 acts as a receptor, directing Cdc34 for rapid and efficient K48-linked Ub chain synthesis that depends on SCF(beta TrCP2) and the substrate's N terminus. The I kappaB alpha-linked fusion Ub appears to mediate direct contacts with Cdc34 and the SCF's RING subcomplex. Taken together, these results suggest a role for the multifaceted interactions between the I kappaB alpha K21/K22-linked receptor Ub, the SCF's RING complex, and Cdc34 approximately S approximately Ub in establishing the optimal orientation of the receptor Ub to drive conjugation.
Molecular cell 03/2010; 37(6):784-96. · 14.61 Impact Factor
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ABSTRACT: Cdc34 is an E2 ubiquitin-conjugating enzyme that functions in conjunction with SCF (Skp1.Cullin 1.F-box) E3 ubiquitin ligase to catalyze covalent attachment of polyubiquitin chains to a target protein. Here we identified direct interactions between the human Cdc34 C terminus and ubiquitin using NMR chemical shift perturbation assays. The ubiquitin binding activity was mapped to two separate Cdc34 C-terminal motifs (UBS1 and UBS2) that comprise residues 206-215 and 216-225, respectively. UBS1 and UBS2 bind to ubiquitin in the proximity of ubiquitin Lys(48) and C-terminal tail, both of which are key sites for conjugation. When bound to ubiquitin in one orientation, the Cdc34 UBS1 aromatic residues (Phe(206), Tyr(207), Tyr(210), and Tyr(211)) are probably positioned in the vicinity of ubiquitin C-terminal residue Val(70). Replacement of UBS1 aromatic residues by glycine or of ubiquitin Val(70) by alanine decreased UBS1-ubiquitin affinity interactions. UBS1 appeared to support the function of Cdc34 in vivo because human Cdc34(1-215) but not Cdc34(1-200) was able to complement the growth defect by yeast Cdc34 mutant strain. Finally, reconstituted IkappaBalpha ubiquitination analysis revealed a role for each adjacent pair of UBS1 aromatic residues (Phe(206)/Tyr(207), Tyr(210)/Tyr(211)) in conjugation, with Tyr(210) exhibiting the most pronounced catalytic function. Intriguingly, Cdc34 Tyr(210) was required for the transfer of the donor ubiquitin to a receptor lysine on either IkappaBalpha or a ubiquitin in a manner that depended on the neddylated RING sub-complex of the SCF. Taken together, our results identified a new ubiquitin binding activity within the human Cdc34 C terminus that contributes to SCF-dependent ubiquitination.
Journal of Biological Chemistry 03/2010; 285(23):17754-62. · 4.77 Impact Factor
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ABSTRACT: The COP9 signalosome (CSN) is a multiprotein complex that plays a critical role in diverse cellular and developmental processes in various eukaryotic organisms. Despite of its significance, current understanding of the biological functions and regulatory mechanisms of the CSN complex is still very limited. To unravel these molecular mechanisms, we have performed a comprehensive proteomic analysis of the human CSN complex using a new purification method and quantitative mass spectrometry. Purification of the human CSN complex from a stable 293 cell line expressing N-terminal HBTH-tagged CSN5 subunit was achieved by high-affinity streptavidin binding with TEV cleavage elution. Mass spectrometric analysis of the purified CSN complex has revealed the identity of its composition as well as N-terminal modification and phosphorylation of the CSN subunits. N-terminal modifications were determined for seven subunits, six of which have not been reported previously, and six novel phosphorylation sites were also identified. Additionally, we have applied the newly developed MAP-SILAC and PAM-SILAC methods to decipher the dynamics of the human CSN interacting proteins. A total of 52 putative human CSN interacting proteins were identified, most of which are reported for the first time. In comparison to PAM-SILAC results, 20 proteins were classified as stable interactors, whereas 20 proteins were identified as dynamic ones. This work presents the first comprehensive characterization of the human CSN complex by mass spectrometry-based proteomic approach, providing valuable information for further understanding of CSN complex structure and biological functions.
Journal of Proteome Research 11/2008; 7(11):4914-25. · 5.11 Impact Factor
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ABSTRACT: Cullin7 (CUL7) is a molecular scaffold that organizes an E3 ubiquitin ligase containing the F-box protein Fbw8, Skp1 and the ROC1 RING finger protein. Dysregulation of the CUL7 E3 Ligase has been directly linked to hereditary human diseases as cul7 germline mutations were found in patients with autosomal-recessive 3-M and Yakuts short stature syndromes, which are characterized by profound pre- and postnatal growth retardation. In addition, genetic ablation of CUL7 in mice resulted in intrauterine growth retardation and perinatal lethality, underscoring its importance for growth regulation. The recent identification of insulin receptor substrate 1, a critical mediator of insulin and insulin-like growth factor-1 signaling, as the proteolytic target of the CUL7 E3 ligase, provided a molecular link between CUL7 and a well-established growth regulatory pathway. This result, coupled with other studies demonstrating interactions between CUL7 and the p53 tumor suppressor protein, as well as the simian virus 40 large T antigen oncoprotein, further implicated CUL7 as a novel player in growth control and suggested pathomechanistic insights into CUL7-linked growth retardation syndromes.
Cell cycle (Georgetown, Tex.) 11/2008; 7(20):3154-61. · 5.36 Impact Factor
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ABSTRACT: SCF (Skp1 x CUL1 x F-box protein x ROC1) E3 ubiquitin ligase and Cdc34 E2-conjugating enzyme catalyze polyubiquitination in a precisely regulated fashion. Here, we describe biochemical evidence suggesting an autoinhibitory role played by the human CUL1 ECTD (extreme C-terminal domain; spanning the C-terminal 50 amino acids), a region that is predicted to contact the ROC1 RING finger protein by structural studies. We showed that ECTD did not contribute to CUL1's stable association with ROC1. Remarkably, deletion of ECTD, or missense mutations designed to disrupt the predicted ECTD x ROC1 interaction, markedly increased the ability of SCF(betaTrCP2) to promote IkappaB alpha polyubiquitination and polyubiquitin chain assembly by Cdc34 in vitro. Thus, disruption of ECTD yields in vitro effects that parallel SCF activation by Nedd8 conjugation to CUL1. We propose that SCF may be subject to autoinhibitory regulation, in which Nedd8 conjugation acts as a molecular switch to drive the E3 into an active state by diminishing the inhibitory ECTD x ROC1 interaction.
Proceedings of the National Academy of Sciences 09/2008; 105(34):12230-5. · 9.68 Impact Factor
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Xinsong Xu,
Antonio Sarikas,
Dora C Dias-Santagata,
Georgia Dolios,
Pascal J Lafontant,
Shih-Chong Tsai,
Wuqiang Zhu,
Hidehiro Nakajima,
Hisako O Nakajima,
Loren J Field,
Rong Wang, Zhen-Qiang Pan
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ABSTRACT: Recent genetic studies have documented a pivotal growth-regulatory role played by the Cullin 7 (CUL7) E3 ubiquitin ligase complex containing the Fbw8-substrate-targeting subunit, Skp1, and the ROC1 RING finger protein. In this report, we identified insulin receptor substrate 1 (IRS-1), a critical mediator of the insulin/insulin-like growth factor 1 signaling, as a proteolytic target of the CUL7 E3 ligase in a manner that depends on mammalian target of rapamycin and the p70 S6 kinase activities. Interestingly, while embryonic fibroblasts of Cul7-/- mice were found to accumulate IRS-1 and exhibit increased activation of IRS-1's downstream Akt and MEK/ERK pathways, these null cells grew poorly and displayed phenotypes reminiscent of those associated with oncogene-induced senescence. Taken together, our findings demonstrate a key role for the CUL7 E3 in targeting IRS-1 for degradation, a process that may contribute to the regulation of cellular senescence.
Molecular cell 06/2008; 30(4):403-14. · 14.61 Impact Factor
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ABSTRACT: Nedd8 is a small ubiquitin-like protein that can be conjugated to substrate-proteins in a process known as neddylation. Although neddylation plays a critical regulatory role in cell proliferation and development, the spectrum of Nedd8 substrates and its interaction network remain poorly understood. To explore the neddylation pathway at the proteome level, we have affinity purified Nedd8 modified and associated proteins from HEK293 cells stably expressing GST-Nedd8 and employed LC-MS/MS for subsequent protein identification. A total of 496 GST-Nedd8 modified and associated proteins have been identified, including all of the eight cullin family members (i.e., Cul-1, -2, -3, -4A, -4B, -5, -7, and Parc) that are involved in the neddylation and ubiquitin-proteasome degradation pathway. In addition, a group of proteins involved in transcription, DNA repair and replication, cell cycle regulation and chromatin organization, and remodeling have been copurified and identified. Apart from protein identification, the neddylation sites of cullins were determined by MS/MS analysis, which agree well with previous mutagenesis studies. Furthermore, MS analyses revealed that Nedd8 K11, K22, K48, and K60 can form chains in vivo, whereas Nedd8 K22 and K48 can be neddylated in vitro. These results present the first molecular evidence for in vitro and in vivo polyneddylation, suggesting that chain formation of ubiquitin and ubiquitin-like proteins may be a general phenomenon for these modifications. Although much remains to be explored for the biological significance of the observations, this work provides critically important information regarding Nedd8 chain assembly and its interaction network. The vast amount of proteomic information obtained here can provide clues on the biological role of Nedd8 and lay the foundation for an in-depth analysis of the regulation of the Nedd8 pathway.
Journal of Proteome Research 04/2008; 7(3):1274-87. · 5.11 Impact Factor
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ABSTRACT: Skp2B, an F-box protein of unknown function, is frequently overexpressed in breast cancer. In order to determine the function of Skp2B and whether it has a role in breast cancer, we performed a two-hybrid screen and established transgenic mice expressing Skp2B in the mammary glands. We found that Skp2B interacts with the repressor of estrogen receptor activity (REA) and that overexpression of Skp2B leads to a reduction in REA levels. In the mammary glands of MMTV-Skp2B mice, REA levels are also low. Our results show that in virgin transgenic females, Skp2B induces lobuloalveolar development and differentiation of the mammary glands normally observed during pregnancy. As this phenotype is identical to what was observed for REA heterozygote mice, our observations suggest that the Skp2B-REA interaction is physiologically relevant. However, in contrast to REA(+/-) mice, MMTV-Skp2B mice develop mammary tumors, suggesting that Skp2B affects additional proteins. These results indicate that the observed expression of Skp2B in breast cancer does contribute to tumorigenesis at least in part by modulating the activity of the estrogen receptor.
Molecular and Cellular Biology 12/2007; 27(21):7615-22. · 5.53 Impact Factor
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ABSTRACT: The resident prokaryotic microflora of the mammalian intestine influences diverse homeostatic functions of the gut, including regulation of cellular growth and immune responses; however, it is unknown how commensal prokaryotic organisms mechanistically influence eukaryotic signaling networks. We have shown that bacterial coculture with intestinal epithelial cells modulates ubiquitin-mediated degradation of important signaling intermediates, including beta-catenin and the NF-kappaB inhibitor IkappaB-alpha. Ubiquitination of these proteins as well as others is catalyzed by the SCF(betaTrCP) ubiquitin ligase, which itself requires regulated modification of the cullin-1 subunit by the ubiquitin-like protein NEDD8. Here we show that epithelia contacted by enteric commensal bacteria in vitro and in vivo rapidly generate reactive oxygen species (ROS). Bacterially induced ROS causes oxidative inactivation of the catalytic cysteine residue of Ubc12, the NEDD8-conjugating enzyme, resulting in complete but transient loss of cullin-1 neddylation and consequent effects on NF-kappaB and beta-catenin signaling. Our results demonstrate that commensal bacteria directly modulate a critical control point of the ubiquitin-proteasome system, and suggest how enteric commensal bacterial flora influences the regulatory pathways of the mammalian intestinal epithelia.
The EMBO Journal 11/2007; 26(21):4457-66. · 9.20 Impact Factor
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ABSTRACT: The Cdc34 E2 ubiquitin (Ub) conjugating enzyme catalyzes polyubiquitination of a substrate recruited by the Skp1-Cullin 1-F-box protein-ROC1 E3 Ub ligase. Using mutagenesis studies, we now show that human Cdc34 employs distinct sites to coordinate the transfer of Ub to a substrate and the assembly of polyubiquitin chains. Mutational disruption of the conserved charged stretch (residues 143 to 153) or the acidic loop residues D102 and D103 led to accumulation of monoubiquitinated IkappaBalpha while failing to yield polyubiquitin chains, due to a catalytic defect in Ub-Ub ligation. These results suggest an ability of human Cdc34 to position the attacking Ub for assembly of polyubiquitin chains. Analysis of Cdc34N85Q and Cdc34S138A revealed severe defects of these mutants in both poly- and monoubiquitination of IkappaBalpha, supporting a role for N85 in stabilizing the oxyanion and in coordinating, along with S138, the attacking lysine for catalysis. Finally, Cdc34S95D and Cdc34(E108A/E112A) abolished both poly- and monoubiquitination of IkappaBalpha. Unexpectedly, the catalytic defects of these mutants in di-Ub synthesis can be rescued by fusion of a glutathione S-transferase moiety at E2's N terminus. These findings support the hypothesis that human Cdc34 S95 and E108/E112 are required to position the donor Ub optimally for catalysis, in a manner that might depend on E2 dimerization.
Molecular and Cellular Biology 11/2007; 27(20):7041-52. · 5.53 Impact Factor
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ABSTRACT: Mdm2, a key negative regulator of the p53 tumor suppressor, is a RING-type E3 ubiquitin ligase. The Mdm2 RING domain can be biochemically fractionated into two discrete species, one of which exists as higher order oligomers that are visible by electron microscopy, whereas the other is a monomer. Both fractions are ATP binding and E3 ligase activity competent, although the oligomeric fraction exhibits lower dependence on the E2 component of ubiquitin polymerization reactions. The extreme C-terminal five amino acids of Mdm2 are essential for E3 ligase activity in vivo and in vitro, as well as for oligomeric assembly of the protein. A single residue (phenylalanine 490) in that sequence is critical for both properties. Interestingly, the C-terminus of the Mdm2 homologue, MdmX (itself inert as an E3 ligase), can fully substitute for the equivalent segment of Mdm2 and restore its E3 activity. We further show that the Mdm2 C-terminus is involved in intramolecular interactions and can set up a platform for direct protein-protein interactions with the E2.
The EMBO Journal 02/2007; 26(1):90-101. · 9.20 Impact Factor
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ABSTRACT: PHD1-3 (prolyl hydroxylases 1-3) catalyse the hydroxylation of HIF (hypoxia-inducible factor)-alpha subunit that triggers the substrate ubiquitination and subsequent degradation. The RING (really interesting new gene) finger E3 ligase Siah2 preferentially targets PHD3 for degradation. Here, we identify the requirements for such selective targeting. Firstly, PHD3 lacks an N-terminal extension found in PHD1 and PHD2; deletion of this domain from PHD1 and PHD2 renders them susceptible to degradation by Siah2. Secondly, PHD3 can homo- and hetero-multimerize with other PHDs. Consequently, PHD3 is found in high-molecular-mass fractions that were enriched in hypoxia. Interestingly, within the lower-molecular-mass complex, PHD3 exhibits higher specific activity towards hydroxylation of HIF-1alpha and co-localizes with Siah2, suggesting that Siah2 limits the availability of the more active form of PHD3. These findings provide new insight into the mechanism underlying the regulation of PHD3 availability and activity in hypoxia by the E3 ligase Siah2.
Biochemical Journal 02/2007; 401(1):217-26. · 4.90 Impact Factor
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Céline Huber,
Dora Dias-Santagata,
Anna Glaser,
James O'Sullivan,
Raja Brauner,
Kenneth Wu,
Xinsong Xu,
Kerra Pearce,
Rong Wang,
Maria Luisa Giovannucci Uzielli, [......],
Ineke Van der Burgt,
Graeme C M Black,
Peter E Clayton,
Andrew Read,
Martine Le Merrer,
Peter J Scambler,
Arnold Munnich, Zhen-Qiang Pan,
Robin Winter,
Valérie Cormier-Daire
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ABSTRACT: Intrauterine growth retardation is caused by maternal, fetal or placental factors that result in impaired endovascular trophoblast invasion and reduced placental perfusion. Although various causes of intrauterine growth retardation have been identified, most cases remain unexplained. Studying 29 families with 3-M syndrome (OMIM 273750), an autosomal recessive condition characterized by severe pre- and postnatal growth retardation, we first mapped the underlying gene to chromosome 6p21.1 and then identified 25 distinct mutations in the gene cullin 7 (CUL7). CUL7 assembles an E3 ubiquitin ligase complex containing Skp1, Fbx29 (also called Fbw8) and ROC1 and promotes ubiquitination. Using deletion analysis, we found that CUL7 uses its central region to interact with the Skp1-Fbx29 heterodimer. Functional studies indicated that the 3-M-associated CUL7 nonsense and missense mutations R1445X and H1464P, respectively, render CUL7 deficient in recruiting ROC1. These results suggest that impaired ubiquitination may have a role in the pathogenesis of intrauterine growth retardation in humans.
Nature Genetics 11/2005; 37(10):1119-24. · 35.53 Impact Factor
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ABSTRACT: Cdc34 is an E2-conjugating enzyme required for catalyzing the polyubiquitination reaction mediated by the Skp1.CUL1.F-box (SCF) protein E3 ubiquitin (Ub) ligase. Here, we show that the activity of human Cdc34 in the Ub-Ub ligation reaction was enhanced dramatically by SCF's core Ub ligase module, composed of a heterodimeric complex formed by the ROC1 RING finger protein and the CUL1 C terminus that contains a Nedd8 moiety covalently conjugated at K720. Unexpectedly, we found that N-terminal fusion of a GST moiety to human Cdc34 generated dimeric GST-Cdc34 that was constitutively active in supporting the assembly of K48-linked polyUb chains independently of SCF. Furthermore, fusion of a FK506-binding protein (FKBP) to the N terminus of human Cdc34 yielded FKBP-Cdc34 that was induced to form a dimer upon treatment with the chemical inducer AP20187. The AP20187-induced dimeric form of FKBP-Cdc34 was substantially more active than the monomer in catalyzing Ub-Ub ligation. Thus, juxtaposition of human Cdc34 activates its catalytic capability, suggesting that the SCF-mediated polyubiquitination reaction may require the conversion of Cdc34 from an inactive monomer to a highly active dimeric form.
Proceedings of the National Academy of Sciences 11/2005; 102(42):15053-8. · 9.68 Impact Factor
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Zhen-Qiang Pan
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ABSTRACT: This Teaching Resource provides lecture notes and slides for a class covering the mechanism and regulation of eukaryotic gene transcription machinery and is part of the course "Cell signaling systems: a course for graduate students." This lecture begins with an overview of eukaryotic gene transcription and provides discussions regarding the current models of mechanisms of transcription by RNA polymerase II (Pol II). Recent views on the action of Mediator will also be discussed. Finally, a detailed study will be presented to provide evidence for a role for enhancer-promoter communication in gene activation.
Science s STKE 07/2005; 2005(289):tr18.
