The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Wilhelm Krek - One of the best experts on this subject based on the ideXlab platform.
-
the f box Protein skp2 is a ubiquitylation target of a cul1 based core Ubiquitin Ligase complex evidence for a role of cul1 in the suppression of skp2 expression in quiescent fibroblasts
The EMBO Journal, 2000Co-Authors: Christiane Wirbelauer, Hedwig Sutterluty, Mathias Gstaiger, F. Reymond, Marc Blondel, Matthias Peter, Wilhelm KrekAbstract:The Ubiquitin Protein Ligase SCFSkp2 is composed of Skp1, Cul1, Roc1/Rbx1 and the F-box Protein Skp2, the substrate-recognition subunit. Levels of Skp2 decrease as cells exit the cell cycle and increase as cells re-enter the cycle. Ectopic expression of Skp2 in quiescent fibroblasts causes mitogen-independent S-phase entry. Hence, mechanisms must exist for limiting Skp2 Protein expression during the G0/G1 phases. Here we show that Skp2 is degraded by the proteasome in G0/G1 and is stabilized when cells re-enter the cell cycle. Rapid degradation of Skp2 in quiescent cells depends on Skp2 sequences that contribute to Cul1 binding and interference with endogenous Cul1 function in serum-deprived cells induces Skp2 expression. Furthermore, recombinant Cul1–Roc1/Rbx1–Skp1 complexes can catalyse Skp2 ubiquitylation in vitro. These results suggest that degradation of Skp2 in G0/G1 is mediated, at least in part, by an autocatalytic mechanism involving a Skp2-bound Cul1-based core Ubiquitin Ligase and imply a role for this mechanism in the suppression of SCFSkp2 Ubiquitin Protein Ligase function during the G0/G1 phases of the cell cycle.
-
the von hippel lindau tumor suppressor Protein is a component of an e3 Ubiquitin Protein Ligase activity
Genes & Development, 1999Co-Authors: Joanna Lisztwan, Christiane Wirbelauer, Mathias Gstaiger, Georges Imbert, Wilhelm KrekAbstract:von Hippel–Lindau (VHL) disease is a hereditary cancer syndrome leading to the development of a variety of tumors, including clear cell carcinomas of the kidney and pheochromocytomas and vascular tumors of the central nervous system and retina (Maher and Kaelin 1997; Kaelin and Maher 1998). The VHL susceptibility gene is a tumor-suppressor gene, and germ-line mutations affecting this gene have been documented in ∼80% of VHL patients (Latif et al. 1993). Functional inactivation of both VHL alleles also occurs in the majority of sporadic clear cell renal carcinomas (Gnarra et al. 1994). Therefore, inactivation of the VHL tumor-suppressor gene appears to be a requisite step in the development of clear cell renal carcinoma. The VHL gene, which contains three exons, encodes a 213-amino-acid polypeptide (Latif et al. 1993; Iliopoulos et al. 1995). The biochemical function of pVHL is as yet unknown. pVHL resides mostly in the cytoplasm and, to a lesser extent, in the nucleus and in association with cell membranes (Duan et al. 1995a; Iliopoulos et al. 1995; Pause et al. 1997; Ohh et al. 1998; Lee et al. 1999). In addition, many cells also produce a shorter variant of pVHL Protein, pVHL19, which appears to arise as a result of alternative translation initiation (Iliopoulos et al. 1998; Schoenfeld et al. 1998). Cell fractionation studies suggest that pVHL19, unlike pVHL, is equally distributed between the nucleus and cytoplasm and is not found in association with membranes (Iliopoulos et al. 1998). Recent evidence suggests that pVHL shuttles between the nuclear and the cytoplasmic compartments in a transcription-dependent manner (Lee et al. 1999). pVHL displays no similarity to other known Proteins, thus giving no clues about its function. Hence, most studies aimed at elucidating pVHL function have focused on the identification and functional analysis of interacting Proteins. pVHL associates with a number of cellular Proteins including Fibronectin (Ohh et al. 1998), Elongin B and Elongin C (Duan et al. 1995b; Kibel et al. 1995; Takagi et al. 1997), Cullin-2 (Cul-2) (Pause et al. 1997; Lonergan et al. 1998) and Rbx1 (Kamura et al. 1999). The interaction of pVHL with Fibronectin is thought to be required for the proper assembly of an extracellular fibronectin matrix and is abolished by all disease-causing mutations analyzed to date, suggesting that the binding of pVHL to fibronectin is likely to contribute to the ability of pVHL to suppress tumor growth (Ohh et al. 1998). Similarly, the ability of pVHL to form a stable complex with Elongin B, Elongin C, and Cul-2 has been linked to pVHL’s capacity to regulate the stability of hypoxia-inducible mRNAs (Lonergan et al. 1998), including mRNAs that encode angiogenic Proteins [e.g., vascular endothelial growth factor (VEGF)] (Gnarra et al. 1996; Iliopoulos et al. 1996; Siemeister et al. 1996), mitogenic factors [e.g., platelet-derived growth factor-B (PDGF-B) and transforming growth factor-α (TGF-α)] (Iliopoulos et al. 1996; Knebelmann et al. 1998), and Proteins involved in cell metabolism (e.g., Glut-1 glucose transporter) (Iliopoulos et al. 1996). The pVHL sequence includes a carboxy-terminal BC-box motif (Aso et al. 1995; Kamura et al. 1998) and GEExE repeats in its amino terminus (Latif et al. 1993). The BC-box motif within pVHL, a short ∼12-amino-acid-long degenerate sequence, is a region frequently mutated in pVHL, suggesting there resides a critical domain for pVHL tumor-suppressor function (Stebbins et al. 1999). The BC-box motif is also present in a number of other cellular Proteins including the F-box Protein elongin A (Aso et al. 1996) and members of the SOCS (suppressors of cytokine signaling) family (Kamura et al. 1998). As in the case of Elongin A and SOCS-1, the BC-box motif in pVHL serves as an interaction site for the elongin BC complex. Interestingly, the BC box of SOCS-family members is embedded within the SOCS-box motif (Kamura et al. 1998). The identification of Cul-2 and Rbx1 as pVHL-associated Proteins prompted speculations about a possible role for pVHL in the process of Ubiquitination (Pause et al. 1997; Lonergan et al. 1998; Kamura et al. 1999). Cul-2 is a member of a recently identified evolutionary-conserved gene family referred to as the Cullins (Kipreos et al. 1996). In higher eukaryotes, six members of the Cullin family have been identified, namely Cul-1, -2, -3, -4A, -4B, and -5. Little is known about their respective gene products and function(s). The best-characterized member of this family is Cul-1, a structural and functional homolog of budding yeast Cdc53, which has a key role in regulated Ubiquitin-mediated proteolysis (Patton et al. 1998; Koepp et al. 1999). Cdc53/Cul-1 assembles with Skp1 and distinct F-box Proteins into E3 Ubiquitin-Protein Ligase complexes, referred to as SCF (for Skp1/Cdc53(Cul-1)/F-box Protein) complexes (Bai et al. 1996; Feldman et al. 1997; Skowyra et al. 1997). In this type of E3 Ligase, the F-box Protein is believed to function as the substrate-specific receptor (Krek 1998; Patton et al. 1998; Koepp et al. 1999). A recently identified evolutionary conserved Protein, Rbx1/Roc1, the fourth subunit of SCF complexes, associates with Cdc53/Cul-1 and is critical for efficient recruitment of an E2 enzyme to Cdc53/Cul-1 and, hence, essential for SCF function (Kamura et al. 1999; Ohta et al. 1999; Skowyra et al. 1999; Tan et al. 1999). Interestingly, the SCF subunit Rbx1/Roc1 has also been identified as a pVHL-associated Protein (Kamura et al. 1999). In addition, the primary sequence of Elongin C, which bridges the interaction of pVHL and Cul-2, displays homology to Skp1 (Bai et al. 1996; Stebbins et al. 1999). Elongin B interacts with Elongin C in the pVHL complex and harbors a Ubiquitin-like domain (Garrett et al. 1995; Stebbins et al. 1999). Taken together, these findings suggest similarities in the overall architecture of the pVHL complex to SCF E3 Ligases. Biochemical evidence supporting a role for pVHL in the process of Ubiquitination is at present lacking. Here we show that recombinant pVHL recruits Ubiquitination-promoting activity from human cell extracts. The appearance of this activity can be specifically blocked by peptides that block pVHL/Elongin BC/Cul-2 association. We also show that antibodies specific for pVHL immunoprecipitate pVHL-associated Ubiquitination-promoting activity from unperturbed VHL+/+ cells. Likewise, anti-hemagglutinin (HA) immunoprecipitates derived from VHL−/− renal cell carcinoma (RCC) cell lines that stably express a HA-tagged version of wild-type pVHL exhibit Ubiquitination activity. In contrast, selected, naturally occurring mutants of pVHL lack this activity. Finally, sequences surrounding and including the BC box in pVHL share sequence homology with an F-box motif. Our data imply that the pVHL may function, at least in part, as a component of an E3 Ubiquitin–Protein Ligase complex.
-
the von hippel lindau tumor suppressor Protein is a component of an e3 Ubiquitin Protein Ligase activity
Genes & Development, 1999Co-Authors: Joanna Lisztwan, Christiane Wirbelauer, Mathias Gstaiger, Georges Imbert, Wilhelm KrekAbstract:pVHL, the product of the VHL tumor suppressor gene, plays an important role in the regulation of cell growth and differentiation of human kidney cells, and inactivation of the VHL gene is the most frequent genetic event in human kidney cancer. The biochemical function of pVHL is unknown. Here we report that pVHL exists in vivo in a complex that displays Ubiquitination-promoting activity in conjunction with the universally required components E1, E2, and Ubiquitin. pVHL-associated Ubiquitination activity requires, at a minimum, pVHL to bind elongin C and Cul-2, relatives of core components of SCF (Skp1-Cdc53/Cul-1-F-box Protein) E3 Ligase complexes. Notably, certain tumor-derived mutants of pVHL demonstrate loss of associated Ubiquitination promoting activity. These results identify pVHL as a component of a potential SCF-like E3 Ubiquitin-Protein Ligase complex and suggest a direct link between pVHL tumor suppressor and the process of Ubiquitination.
-
interaction between Ubiquitin Protein Ligase scf skp2 and e2f 1 underlies the regulation of e2f 1 degradation
Nature Cell Biology, 1999Co-Authors: Alain Marti, Christiane Wirbelauer, Martin Scheffner, Wilhelm KrekAbstract:Interaction between Ubiquitin–Protein Ligase SCF SKP2 and E2F-1 underlies the regulation of E2F-1 degradation
-
association of human cul 1 and Ubiquitin conjugating enzyme cdc34 with the f box Protein p45skp2 evidence for evolutionary conservation in the subunit composition of the cdc34 scf pathway
The EMBO Journal, 1998Co-Authors: Joanna Lisztwan, Christiane Wirbelauer, Hedwig Sutterluty, Mathias Gstaiger, Alain Marti, Wilhelm KrekAbstract:In normal and transformed cells, the F-box Protein p45(SKP2) is required for S phase and forms stable complexes with p19(SKP1) and cyclin A-cyclin-dependent kinase (CDK)2. Here we identify human CUL-1, a member of the cullin family, and the Ubiquitin-conjugating enzyme CDC34 as additional partners of p45(SKP2) in vivo. CUL-1 also associates with cyclin A and p19(SKP1) in vivo and, with p45(SKP2), they assemble into a large multiProtein complex. In Saccharomyces cerevisiae, a complex of similar molecular composition (an F-box Protein, a member of the cullin family and a homolog of p19(SKP1)) forms a functional E3 Ubiquitin Protein Ligase complex, designated SCFCDC4, that facilitates Ubiquitination of a CDK inhibitor by CDC34. The data presented here imply that the p45(SKP2)-CUL-1-p19(SKP1) complex may be a human representative of an SCF-type E3 Ubiquitin Protein Ligase. We propose that all eukaryotic cells may use a common Ubiquitin conjugation apparatus to promote S phase. Finally, we show that multiProtein complex formation involving p45(SKP2)-CUL-1 and p19(SKP1) is governed, in part, by periodic, S phase-specific accumulation of the p45(SKP2) subunit and by the p45(SKP2)-bound cyclin A-CDK2. The dependency of p45(SKP2)-p19(SKP1) complex formation on cyclin A-CDK2 may ensure tight coordination of the activities of the cell cycle clock with those of a potential Ubiquitin conjugation pathway.
Jon M Huibregtse - One of the best experts on this subject based on the ideXlab platform.
-
human scribble vartul is targeted for Ubiquitin mediated degradation by the high risk papillomavirus e6 Proteins and the e6ap Ubiquitin Protein Ligase
Molecular and Cellular Biology, 2000Co-Authors: Shunsuke Nakagawa, Jon M HuibregtseAbstract:The high-risk human papillomavirus (HPV) E6 Proteins stimulate the Ubiquitination and degradation of p53, dependent on the E6AP Ubiquitin-Protein Ligase. Other Proteins have also been shown to be targeted for degradation by E6, including hDlg, the human homolog of the Drosophila melanogaster Discs large (Dlg) tumor suppressor. We show here that the human homolog of the Drosophila Scribble (Vartul) (hScrib) tumor suppressor Protein is also targeted for Ubiquitination by the E6-E6AP complex in vitro and that expression of E6 induces degradation of hScrib in vivo. Characterization of the E6AP-E6-hScrib complex indicated that hScrib binds directly to E6 and that the binding is mediated by the PDZ domains of hScrib and a carboxyl-terminal epitope conserved among the high-risk HPV E6 Proteins. Green fluorescent Protein-hScrib was localized to the periphery of MDCK cells, where it colocalized with ZO-1, a component of tight junctions. E6 expression resulted in loss of integrity of tight junctions, as measured by ZO-1 localization, and this effect was dependent on the PDZ binding epitope of E6. Thus, the high-risk HPV E6 Proteins induce the degradation of the human homologs of two Drosophila PDZ domain-containing tumor suppressor Proteins, hDlg and hScrib, both of which are associated with cell junction complexes. The fact that Scrib/Vart and Dlg appear to cooperate in a pathway that controls Drosophila epithelial cell growth suggests that the combined targeting of hScrib and hDlg is an important component of the biologic activity of high-risk HPV E6 Proteins.
-
human papillomavirus type 16 e6 induces self Ubiquitination of the e6ap Ubiquitin Protein Ligase
Journal of Virology, 2000Co-Authors: Sylvie Beaudenon, Jon M Huibregtse, Andrea L Talis, Peter M HowleyAbstract:The E6 Protein of the high-risk human papillomaviruses (HPVs) and the cellular Ubiquitin-Protein Ligase E6AP form a complex which causes the Ubiquitination and degradation of p53. We show here that HPV16 E6 promotes the Ubiquitination and degradation of E6AP itself. The half-life of E6AP is shorter in HPV-positive cervical cancer cells than in HPV-negative cervical cancer cells, and E6AP is stabilized in HPV-positive cancer cells when expression of the viral oncoProteins is repressed. Expression of HPV16 E6 in cells results in a threefold decrease in the half-life of transfected E6AP. E6-mediated degradation of E6AP requires (i) the binding of E6 to E6AP, (ii) the catalytic activity of E6AP, and (iii) activity of the 26S proteasome, suggesting that E6-E6AP interaction results in E6AP self-Ubiquitination and degradation. In addition, both in vitro and in vivo experiments indicate that E6AP self-Ubiquitination results primarily from an intramolecular transfer of Ubiquitin from the active-site cysteine to one or more lysine residues; however, intermolecular transfer can also occur in the context of an E6-mediated E6AP multimer. Finally, we demonstrate that an E6 mutant that is able to immortalize human mammary epithelial cells but is unable to degrade p53 retains its ability to bind and degrade E6AP, raising the possibility that E6-mediated degradation of E6AP contributes to its ability to transform mammalian cells.
-
rsp5 Ubiquitin Protein Ligase mediates dna damage induced degradation of the large subunit of rna polymerase ii in saccharomyces cerevisiae
Molecular and Cellular Biology, 1999Co-Authors: Sylvie Beaudenon, Guangli Wang, Maria R Huacani, Donald P Mcdonnell, Jon M HuibregtseAbstract:Rsp5 is an E3 Ubiquitin-Protein Ligase of Saccharomyces cerevisiae that belongs to the hect domain family of E3 Proteins. We have previously shown that Rsp5 binds and Ubiquitinates the largest subunit of RNA polymerase II, Rpb1, in vitro. We show here that Rpb1 Ubiquitination and degradation are induced in vivo by UV irradiation and by the UV-mimetic compound 4-nitroquinoline-1-oxide (4-NQO) and that a functional RSP5 gene product is required for this effect. The 26S proteasome is also required; a mutation of SEN3/RPN2 (sen3-1), which encodes an essential regulatory subunit of the 26S proteasome, partially blocks 4-NQO-induced degradation of Rpb1. These results suggest that Rsp5-mediated Ubiquitination and degradation of Rpb1 are components of the response to DNA damage. A human WW domain-containing hect (WW-hect) E3 Protein closely related to Rsp5, Rpf1/hNedd4, also binds and Ubiquitinates both yeast and human Rpb1 in vitro, suggesting that Rpf1 and/or another WW-hect E3 Protein mediates UV-induced degradation of the large subunit of polymerase II in human cells.
-
functional domains of the rsp5 Ubiquitin Protein Ligase
Molecular and Cellular Biology, 1999Co-Authors: Guangli Wang, Joyce C Yang, Jon M HuibregtseAbstract:RSP5, an essential gene of Saccharomyces cerevisiae, encodes a hect domain E3 Ubiquitin-Protein Ligase. Hect E3 Proteins have been proposed to consist of two broad functional domains: a conserved catalytic carboxyl-terminal domain of approximately 350 amino acids (the hect domain) and a large, nonconserved amino-terminal domain containing determinants of substrate specificity. We report here the mapping of the minimal region of Rsp5 necessary for its essential in vivo function, the minimal region necessary to stably interact with a substrate of Rsp5 (Rpb1, the large subunit of RNA polymerase II), and the finding that the hect domain, by itself, is sufficient for formation of the Ubiquitin-thioester intermediate. Mutations within the hect domain that affect either the ability to form a Ubiquitin-thioester or to catalyze substrate Ubiquitination abrogate in vivo function, strongly suggesting that the Ubiquitin-Protein Ligase activity of Rsp5 is intrinsically linked to its essential function. The amino-terminal region of Rsp5 contains three WW domains and a C2 calcium-binding domain. Two of the three WW domains are required for the essential in vivo function, while the C2 domain is not, and requirements for Rpb1 binding and Ubiquitination lie within the region required for in vivo function. Together, these results support the two-domain model for hect E3 function and indicate that the WW domains play a role in the recognition of at least some of the substrates of Rsp5, including those related to its essential function. In addition, we show that haploid yeast strains bearing complete disruptions of either of two other hect E3 genes of yeast, designated HUL4 (YJR036C) and HUL5 (YGL141W), are viable.
-
the humane6 apgene ube3a encodes three potential Protein isoforms generated by differential splicing
Genomics, 1997Co-Authors: Yoshiki Yamamoto, Jon M Huibregtse, Peter M HowleyAbstract:The E6-AP gene (UBE3A) encodes an E3 Ubiquitin-Protein Ligase that binds the human papillomavirus E6 oncoProtein and catalyzes the Ubiquitination of p53. Recent studies have also established that mutations in E6-AP are the genetic basis of the Angelman syndrome in humans. In this study we present the genomic structure of the coding region of E6-AP and an analysis of a set of five E6-AP mRNAs with the potential to encode three Protein isoforms of the E6-AP Protein (isoforms I, II, and III) that differ at their extreme amino-termini. These transcripts were expressed in a variety of different cell lines examined.
Jonathan D Ashwell - One of the best experts on this subject based on the ideXlab platform.
-
non canonical nf κb activation and abnormal b cell accumulation in mice expressing Ubiquitin Protein Ligase inactive c iap2
PLOS Biology, 2010Co-Authors: Dietrich B Conze, Yongge Zhao, Jonathan D AshwellAbstract:Chromosomal translocations between loci encoding MALT1 and c-IAP2 are common in MALT lymphomas. The resulting fusion Proteins lack the c-IAP2 RING domain, the region responsible for its Ubiquitin Protein Ligase (E3) activity. Ectopic expression of the fusion Protein activates the canonical NF-κB signaling cascade, but how it does so is controversial and how it promotes MALT lymphoma is unknown. Considering recent reports implicating c-IAP1 and c-IAP2 E3 activity in repression of non-canonical NF-κB signaling, we asked if the c-IAP2/MALT fusion Protein can initiate non-canonical NF-κB activation. Here we show that in addition to canonical activation, the fusion Protein stabilizes NIK and activates non-canonical NF-κB. Canonical but not non-canonical activation depended on MALT1 paracaspase activity, and expression of E3-inactive c-IAP2 activated non-canonical NF-κB. Mice in which endogenous c-IAP2 was replaced with an E3-inactive mutant accumulated abnormal B cells with elevated non-canonical NF-κB and had increased numbers of B cells with a marginal zone phenotype, gut-associated lymphoid hyperplasia, and other features of MALT lymphoma. Thus, the c-IAP2/MALT1 fusion Protein activates NF-κB by two distinct mechanisms, and loss of c-IAP2 E3 activity in vivo is sufficient to induce abnormalities common to MALT lymphoma.
-
non canonical nf κb activation and abnormal b cell accumulation in mice expressing Ubiquitin Protein Ligase inactive c iap2
PLOS Biology, 2010Co-Authors: Dietrich B Conze, Yongge Zhao, Jonathan D AshwellAbstract:Chromosomal translocations between loci encoding MALT1 and c-IAP2 are common in MALT lymphomas. The resulting fusion Proteins lack the c-IAP2 RING domain, the region responsible for its Ubiquitin Protein Ligase (E3) activity. Ectopic expression of the fusion Protein activates the canonical NF-κB signaling cascade, but how it does so is controversial and how it promotes MALT lymphoma is unknown. Considering recent reports implicating c-IAP1 and c-IAP2 E3 activity in repression of non-canonical NF-κB signaling, we asked if the c-IAP2/MALT fusion Protein can initiate non-canonical NF-κB activation. Here we show that in addition to canonical activation, the fusion Protein stabilizes NIK and activates non-canonical NF-κB. Canonical but not non-canonical activation depended on MALT1 paracaspase activity, and expression of E3-inactive c-IAP2 activated non-canonical NF-κB. Mice in which endogenous c-IAP2 was replaced with an E3-inactive mutant accumulated abnormal B cells with elevated non-canonical NF-κB and had increased numbers of B cells with a marginal zone phenotype, gut-associated lymphoid hyperplasia, and other features of MALT lymphoma. Thus, the c-IAP2/MALT1 fusion Protein activates NF-κB by two distinct mechanisms, and loss of c-IAP2 E3 activity in vivo is sufficient to induce abnormalities common to MALT lymphoma.
-
posttranscriptional downregulation of c iap2 by the Ubiquitin Protein Ligase c iap1 in vivo
Molecular and Cellular Biology, 2005Co-Authors: Dietrich B Conze, Lori Albert, David A Ferrick, David V Goeddel, Jonathan D AshwellAbstract:Inhibitor of apoptosis Proteins (IAPs) c-IAP1 and c-IAP2 were identified as part of the tumor necrosis factor receptor 2 (TNFR2) signaling complex and have been implicated as intermediaries in tumor necrosis factor alpha signaling. Like all RING domain-containing IAPs, c-IAP1 and c-IAP2 have Ubiquitin Protein Ligase (E3) activity. To explore the function of c-IAP1 in a physiologic setting, c-IAP1-deficient mice were generated by homologous gene recombination. These animals are viable and have no obvious sensitization to proapoptotic stimuli. Cells from c-IAP1 / mice do, however, express markedly elevated levels of c-IAP2 Protein in the absence of increased c-IAP2 mRNA. In contrast to reports implicating c-IAPs in the activation of NF-B, resting and cytokine-induced NF-B activation was not impaired in c-IAP1-deficient cells. Transient transfection studies with wild-type and E3-defective c-IAP1 revealed that c-IAP2 is a direct target for c-IAP1mediated Ubiquitination and subsequent degradation, which are potentiated by the adaptor function of TRAF2. Thus, the c-IAPs represent a pair of TNFR-associated Ubiquitin Protein Ligases in which one regulates the expression of the other by a posttranscriptional and E3-dependent mechanism.
-
tnf rii and c iap1 mediate Ubiquitination and degradation of traf2
Nature, 2002Co-Authors: Yili Yang, Jonathan D AshwellAbstract:Tumour necrosis factor-α (TNF-α) is a proinflammatory mediator that exerts its biological functions by binding two TNF receptors (TNF-RI and TNF-RII), which initiate biological responses by interacting with adaptor and signalling Proteins. Among the signalling components that associate with TNF receptors are members of the TNF-R-associated factor (TRAF) family1,2. TRAF2 is required for TNF-α-mediated activation of c-Jun N-terminal kinase (JNK), contributes to activation of NF-κB, and mediates anti-apoptotic signals3, 4. TNF-RI and TNF-RII signalling complexes also contain the anti-apoptotic (‘inhibitor of apoptosis’) molecules c-IAP1 and c-IAP2 (refs 5, 6), which also have RING domain-dependent Ubiquitin Protein Ligase (E3) activity7. The function of IAPs in TNF-R signalling is unknown. Here we show that binding of TNF-α to TNF-RII induces Ubiquitination and proteasomal degradation of TRAF2. Although c-IAP1 bound TRAF2 and TRAF1 in vitro, it Ubiquitinated only TRAF2. Expression of wild-type c-IAP1, but not an E3-defective mutant, resulted in TRAF2 Ubiquitination and degradation. Moreover, E3-defective c-IAP1 prevented TNF-α-induced TRAF2 degradation and inhibited apoptosis. These findings identify a physiologic role for c-IAP1 and define a mechanism by which TNF-RII-regulated Ubiquitin Protein Ligase activity can potentiate TNF-induced apoptosis.
Tanjun Tong - One of the best experts on this subject based on the ideXlab platform.
-
ww domain containing e3 Ubiquitin Protein Ligase 1 wwp1 delays cellular senescence by promoting p27 kip1 degradation in human diploid fibroblasts
Journal of Biological Chemistry, 2011Co-Authors: Tianda Chen, Tanjun TongAbstract:WW domain-containing E3 Ubiquitin Protein Ligase 1 (WWP1) plays an important role in the proliferation of tumor cells and the lifespan of Caenorhabditis elegans. However, the role of WWP1 in cellular senescence is still unknown. Here, we show that the expression patterns of p27Kip1 and WWP1 are inversely correlated during cellular senescence. Moreover, the overexpression of WWP1 delayed senescence, whereas the knockdown of WWP1 led to premature senescence in human fibroblasts. Furthermore, we demonstrate that WWP1 repressed endogenous p27Kip1 expression through Ubiquitin-proteasome-mediated degradation. Additionally, WWP1 had a strong preference for catalyzing the Lys-48-linked polyUbiquitination of p27Kip1 in vitro. Finally, we demonstrate that WWP1 markedly inhibited the replicative senescence induced by p27Kip1 by promoting p27Kip1 degradation. Therefore, our study provides a new molecular mechanism for the regulation of cellular senescence.
-
ww domain containing e3 Ubiquitin Protein Ligase 1 wwp1 delays cellular senescence by promoting p27kip1 degradation in human diploid fibroblasts
Journal of Biological Chemistry, 2011Co-Authors: Xiaoxiao Cao, Tianda Chen, Lixiang Xue, Limin Han, Tanjun TongAbstract:WW domain-containing E3 Ubiquitin Protein Ligase 1 (WWP1) plays an important role in the proliferation of tumor cells and the lifespan of Caenorhabditis elegans. However, the role of WWP1 in cellular senescence is still unknown. Here, we show that the expression patterns of p27Kip1 and WWP1 are inversely correlated during cellular senescence. Moreover, the overexpression of WWP1 delayed senescence, whereas the knockdown of WWP1 led to premature senescence in human fibroblasts. Furthermore, we demonstrate that WWP1 repressed endogenous p27Kip1 expression through Ubiquitin-proteasome-mediated degradation. Additionally, WWP1 had a strong preference for catalyzing the Lys-48-linked polyUbiquitination of p27Kip1 in vitro. Finally, we demonstrate that WWP1 markedly inhibited the replicative senescence induced by p27Kip1 by promoting p27Kip1 degradation. Therefore, our study provides a new molecular mechanism for the regulation of cellular senescence.
Peter M Howley - One of the best experts on this subject based on the ideXlab platform.
-
human papillomavirus type 16 e6 induces self Ubiquitination of the e6ap Ubiquitin Protein Ligase
Journal of Virology, 2000Co-Authors: Sylvie Beaudenon, Jon M Huibregtse, Andrea L Talis, Peter M HowleyAbstract:The E6 Protein of the high-risk human papillomaviruses (HPVs) and the cellular Ubiquitin-Protein Ligase E6AP form a complex which causes the Ubiquitination and degradation of p53. We show here that HPV16 E6 promotes the Ubiquitination and degradation of E6AP itself. The half-life of E6AP is shorter in HPV-positive cervical cancer cells than in HPV-negative cervical cancer cells, and E6AP is stabilized in HPV-positive cancer cells when expression of the viral oncoProteins is repressed. Expression of HPV16 E6 in cells results in a threefold decrease in the half-life of transfected E6AP. E6-mediated degradation of E6AP requires (i) the binding of E6 to E6AP, (ii) the catalytic activity of E6AP, and (iii) activity of the 26S proteasome, suggesting that E6-E6AP interaction results in E6AP self-Ubiquitination and degradation. In addition, both in vitro and in vivo experiments indicate that E6AP self-Ubiquitination results primarily from an intramolecular transfer of Ubiquitin from the active-site cysteine to one or more lysine residues; however, intermolecular transfer can also occur in the context of an E6-mediated E6AP multimer. Finally, we demonstrate that an E6 mutant that is able to immortalize human mammary epithelial cells but is unable to degrade p53 retains its ability to bind and degrade E6AP, raising the possibility that E6-mediated degradation of E6AP contributes to its ability to transform mammalian cells.
-
the humane6 apgene ube3a encodes three potential Protein isoforms generated by differential splicing
Genomics, 1997Co-Authors: Yoshiki Yamamoto, Jon M Huibregtse, Peter M HowleyAbstract:The E6-AP gene (UBE3A) encodes an E3 Ubiquitin-Protein Ligase that binds the human papillomavirus E6 oncoProtein and catalyzes the Ubiquitination of p53. Recent studies have also established that mutations in E6-AP are the genetic basis of the Angelman syndrome in humans. In this study we present the genomic structure of the coding region of E6-AP and an analysis of a set of five E6-AP mRNAs with the potential to encode three Protein isoforms of the E6-AP Protein (isoforms I, II, and III) that differ at their extreme amino-termini. These transcripts were expressed in a variety of different cell lines examined.
-
a family of Proteins structurally and functionally related to the e6 ap Ubiquitin Protein Ligase
Proceedings of the National Academy of Sciences of the United States of America, 1995Co-Authors: Sylvie Beaudenon, Peter M HowleyAbstract:Abstract E6-AP is a 100-kDa cellular Protein that interacts with the E6 Protein of the cancer-associated human papillomavirus types 16 and 18. The E6/E6-AP complex binds to and targets the p53 tumor-suppressor Protein for Ubiquitin-mediated proteolysis. E6-AP is an E3 Ubiquitin-Protein Ligase which accepts Ubiquitin from an E2 Ubiquitin-conjugating enzyme in the form of a thioester and then directly transfers the Ubiquitin to targeted substrates. The amino acid sequence of E6-AP shows similarity to a number of Protein sequences over an approximately 350-aa region corresponding to the carboxyl termini of both E6-AP and the E6-AP-related Proteins. Of particular note is a conserved cysteine residue within the last 32-34 aa, which in E6-AP is likely to be the site of Ubiquitin thioester formation. Two of the E6-AP-related Proteins, a rat 100-kDa Protein and a yeast 95-kDa Protein (RSP5), both of previously unknown function, are shown here to form thioesters with Ubiquitin. Mutation of the conserved cysteine residue of these Proteins destroys their ability to accept Ubiquitin. These data strongly suggest that the rat 100-kDa Protein and RSP5, as well as the other E6-AP-related Proteins, belong to a class of functionally related E3 Ubiquitin-Protein Ligases, defined by a domain homologous to the E6-AP carboxyl terminus (hect domain).
-
the hpv 16 e6 and e6 ap complex functions as a Ubiquitin Protein Ligase in the Ubiquitination of p53
Cell, 1993Co-Authors: Martin Scheffner, Jon M Huibregtse, Richard David Vierstra, Peter M HowleyAbstract:Abstract The Ubiquitin-dependent proteolytic pathway plays a major role in selective Protein degradation. Ubiquitination of Proteins requires the sequential action of the Ubiquitin-activating enzyme (E1), Ubiquitin-conjugating enzymes (E2), and in some cases Ubiquitin-Protein Ligases (E3s). The oncogenic human papillomavirus (HPV) types 16 and 18 utilize this cellular proteolytic system to target the tumor suppressor Protein p53. The HPV E6 oncoProtein binds to a cellular Protein of 100 kd, termed E6-associated Protein (E6-AP). The E6-E6-AP complex specifically interacts with p53, resulting in the rapid Ubiquitin-dependent degradation of p53. Here we report the purification and identification of the factors necessary for the E6-E6-AP-mediated Ubiquitination of p53. The Ubiquitination of p53 requires the E1 enzyme and a novel E2 in mammalian cells, while E3 activity is conferred by the E6-E6-AP complex. Furthermore, E6-AP appears to have Ubiquitin-Protein Ligase activity in the absence of E6.
