scholarly journals Mammalian DET1 Regulates Cul4A Activity and Forms Stable Complexes with E2 Ubiquitin-Conjugating Enzymes

2007 ◽  
Vol 27 (13) ◽  
pp. 4708-4719 ◽  
Author(s):  
Elah Pick ◽  
On-Sun Lau ◽  
Tomohiko Tsuge ◽  
Suchithra Menon ◽  
Yingchun Tong ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Associated Factors ◽  
Cultured Cells ◽  
Negative Regulator ◽  
Polyubiquitin Chain ◽  
Light Responses ◽  
E3 Ligases ◽  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes

ABSTRACT DET1 (de-etiolated 1) is an essential negative regulator of plant light responses, and it is a component of the Arabidopsis thaliana CDD complex containing DDB1 and COP10 ubiquitin E2 variant. Human DET1 has recently been isolated as one of the DDB1- and Cul4A-associated factors, along with an array of WD40-containing substrate receptors of the Cul4A-DDB1 ubiquitin ligase. However, DET1 differs from conventional substrate receptors of cullin E3 ligases in both biochemical behavior and activity. Here we report that mammalian DET1 forms stable DDD-E2 complexes, consisting of DDB1, DDA1 (DET1, DDB1 associated 1), and a member of the UBE2E group of canonical ubiquitin-conjugating enzymes. DDD-E2 complexes interact with multiple ubiquitin E3 ligases. We show that the E2 component cannot maintain the ubiquitin thioester linkage once bound to the DDD core, rendering mammalian DDD-E2 equivalent to the Arabidopsis CDD complex. While free UBE2E-3 is active and able to enhance UbcH5/Cul4A activity, the DDD core specifically inhibits Cul4A-dependent polyubiquitin chain assembly in vitro. Overexpression of DET1 inhibits UV-induced CDT1 degradation in cultured cells. These findings demonstrate that the conserved DET1 complex modulates Cul4A functions by a novel mechanism.

Two different classes of E2 ubiquitin-conjugating enzymes are required for the mono-ubiquitination of proteins and elongation by polyubiquitin chains with a specific topology

2008 ◽  
Vol 409 (3) ◽  
pp. 723-729 ◽  
Author(s):  
Mark Windheim ◽  
Mark Peggie ◽  
Philip Cohen
Keyword(s):  
E3 Ligase ◽  
Lysine Residue ◽  
Chain Elongation ◽  
Tumour Necrosis Factor Receptor ◽  
Polyubiquitin Chain ◽  
Polyubiquitin Chains ◽  
E3 Ligases ◽  
Ring E3 Ligase ◽  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes

RING (really interesting new gene) and U-box E3 ligases bridge E2 ubiquitin-conjugating enzymes and substrates to enable the transfer of ubiquitin to a lysine residue on the substrate or to one of the seven lysine residues of ubiquitin for polyubiquitin chain elongation. Different polyubiquitin chains have different functions. Lys48-linked chains target proteins for proteasomal degradation, and Lys63-linked chains function in signal transduction, endocytosis and DNA repair. For this reason, chain topology must be tightly controlled. Using the U-box E3 ligase CHIP [C-terminus of the Hsc (heat-shock cognate) 70-interacting protein] and the RING E3 ligase TRAF6 (tumour-necrosis-factor-receptor-associated factor 6) with the E2s Ubc13 (ubiquitin-conjugating enzyme 13)–Uev1a (ubiquitin E2 variant 1a) and UbcH5a, in the present study we demonstrate that Ubc13–Uev1a supports the formation of free Lys63-linked polyubiquitin chains not attached to CHIP or TRAF6, whereas UbcH5a catalyses the formation of polyubiquitin chains linked to CHIP and TRAF6 that lack specificity for any lysine residue of ubiquitin. Therefore the abilities of these E2s to ubiquitinate a substrate and to elongate polyubiquitin chains of a specific topology appear to be mutually exclusive. Thus two different classes of E2 may be required to attach a polyubiquitin chain of a particular topology to a substrate: the properties of one E2 are designed to mono-ubiquitinate a substrate with no or little inherent specificity for an acceptor lysine residue, whereas the properties of the second E2 are tailored to the elongation of a polyubiquitin chain using a defined lysine residue of ubiquitin.

scholarly journals UBE2G1 Governs the Destruction of Cereblon Neomorphic Substrates

2018 ◽  
Author(s):  
Gang Lu ◽  
Stephanie Weng ◽  
Mary Matyskiela ◽  
Xinde Zheng ◽  
Wei Fang ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Resistance Mechanism ◽  
Chain Extension ◽  
Dependent Manner ◽  
The Novel ◽  
Polyubiquitin Chain ◽  
Antitumor Activities ◽  
Ubiquitin Ligase Complex ◽  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes

AbstractThe immunomodulatory drugs (IMiDs) thalidomide, lenalidomide, and pomalidomide as well as the novel cereblon modulating agents (CMs) including CC-122, CC-220 and cereblon-based proteolysis-targeting chimaeras (PROTACs) repurpose the Cul4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase complex to induce the degradation of specific neomorphic substrates via polyubiquitination in conjunction with an E1 ubiquitin-activating enzyme and E2 ubiquitin-conjugating enzymes, which have until now remained elusive. Here we show that the ubiquitin-conjugating enzymes UBE2G1 and UBE2D3 cooperatively promote the polyubiquitination of CRL4CRBN neomorphic substrates in a cereblon- and CM-dependent manner via a sequential ubiquitination mechanism: UBE2D3 transforms the neomorphic substrates into mono-ubiquitinated forms, upon which UBE2G1 catalyzes K48-linked polyubiquitin chain extension. Blockade of UBE2G1 diminishes the ubiquitination and degradation of neomorphic substrates, and consequent antitumor activities elicited by all tested CMs. For example, UBE2G1 inactivation significantly attenuated the degradation of myeloma survival factors IKZF1 and IKZF3 induced by lenalidomide and pomalidomide, hence conferring drug resistance. UBE2G1-deficient myeloma cells, however, remained sensitive to a more potent IKZF1/3 degrader CC-220. Collectively, these findings suggest that loss of UBE2G1 activity might be a resistance mechanism to drugs that hijack the CRL4CRBN to eliminate disease-driving proteins, and that this resistance mechanism can be overcome by next-generation CMs that destroy the same targeted protein more effectively.

scholarly journals E3 Ubiquitin Ligase SPL2 Is a Lanthanide-Binding Protein

2021 ◽  
Vol 22 (11) ◽  
pp. 5712
Author(s):  
Michał Tracz ◽  
Ireneusz Górniak ◽  
Andrzej Szczepaniak ◽  
Wojciech Białek
Keyword(s):  
Ubiquitin Ligase ◽  
Conformational Changes ◽  
Protein Interactions ◽  
E3 Ubiquitin Ligase ◽  
Lanthanide Ions ◽  
E3 Ligases ◽  
Fluorescence Measurements ◽  
Partial Unfolding

The SPL2 protein is an E3 ubiquitin ligase of unknown function. It is one of only three types of E3 ligases found in the outer membrane of plant chloroplasts. In this study, we show that the cytosolic fragment of SPL2 binds lanthanide ions, as evidenced by fluorescence measurements and circular dichroism spectroscopy. We also report that SPL2 undergoes conformational changes upon binding of both Ca2+ and La3+, as evidenced by its partial unfolding. However, these structural rearrangements do not interfere with SPL2 enzymatic activity, as the protein retains its ability to auto-ubiquitinate in vitro. The possible applications of lanthanide-based probes to identify protein interactions in vivo are also discussed. Taken together, the results of this study reveal that the SPL2 protein contains a lanthanide-binding site, showing for the first time that at least some E3 ubiquitin ligases are also capable of binding lanthanide ions.

scholarly journals SLAP2 adaptor binding disrupts c-CBL autoinhibition to activate ubiquitin ligase function

2020 ◽  
Author(s):  
Leanne E. Wybenga-Groot ◽  
Andrea J. Tench ◽  
Craig D. Simpson ◽  
Jonathan St. Germain ◽  
Brian Raught ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Binding Site ◽  
Ubiquitin Ligase ◽  
Adaptor Proteins ◽  
Negative Regulator ◽  
Alternative Mechanism ◽  
Kinase Signaling ◽  
Tyrosine Kinase Signaling ◽  
Ligase Function

AbstractCBL is a RING type E3 ubiquitin ligase that functions as a negative regulator of tyrosine kinase signaling and loss of CBL E3 function is implicated in several forms of leukemia. The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL and are required for CBL-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling. Despite the established role of SLAP/SLAP2 in regulating CBL activity, the nature of the interaction and the mechanisms involved are not known. To understand the molecular basis of the interaction between SLAP/SLAP2 and CBL, we solved the crystal structure of CBL tyrosine kinase binding domain (TKBD) in complex with SLAP2. The carboxy-terminal region of SLAP2 adopts an α-helical structure which binds in a cleft between the 4H, EF-hand, and SH2 domains of the TKBD. This SLAP2 binding site is remote from the canonical TKBD phospho-tyrosine peptide binding site but overlaps with a region important for stabilizing CBL in its autoinhibited conformation. In addition, binding of SLAP2 to CBL in vitro activates the ubiquitin ligase function of autoinhibited CBL. Disruption of the CBL/SLAP2 interface through mutagenesis demonstrated a role for this protein-protein interaction in regulation of CBL E3 ligase activity in cells. Our results reveal that SLAP2 binding to a regulatory cleft of the TKBD provides an alternative mechanism for activation of CBL ubiquitin ligase function.

scholarly journals OTUB1 non-catalytically stabilizes the E2 ubiquitin-conjugating enzyme UBE2E1 by preventing its autoubiquitination

2018 ◽  
Vol 293 (47) ◽  
pp. 18285-18295 ◽  
Author(s):  
Nagesh Pasupala ◽  
Marie E. Morrow ◽  
Lauren T. Que ◽  
Barbara A. Malynn ◽  
Averil Ma ◽  
...  
Keyword(s):  
Polyubiquitin Chains ◽  
Protein Ubiquitination ◽  
Ubiquitin Conjugating Enzyme ◽  
Conjugating Enzymes ◽  
E2 Enzymes ◽  
Ubiquitin Conjugating Enzymes ◽  
In Cells ◽  
Conjugating Enzyme

OTUB1 is a deubiquitinating enzyme that cleaves Lys-48–linked polyubiquitin chains and also regulates ubiquitin signaling through a unique, noncatalytic mechanism. OTUB1 binds to a subset of E2 ubiquitin-conjugating enzymes and inhibits their activity by trapping the E2∼ubiquitin thioester and preventing ubiquitin transfer. The same set of E2s stimulate the deubiquitinating activity of OTUB1 when the E2 is not charged with ubiquitin. Previous studies have shown that, in cells, OTUB1 binds to E2-conjugating enzymes of the UBE2D (UBCH5) and UBE2E families, as well as to UBE2N (UBC13). Cellular roles have been identified for the interaction of OTUB1 with UBE2N and members of the UBE2D family, but not for interactions with UBE2E E2 enzymes. We report here a novel role for OTUB1–E2 interactions in modulating E2 protein ubiquitination. We observe that Otub1−/− knockout mice exhibit late-stage embryonic lethality. We find that OTUB1 depletion dramatically destabilizes the E2-conjugating enzyme UBE2E1 (UBCH6) in both mouse and human OTUB1 knockout cell lines. Of note, this effect is independent of the catalytic activity of OTUB1, but depends on its ability to bind to UBE2E1. We show that OTUB1 suppresses UBE2E1 autoubiquitination in vitro and in cells, thereby preventing UBE2E1 from being targeted to the proteasome for degradation. Taken together, we provide evidence that OTUB1 rescues UBE2E1 from degradation in vivo.

scholarly journals A Context-Dependent Role for the RNF146 Ubiquitin Ligase in Wingless/Wnt Signaling in Drosophila

Genetics ◽  
2018 ◽  
Vol 211 (3) ◽  
pp. 913-923 ◽  
Author(s):  
Zhenghan Wang ◽  
Ofelia Tacchelly-Benites ◽  
Geoffrey P. Noble ◽  
Megan K. Johnson ◽  
Jean-Philippe Gagné ◽  
...  
Keyword(s):  
Wnt Signaling ◽  
Ubiquitin Ligase ◽  
Wnt Pathway ◽  
Gene Activation ◽  
Negative Regulator ◽  
Compensatory Mechanisms ◽  
E3 Ligases ◽  
Pathway Activation ◽  
Wingless Signaling

Aberrant activation of the Wnt signal transduction pathway triggers the development of colorectal cancer. The ADP-ribose polymerase Tankyrase (TNKS) mediates proteolysis of Axin—a negative regulator of Wnt signaling—and provides a promising therapeutic target for Wnt-driven diseases. Proteolysis of TNKS substrates is mediated through their ubiquitination by the poly-ADP-ribose (pADPr)-dependent RING-domain E3 ubiquitin ligase RNF146/Iduna. Like TNKS, RNF146 promotes Axin proteolysis and Wnt pathway activation in some cultured cell lines, but in contrast with TNKS, RNF146 is dispensable for Axin degradation in colorectal carcinoma cells. Thus, the contexts in which RNF146 is essential for TNKS-mediated Axin destabilization and Wnt signaling remain uncertain. Herein, we tested the requirement for RNF146 in TNKS-mediated Axin proteolysis and Wnt pathway activation in a range of in vivo settings. Using null mutants in Drosophila, we provide genetic and biochemical evidence that Rnf146 and Tnks function in the same proteolysis pathway in vivo. Furthermore, like Tnks, Drosophila Rnf146 promotes Wingless signaling in multiple developmental contexts by buffering Axin levels to ensure they remain below the threshold at which Wingless signaling is inhibited. However, in contrast with Tnks, Rnf146 is dispensable for Wingless target gene activation and the Wingless-dependent control of intestinal stem cell proliferation in the adult midgut during homeostasis. Together, these findings demonstrate that the requirement for Rnf146 in Tnks-mediated Axin proteolysis and Wingless pathway activation is dependent on physiological context, and suggest that, in some cell types, functionally redundant pADPr-dependent E3 ligases or other compensatory mechanisms promote the Tnks-dependent proteolysis of Axin in both mammalian and Drosophila cells.

scholarly journals UBE2G1 governs the destruction of cereblon neomorphic substrates

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Gang Lu ◽  
Stephanie Weng ◽  
Mary Matyskiela ◽  
Xinde Zheng ◽  
Wei Fang ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Antitumor Activities ◽  
Fundamental Interest ◽  
Myeloma Cells ◽  
Ubiquitin Ligase Complex ◽  
Clinical Resistance ◽  
Conjugating Enzymes ◽  
Ubiquitin Conjugating Enzymes

The cereblon modulating agents (CMs) including lenalidomide, pomalidomide and CC-220 repurpose the Cul4-RBX1-DDB1-CRBN (CRL4CRBN) E3 ubiquitin ligase complex to induce the degradation of specific neomorphic substrates via polyubiquitination in conjunction with E2 ubiquitin-conjugating enzymes, which have until now remained elusive. Here we show that the ubiquitin-conjugating enzymes UBE2G1 and UBE2D3 cooperatively promote the K48-linked polyubiquitination of CRL4CRBN neomorphic substrates via a sequential ubiquitination mechanism. Blockade of UBE2G1 diminishes the ubiquitination and degradation of neomorphic substrates, and consequent antitumor activities elicited by all tested CMs. For example, UBE2G1 inactivation significantly attenuated the degradation of myeloma survival factors IKZF1 and IKZF3 induced by lenalidomide and pomalidomide, hence conferring drug resistance. UBE2G1-deficient myeloma cells, however, remained sensitive to a more potent IKZF1/3 degrader CC-220. Collectively, it will be of fundamental interest to explore if loss of UBE2G1 activity is linked to clinical resistance to drugs that hijack the CRL4CRBN to eliminate disease-driving proteins.

scholarly journals Senescence-inducing stress promotes proteolysis of phosphoglycerate mutase via ubiquitin ligase Mdm2

2014 ◽  
Vol 204 (5) ◽  
pp. 729-745 ◽  
Author(s):  
Takumi Mikawa ◽  
Takeshi Maruyama ◽  
Koji Okamoto ◽  
Hitoshi Nakagama ◽  
Matilde E. Lleonart ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Warburg Effect ◽  
Cultured Cells ◽  
Glycolytic Enzyme ◽  
Phosphoglycerate Mutase ◽  
Proliferative Potential ◽  
Downstream Effector ◽  
Direct Binding ◽  
The Warburg Effect

Despite the well-documented clinical significance of the Warburg effect, it remains unclear how the aggressive glycolytic rates of tumor cells might contribute to other hallmarks of cancer, such as bypass of senescence. Here, we report that, during oncogene- or DNA damage–induced senescence, Pak1-mediated phosphorylation of phosphoglycerate mutase (PGAM) predisposes the glycolytic enzyme to ubiquitin-mediated degradation. We identify Mdm2 as a direct binding partner and ubiquitin ligase for PGAM in cultured cells and in vitro. Mutations in PGAM and Mdm2 that abrogate ubiquitination of PGAM restored the proliferative potential of primary cells under stress conditions and promoted neoplastic transformation. We propose that Mdm2, a downstream effector of p53, attenuates the Warburg effect via ubiquitination and degradation of PGAM.

scholarly journals The APC11 RING-H2 Finger Mediates E2-Dependent Ubiquitination

2000 ◽  
Vol 11 (7) ◽  
pp. 2315-2325 ◽  
Author(s):  
Joel D. Leverson ◽  
Claudio A.P. Joazeiro ◽  
Andrew M. Page ◽  
Han-kuei Huang ◽  
Philip Hieter ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
26S Proteasome ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Protein Substrates ◽  
Ubiquitin Ligase Activity ◽  
Ubiquitin Conjugating Enzyme ◽  
Ubiquitin Conjugating Enzymes

Polyubiquitination marks proteins for degradation by the 26S proteasome and is carried out by a cascade of enzymes that includes ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s). The anaphase-promoting complex or cyclosome (APC/C) comprises a multisubunit ubiquitin ligase that mediates mitotic progression. Here, we provide evidence that theSaccharomyces cerevisiae RING-H2 finger protein Apc11 defines the minimal ubiquitin ligase activity of the APC. We found that the integrity of the Apc11p RING-H2 finger was essential for budding yeast cell viability, Using purified, recombinant proteins we showed that Apc11p interacted directly with the Ubc4 ubiquitin conjugating enzyme (E2). Furthermore, purified Apc11p was capable of mediating E1- and E2-dependent ubiquitination of protein substrates, including Clb2p, in vitro. The ability of Apc11p to act as an E3 was dependent on the integrity of the RING-H2 finger, but did not require the presence of the cullin-like APC subunit Apc2p. We suggest that Apc11p is responsible for recruiting E2s to the APC and for mediating the subsequent transfer of ubiquitin to APC substrates in vivo.

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