scholarly journals The Abundance of Met30p Limits SCFMet30p Complex Activity and Is Regulated by Methionine Availability

2000 ◽  
Vol 20 (21) ◽  
pp. 7845-7852 ◽  
Author(s):  
Dechelle B. Smothers ◽  
Lukasz Kozubowski ◽  
Cheryl Dixon ◽  
Mark G. Goebl ◽  
Neal Mathias
Keyword(s):  
Ubiquitin Ligase ◽  
Environmental Conditions ◽  
Cellular Responses ◽  
Biological Pathways ◽  
Variable Component ◽  
Complex Activity ◽  
Scf Complex ◽  
Ubiquitin Ligase Activity ◽  
F Box Protein

ABSTRACT Ubiquitin-mediated degradation plays a crucial role in many fundamental biological pathways, including the mediation of cellular responses to changes in environmental conditions. A family of ubiquitin ligase complexes, called SCF complexes, found throughout eukaryotes, is involved in a variety of biological pathways. In Saccharomyces cerevisiae, an SCF complex contains a common set of components, namely, Cdc53p, Skp1p, and Hrt1p. Substrate specificity is defined by a variable component called an F-box protein. The F- box is a ∼40-amino-acid motif that allows the F-box protein to bind Skp1p. Each SCF complex recognizes different substrates according to which F-box protein is associated with the complex. In yeasts, three SCF complexes have been demonstrated to associate with the ubiquitin-conjugating enzyme Cdc34p and have ubiquitin ligase activity. F-box proteins are not abundant and are unstable. As part of the SCFMet30p complex, the F-box protein Met30p represses methionine biosynthetic gene expression when availability ofl-methionine is high. Here we demonstrate that in vivo SCFMet30p complex activity can be regulated by the abundance of Met30p. Furthermore, we provide evidence that Met30p abundance is regulated by the availability of l-methionine. We propose that the cellular responses mediated by an SCF complex are directly regulated by environmental conditions through the control of F-box protein stability.

scholarly journals Overproduction of Polypeptides Corresponding to the Amino Terminus of the F-Box Proteins Cdc4p and Met30p Inhibits Ubiquitin Ligase Activities of Their SCF Complexes

2003 ◽  
Vol 2 (1) ◽  
pp. 123-133 ◽  
Author(s):  
Cheryl Dixon ◽  
Lee Ellen Brunson ◽  
Mary Margaret Roy ◽  
Dechelle Smothers ◽  
Michael G. Sehorn ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Ubiquitin Ligases ◽  
Cellular Responses ◽  
Amino Terminus ◽  
Variable Component ◽  
Amino Terminal ◽  
F Box Protein ◽  
Substrate Proteins

ABSTRACT Ubiquitin ligases direct the transfer of ubiquitin onto substrate proteins and thus target the substrate for proteasome-dependent degradation. SCF complexes are a family of ubiquitin ligases composed of a common core of components and a variable component called an F-box protein that defines substrate specificity. Distinct SCF complexes, defined by a particular F-box protein, target different substrate proteins for degradation. Although a few have been identified to be involved in important biological pathways, such as the cell division cycle and coordinating cellular responses to changes in environmental conditions, the role of the overwhelming majority of F-box proteins is not clear. Creating inhibitors that will block the in vivo activities of specific SCF ubiquitin ligases may provide identification of substrates of these uncharacterized F-box proteins. Using Saccharomyces cerevisiae as a model system, we demonstrate that overproduction of polypeptides corresponding to the amino terminus of the F-box proteins Cdc4p and Met30p results in specific inhibition of their SCF complexes. Analyses of mutant amino-terminal alleles demonstrate that the interaction of these polypeptides with their full-length counterparts is an important step in the inhibitory process. These results suggest a common means to inhibit specific SCF complexes in vivo.

scholarly journals Diverse Effects of Mutations in Exon II of the von Hippel-Lindau (VHL) Tumor Suppressor Gene on the Interaction of pVHL with the Cytosolic Chaperonin and pVHL-Dependent Ubiquitin Ligase Activity

2002 ◽  
Vol 22 (6) ◽  
pp. 1947-1960 ◽  
Author(s):  
William J. Hansen ◽  
Michael Ohh ◽  
Javid Moslehi ◽  
Keiichi Kondo ◽  
William G. Kaelin ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Ubiquitin Ligase ◽  
Hypoxia Inducible Factor ◽  
Detectable Effect ◽  
Missense Mutations ◽  
Subsequent Formation ◽  
Von Hippel Lindau ◽  
Ubiquitin Ligase Activity

ABSTRACT We examined the biogenesis of the von Hippel-Lindau (VHL) tumor suppressor protein (pVHL) in vitro and in vivo. pVHL formed a complex with the cytosolic chaperonin containing TCP-1 (CCT or TRiC) en route to assembly with elongin B/C and the subsequent formation of the VCB-Cul2 ubiquitin ligase. Blocking the interaction of pVHL with elongin B/C resulted in accumulation of pVHL within the CCT complex. pVHL present in purified VHL-CCT complexes, when added to rabbit reticulocyte lysate, proceeded to form VCB and VCB-Cul2. Thus, CCT likely functions, at least in part, by retaining VHL chains pending the availability of elongin B/C for final folding and/or assembly. Tumor-associated mutations within exon II of the VHL syndrome had diverse effects upon the stability and/or function of pVHL-containing complexes. First, a pVHL mutant lacking the entire region encoded by exon II did not bind to CCT and yet could still assemble into complexes with elongin B/C and elongin B/C-Cul2. Second, a number of tumor-derived missense mutations in exon II did not decrease CCT binding, and most had no detectable effect upon VCB-Cul2 assembly. Many exon II mutants, however, were found to be defective in the binding to and subsequent ubiquitination of hypoxia-inducible factor 1α (HIF-1α), a substrate of the VCB-Cul2 ubiquitin ligase. We conclude that the selection pressure to mutate VHL exon II during tumorigenesis does not relate to loss of CCT binding but may reflect quantitative or qualitative defects in HIF binding and/or in pVHL-dependent ubiquitin ligase activity.

scholarly journals Lys29-linkage of ASK1 by Skp1−Cullin 1−Fbxo21 ubiquitin ligase complex is required for antiviral innate response

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Zhou Yu ◽  
Taoyong Chen ◽  
Xuelian Li ◽  
Mingjin Yang ◽  
Songqing Tang ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Type I Interferon ◽  
Innate Immune ◽  
Type I ◽  
Innate Response ◽  
Scf Complex ◽  
Protein Ubiquitination ◽  
Ubiquitin Ligase Complex ◽  
F Box Protein ◽  
Unknown Component

Protein ubiquitination regulated by ubiquitin ligases plays important roles in innate immunity. However, key regulators of ubiquitination during innate response and roles of new types of ubiquitination (apart from Lys48- and Lys63-linkage) in control of innate signaling have not been clearly understood. Here we report that F-box only protein Fbxo21, a functionally unknown component of SCF (Skp1–Cul1–F-box protein) complex, facilitates Lys29-linkage and activation of ASK1 (apoptosis signal-regulating kinase 1), and promotes type I interferon production upon viral infection. Fbxo21 deficiency in mice cells impairs virus-induced Lys29-linkage and activation of ASK1, attenuates c-Jun N-terminal kinase (JNK) and p38 signaling pathway, and decreases the production of proinflammatory cytokines and type I interferon, resulting in reduced antiviral innate response and enhanced virus replication. Therefore Fbxo21 is required for ASK1 activation via Lys29-linkage of ASK1 during antiviral innate response, providing mechanistic insights into non-proteolytic roles of SCF complex in innate immune response.

scholarly journals Protein Phosphatase 2A Stabilizes Human Securin, Whose Phosphorylated Forms Are Degraded via the SCF Ubiquitin Ligase

2006 ◽  
Vol 26 (11) ◽  
pp. 4017-4027 ◽  
Author(s):  
Ana M. Gil-Bernabé ◽  
Francisco Romero ◽  
M. Cristina Limón-Mortés ◽  
María Tortolero
Keyword(s):  
Ubiquitin Ligase ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Anaphase Promoting Complex ◽  
Anaphase Onset ◽  
Chromatid Segregation ◽  
Phosphatase 2A ◽  
F Box Protein

ABSTRACT Sister chromatid segregation is triggered at the metaphase-to-anaphase transition by the activation of the protease separase. For most of the cell cycle, separase activity is kept in check by its association with the inhibitory chaperone securin. Activation of separase occurs at anaphase onset, when securin is targeted for destruction by the anaphase-promoting complex or cyclosome E3 ubiquitin protein ligase. This results in the release of the cohesins from chromosomes, which in turn allows the segregation of sister chromatids to opposite spindle poles. Here we show that human securin (hSecurin) forms a complex with enzymatically active protein phosphatase 2A (PP2A) and that it is a substrate of the phosphatase, both in vitro and in vivo. Treatment of cells with okadaic acid, a potent inhibitor of PP2A, results in various hyperphosphorylated forms of hSecurin which are extremely unstable, due to the action of the Skp1/Cul1/F-box protein complex ubiquitin ligase. We propose that PP2A regulates hSecurin levels by counteracting its phosphorylation, which promotes its degradation. Misregulation of this process may lead to the formation of tumors, in which overproduction of hSecurin is often observed.

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.

scholarly journals The CUL1 C-Terminal Sequence and ROC1 Are Required for Efficient Nuclear Accumulation, NEDD8 Modification, and Ubiquitin Ligase Activity of CUL1

2000 ◽  
Vol 20 (21) ◽  
pp. 8185-8197 ◽  
Author(s):  
Manabu Furukawa ◽  
Yanping Zhang ◽  
Joseph McCarville ◽  
Tomohiko Ohta ◽  
Yue Xiong
Keyword(s):  
Nuclear Localization ◽  
Ubiquitin Ligase ◽  
Nuclear Import ◽  
Ring Finger ◽  
Nuclear Accumulation ◽  
Protein Families ◽  
Terminal Sequence ◽  
Ubiquitin Ligase Activity

ABSTRACT Members of the cullin and RING finger ROC protein families form heterodimeric complexes to constitute a potentially large number of distinct E3 ubiquitin ligases. We report here that the highly conserved C-terminal sequence in CUL1 is dually required, both for nuclear localization and for modification by NEDD8. Disruption of ROC1 binding impaired nuclear accumulation of CUL1 and decreased NEDD8 modification in vivo but had no effect on NEDD8 modification of CUL1 in vitro, suggesting that ROC1 promotes CUL1 nuclear accumulation to facilitate its NEDD8 modification. Disruption of NEDD8 binding had no effect on ROC1 binding, nor did it affect nuclear localization of CUL1, suggesting that nuclear localization and NEDD8 modification of CUL1 are two separable steps, with nuclear import preceding and required for NEDD8 modification. Disrupting NEDD8 modification diminishes the IκBα ubiquitin ligase activity of CUL1. These results identify a pathway for regulation of CUL1 activity—ROC1 and the CUL1 C-terminal sequence collaboratively mediate nuclear accumulation and NEDD8 modification, facilitating assembly of active CUL1 ubiquitin ligase. This pathway may be commonly utilized for the assembly of other cullin ligases.

scholarly journals SUSA2 is an F-box protein required for autoimmunity mediated by paired NLRs SOC3-CHS1 and SOC3-TN2

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Wanwan Liang ◽  
Meixuezi Tong ◽  
Xin Li
Keyword(s):  
Ubiquitin Ligase ◽  
Higher Plants ◽  
Interaction Analysis ◽  
E3 Ubiquitin Ligase ◽  
E3 Ligase ◽  
Pairwise Interaction ◽  
Leucine Rich Repeat ◽  
Scf Complex ◽  
F Box Protein ◽  
Effector Recognition

Abstract Both higher plants and mammals rely on nucleotide-binding leucine-rich repeat (NLR) immune receptors to detect pathogens and initiate immunity. Upon effector recognition, plant NLRs oligomerize for defense activation, the mechanism of which is poorly understood. We previously showed that disruption of the E3 ligase, Senescence-Associated E3 Ubiquitin Ligase 1 (SAUL1) leads to the activation of the NLR SOC3. Here, we report the identification of suppressor of saul1 2 (susa2) and susa3 from the saul1-1 suppressor screen. Pairwise interaction analysis suggests that both SUSA proteins interact with components of an SCFSUSA2 E3 ligase complex as well as CHS1 or TN2, truncated NLRs that pair with SOC3. susa2-2 only suppresses the autoimmunity mediated by either CHS1 or TN2, suggesting its specific involvement in SOC3-mediated immunity. In summary, our study indicates links between plant NLRs and an SCF complex that may enable ubiquitination and degradation of unknown downstream components to activate defense.

scholarly journals Np95 Is a Histone-Binding Protein Endowed with Ubiquitin Ligase Activity

2004 ◽  
Vol 24 (6) ◽  
pp. 2526-2535 ◽  
Author(s):  
Elisabetta Citterio ◽  
Roberto Papait ◽  
Francesco Nicassio ◽  
Manuela Vecchi ◽  
Paola Gomiero ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Ubiquitin Ligase ◽  
Molecular Mechanisms ◽  
Important Determinant ◽  
Histone H3 ◽  
Ring Finger ◽  
Protein Domain ◽  
Ubiquitin Ligase Activity

ABSTRACT Np95 is an important determinant in cell cycle progression. Its expression is tightly regulated and becomes detectable shortly before the entry of cells into S phase. Accordingly, Np95 is absolutely required for the G1/S transition. Its continued expression throughout the S/G2/M phases further suggests additional roles. Indeed, Np95 has been implicated in DNA damage response. Here, we show that Np95 is tightly bound to chromatin in vivo and that it binds to histones in vivo and in vitro. The binding to histones is direct and shows a remarkable preference for histone H3 and its N-terminal tail. A novel protein domain, the SRA-YDG domain, contained in Np95 is indispensable both for the interaction with histones and for chromatin binding in vivo. Np95 contains a RING finger. We show that this domain confers E3 ubiquitin ligase activity on Np95, which is specific for core histones, in vitro. Finally, Np95 shows specific E3 activity for histone H3 when the endogenous core octamer, coimmunoprecipitating with Np95, is used as a substrate. Histone ubiquitination is an important determinant in the regulation of chromatin structure and gene transcription. Thus, the demonstration that Np95 is a chromatin-associated ubiquitin ligase suggests possible molecular mechanisms for its action as a cell cycle regulator.

scholarly journals The E3 Ubiquitin Ligase TBK1 Mediates the Degradation of Multiple Picornavirus VP3 Proteins by Phosphorylation and Ubiquitination

2019 ◽  
Vol 93 (23) ◽  
Author(s):  
Dan Li ◽  
Wenping Yang ◽  
Jingjing Ren ◽  
Yi Ru ◽  
Keshan Zhang ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Molecular Mechanisms ◽  
E3 Ubiquitin Ligase ◽  
Adaptor Protein ◽  
Ubiquitin Ligase Activity ◽  
Lysine Residues ◽  
Innate Antiviral Immunity

ABSTRACT TANK-binding kinase 1 (TBK1) is essential for interferon beta (IFN-β) production and innate antiviral immunity. However, other, additional functions of TBK1 have remained elusive. Here, we showed that TBK1 is an E3 ubiquitin ligase that undergoes self-ubiquitylation in vitro in the presence of the E2 enzyme UbcH5c. Further evidence showed that TBK1 could also be self-ubiquitylated in vivo. Importantly, multiple picornavirus VP3 proteins were degraded by TBK1 through its kinase and E3 ubiquitin ligase activity. Mechanistically, TBK1 phosphorylated multiple picornavirus VP3 proteins at serine residues and ubiquitinated them via K63-linked ubiquitination at lysine residues. In addition, the C426 and C605 residues of TBK1 were not essential for TBK1 innate immunity activity; however, these residues were required for degradation of multiple picornavirus VP3 proteins and for its E3 ubiquitin ligase activity. Hence, our findings identified a novel role of TBK1 in regulating the virus life cycle and provided new insights into the molecular mechanisms of TBK1-mediated antiviral response. IMPORTANCE TBK1 is an important adaptor protein required for innate immune response to viruses, but its other functions were unknown. In this study, we found that TBK1 is an E3 ubiquitin ligase that undergoes self-ubiquitylation in vitro in the presence of the E2 enzyme UbcH5c. In addition, multiple picornavirus VP3 proteins were degraded by TBK1 through its kinase and E3 ubiquitin ligase activity. Our report provides evidence that TBK1 plays a role in viral protein degradation.

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