scholarly journals The multi-subunit GID/CTLH E3 ubiquitin ligase promotes cell proliferation and targets the transcription factor Hbp1 for degradation

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Fabienne Lampert ◽  
Diana Stafa ◽  
Algera Goga ◽  
Martin Varis Soste ◽  
Samuel Gilberto ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Hierarchical Organization ◽  
Negative Regulator ◽  
Cell Cycle Exit ◽  
Cellular Analysis ◽  
Ubiquitin Ligase Activity

In yeast, the glucose-induced degradation-deficient (GID) E3 ligase selectively degrades superfluous gluconeogenic enzymes. Here, we identified all subunits of the mammalian GID/CTLH complex and provide a comprehensive map of its hierarchical organization and step-wise assembly. Biochemical reconstitution demonstrates that the mammalian complex possesses inherent E3 ubiquitin ligase activity, using Ube2H as its cognate E2. Deletions of multiple GID subunits compromise cell proliferation, and this defect is accompanied by deregulation of critical cell cycle markers such as the retinoblastoma (Rb) tumor suppressor, phospho-Histone H3 and Cyclin A. We identify the negative regulator of pro-proliferative genes Hbp1 as a bonafide GID/CTLH proteolytic substrate. Indeed, Hbp1 accumulates in cells lacking GID/CTLH activity, and Hbp1 physically interacts and is ubiquitinated in vitro by reconstituted GID/CTLH complexes. Our biochemical and cellular analysis thus demonstrates that the GID/CTLH complex prevents cell cycle exit in G1, at least in part by degrading Hbp1.

scholarly journals Liganded ERα Stimulates the E3 Ubiquitin Ligase Activity of UBE3C to Facilitate Cell Proliferation

2015 ◽  
Vol 29 (11) ◽  
pp. 1646-1657 ◽  
Author(s):  
Maiko Okada ◽  
Fumiaki Ohtake ◽  
Hiroyuki Nishikawa ◽  
Wenwen Wu ◽  
Yasushi Saeki ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Cyclin B1 ◽  
Dependent Manner ◽  
Ubiquitin Ligase Activity ◽  
Dependent Cell ◽  
Estrogen Antagonist

Abstract Estrogen receptor (ER)α is a well-characterized ligand-dependent transcription factor. However, the global picture of its nongenomic functions remains to be illustrated. Here, we demonstrate a novel function of ERα during mitosis that facilitates estrogen-dependent cell proliferation. An E3 ubiquitin ligase, UBE3C, was identified in an ERα complex from estrogen-treated MCF-7 breast cancer cells arrested at mitosis. UBE3C interacts with ERα during mitosis in an estrogen-dependent manner. In vitro, estrogen dramatically stimulates the E3 activity of UBE3C in the presence of ERα. This effect was inhibited by the estrogen antagonist tamoxifen. Importantly, estrogen enhances the ubiquitination of cyclin B1 (CCNB1) and destabilizes CCNB1 during mitosis in a manner dependent on endogenous UBE3C. ERα, UBE3C, and CCNB1 colocalize in prophase nuclei and at metaphase spindles before CCNB1 is degraded in anaphase. Depletion of UBE3C attenuates estrogen-dependent cell proliferation without affecting the transactivation function of ERα. Collectively, these results demonstrate a novel ligand-dependent action of ERα that stimulates the activity of an E3 ligase. The mitotic role of estrogen may contribute to its effects on proliferation in addition to its roles in target gene expression.

scholarly journals Virion-Associated Vpr Alleviates a Postintegration Block to HIV-1 Infection of Dendritic Cells

2017 ◽  
Vol 91 (13) ◽  
Author(s):  
Caitlin M. Miller ◽  
Hisashi Akiyama ◽  
Luis M. Agosto ◽  
Ann Emery ◽  
Chelsea R. Ettinger ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Culture ◽  
Dendritic Cells ◽  
Ubiquitin Ligase ◽  
Culture System ◽  
E3 Ubiquitin Ligase ◽  
Cell Culture System ◽  
Ubiquitin Ligase Complex ◽  
Hiv 1

ABSTRACT Viral protein R (Vpr) is an HIV-1 accessory protein whose function remains poorly understood. In this report, we sought to determine the requirement of Vpr for facilitating HIV-1 infection of monocyte-derived dendritic cells (MDDCs), one of the first cell types to encounter virus in the peripheral mucosal tissues. In this report, we characterize a significant restriction of Vpr-deficient virus replication and spread in MDDCs alone and in cell-to-cell spread in MDDC-CD4+ T cell cocultures. This restriction of HIV-1 replication in MDDCs was observed in a single round of virus replication and was rescued by the expression of Vpr in trans in the incoming virion. Interestingly, infections of MDDCs with viruses that encode Vpr mutants unable to interact with either the DCAF1/DDB1 E3 ubiquitin ligase complex or a host factor hypothesized to be targeted for degradation by Vpr also displayed a significant replication defect. While the extent of proviral integration in HIV-1-infected MDDCs was unaffected by the absence of Vpr, the transcriptional activity of the viral long terminal repeat (LTR) from Vpr-deficient proviruses was significantly reduced. Together, these results characterize a novel postintegration restriction of HIV-1 replication in MDDCs and show that the interaction of Vpr with the DCAF1/DDB1 E3 ubiquitin ligase complex and the yet-to-be-identified host factor might alleviate this restriction by inducing transcription from the viral LTR. Taken together, these findings identify a robust in vitro cell culture system that is amenable to addressing mechanisms underlying Vpr-mediated enhancement of HIV-1 replication. IMPORTANCE Despite decades of work, the function of the HIV-1 protein Vpr remains poorly understood, primarily due to the lack of an in vitro cell culture system that demonstrates a deficit in replication upon infection with viruses in the absence of Vpr. In this report, we describe a novel cell infection system that utilizes primary human dendritic cells, which display a robust decrease in viral replication upon infection with Vpr-deficient HIV-1. We show that this replication difference occurs in a single round of infection and is due to decreased transcriptional output from the integrated viral genome. Viral transcription could be rescued by virion-associated Vpr. Using mutational analysis, we show that domains of Vpr involved in binding to the DCAF1/DDB1/E3 ubiquitin ligase complex and prevention of cell cycle progression into mitosis are required for LTR-mediated viral expression, suggesting that the evolutionarily conserved G2 cell cycle arrest function of Vpr is essential for HIV-1 replication.

scholarly journals Linear Ubiquitination Mediates EGFR-Induced NF-κB Pathway and Tumor Development

2021 ◽  
Vol 22 (21) ◽  
pp. 11875
Author(s):  
Fang Hua ◽  
Wenzhuo Hao ◽  
Lingyan Wang ◽  
Shitao Li
Keyword(s):  
Cell Proliferation ◽  
Ubiquitin Ligase ◽  
Tumor Development ◽  
E3 Ubiquitin Ligase ◽  
Growth Factor Receptor ◽  
Ubiquitin Chain ◽  
Plakophilin 2 ◽  
Ubiquitin Ligase Activity ◽  
Signaling Axis ◽  
Differentiation And Proliferation

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that instigates several signaling cascades, including the NF-κB signaling pathway, to induce cell differentiation and proliferation. Overexpression and mutations of EGFR are found in up to 30% of solid tumors and correlate with a poor prognosis. Although it is known that EGFR-mediated NF-κB activation is involved in tumor development, the signaling axis is not well elucidated. Here, we found that plakophilin 2 (PKP2) and the linear ubiquitin chain assembly complex (LUBAC) were required for EGFR-mediated NF-κB activation. Upon EGF stimulation, EGFR recruited PKP2 to the plasma membrane, and PKP2 bridged HOIP, the catalytic E3 ubiquitin ligase in the LUBAC, to the EGFR complex. The recruitment activated the LUBAC complex and the linear ubiquitination of NEMO, leading to IκB phosphorylation and subsequent NF-κB activation. Furthermore, EGF-induced linear ubiquitination was critical for tumor cell proliferation and tumor development. Knockout of HOIP impaired EGF-induced NF-κB activity and reduced cell proliferation. HOIP knockout also abrogated the growth of A431 epidermal xenograft tumors in nude mice by more than 70%. More importantly, the HOIP inhibitor, HOIPIN-8, inhibited EGFR-mediated NF-κB activation and cell proliferation of A431, MCF-7, and MDA-MB-231 cancer cells. Overall, our study reveals a novel linear ubiquitination signaling axis of EGFR and that perturbation of HOIP E3 ubiquitin ligase activity is potential targeted cancer therapy.

scholarly journals Cyclin G1 inhibits the proliferation of mouse endometrial stromal cell in decidualization

2017 ◽  
Vol 69 (1) ◽  
pp. 71-81
Author(s):  
Qian Xu ◽  
Dong-zhi Yuan ◽  
Sheng Zhang ◽  
Ting Qu ◽  
Shi-mao Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Stromal Cells ◽  
Stromal Cell ◽  
Embryo Implantation ◽  
Negative Regulator ◽  
Cell Cycle Regulators ◽  
Cyclin G1 ◽  
Decidual Cells

Uterine stromal cell decidualization is a dynamic physiological process in which cell proliferation, differentiation and apoptosis are orchestrated and occur in a temporal and cell-specific manner. This process is important for successful embryo implantation. Many cell-cycle regulators are involved in decidualization. The protein cyclin G1 is a unique regulator of the cell cycle with dual functions in cell proliferation. It was reported that cyclin G1 is expressed in mouse uterine stromal cells during the period of peri-implantation. To prove the function of cyclin G1 in mouse uterine stromal cells during this period, immunohistochemistry was used to stain mouse uterine tissues on days 4-8 of pregnancy. The results showed obvious spatial and temporal expression of cyclin G1 in uterine stromal cells, and that it is expressed in the cells of the primary decidual zone (PDZ) on day 5 and secondary decidual zone (SDZ) on days 6 and 7, when the stromal cells experienced active proliferation and differentiation was initiated. Applying the decidualization model of cultured primary stromal cells in vitro, we further revealed that the expression of cyclin G1 is associated with decidualization of stromal cells induced by medroxyprogesterone acetate (MPA) and estradiol-17? (E2). RNA interference was used for the knockdown of cyclin G1 in the induced decidual cells. Flow cytometry analysis indicated that the proportion of cells in the S stage was increased, and decreased in the G2/M phase. Our study indicates that cyclin G1, as a negative regulator of the cell cycle, plays an important role in the process of decidualization in mouse uterine stromal cells by inhibiting cell-cycle progression.

scholarly journals Spotted leaf11, a Negative Regulator of Plant Cell Death and Defense, Encodes a U-Box/Armadillo Repeat Protein Endowed with E3 Ubiquitin Ligase Activity

2004 ◽  
Vol 16 (10) ◽  
pp. 2795-2808 ◽  
Author(s):  
Li-Rong Zeng ◽  
Shaohong Qu ◽  
Alicia Bordeos ◽  
Chengwei Yang ◽  
Marietta Baraoidan ◽  
...  
Keyword(s):  
Cell Death ◽  
Plant Cell ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Negative Regulator ◽  
Repeat Protein ◽  
Ubiquitin Ligase Activity ◽  
Armadillo Repeat

scholarly journals Linear Ubiquitination Mediates EGFR-Induced NF-kB Pathway and Tumor Development

2021 ◽  
Author(s):  
Fang Hua ◽  
Wenzhuo Hap ◽  
Lingyan Wang ◽  
Shitao Li
Keyword(s):  
Cell Proliferation ◽  
Ubiquitin Ligase ◽  
Tumor Development ◽  
E3 Ubiquitin Ligase ◽  
Growth Factor Receptor ◽  
Signaling Cascades ◽  
Ubiquitin Ligase Activity ◽  
Signaling Axis ◽  
Epidermal Growth ◽  
Differentiation And Proliferation

Epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase that instigates several signaling cascades, including the NF-kB signaling pathway, to induce cell differentiation and proliferation. Overexpression and mutations of EGFR are found in up to 30% of solid tumors and correlate with a poor prognosis. Although it is known that EGFR-mediated NF-kB activation is involved in tumor development, the signaling axis is not well elucidated. Here, we found that PKP2 and the LUBAC complex were required for EGFR-mediated NF-kB activation. Upon EGF stimulation, EGFR recruited PKP2 to the plasma membrane, and PKP2 bridged HOIP, the catalytic E3 ubiquitin ligase in the LUBAC, to the EGFR complex. The recruitment activated the LUBAC complex and the linear ubiquitination of NEMO, leading to IkB phosphorylation and subsequent NF-kB activation. Furthermore, EGF-induced linear ubiquitination was critical for tumor cell proliferation and tumor development. Knockout of HOIP impaired EGF-induced NF-kB activity and reduced cell proliferation. HOIP knockout also abrogated the growth of A431 epidermal xenograft tumors in nude mice by more than 70%. More importantly, the HOIP inhibitor, HOIPIN-8, inhibited EGFR-mediated NF-kB activation and cell proliferation of A431, MCF-7, and MDA-MB-231 cancer cells. Overall, our study reveals a novel linear ubiquitination signaling axis of EGFR, and perturbation of HOIP E3 ubiquitin ligase activity is potential targeted cancer therapy.

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.

scholarly journals Only Akt1 Is Required for Proliferation, while Akt2 Promotes Cell Cycle Exit through p21 Binding

2006 ◽  
Vol 26 (22) ◽  
pp. 8267-8280 ◽  
Author(s):  
Lisa Héron-Milhavet ◽  
Celine Franckhauser ◽  
Vanessa Rana ◽  
Cyril Berthenet ◽  
Daniel Fisher ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Mammalian Cells ◽  
Cell Cycle Progression ◽  
Specific Interaction ◽  
Cyclin A ◽  
Cell Cycle Exit ◽  
Cycle Progression ◽  
Negative Effect

ABSTRACT Protein kinase B (PKB/Akt) is an important modulator of insulin signaling, cell proliferation, and survival. Using small interfering RNA duplexes in nontransformed mammalian cells, we show that only Akt1 is essential for cell proliferation, while Akt2 promotes cell cycle exit. Silencing Akt1 resulted in decreased cyclin A levels and inhibition of S-phase entry, effects not seen with Akt2 knockdown and specifically rescued by microinjection of Akt1, not Akt2. In differentiating myoblasts, Akt2 knockout prevented myoblasts from exiting the cell cycle and showed sustained cyclin A expression. In contrast, overexpression of Akt2 reduced cyclin A and hindered cell cycle progression in M-G1 with increased nuclear p21. p21 is a major target in the differential effects of Akt isoforms, with endogenous Akt2 and not Akt1 binding p21 in the nucleus and increasing its level. Accordingly, Akt2 knockdown cells, and not Akt1 knockdown cells, showed reduced levels of p21. A specific Akt2/p21 interaction can be reproduced in vitro, and the Akt2 binding site on p21 is similar to that in cyclin A spanning T145 to T155, since (i) prior incubation with cyclin A prevents Akt2 binding, (ii) T145 phosphorylation on p21 by Akt1 prevents Akt2 binding, and (iii) binding Akt2 prevents phosphorylation of p21 by Akt1. These data show that specific interaction of the Akt2 isoform with p21 is key to its negative effect on normal cell cycle progression.

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