The ubiquitin conjugating enzyme UbcH10 competes with UbcH3 for binding to the SCF complex, a ubiquitin ligase involved in cell cycle progression

Present in organisms ranging from yeast to man, homologues of the Saccharomyces cerevisiae ubiquitin-conjugating enzyme CDC34 have been shown to play important roles in the regulation of cell cycle progression and checkpoint function. Here we analyze the expression and intracellular localization of endogenous CDC34 during mammalian cell cycle progression. We find that CDC34 protein is constitutively expressed during all stages of the cell cycle. Immunofluorescence experiments reveal that during interphase, endogenous CDC34 is localized to distinct speckles in both the nucleus and the cytoplasm. The presence of CDC34 in these compartments has also been established by biochemical fractionation experiments. Interestingly, nuclear localization depends on the presence of specific carboxy-terminal CDC34 sequences that have previously been shown to be required for CDC34's cell cycle function in Saccharomyces cerevisiae. Finally, we find that in anaphase and not during early stages of mitosis, CDC34 colocalizes with (beta)-tubulin at the mitotic spindle, implying that it may contribute to spindle function at later stages of mitosis. Taken together, these results support a model in which CDC34 ubiquitin-conjugating enzyme functions in the regulation of nuclear and cytoplasmic activities as well as in the process of chromosome segregation at the onset of anaphase in mammalian cells.

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The Ubc3 (Cdc34) ubiquitin-conjugating enzyme is ubiquitinated and phosphorylated in vivo.

Molecular and Cellular Biology ◽

10.1128/mcb.14.5.3022 ◽

1994 ◽

Vol 14 (5) ◽

pp. 3022-3029 ◽

Cited By ~ 49

Author(s):

M G Goebl ◽

L Goetsch ◽

B Byers

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Protein S ◽

S Phase ◽

Cycle Progression ◽

Endogenous Protein ◽

Ubiquitin Conjugating Enzyme ◽

Conjugating Enzyme

The transition from G1 to S phase of the cell cycle in Saccharomyces cerevisiae requires the activity of the Ubc3 (Cdc34) ubiquitin-conjugating enzyme. S. cerevisiae cells lacking a functional UBC3 (CDC34) gene are able to execute the Start function that initiates the cell cycle but fail to form a mitotic spindle or enter S phase. The Ubc3 (Cdc34) enzyme has previously been shown to catalyze the attachment of multiple ubiquitin molecules to model substrates, suggesting that the role of this enzyme in cell cycle progression depends on its targeting an endogenous protein(s) for degradation. In this report, we demonstrate that the Ubc3 (Cdc34) protein is itself a substrate for both ubiquitination and phosphorylation. Immunochemical localization of the gene product to the nucleus renders it likely that the relevant substrates similarly reside within the nucleus.

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SENP8 limits aberrant neddylation of NEDD8 pathway components to promote cullin-RING ubiquitin ligase function

eLife ◽

10.7554/elife.24325 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 21

Author(s):

Kate E Coleman ◽

Miklós Békés ◽

Jessica R Chapman ◽

Sarah B Crist ◽

Mathew JK Jones ◽

...

Keyword(s):

Cell Cycle ◽

Ubiquitin Ligase ◽

Cell Cycle Progression ◽

Cycle Progression ◽

E3 Ligases ◽

Resistant Form ◽

Ligase Function ◽

Conjugating Enzyme

NEDD8 is a ubiquitin-like modifier most well-studied for its role in activating the largest family of ubiquitin E3 ligases, the cullin-RING ligases (CRLs). While many non-cullin neddylation substrates have been proposed over the years, validation of true NEDD8 targets has been challenging, as overexpression of exogenous NEDD8 can trigger NEDD8 conjugation through the ubiquitylation machinery. Here, we developed a deconjugation-resistant form of NEDD8 to stabilize the neddylated form of cullins and other non-cullin substrates. Using this strategy, we identified Ubc12, a NEDD8-specific E2 conjugating enzyme, as a substrate for auto-neddylation. Furthermore, we characterized SENP8/DEN1 as the protease that counteracts Ubc12 auto-neddylation, and observed aberrant neddylation of Ubc12 and other NEDD8 conjugation pathway components in SENP8-deficient cells. Importantly, loss of SENP8 function contributes to accumulation of CRL substrates and defective cell cycle progression. Thus, our study highlights the importance of SENP8 in maintaining proper neddylation levels for CRL-dependent proteostasis.

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Cdc53p acts in concert with Cdc4p and Cdc34p to control the G1-to-S-phase transition and identifies a conserved family of proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.16.12.6634 ◽

1996 ◽

Vol 16 (12) ◽

pp. 6634-6643 ◽

Cited By ~ 127

Author(s):

N Mathias ◽

S L Johnson ◽

M Winey ◽

A E Adams ◽

L Goetsch ◽

...

Keyword(s):

Cell Cycle ◽

Cell Cycle Progression ◽

Large Family ◽

S Phase ◽

Genetic Interactions ◽

Cycle Progression ◽

Cyclin Dependent Kinases ◽

Ubiquitin Conjugating Enzyme ◽

Regulation Of Cell Cycle

Regulation of cell cycle progression occurs in part through the targeted degradation of both activating and inhibitory subunits of the cyclin-dependent kinases. During G1, CDC4, encoding a WD-40 repeat protein, and CDC34, encoding a ubiquitin-conjugating enzyme, are involved in the destruction of these regulators. Here we describe evidence indicating that CDC53 also is involved in this process. Mutations in CDC53 cause a phenotype indistinguishable from those of cdc4 and cdc34 mutations, numerous genetic interactions are seen between these genes, and the encoded proteins are found physically associated in vivo. Cdc53p defines a large family of proteins found in yeasts, nematodes, and humans whose molecular functions are uncharacterized. These results suggest a role for this family of proteins in regulating cell cycle proliferation through protein degradation.

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The CRL2LRR-1 ubiquitin ligase regulates cell cycle progression during C. elegans development.

Journal of Cell Science ◽

10.1242/jcs.083006 ◽

2010 ◽

Vol 123 (22) ◽

pp. e1-e1

Author(s):

J. Merlet ◽

J. Burger ◽

N. Tavernier ◽

B. Richaudeau ◽

J.-E. Gomes ◽

...

Keyword(s):

Cell Cycle ◽

Ubiquitin Ligase ◽

Cell Cycle Progression ◽

Cycle Progression ◽

C Elegans

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The E3 ubiquitin ligase HECTD1 contributes to cell cycle progression through an effect on mitosis

10.1101/2021.12.17.473173 ◽

2021 ◽

Author(s):

Natalie Vaughan ◽

Nico Scholz ◽

Catherine Lindon ◽

Julien D, F Licchesi

Keyword(s):

Cell Cycle ◽

Ubiquitin Ligase ◽

Cell Cycle Progression ◽

Spindle Assembly Checkpoint ◽

Time Lapse ◽

Ubiquitin Chain ◽

Cycle Progression ◽

Anaphase Onset ◽

Ubiquitin Ligase Activity ◽

Complex Protein

Mechanistic studies of how protein ubiquitylation regulates the cell cycle, in particular during mitosis, has provided unique insights which have contributed to the emergence of the Ubiquitin code. In contrast to RING E3 ubiquitin ligases such as the APC/c ligase complex, the contribution of other E3 ligase families during cell cycle progression remains less well understood. Similarly, the contribution of ubiquitin chain types beyond homotypic K48 chains in S-phase or branched K11/K48 chains assembled by APC/c during mitosis, also remains to be fully determined. Our recent findings that HECTD1 ubiquitin ligase activity assembles branched K29/K48 ubiquitin linkages prompted us to evaluate its function during the cell cycle. We used transient knockdown and genetic knockout to show that HECTD1 depletion in HEK293T and HeLa cells decreases cell proliferation and we established that this is mediated through loss of its ubiquitin ligase activity. Interestingly, we found that HECTD1 depletion increases the proportion of cells with aligned chromosomes (Prometa/Metaphase). We confirmed this molecularly using phospho-Histone H3 (Ser28) as a marker of mitosis. Time-lapse microscopy of NEBD to anaphase onset established that HECTD1-depleted cells take on average longer to go through mitosis. To explore the mechanisms involved, we used proteomics to explore the endogenous HECTD1 interactome in mitosis and validated the Mitosis Checkpoint Complex protein BUB3 as a novel HECTD1 interactor. In line with this, we found that HECTD1 depletion reduces the activity of the Spindle Assembly Checkpoint. Overall, our data suggests a novel role for HECTD1 ubiquitin ligase activity in mitosis.

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NEK7 is required for G1 progression and procentriole formation

Molecular Biology of the Cell ◽

10.1091/mbc.e16-09-0643 ◽

2017 ◽

Vol 28 (15) ◽

pp. 2123-2134 ◽

Cited By ~ 12

Author(s):

Akshari Gupta ◽

Yuki Tsuchiya ◽

Midori Ohta ◽

Gen Shiratsuchi ◽

Daiju Kitagawa

Keyword(s):

Cell Cycle ◽

Ubiquitin Ligase ◽

Cell Cycle Progression ◽

Primary Cilia ◽

S Phase ◽

Cycle Progression ◽

Restriction Point ◽

Centriole Duplication ◽

Abnormal Accumulation ◽

Phase Entry

The decision to commit to the cell cycle is made during G1 through the concerted action of various cyclin–CDK complexes. Not only DNA replication, but also centriole duplication is initiated as cells enter the S-phase. The NIMA-related kinase NEK7 is one of many factors required for proper centriole duplication, as well as for timely cell cycle progression. However, its specific roles in these events are poorly understood. In this study, we find that depletion of NEK7 inhibits progression through the G1 phase in human U2OS cells via down-regulation of various cyclins and CDKs and also inhibits the earliest stages of procentriole formation. Depletion of NEK7 also induces formation of primary cilia in human RPE1 cells, suggesting that NEK7 acts at least before the restriction point during G1. G1-arrested cells in the absence of NEK7 exhibit abnormal accumulation of the APC/C cofactor Cdh1 at the vicinity of centrioles. Furthermore, the ubiquitin ligase APC/CCdh1continuously degrades the centriolar protein STIL in these cells, thus inhibiting centriole assembly. Collectively our results demonstrate that NEK7 is involved in the timely regulation of G1 progression, S-phase entry, and procentriole formation.

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The E3 ubiquitin ligase TRIP12 participates in cell cycle progression and chromosome stability

Scientific Reports ◽

10.1038/s41598-020-57762-9 ◽

2020 ◽

Vol 10 (1) ◽

Cited By ~ 6

Author(s):

D. Larrieu ◽

M. Brunet ◽

C. Vargas ◽

N. Hanoun ◽

L. Ligat ◽

...

Keyword(s):

Cell Cycle ◽

Ubiquitin Ligase ◽

Cell Cycle Progression ◽

E3 Ubiquitin Ligase ◽

Chromosome Stability ◽

Cycle Progression

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Cell adhesion induces p27Kip1-associated cell-cycle arrest through down-regulation of the SCFSkp2 ubiquitin ligase pathway in mantle-cell and other non-Hodgkin B-cell lymphomas

Blood ◽

10.1182/blood-2006-11-060350 ◽

2007 ◽

Vol 110 (5) ◽

pp. 1631-1638 ◽

Cited By ~ 71

Author(s):

Tint Lwin ◽

Lori A. Hazlehurst ◽

Sophie Dessureault ◽

Raymond Lai ◽

Wenlong Bai ◽

...

Keyword(s):

Cell Cycle ◽

Cell Adhesion ◽

Tumor Cell ◽

Ubiquitin Ligase ◽

Cell Cycle Progression ◽

Response To Therapy ◽

G1 Arrest ◽

Down Regulation ◽

Cycle Progression ◽

Mantle Cell

Abstract Mounting evidence suggests that dynamic interactions between a tumor and its microenvironment play a critical role in tumor development, cell-cycle progression, and response to therapy. In this study, we used mantle cell lymphoma (MCL) as a model to characterize the mechanisms by which stroma regulate cell-cycle progression. We demonstrated that adhesion of MCL and other non-Hodgkin lymphoma (NHL) cells to bone marrow stromal cells resulted in a reversible G1 arrest associated with elevated p27Kip1 and p21 (WAF1) proteins. The adhesion-mediated p27Kip1 and p21 increases were posttranslationally regulated via the down-regulation of Skp2, a subunit of SCFSkp2 ubiquitin ligase. Overexpression of Skp2 in MCL decreased p27Kip1, whereas inhibition of Skp2 by siRNA increased p27Kip1 and p21 levels. Furthermore, we found cell adhesion up-regulated Cdh1 (an activating subunit of anaphase-promoting complex [APC] ubiquitin ligase), and reduction of Cdh1 by siRNA induced Skp2 accumulation and hence p27Kip1 degradation, thus implicating Cdh1 as an upstream effector of the Skp2/p27Kip1 signaling pathway. Overall, this report, for the first time, demonstrates that cell-cell contact controls the tumor cell cycle via ubiquitin-proteasome proteolytic pathways in MCL and other NHLs. The understanding of this novel molecular pathway may prove valuable in designing new therapeutic approaches for modifying tumor cell growth and response to therapy.

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Cell cycle oscillators underlying orderly proteolysis of E2F8

10.1101/672964 ◽

2019 ◽

Author(s):

Danit Wasserman ◽

Sapir Nachum ◽

Meital Cohen ◽

Taylor P Enrico ◽

Meirav Noach-Hirsh ◽

...

Keyword(s):

Cell Cycle ◽

Ubiquitin Ligase ◽

Cell Cycle Progression ◽

Cycle Progression ◽

Dynamic Cell ◽

G2 Phase ◽

Differential Stability ◽

Complementary Set ◽

Cyclin F ◽

Transcription Factor E2f1

AbstractE2F8 is a transcriptional repressor that antagonizes the canonical cell cycle transcription factor E2F1. Despite the importance of this atypical E2F family member in cell cycle, apoptosis and cancer, we lack a complete description of the mechanisms that control its dynamics. To address this question, we developed a complementary set of static and dynamic cell-free systems of human origin, which recapitulate inter-mitotic and G1 phases, and a full transition from pro-metaphase to G1. This revealed an interlocking molecular switch controlling E2F8 degradation at mitotic exit, involving dephosphorylation of Cdk1 sites in E2F8 and the activation of APC/CCdh1, but not APC/CCdc20. Further, we revealed a differential stability of E2F8, accounting for its accumulation in late G1 while APC/CCdh1 is still active and suggesting a key role for APC/C in controlling G1-S transcription. Finally, we identified SCF-Cyclin F as the ubiquitin ligase controlling E2F8 in G2-phase. Altogether, our data provide new insights into the regulation of E2F8 throughout the cell cycle, illuminating an extensive coordination between phosphorylation, ubiquitination and transcription in promoting orderly cell cycle progression.

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