scholarly journals Fission Yeast F-box Protein Pof3 Is Required for Genome Integrity and Telomere Function

2002 ◽  
Vol 13 (1) ◽  
pp. 211-224 ◽  
Author(s):  
Satoshi Katayama ◽  
Kenji Kitamura ◽  
Anna Lehmann ◽  
Osamu Nikaido ◽  
Takashi Toda
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Ubiquitin Ligase ◽  
Repeat Motif ◽  
High Rate ◽  
Genome Integrity ◽  
Gene Encoding ◽  
Cell Cycle Delay ◽  
C Terminus ◽  
F Box Protein

The Skp1-Cullin-1/Cdc53-F-box protein (SCF) ubiquitin ligase plays an important role in various biological processes. In this enzyme complex, a variety of F-box proteins act as receptors that recruit substrates. We have identified a fission yeast gene encoding a novel F-box protein Pof3, which contains, in addition to the F-box, a tetratricopeptide repeat motif in its N terminus and a leucine-rich-repeat motif in the C terminus, two ubiquitous protein–protein interaction domains. Pof3 forms a complex with Skp1 and Pcu1 (fission yeast cullin-1), suggesting that Pof3 functions as an adaptor for specific substrates. In the absence of Pof3, cells exhibit a number of phenotypes reminiscent of genome integrity defects. These include G2 cell cycle delay, hypersensitivity to UV, appearance of lagging chromosomes, and a high rate of chromosome loss.pof3 deletion strains are viable because the DNA damage checkpoint is continuously activated in the mutant, and this leads to G2 cell cycle delay, thereby preventing the mutant from committing lethal mitosis. Pof3 localizes to the nucleus during the cell cycle. Molecular analysis reveals that in this mutant the telomere is substantially shortened and furthermore transcriptional silencing at the telomere is alleviated. The results highlight a role of the SCFPof3 ubiquitin ligase in genome integrity via maintaining chromatin structures.

scholarly journals Binding of STIL to Plk4 activates kinase activity to promote centriole assembly

2015 ◽  
Vol 209 (6) ◽  
pp. 863-878 ◽  
Author(s):  
Tyler C. Moyer ◽  
Kevin M. Clutario ◽  
Bramwell G. Lambrus ◽  
Vikas Daggubati ◽  
Andrew J. Holland
Keyword(s):  
Cell Cycle ◽  
Gene Editing ◽  
Genetic System ◽  
Genome Integrity ◽  
Chemical Genetic ◽  
Centriole Duplication ◽  
C Terminus ◽  
A Cell ◽  
Direct Binding ◽  
Centriole Assembly

Centriole duplication occurs once per cell cycle in order to maintain control of centrosome number and ensure genome integrity. Polo-like kinase 4 (Plk4) is a master regulator of centriole biogenesis, but how its activity is regulated to control centriole assembly is unclear. Here we used gene editing in human cells to create a chemical genetic system in which endogenous Plk4 can be specifically inhibited using a cell-permeable ATP analogue. Using this system, we demonstrate that STIL localization to the centriole requires continued Plk4 activity. Most importantly, we show that direct binding of STIL activates Plk4 by promoting self-phosphorylation of the activation loop of the kinase. Plk4 subsequently phosphorylates STIL to promote centriole assembly in two steps. First, Plk4 activity promotes the recruitment of STIL to the centriole. Second, Plk4 primes the direct binding of STIL to the C terminus of SAS6. Our findings uncover a molecular basis for the timing of Plk4 activation through the cell cycle–regulated accumulation of STIL.

scholarly journals A fission yeast homolog of CDC20/p55CDC/Fizzy is required for recovery from DNA damage and genetically interacts with p34cdc2.

1997 ◽  
Vol 17 (2) ◽  
pp. 742-750 ◽  
Author(s):  
T Matsumoto
Keyword(s):  
Cell Cycle ◽  
Dna Damage ◽  
Dna Repair ◽  
Fission Yeast ◽  
Cell Cycle Progression ◽  
Eukaryotic Cell ◽  
Cycle Progression ◽  
Cell Cycle Delay ◽  
Successful Recovery ◽  
Synthetically Lethal

Successful recovery from DNA damage requires coordination of several biological processes. Eukaryotic cell cycle progression is delayed when the cells encounter DNA-damaging agents. This cell cycle delay allows the cells to cope with DNA damage by utilizing DNA repair enzymes. Thus, at least two processes, induction of the cell cycle delay and repair of damaged DNA, are coordinately required for recovery. In this study, a fission yeast rad mutant (slp1-362) was genetically investigated. In response to radiation, slp1 stops cell division; however, it does not restart it. This defect is suppressed when slp1-362 is combined with wee1-50 or cdc2-3w; in these mutants, the onset of mitosis is advanced due to the premature activation of p34cdc2. In contrast, slp1 is synthetically lethal with cdc25, nim1/cdr1, or cdr2, all of which are unable to activate the p34cdc2 kinase correctly. These genetic interactions of slp1 with cdc2 and its modulators imply that slp1 is not defective in either "induction of cell cycle delay" or "DNA repair." slp1+ may be involved in a critical process which restarts cell cycle progression after the completion of DNA repair. Molecular cloning of slp1+ revealed that slp1+ encodes a putative 488-amino-acid polypeptide exhibiting significant homology to WD-domain proteins, namely, CDC20 (budding yeast), p55CDC (human), and Fizzy (fly). A possible role of slp1+ is proposed.

scholarly journals The ETS Protein MEF Is Regulated by Phosphorylation-Dependent Proteolysis via the Protein-Ubiquitin Ligase SCFSkp2

2006 ◽  
Vol 26 (8) ◽  
pp. 3114-3123 ◽  
Author(s):  
Yan Liu ◽  
Cyrus V. Hedvat ◽  
Shifeng Mao ◽  
Xin-Hua Zhu ◽  
Jinjuan Yao ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Transcription Factor ◽  
Ubiquitin Ligase ◽  
Nk Cell ◽  
Retinoic Acid Receptor ◽  
Hematopoietic Stem ◽  
C Terminus ◽  
Related Transcription Factor ◽  
Retinoic Acid Receptor Α

ABSTRACT MEF is an ETS-related transcription factor with strong transcriptional activating activity that affects hematopoietic stem cell behavior and is required for normal NK cell and NK T-cell development. The MEF (also known as ELF4) gene is repressed by several leukemia-associated fusion transcription factor proteins (PML-retinoic acid receptor α and AML1-ETO), but it is also activated by retroviral insertion in several cancer models. We have previously shown that cyclin A-dependent phosphorylation of MEF largely restricts its activity to the G1 phase of the cell cycle; we now show that MEF is a short-lived protein whose expression level also peaks during late G1 phase. Mutagenesis studies show that the rapid turnover of MEF in S phase is dependent on the specific phosphorylation of threonine 643 and serine 648 at the C terminus of MEF by cdk2 and on the Skp1/Cul1/F-box (SCF) E3 ubiquitin ligase complex SCFSkp2, which targets MEF for ubiquitination and proteolysis. Overexpression of MEF drives cells through the G1/S transition, thereby promoting cell proliferation. The tight regulation of MEF levels during the cell cycle contributes to its effects on regulating cell cycle entry and cell proliferation.

scholarly journals Human Immunodeficiency Virus Type 1 Vif Induces Cell Cycle Delay via Recruitment of the Same E3 Ubiquitin Ligase Complex That Targets APOBEC3 Proteins for Degradation

2008 ◽  
Vol 82 (18) ◽  
pp. 9265-9272 ◽  
Author(s):  
Jason L. DeHart ◽  
Alberto Bosque ◽  
Reuben S. Harris ◽  
Vicente Planelles
Keyword(s):  
Cell Cycle ◽  
Human Immunodeficiency Virus ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Ubiquitin Ligase ◽  
Cell Cycle Delay ◽  
Immunodeficiency Virus ◽  
Apobec3 Proteins ◽  
Hiv 1

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Vif recruits a Cullin 5 ubiquitin ligase that targets APOBEC3 proteins for degradation. Recently, Vif has also been shown to induce cell cycle disturbance in G2. We show that in contrast to the expression of Vpr, the expression of Vif does not preclude cell division, and therefore, Vif causes delay and not arrest in G2. We also demonstrate that the interaction of Vif with the ubiquitin ligase is required for cell cycle disruption, as was previously shown for HIV-1 Vpr. The presence of APOBEC3 D/E, F, and G had no influence on Vif-induced alteration of the cell cycle. We conclude that cell cycle delay by Vif is a result of ubiquitination and degradation of a cellular protein that is different from the known APOBEC3 family members.

scholarly journals Functional Domains of Rep2, a Transcriptional Activator Subunit for Res2–Cdc10, Controlling the Cell Cycle “Start”

1998 ◽  
Vol 9 (6) ◽  
pp. 1577-1588 ◽  
Author(s):  
Sayaka Tahara ◽  
Koichi Tanaka ◽  
Yasuhito Yuasa ◽  
Hiroto Okayama
Keyword(s):  
Cell Cycle ◽  
Amino Acid ◽  
Fission Yeast ◽  
Transcriptional Activation ◽  
Transcriptional Activator ◽  
S Phase ◽  
Activation Domains ◽  
Multicopy Suppressor ◽  
C Terminus

In the fission yeast Schizosaccharomyces pombe, passage from G1 to S-phase requires the execution of the transcriptional factor complex that consists of the Cdc10 and Res1/2 molecules. This complex activates the MluI cell cycle box cis-element contained in genes essential for S-phase onset and progression. The rep2 + gene, isolated as a multicopy suppressor of a temperature-sensitivecdc10 mutant, has been postulated to encode a putative transcriptional activator subunit for the Res2–Cdc10 complex. To identify the rep2 + function and molecularly define its domain organization, we reconstituted the Res2–Cdc10 complex-dependent transcriptional activation in Saccharomyces cerevisiae. Reconstitution experiments, deletion analyses using one and two hybrid systems, and in vivo Res2 coimmunoprecipitation assays show that the Res2–Cdc10 complex itself can recognize but cannot activate MluI cell cycle box without Rep2, and that consistent with its postulated function, Rep2 contains 45-amino acid Res2 binding and 22-amino acid transcriptional activation domains in the middle and C terminus of the molecule, respectively. The functional essentiality of these domains is also demonstrated by their requirement for rescue of the cold-sensitive rep2deletion mutant of fission yeast.

The ASK1 gene regulates B function gene expression in cooperation with UFO and LEAFY in Arabidopsis

Development ◽  
2001 ◽  
Vol 128 (14) ◽  
pp. 2735-2746
Author(s):  
Dazhong Zhao ◽  
Qilu Yu ◽  
Min Chen ◽  
Hong Ma
Keyword(s):  
Ubiquitin Ligase ◽  
Floral Organ ◽  
Abc Model ◽  
Gene Encoding ◽  
Genetic Studies ◽  
Organ Identity ◽  
F Box Protein ◽  
In Situ Hybridizations ◽  
Floral Regulatory Genes

The Arabidopsis floral regulatory genes APETALA3 (AP3) and PISTILLATA (PI) are required for the B function according to the ABC model for floral organ identity. AP3 and PI expression are positively regulated by the LEAFY (LFY) and UNUSUAL FLORAL ORGANS (UFO) genes. UFO encodes an F-box protein, and we have shown previously that UFO genetically interacts with the ASK1 gene encoding a SKP1 homologue; both the F-box containing protein and SKP1 are subunits of ubiquitin ligases. We show here that the ask1-1 mutation can enhance the floral phenotypes of weak lfy and ap3 mutants; therefore, like UFO, ASK1 also interacts with LFY and AP3 genetically. Furthermore, our results from RNA in situ hybridizations indicate that ASK1 regulates early AP3 and PI expression. These results support the idea that UFO and ASK1 together positively regulate AP3 and PI expression. We propose that the UFO and ASK1 proteins are components of a ubiquitin ligase that mediates the proteolysis of a repressor of AP3 and PI expression. Our genetic studies also indicate that ASK1 and UFO play a role in regulating the number of floral organ primordia, and we discuss possible mechanisms for such a regulation.

A NIMA homologue promotes chromatin condensation in fission yeast

1998 ◽  
Vol 111 (7) ◽  
pp. 967-976 ◽  
Author(s):  
M.J. Krien ◽  
S.J. Bugg ◽  
M. Palatsides ◽  
G. Asouline ◽  
M. Morimyo ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Chromatin Condensation ◽  
Cell Types ◽  
Cell Cycle Delay ◽  
Downstream Target ◽  
A Cell ◽  
Chromatin Organisation ◽  
Bona Fide ◽  
P34 Cdc2

Entry into mitosis requires p34(cdc2), which activates downstream mitotic events through phosphorylation of key target proteins. In Aspergillus nidulans, the NIMA protein kinase has been identified as a potential downstream target and plays a role in regulating chromatin condensation at mitosis. nimA- mutants arrest in a state that physically resembles interphase even though p34(cdc2) is fully active. Despite evidence for the existence of NIMA-like activities in a variety of cell types, the only bona fide NIMA homologue that has been identified is the nim-1 gene of Neurospora crassa. We report here the isolation of a fission yeast NIMA homologue, and have designated this gene fin1 and the 83 kDa predicted protein p83(fin1). Overexpression of fin1 promotes premature chromatin condensation from any point in the cell cycle independently of p34(cdc2) function. Like NIMA, p83(fin1) levels fluctuate through the cell cycle, peaking in mitosis and levels are greatly elevated by removal of C-terminal PEST sequences. Deletion of fin1 results in viable but elongated cells, indicative of a cell cycle delay. Genetic analysis has placed this delay in G2 but, unlike in nimA mutants of Aspergillus, p34(cdc2) activation appears to be delayed. Interaction of fin1 mutants with other strains defective in chromatin organisation also support the hypothesis of p83(fin1) playing a role in this process at the onset of mitosis. These data indicate that NIMA-related kinases may be a general feature of the cell cycle and chromatin organisation at mitosis.

scholarly journals The SCFSlimb ubiquitin ligase regulates Plk4/Sak levels to block centriole reduplication

2009 ◽  
Vol 184 (2) ◽  
pp. 225-239 ◽  
Author(s):  
Gregory C. Rogers ◽  
Nasser M. Rusan ◽  
David M. Roberts ◽  
Mark Peifer ◽  
Stephen L. Rogers
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Genomic Stability ◽  
Ubiquitin Ligases ◽  
S Phase ◽  
Proteolytic Degradation ◽  
Centriole Duplication ◽  
F Box Protein

Restricting centriole duplication to once per cell cycle is critical for chromosome segregation and genomic stability, but the mechanisms underlying this block to reduplication are unclear. Genetic analyses have suggested an involvement for Skp/Cullin/F box (SCF)-class ubiquitin ligases in this process. In this study, we describe a mechanism to prevent centriole reduplication in Drosophila melanogaster whereby the SCF E3 ubiquitin ligase in complex with the F-box protein Slimb mediates proteolytic degradation of the centrosomal regulatory kinase Plk4. We identified SCFSlimb as a regulator of centriole duplication via an RNA interference (RNAi) screen of Cullin-based ubiquitin ligases. We found that Plk4 binds to Slimb and is an SCFSlimb target. Both Slimb and Plk4 localize to centrioles, with Plk4 levels highest at mitosis and absent during S phase. Using a Plk4 Slimb-binding mutant and Slimb RNAi, we show that Slimb regulates Plk4 localization to centrioles during interphase, thus regulating centriole number and ensuring the block to centriole reduplication.

scholarly journals Cdc6 degradation requires phosphodegron created by GSK-3 and Cdk1 for SCFCdc4 recognition in Saccharomyces cerevisiae

2015 ◽  
Vol 26 (14) ◽  
pp. 2609-2619 ◽  
Author(s):  
Amr Al-Zain ◽  
Lea Schroeder ◽  
Alina Sheglov ◽  
Amy E. Ikui
Keyword(s):  
Dna Damage ◽  
Dna Replication ◽  
Ubiquitin Ligase ◽  
Genome Integrity ◽  
Binding Motif ◽  
Dependent Manner ◽  
Cyclin Dependent Kinase ◽  
Origin Firing ◽  
C Terminus ◽  
Prereplicative Complex

To ensure genome integrity, DNA replication takes place only once per cell cycle and is tightly controlled by cyclin-dependent kinase (Cdk1). Cdc6p is part of the prereplicative complex, which is essential for DNA replication. Cdc6 is phosphorylated by cyclin-Cdk1 to promote its degradation after origin firing to prevent DNA rereplication. We previously showed that a yeast GSK-3 homologue, Mck1 kinase, promotes Cdc6 degradation in a SCFCdc4-dependent manner, therefore preventing rereplication. Here we present evidence that Mck1 directly phosphorylates a GSK-3 consensus site in the C-terminus of Cdc6. The Mck1-dependent Cdc6 phosphorylation required priming by cyclin/Cdk1 at an adjacent CDK consensus site. The sequential phosphorylation by Mck1 and Clb2/Cdk1 generated a Cdc4 E3 ubiquitin ligase–binding motif to promote Cdc6 degradation during mitosis. We further revealed that Cdc6 degradation triggered by Mck1 kinase was enhanced upon DNA damage caused by the alkylating agent methyl methanesulfonate and that the resulting degradation was mediated through Cdc4. Thus, Mck1 kinase ensures proper DNA replication, prevents DNA damage, and maintains genome integrity by inhibiting Cdc6.

scholarly journals Wobble Inosine tRNA Modification Is Essential to Cell Cycle Progression in G1/S and G2/M Transitions in Fission Yeast

2007 ◽  
Vol 282 (46) ◽  
pp. 33459-33465 ◽  
Author(s):  
Satoshi Tsutsumi ◽  
Reiko Sugiura ◽  
Yan Ma ◽  
Hideki Tokuoka ◽  
Kazuki Ohta ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Fission Yeast ◽  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Temperature Sensitive ◽  
Trna Modification ◽  
Gene Encoding ◽  
Cycle Progression ◽  
Catalytically Active ◽  
Mutant Cells

Inosine (I) at position 34 (wobble position) of tRNA is formed by the hydrolytic deamination of a genomically encoded adenosine (A). The enzyme catalyzing this reaction, termed tRNA A:34 deaminase, is the heterodimeric Tad2p/ADAT2·Tad3p/ADAT3 complex in eukaryotes. In budding yeast, deletion of each subunit is lethal, indicating that the wobble inosine tRNA modification is essential for viability; however, most of its physiological roles remain unknown. To identify novel cell cycle mutants in fission yeast, we isolated the tad3-1 mutant that is allelic to the tad3+ gene encoding a homolog of budding yeast Tad3p. Interestingly, the tad3-1 mutant cells principally exhibited cell cycle-specific phenotype, namely temperature-sensitive and irreversible cell cycle arrest both in G1 and G2. Further analyses revealed that in the tad3-1 mutant cells, the S257N mutation that occurred in the catalytically inactive Tad3 subunit affected its association with catalytically active Tad2 subunit, leading to an impairment in the A to I conversion at position 34 of tRNA. In tad3-1 mutant cells, the overexpression of the tad3+ gene completely suppressed the decreased tRNA inosine content. Notably, the overexpression of the tad2+ gene partially suppressed the temperature-sensitive phenotype and the decreased tRNA inosine content, indicating that the tad3-1 mutant phenotype is because of the insufficient I34 formation of tRNA. These results suggest that the wobble inosine tRNA modification is essential for cell cycle progression in the G1/S and G2/M transitions in fission yeast.

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