scholarly journals Distinct Mechanisms Control the Stability of the Related S-Phase Cyclins Clb5 and Clb6

2006 ◽  
Vol 26 (6) ◽  
pp. 2456-2466 ◽  
Author(s):  
Leisa P. Jackson ◽  
Steven I. Reed ◽  
Steven B. Haase
Keyword(s):  
Ubiquitin Ligase ◽  
S Phase ◽  
Phosphorylation Sites ◽  
Anaphase Promoting Complex ◽  
Functional Differences ◽  
Ubiquitin Ligase Complex ◽  
N Terminus ◽  
Phase Progression ◽  
The Stability ◽  
Related Proteins

ABSTRACT The yeast S-phase cyclins Clb5 and Clb6 are closely related proteins that are synthesized late in G1. Although often grouped together with respect to function, Clb5 and Clb6 exhibit differences in their ability to promote S-phase progression. DNA replication is significantly slowed in clb5Δ mutants but not in clb6Δ mutants. We have examined the basis for the differential functions of Clb5 and Clb6 and determined that unlike Clb5, which is stable until mitosis, Clb6 is degraded rapidly at the G1/S border. N-terminal deletions of CLB6 were hyperstabilized, suggesting that the sequences responsible for directing the destruction of Clb6 reside in the N terminus. Clb6 lacks the destruction box motif responsible for the anaphase promoting complex-mediated destruction of Clb5 but contains putative Cdc4 degron motifs in the N terminus. Clb6 was hyperstabilized in cdc34-3 and cdc4-3 mutants at restrictive temperatures and when S/T-P phosphorylation sites in the N terminus were mutated to nonphosphorylatable residues. Efficient degradation of Clb6 requires the activities of both Cdc28 and Pho85. Finally, hyperstabilized Clb6 expressed from the CLB6 promoter rescued the slow S-phase defect exhibited by clb5Δ cells. Taken together, these findings suggest that the SCFCdc4 ubiquitin ligase complex regulates Clb6 turnover and that the functional differences exhibited by Clb5 and Clb6 arise from the distinct mechanisms controlling their stability.

scholarly journals Hect E3 ubiquitin ligase Tom1 controls Dia2 degradation during the cell cycle

2012 ◽  
Vol 23 (21) ◽  
pp. 4203-4211 ◽  
Author(s):  
Dong-Hwan Kim ◽  
Deanna M. Koepp
Keyword(s):  
Cell Cycle ◽  
Protein Degradation ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
S Phase ◽  
Temperature Sensitive ◽  
Dependent Manner ◽  
Charged Residues ◽  
Phase Progression ◽  
Positively Charged

The ubiquitin proteasome system plays a pivotal role in controlling the cell cycle. The budding yeast F-box protein Dia2 is required for genomic stability and is targeted for ubiquitin-dependent degradation in a cell cycle–dependent manner, but the identity of the ubiquitination pathway is unknown. We demonstrate that the Hect domain E3 ubiquitin ligase Tom1 is required for Dia2 protein degradation. Deletion of DIA2 partially suppresses the temperature-sensitive phenotype of tom1 mutants. Tom1 is required for Dia2 ubiquitination and degradation during G1 and G2/M phases of the cell cycle, whereas the Dia2 protein is stabilized during S phase. We find that Tom1 binding to Dia2 is enhanced in G1 and reduced in S phase, suggesting a mechanism for this proteolytic switch. Tom1 recognizes specific, positively charged residues in a Dia2 degradation/NLS domain. Loss of these residues blocks Tom1-mediated turnover of Dia2 and causes a delay in G1–to–S phase progression. Deletion of DIA2 rescues a delay in the G1–to–S phase transition in the tom1Δ mutant. Together our results suggest that Tom1 targets Dia2 for degradation during the cell cycle by recognizing positively charged residues in the Dia2 degradation/NLS domain and that Dia2 protein degradation contributes to G1–to–S phase progression.

scholarly journals Degradation of the Transcription Factor Gcn4 Requires the Kinase Pho85 and the SCFCDC4 Ubiquitin–Ligase Complex

2000 ◽  
Vol 11 (3) ◽  
pp. 915-927 ◽  
Author(s):  
Ariella Meimoun ◽  
Tsvi Holtzman ◽  
Ziva Weissman ◽  
Helen J. McBride ◽  
David J. Stillman ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Ubiquitin Ligase ◽  
Protein A ◽  
Purine Biosynthesis ◽  
Cell Cycle Regulators ◽  
Rich Medium ◽  
Ubiquitin Ligase Complex ◽  
Ubiquitin Conjugating Enzyme ◽  
The Stability

Gcn4, a yeast transcriptional activator that promotes the expression of amino acid and purine biosynthesis genes, is rapidly degraded in rich medium. Here we report that SCFCDC4, a recently characterized protein complex that acts in conjunction with the ubiquitin-conjugating enzyme Cdc34 to degrade cell cycle regulators, is also necessary for the degradation of the transcription factor Gcn4. Degradation of Gcn4 occurs throughout the cell cycle, whereas degradation of the known cell cycle substrates of Cdc34/SCFCDC4 is cell cycle regulated. Gcn4 ubiquitination and degradation are regulated by starvation for amino acids, whereas the degradation of the cell cycle substrates of Cdc34/SCFCDC4 is unaffected by starvation. We further show that unlike the cell cycle substrates of Cdc34/SCFCDC4, which require phosphorylation by the kinase Cdc28, Gcn4 degradation requires the kinase Pho85. We identify the critical target site of Pho85 on Gcn4; a mutation of this site stabilizes the protein. A specific Pho85-Pcl complex that is able to phosphorylate Gcn4 on that site is inactive under conditions under which Gcn4 is stable. Thus, Cdc34/SCFCDC4 activity is constitutive, and regulation of the stability of its various substrates occurs at the level of their phosphorylation.

scholarly journals Nonperiodic Activity of the Human Anaphase-Promoting Complex–Cdh1 Ubiquitin Ligase Results in Continuous DNA Synthesis Uncoupled from Mitosis

2000 ◽  
Vol 20 (20) ◽  
pp. 7613-7623 ◽  
Author(s):  
Claus Storgaard Sørensen ◽  
Claudia Lukas ◽  
Edgar R. Kramer ◽  
Jan-Michael Peters ◽  
Jiri Bartek ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Division ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Kinase Inhibitor ◽  
Cyclin B1 ◽  
S Phase ◽  
Anaphase Promoting Complex

ABSTRACT Ubiquitin-proteasome-mediated destruction of rate-limiting proteins is required for timely progression through the main cell cycle transitions. The anaphase-promoting complex (APC), periodically activated by the Cdh1 subunit, represents one of the major cellular ubiquitin ligases which, in Saccharomyces cerevisiae andDrosophila spp., triggers exit from mitosis and during G1 prevents unscheduled DNA replication. In this study we investigated the importance of periodic oscillation of the APC-Cdh1 activity for the cell cycle progression in human cells. We show that conditional interference with the APC-Cdh1 dissociation at the G1/S transition resulted in an inability to accumulate a surprisingly broad range of critical mitotic regulators including cyclin B1, cyclin A, Plk1, Pds1, mitosin (CENP-F), Aim1, and Cdc20. Unexpectedly, although constitutively assembled APC-Cdh1 also delayed G1/S transition and lowered the rate of DNA synthesis during S phase, some of the activities essential for DNA replication became markedly amplified, mainly due to a progressive increase of E2F-dependent cyclin E transcription and a rapid turnover of the p27Kip1 cyclin-dependent kinase inhibitor. Consequently, failure to inactivate APC-Cdh1 beyond the G1/S transition not only inhibited productive cell division but also supported slow but uninterrupted DNA replication, precluding S-phase exit and causing massive overreplication of the genome. Our data suggest that timely oscillation of the APC-Cdh1 ubiquitin ligase activity represents an essential step in coordinating DNA replication with cell division and that failure of mechanisms regulating association of APC with the Cdh1 activating subunit can undermine genomic stability in mammalian cells.

The anaphase-promoting complex (APC): the sum of its parts?

2004 ◽  
Vol 32 (5) ◽  
pp. 724-727 ◽  
Author(s):  
L.A. Passmore
Keyword(s):  
Cell Cycle ◽  
Ubiquitin Ligase ◽  
E3 Ubiquitin Ligase ◽  
Ubiquitin Ligases ◽  
Anaphase Promoting Complex ◽  
Activator Proteins ◽  
Related Proteins ◽  
Single Subunit ◽  
Insight Into

The APC (anaphase-promoting complex) is a multisubunit E3 ubiquitin ligase that targets cell-cycle-related proteins for degradation by the 26 S proteasome. The APC contains at least 13 subunits and is regulated by the binding of co-activator proteins and by phosphorylation. It is not known why the APC contains 13 subunits when many other ubiquitin ligases are small single-subunit enzymes. In the present study, the structures and functions of individual APC subunits are discussed. By dissecting the roles of its parts, we hope to gain insight into the mechanism of the intact APC.

Fulfilling the metabolic requirements for cell proliferation

2012 ◽  
Vol 446 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Salvador Moncada ◽  
E. Annie Higgs ◽  
Sergio L. Colombo
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Ubiquitin Ligase ◽  
Cell Cycle Progression ◽  
Ubiquitin Ligases ◽  
S Phase ◽  
Simultaneous Increase ◽  
Anaphase Promoting Complex ◽  
Restriction Point ◽  
Specific Degradation

The activity of key metabolic enzymes is regulated by the ubiquitin ligases that control the function of the cyclins; therefore the activity of these ubiquitin ligases explains the coordination of cell-cycle progression with the supply of substrates necessary for cell duplication. APC/C (anaphase-promoting complex/cyclosome)-Cdh1, the ubiquitin ligase that controls G1- to S-phase transition by targeting specific degradation motifs in cell-cycle proteins, also regulates the glycolysis-promoting enzyme PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3) and GLS1 (glutaminase 1), a critical enzyme in glutaminolysis. A decrease in the activity of APC/C-Cdh1 in mid-to-late G1 releases both proteins, thus explaining the simultaneous increase in the utilization of glucose and glutamine during cell proliferation. This occurs at a time consistent with the point in G1 that has been described as the nutrient-sensitive restriction point and is responsible for the transition from G1 to S. PFKFB3 is also a substrate at the onset of S-phase for the ubiquitin ligase SCF (Skp1/cullin/F-box)-β-TrCP (β-transducin repeat-containing protein), so that the activity of PFKFB3 is short-lasting, coinciding with a peak in glycolysis in mid-to-late G1, whereas the activity of GLS1 remains high throughout S-phase. The differential regulation of the activity of these proteins indicates that a finely-tuned set of mechanisms is activated to fulfil specific metabolic demands at different stages of the cell cycle. These findings have implications for the understanding of cell proliferation in general and, in particular, of cancer, its prevention and treatment.

scholarly journals Dbf4p, an Essential S Phase-Promoting Factor, Is Targeted for Degradation by the Anaphase-Promoting Complex

2000 ◽  
Vol 20 (1) ◽  
pp. 242-248 ◽  
Author(s):  
Miguel Godinho Ferreira ◽  
Corrado Santocanale ◽  
Lucy S. Drury ◽  
John F. X. Diffley
Keyword(s):  
Cell Cycle ◽  
Chromosome Segregation ◽  
S Phase ◽  
Regulatory Subunit ◽  
Anaphase Promoting Complex ◽  
Rapid Degradation ◽  
N Terminus ◽  
Chromosome Separation ◽  
Apc Mutation ◽  
Redundant Role

ABSTRACT The Dbf4p/Cdc7p protein kinase is essential for the activation of replication origins during S phase. The catalytic subunit, Cdc7p, is present at constant levels throughout the cell cycle. In contrast, we show here that the levels of the regulatory subunit, Dbf4p, oscillate during the cell cycle. Dbf4p is absent from cells during G1and accumulates during the S and G2 phases. Dbf4p is rapidly degraded at the time of chromosome segregation and remains highly unstable during pre-Start G1 phase. The rapid degradation of Dbf4p during G1 requires a functional anaphase-promoting complex (APC). Mutation of a sequence in the N terminus of Dbf4p which resembles the cyclin destruction box eliminates this APC-dependent degradation of Dbf4p. We suggest that the coupling of Dbf4p degradation to chromosome separation may play a redundant role in ensuring that prereplicative complexes, which assemble after chromosome segregation, do not immediately refire.

scholarly journals How cyclin A destruction escapes the spindle assembly checkpoint

2010 ◽  
Vol 190 (4) ◽  
pp. 501-509 ◽  
Author(s):  
Barbara Di Fiore ◽  
Jonathon Pines
Keyword(s):  
Ubiquitin Ligase ◽  
Spindle Assembly Checkpoint ◽  
Spindle Assembly ◽  
Cyclin A ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
N Terminus ◽  
Specific Proteins ◽  
Necessary And Sufficient

The anaphase-promoting complex/cyclosome (APC/C) is the ubiquitin ligase essential to mitosis, which ensures that specific proteins are degraded at specific times to control the order of mitotic events. The APC/C coactivator, Cdc20, is targeted by the spindle assembly checkpoint (SAC) to restrict APC/C activity until metaphase, yet early substrates, such as cyclin A, are degraded in the presence of the active checkpoint. Cdc20 and the cyclin-dependent kinase cofactor, Cks, are required for cyclin A destruction, but how they enable checkpoint-resistant destruction has not been elucidated. In this study, we answer this problem: we show that the N terminus of cyclin A binds directly to Cdc20 and with sufficient affinity that it can outcompete the SAC proteins. Subsequently, the Cks protein is necessary and sufficient to promote cyclin A degradation in the presence of an active checkpoint by binding cyclin A–Cdc20 to the APC/C.

scholarly journals A Subunit of the Anaphase-Promoting Complex Is a Centromere-Associated Protein in Mammalian Cells

1998 ◽  
Vol 18 (1) ◽  
pp. 468-476 ◽  
Author(s):  
Pia-Marie Jörgensen ◽  
Eva Brundell ◽  
Maria Starborg ◽  
Christer Höög
Keyword(s):  
Ubiquitin Ligase ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Separation Process ◽  
Soluble Form ◽  
Anaphase Promoting Complex ◽  
Mitotic Chromosomes ◽  
Sister Chromatids ◽  
Ubiquitin Ligase Complex ◽  
A Subunit

ABSTRACT Sister chromatids in early mitotic cells are held together mainly by interactions between centromeres. The separation of sister chromatids at the transition between the metaphase and the anaphase stages of mitosis depends on the anaphase-promoting complex (APC), a 20S ubiquitin-ligase complex that targets proteins for destruction. A subunit of the APC, called APC-α in Xenopus (and whose homologs are APC-1, Cut4, BIME, and Tsg24), has recently been identified and shown to be required for entry into anaphase. We now show that the mammalian APC-α homolog, Tsg24, is a centromere-associated protein. While this protein is detected only during the prophase to the anaphase stages of mitosis in Chinese hamster cells, it is constitutively associated with the centromeres in murine cells. We show that there are two forms of this protein in mammalian cells, a soluble form associated with other components of the APC and a centromere-bound form. We also show that both the Tsg24 protein and the Cdc27 protein, another APC component, are bound to isolated mitotic chromosomes. These results therefore support a model in which the APC by ubiquitination of a centromere protein regulates the sister chromatid separation process.

scholarly journals Regulation of FLIP(L) and TRAIL-R2 signalling by the SCFSkp2 Ubiquitin Ligase Complex

2019 ◽  
Author(s):  
JZ Roberts ◽  
C Holohan ◽  
T Sessler ◽  
J Fox ◽  
C. Higgins ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Large Subunit ◽  
Caspase 8 ◽  
Apoptosis Induction ◽  
Splice Form ◽  
Expression Levels ◽  
Ubiquitin Ligase Complex ◽  
Direct Binding ◽  
The Stability ◽  
Ring E3

AbstractDepending on its expression levels, the long splice form of the pseudo-caspase FLIP (FLIP(L)) can act as an inhibitor (high expression) or activator (low expression) of apoptosis induction by the TRAIL-R2 death-inducing signalling complex (DISC); its expression levels are therefore tightly regulated. Here, we demonstrate that the Skp1-Cullin-1-F-box (SCF) Cullin-Ring E3 Ubiquitin Ligase complex containing Skp2 (SCFSkp2) regulates the stability of FLIP(L) (but not the short splice form FLIP(S)), and, unusually, this is mediated by direct binding of FLIP(L) to Cullin-1 rather than via Skp2. By fine mapping the interaction of FLIP(L) with Cullin-1 to the large subunit of its pseudo-caspase domain, we found that the interaction is significantly stronger with FLIP(L)’s DISC-processed p43-form. Importantly, this interaction disrupts the ability of p43-FLIP to interact with FADD, caspase-8 and another DISC component, TRAF2. Moreover, we find that SCFSkp2 associates with TRAIL-R2 constitutively and does so independently of FLIP(L) and other canonical DISC components. Inhibition of Cullin-1 expression (using siRNA) or activity (using a NEDDylation inhibitor, MLN4924) enhanced FLIP(L) and TRAF2 levels at the TRAIL-R2 DISC and enhanced caspase-8 processing. This suggests that processing of FLIP(L) to p43-FLIP at the TRAIL-R2 DISC enhances its interaction with co-localised SCFSkp2, leading to disruption of p43-FLIP’s association with the DISC thereby altering caspase-8 processing. These findings provide important new insights into how FLIP(L) expression and TRAIL-R2 signaling is controlled.

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