scholarly journals Characterization of Vertebrate Cohesin Complexes and Their Regulation in Prophase

2000 ◽  
Vol 151 (4) ◽  
pp. 749-762 ◽  
Author(s):  
Izabela Sumara ◽  
Elisabeth Vorlaufer ◽  
Christian Gieffers ◽  
Beate H. Peters ◽  
Jan-Michael Peters
Keyword(s):  
Budding Yeast ◽  
Human Cells ◽  
Cyclin B ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Sister Chromatids ◽  
A Subunit ◽  
Mitotic Regulation

In eukaryotes, sister chromatids remain connected from the time of their synthesis until they are separated in anaphase. This cohesion depends on a complex of proteins called cohesins. In budding yeast, the anaphase-promoting complex (APC) pathway initiates anaphase by removing cohesins from chromosomes. In vertebrates, cohesins dissociate from chromosomes already in prophase. To study their mitotic regulation we have purified two 14S cohesin complexes from human cells. Both complexes contain SMC1, SMC3, SCC1, and either one of the yeast Scc3p orthologs SA1 and SA2. SA1 is also a subunit of 14S cohesin in Xenopus. These complexes interact with PDS5, a protein whose fungal orthologs have been implicated in chromosome cohesion, condensation, and recombination. The bulk of SA1- and SA2-containing complexes and PDS5 are chromatin-associated until they become soluble from prophase to telophase. Reconstitution of this process in mitotic Xenopus extracts shows that cohesin dissociation does neither depend on cyclin B proteolysis nor on the presence of the APC. Cohesins can also dissociate from chromatin in the absence of cyclin-dependent kinase 1 activity. These results suggest that vertebrate cohesins are regulated by a novel prophase pathway which is distinct from the APC pathway that controls cohesins in yeast.

scholarly journals Cell cycle regulation in Aspergillus by two protein kinases

1996 ◽  
Vol 317 (3) ◽  
pp. 633-641 ◽  
Author(s):  
Stephen A. OSMANI ◽  
Xiang S. YE
Keyword(s):  
Cell Cycle ◽  
Protein Kinases ◽  
Cell Cycle Regulation ◽  
Cell Cycle Progression ◽  
Human Cells ◽  
Dominant Negative ◽  
Cyclin B ◽  
Cyclin Dependent Kinases ◽  
Cycle Regulation ◽  
Mitotic Regulation

Great progress has recently been made in our understanding of the regulation of the eukaryotic cell cycle, and the central role of cyclin-dependent kinases is now clear. In Aspergillus nidulans it has been established that a second class of cell-cycle-regulated protein kinases, typified by NIMA (encoded by the nimA gene), is also required for cell cycle progression into mitosis. Indeed, both p34cdc2/cyclin B and NIMA have to be correctly activated before mitosis can be initiated in this species, and p34cdc2/cyclin B plays a role in the mitosis-specific activation of NIMA. In addition, both kinases have to be proteolytically destroyed before mitosis can be completed. NIMA-related kinases may also regulate the cell cycle in other eukaryotes, as expression of NIMA can promote mitotic events in yeast, frog or human cells. Moreover, dominant-negative versions of NIMA can adversely affect the progression of human cells into mitosis, as they do in A. nidulans. The ability of NIMA to influence mitotic regulation in human and frog cells strongly suggests the existence of a NIMA pathway of mitotic regulation in higher eukaryotes. A growing number of NIMA-related kinases have been isolated from organisms ranging from fungi to humans, and some of these kinases are also cell-cycle-regulated. How NIMA-related kinases and cyclin-dependent kinases act in concert to promote cell cycle transitions is just beginning to be understood. This understanding is the key to a full knowledge of cell cycle regulation.

scholarly journals Cdc28 Activates Exit from Mitosis in Budding Yeast

2000 ◽  
Vol 149 (7) ◽  
pp. 1361-1376 ◽  
Author(s):  
Adam D. Rudner ◽  
Kevin G. Hardwick ◽  
Andrew W. Murray
Keyword(s):  
Cell Cycle ◽  
Kinase Activity ◽  
Budding Yeast ◽  
G1 Phase ◽  
Phosphorylation Sites ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Reduced Activity

The activity of the cyclin-dependent kinase 1 (Cdk1), Cdc28, inhibits the transition from anaphase to G1 in budding yeast. CDC28-T18V, Y19F (CDC28-VF), a mutant that lacks inhibitory phosphorylation sites, delays the exit from mitosis and is hypersensitive to perturbations that arrest cells in mitosis. Surprisingly, this behavior is not due to a lack of inhibitory phosphorylation or increased kinase activity, but reflects reduced activity of the anaphase-promoting complex (APC), a defect shared with other mutants that lower Cdc28/Clb activity in mitosis. CDC28-VF has reduced Cdc20- dependent APC activity in mitosis, but normal Hct1- dependent APC activity in the G1 phase of the cell cycle. The defect in Cdc20-dependent APC activity in CDC28-VF correlates with reduced association of Cdc20 with the APC. The defects of CDC28-VF suggest that Cdc28 activity is required to induce the metaphase to anaphase transition and initiate the transition from anaphase to G1 in budding yeast.

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 Biochemical and functional characterization of a meiosis-specific Pch2/ORC AAA+ assembly

2020 ◽  
Vol 3 (11) ◽  
pp. e201900630
Author(s):  
María Ascensión Villar-Fernández ◽  
Richard Cardoso da Silva ◽  
Magdalena Firlej ◽  
Dongqing Pan ◽  
Elisabeth Weir ◽  
...  
Keyword(s):  
Biochemical Analysis ◽  
Budding Yeast ◽  
Functional Characterization ◽  
Efficient Removal ◽  
Terminal Domain ◽  
Origins Of Replication ◽  
Mcm Helicase ◽  
A Subunit ◽  
Aaa Protein

Pch2 is a meiosis-specific AAA+ protein that controls several important chromosomal processes. We previously demonstrated that Orc1, a subunit of the ORC, functionally interacts with budding yeast Pch2. The ORC (Orc1-6) AAA+ complex loads the AAA+ MCM helicase to origins of replication, but whether and how ORC collaborates with Pch2 remains unclear. Here, we show that a Pch2 hexamer directly associates with ORC during the meiotic G2/prophase. Biochemical analysis suggests that Pch2 uses its non-enzymatic NH2-terminal domain and AAA+ core and likely engages the interface of ORC that also binds to Cdc6, a factor crucial for ORC-MCM binding. Canonical ORC function requires association with origins, but we show here that despite causing efficient removal of Orc1 from origins, nuclear depletion of Orc2 and Orc5 does not trigger Pch2/Orc1-like meiotic phenotypes. This suggests that the function for Orc1/Pch2 in meiosis can be executed without efficient association of ORC with origins of replication. In conclusion, we uncover distinct functionalities for Orc1/ORC that drive the establishment of a non-canonical, meiosis-specific AAA+ assembly with Pch2.

scholarly journals Budding yeast PAK kinases regulate mitotic exit by two different mechanisms

2003 ◽  
Vol 160 (6) ◽  
pp. 857-874 ◽  
Author(s):  
Elena Chiroli ◽  
Roberta Fraschini ◽  
Alessia Beretta ◽  
Mariagrazia Tonelli ◽  
Giovanna Lucchini ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Budding Yeast ◽  
Mitotic Exit ◽  
Dominant Negative ◽  
Anaphase Promoting Complex ◽  
Gene Encoding ◽  
Cycle Progression ◽  
Mitotic Exit Network ◽  
P21 Activated Kinase

We report the characterization of the dominant-negative CLA4t allele of the budding yeast CLA4 gene, encoding a member of the p21-activated kinase (PAK) family of protein kinases, which, together with its homologue STE20, plays an essential role in promoting budding and cytokinesis. Overproduction of the Cla4t protein likely inhibits both endogenous Cla4 and Ste20 and causes a delay in the onset of anaphase that correlates with inactivation of Cdc20/anaphase-promoting complex (APC)–dependent proteolysis of both the cyclinB Clb2 and securin. Although the precise mechanism of APC inhibition by Cla4t remains to be elucidated, our results suggest that Cla4 and Ste20 may regulate the first wave of cyclinB proteolysis mediated by Cdc20/APC, which has been shown to be crucial for activation of the mitotic exit network (MEN). We show that the Cdk1-inhibitory kinase Swe1 is required for the Cla4t-dependent delay in cell cycle progression, suggesting that it might be required to prevent full Cdc20/APC and MEN activation. In addition, inhibition of PAK kinases by Cla4t prevents mitotic exit also by a Swe1-independent mechanism impinging directly on the MEN activator Tem1.

scholarly journals Punctuated cyclin synthesis drives early embryonic cell cycle oscillations

2012 ◽  
Vol 23 (2) ◽  
pp. 284-296 ◽  
Author(s):  
Qing Kang ◽  
Joseph R. Pomerening
Keyword(s):  
Feedback Loop ◽  
Proteasome Inhibition ◽  
Cyclin B1 ◽  
Embryonic Cell ◽  
Cyclin B ◽  
Synthesis Rate ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Effective Removal ◽  
Embryonic Cell Cycle

Cyclin B activates cyclin-dependent kinase 1 (CDK1) at mitosis, but conflicting views have emerged on the dynamics of its synthesis during embryonic cycles, ranging from continuous translation to rapid synthesis during mitosis. Here we show that a CDK1-mediated negative-feedback loop attenuates cyclin production before mitosis. Cyclin B plateaus before peak CDK1 activation, and proteasome inhibition caused minimal accumulation during mitosis. Inhibiting CDK1 permitted continual cyclin B synthesis, whereas adding nondegradable cyclin stalled it. Cycloheximide treatment before mitosis affected neither cyclin levels nor mitotic entry, corroborating this repression. Attenuated cyclin production collaborates with its destruction, since excess cyclin B1 mRNA accelerated cyclin synthesis and caused incomplete proteolysis and mitotic arrest. This repression involved neither adenylation nor the 3′ untranslated region, but it corresponded with a shift in cyclin B1 mRNA from polysome to nonpolysome fractions. A pulse-driven CDK1–anaphase-promoting complex (APC) model corroborated these results, revealing reduced cyclin levels during an oscillation and permitting more effective removal. This design also increased the robustness of the oscillator, with lessened sensitivity to changes in cyclin synthesis rate. Taken together, the results of this study underscore that attenuating cyclin synthesis late in interphase improves both the efficiency and robustness of the CDK1-APC oscillator.

scholarly journals Germline Missense Variants in CDC20 Result in Aberrant Mitotic Progression and Familial Cancer

2021 ◽  
Author(s):  
Owen J. Chen ◽  
Ester Castellsagué ◽  
Mohamed Moustafa-Kamal ◽  
Javad Nadaf ◽  
Barbara Rivera ◽  
...  
Keyword(s):  
Spindle Assembly Checkpoint ◽  
Familial Cancer ◽  
Anaphase Promoting Complex ◽  
Mitotic Progression ◽  
Daughter Cells ◽  
Sister Chromatids ◽  
Mitotic Slippage ◽  
Oncogenic Mutations ◽  
Whole Exome

SUMMARYCDC20 is a co-activator of the anaphase promoting complex/cyclosome (APC/C) and is essential for mitotic progression. APC/CCDC20 is inhibited by the spindle assembly checkpoint (SAC), which prevents premature separation of sister chromatids and aneuploidy in daughter cells. Although overexpression of CDC20 is common in many cancers, oncogenic mutations have never been identified in humans. Using whole exome sequencing, we identified heterozygous missense CDC20 variants (L151R and N331K) that segregate with cancer in two families. Characterization of these mutants showed they retain APC/C activation activity but show reduced binding to BUBR1, a component of the SAC. Expression of L151R and N331K promoted mitotic slippage in HeLa cells and primary skin fibroblasts derived from carriers. CRISPR/Cas9 was used to generate mice carrying N331K. Homozygous mice carrying N331K were non-viable, however, heterozygotes displayed accelerated oncogenicity in Myc-driven cancers. These findings highlight an unappreciated role for CDC20 variants as tumor promoting genes in humans.

scholarly journals Correct spindle elongation at the metaphase/anaphase transition is an APC-dependent event in budding yeast

2001 ◽  
Vol 155 (5) ◽  
pp. 711-718 ◽  
Author(s):  
Fedor Severin ◽  
Anthony A. Hyman ◽  
Simonetta Piatti
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Chromosome Segregation ◽  
Sister Chromatid ◽  
Budding Yeast ◽  
Sister Chromatid Cohesion ◽  
Anaphase Promoting Complex ◽  
Sister Chromatids ◽  
Chromatid Cohesion ◽  
Spindle Elongation

At the metaphase to anaphase transition, chromosome segregation is initiated by the splitting of sister chromatids. Subsequently, spindles elongate, separating the sister chromosomes into two sets. Here, we investigate the cell cycle requirements for spindle elongation in budding yeast using mutants affecting sister chromatid cohesion or DNA replication. We show that separation of sister chromatids is not sufficient for proper spindle integrity during elongation. Rather, successful spindle elongation and stability require both sister chromatid separation and anaphase-promoting complex activation. Spindle integrity during elongation is dependent on proteolysis of the securin Pds1 but not on the activity of the separase Esp1. Our data suggest that stabilization of the elongating spindle at the metaphase to anaphase transition involves Pds1-dependent targets other than Esp1.

scholarly journals Members of the NAP/SET family of proteins interact specifically with B-type cyclins.

1995 ◽  
Vol 130 (3) ◽  
pp. 661-673 ◽  
Author(s):  
D R Kellogg ◽  
A Kikuchi ◽  
T Fujii-Nakata ◽  
C W Turck ◽  
A W Murray
Keyword(s):  
Cell Cycle ◽  
Fusion Protein ◽  
Affinity Chromatography ◽  
Budding Yeast ◽  
Cyclin A ◽  
Xenopus Embryo ◽  
Catalytic Subunit ◽  
Cyclin B ◽  
Cyclin Dependent Kinase ◽  
Do So

Cyclin-dependent kinase complexes that contain the same catalytic subunit are able to induce different events at different times during the cell cycle, but the mechanisms by which they do so remain largely unknown. To address this problem, we have used affinity chromatography to identify proteins that bind specifically to mitotic cyclins, with the goal of finding proteins that interact with mitotic cyclins to carry out the events of mitosis. This approach has led to the identification of a 60-kD protein called NAP1 that interacts specifically with members of the cyclin B family. This interaction has been highly conserved during evolution: NAP1 in the Xenopus embryo interacts with cyclins B1 and B2, but not with cyclin A, and the S. cerevisiae homolog of NAP1 interacts with Clb2 but not with Clb3. Genetic experiments in budding yeast indicate that NAP1 plays an important role in the function of Clb2, while biochemical experiments demonstrate that purified NAP1 can be phosphorylated by cyclin B/p34cdc2 kinase complexes, but not by cyclin A/p34cdc2 kinase complexes. These results suggest that NAP1 is a protein involved in the specific functions of cyclin B/p34cdc2 kinase complexes. In addition to NAP1, we found a 43-kD protein in Xenopus that is homologous to NAP1 and also interacts specifically with B-type cyclins. This protein is the Xenopus homolog of the human SET protein, which was previously identified as part of a putative oncogenic fusion protein (Von Lindern et al., 1992).

scholarly journals Study of Cyclin Proteolysis in Anaphase-Promoting Complex (APC) Mutant Cells Reveals the Requirement for APC Function in the Final Steps of the Fission Yeast Septation Initiation Network

2001 ◽  
Vol 21 (19) ◽  
pp. 6681-6694 ◽  
Author(s):  
Louise Chang ◽  
Jennifer L. Morrell ◽  
Anna Feoktistova ◽  
Kathleen L. Gould
Keyword(s):  
Kinase Activity ◽  
Nuclear Division ◽  
Cyclin B ◽  
Cyclin Dependent Kinase ◽  
Anaphase Promoting Complex ◽  
Wild Type ◽  
Mitotic Cyclin ◽  
Septation Initiation Network ◽  
Mutant Cells ◽  
Null Mutants

ABSTRACT Cytokinesis in eukaryotic cells requires the inactivation of mitotic cyclin-dependent kinase complexes. An apparent exception to this relationship is found in Schizosaccharomyces pombemutants with mutations of the anaphase-promoting complex (APC). These conditional lethal mutants arrest with unsegregated chromosomes because they cannot degrade the securin, Cut2p. Although failing at nuclear division, these mutants septate and divide. Since septation requires Cdc2p inactivation in wild-type S. pombe, it has been suggested that Cdc2p inactivation occurs in these mutants by a mechanism independent of cyclin degradation. In contrast to this prediction, we show that Cdc2p kinase activity fluctuates in APCcut mutants due to Cdc13/cyclin B destruction. In APC-null mutants, however, septation and cutting do not occur and Cdc13p is stable. We conclude that APC cut mutants are hypomorphic with respect to Cdc13p degradation. Indeed, overproduction of nondestructible Cdc13p prevents septation in APC cutmutants and the normal reorganization of septation initiation network components during anaphase.

Sign in / Sign up

Export Citation Format

Share Document