scholarly journals Plk1 Regulates Activation of the Anaphase Promoting Complex by Phosphorylating and Triggering SCFβTrCP-dependent Destruction of the APC Inhibitor Emi1

2004 ◽  
Vol 15 (12) ◽  
pp. 5623-5634 ◽  
Author(s):  
David V. Hansen ◽  
Alexander V. Loktev ◽  
Kenneth H. Ban ◽  
Peter K. Jackson
Keyword(s):  
Ubiquitin Ligase ◽  
Mitotic Spindle ◽  
Cyclin B ◽  
Anaphase Promoting Complex ◽  
Downstream Targets ◽  
Spindle Poles ◽  
Data Support

Progression through mitosis requires activation of cyclin B/Cdk1 and its downstream targets, including Polo-like kinase and the anaphase-promoting complex (APC), the ubiquitin ligase directing degradation of cyclins A and B. Recent evidence shows that APC activation requires destruction of the APC inhibitor Emi1. In prophase, phosphorylation of Emi1 generates a D-pS-G-X-X-pS degron to recruit the SCFβTrCP ubiquitin ligase, causing Emi1 destruction and allowing progression beyond prometaphase, but the kinases directing this phosphorylation remain undefined. We show here that the polo-like kinase Plk1 is strictly required for Emi1 destruction and that overexpression of Plk1 is sufficient to trigger Emi1 destruction. Plk1 stimulates Emi1 phosphorylation, βTrCP binding, and ubiquitination in vitro and cyclin B/Cdk1 enhances these effects. Plk1 binds to Emi1 in mitosis and the two proteins colocalize on the mitotic spindle poles, suggesting that Plk1 may spatially control Emi1 destruction. These data support the hypothesis that Plk1 activates the APC by directing the SCF-dependent destruction of Emi1 in prophase.

scholarly journals A Specific Form of Phospho Protein Phosphatase 2 Regulates Anaphase-promoting Complex/Cyclosome Association with Spindle Poles

2010 ◽  
Vol 21 (6) ◽  
pp. 897-904 ◽  
Author(s):  
Jorge Z. Torres ◽  
Kenneth H. Ban ◽  
Peter K. Jackson
Keyword(s):  
Mitotic Spindle ◽  
Protein Phosphatase ◽  
Spindle Pole ◽  
Specific Form ◽  
Cyclin B ◽  
Anaphase Promoting Complex ◽  
Spindle Poles ◽  
Protein Phosphatase 2 ◽  
Mitotic Spindle Pole ◽  
Early Mitosis

In early mitosis, the END (Emi1/NuMA/Dynein-dynactin) network anchors the anaphase-promoting complex/cyclosome (APC/C) to the mitotic spindle and poles. Spindle anchoring restricts APC/C activity, thereby limiting the destruction of spindle-associated cyclin B and ensuring maintenance of spindle integrity. Emi1 binds directly to hypophosphorylated APC/C, linking the APC/C to the spindle via NuMA. However, whether the phosphorylation state of the APC/C is important for its association with the spindle and what kinases and phosphatases are necessary for regulating this event remain unknown. Here, we describe the regulation of APC/C-mitotic spindle pole association by phosphorylation. We find that only hypophosphorylated APC/C associates with microtubule asters, suggesting that phosphatases are important. Indeed, a specific form of PPP2 (CA/R1A/R2B) binds APC/C, and PPP2 activity is necessary for Cdc27 dephosphorylation. Screening by RNA interference, we find that inactivation of CA, R1A, or R2B leads to delocalization of APC/C from spindle poles, early mitotic spindle defects, a failure to congress chromosomes, and decreased levels of cyclin B on the spindle. Consistently, inhibition of cyclin B/Cdk1 activity increased APC/C binding to microtubules. Thus, cyclin B/Cdk1 and PPP2 regulate the dynamic association of APC/C with spindle poles in early mitosis, a step necessary for proper spindle formation.

scholarly journals Global phosphoproteomics reveals DYRK1A regulates CDK1 activity in glioblastoma cells

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Ariadna Recasens ◽  
Sean J. Humphrey ◽  
Michael Ellis ◽  
Monira Hoque ◽  
Ramzi H. Abbassi ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Cyclin B ◽  
Anaphase Promoting Complex ◽  
Mechanistic Studies ◽  
Glioblastoma Cells ◽  
Cycle Arrest ◽  
Dual Specificity ◽  
Rb Pathway ◽  
High Doses

AbstractBoth tumour suppressive and oncogenic functions have been reported for dual-specificity tyrosine phosphorylation-regulated kinase 1A (DYRK1A). Herein, we performed a detailed investigation to delineate the role of DYRK1A in glioblastoma. Our phosphoproteomic and mechanistic studies show that DYRK1A induces degradation of cyclin B by phosphorylating CDC23, which is necessary for the function of the anaphase-promoting complex, a ubiquitin ligase that degrades mitotic proteins. DYRK1A inhibition leads to the accumulation of cyclin B and activation of CDK1. Importantly, we established that the phenotypic response of glioblastoma cells to DYRK1A inhibition depends on both retinoblastoma (RB) expression and the degree of residual DYRK1A activity. Moderate DYRK1A inhibition leads to moderate cyclin B accumulation, CDK1 activation and increased proliferation in RB-deficient cells. In RB-proficient cells, cyclin B/CDK1 activation in response to DYRK1A inhibition is neutralized by the RB pathway, resulting in an unchanged proliferation rate. In contrast, complete DYRK1A inhibition with high doses of inhibitors results in massive cyclin B accumulation, saturation of CDK1 activity and cell cycle arrest, regardless of RB status. These findings provide new insights into the complexity of context-dependent DYRK1A signalling in cancer cells.

A centrosomal antigen localized on intermediate filaments and mitotic spindle poles

1990 ◽  
Vol 97 (2) ◽  
pp. 259-271
Author(s):  
B. Buendia ◽  
C. Antony ◽  
F. Verde ◽  
M. Bornens ◽  
E. Karsenti
Keyword(s):  
Monoclonal Antibody ◽  
Intermediate Filaments ◽  
Mitotic Spindle ◽  
Mdck Cells ◽  
Large Fraction ◽  
Human Lymphocytes ◽  
Spindle Poles ◽  
Pericentriolar Material ◽  
Xenopus Egg

A monoclonal antibody (CTR2611) raised against centrosomes isolated from human lymphocytes (KE37) stains the pericentriolar material and intermediate filaments in the same cells. In MDCK cells, where most of the microtubules do not originate from the pericentriolar region during interphase, the antigen is distributed along intermediate filaments. At the onset of mitosis, a large fraction of the CTR2611 antigen associates with the minus-end domain of the microtubules of the mitotic spindle but not with the pericentriolar region itself. Treatment of mitotic MDCK cells with taxol leads to the assembly of many microtubule asters in the cytoplasm at the expense of the mitotic spindle. The CTR2611 antigen is present in the center of each of these asters. Similar asters can also be produced in vitro by adding taxol to concentrated Xenopus egg mitotic cytoplasm. Again, the antigen is found close to the center of the asters. These results suggest that CTR2611 antigen is associated with a material involved in microtubule nucleation or microtubule minus-end stabilization. The monoclonal antibody recognizes a 74 × 10(3) Mr polypeptide and other polypeptides at 120 × 10(3) Mr and 170 × 10(3) Mr. The 74 × 10(3) Mr polypeptide is found in all species examined so far, suggesting that it contains a highly conserved epitope.

scholarly journals Stu1p Is Physically Associated with β-Tubulin and Is Required for Structural Integrity of the Mitotic Spindle

2002 ◽  
Vol 13 (6) ◽  
pp. 1881-1892 ◽  
Author(s):  
Hongwei Yin ◽  
Liru You ◽  
Danielle Pasqualone ◽  
Kristen M. Kopski ◽  
Tim C. Huffaker
Keyword(s):  
Mitotic Spindle ◽  
Structural Integrity ◽  
Spindle Pole ◽  
Temperature Sensitive ◽  
Yeast Saccharomyces Cerevisiae ◽  
Spindle Poles ◽  
Balance Of Forces ◽  
Related Proteins ◽  
Β Tubulin

Formation of the bipolar mitotic spindle relies on a balance of forces acting on the spindle poles. The primary outward force is generated by the kinesin-related proteins of the BimC family that cross-link antiparallel interpolar microtubules and slide them past each other. Here, we provide evidence that Stu1p is also required for the production of this outward force in the yeast Saccharomyces cerevisiae. In the temperature-sensitive stu1–5mutant, spindle pole separation is inhibited, and preanaphase spindles collapse, with their previously separated poles being drawn together. The temperature sensitivity of stu1–5 can be suppressed by doubling the dosage of Cin8p, a yeast BimC kinesin–related protein. Stu1p was observed to be a component of the mitotic spindle localizing to the midregion of anaphase spindles. It also binds to microtubules in vitro, and we have examined the nature of this interaction. We show that Stu1p interacts specifically with β-tubulin and identify the domains required for this interaction on both Stu1p and β-tubulin. Taken together, these findings suggest that Stu1p binds to interpolar microtubules of the mitotic spindle and plays an essential role in their ability to provide an outward force on the spindle poles.

scholarly journals Role of Polo-like kinase 1 in the regulation of the action of p31comet in the disassembly of mitotic checkpoint complexes

2019 ◽  
pp. 201902970 ◽  
Author(s):  
Sharon Kaisari ◽  
Pnina Shomer ◽  
Tamar Ziv ◽  
Danielle Sitry-Shevah ◽  
Shirly Miniowitz-Shemtov ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Hela Cells ◽  
Mitotic Spindle ◽  
Joint Action ◽  
Mitotic Checkpoint ◽  
Anaphase Promoting Complex ◽  
Futile Cycle ◽  
Mitotic Checkpoint Complex ◽  
Bi 2536

The Mad2-binding protein p31comet has important roles in the inactivation of the mitotic checkpoint system, which delays anaphase until chromosomes attach correctly to the mitotic spindle. The activation of the checkpoint promotes the assembly of a Mitotic Checkpoint Complex (MCC), which inhibits the action of the ubiquitin ligase APC/C (Anaphase-Promoting Complex/Cyclosome) to degrade inhibitors of anaphase initiation. The inactivation of the mitotic checkpoint requires the disassembly of MCC. p31comet promotes the disassembly of mitotic checkpoint complexes by liberating their Mad2 component in a joint action with the ATPase TRIP13. Here, we investigated the regulation of p31comet action. The release of Mad2 from checkpoint complexes in extracts from nocodazole-arrested HeLa cells was inhibited by Polo-like kinase 1 (Plk1), as suggested by the effects of selective inhibitors of Plk1. Purified Plk1 bound to p31comet and phosphorylated it, resulting in the suppression of its activity (with TRIP13) to disassemble checkpoint complexes. Plk1 phosphorylated p31comet on S102, as suggested by the prevention of the phosphorylation of this residue in checkpoint extracts by the selective Plk1 inhibitor BI-2536 and by the phosphorylation of S102 with purified Plk1. An S102A mutant of p31comet had a greatly decreased sensitivity to inhibition by Plk1 of its action to disassemble mitotic checkpoint complexes. We propose that the phosphorylation of p31comet by Plk1 prevents a futile cycle of MCC assembly and disassembly during the active mitotic checkpoint.

scholarly journals The HECT E3 ligase Smurf2 is required for Mad2-dependent spindle assembly checkpoint

2008 ◽  
Vol 183 (2) ◽  
pp. 267-277 ◽  
Author(s):  
Evan C. Osmundson ◽  
Dipankar Ray ◽  
Finola E. Moore ◽  
Qingshen Gao ◽  
Gerald H. Thomsen ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Spindle Assembly Checkpoint ◽  
Spindle Checkpoint ◽  
E3 Ligase ◽  
Spindle Assembly ◽  
Cyclin B ◽  
Carboxyl Terminus ◽  
Anaphase Promoting Complex ◽  
Mitotic Spindles ◽  
Anaphase Onset

Activation of the anaphase-promoting complex/cyclosome (APC/C) by Cdc20 is critical for the metaphase–anaphase transition. APC/C-Cdc20 is required for polyubiquitination and degradation of securin and cyclin B at anaphase onset. The spindle assembly checkpoint delays APC/C-Cdc20 activation until all kinetochores attach to mitotic spindles. In this study, we demonstrate that a HECT (homologous to the E6-AP carboxyl terminus) ubiquitin ligase, Smurf2, is required for the spindle checkpoint. Smurf2 localizes to the centrosome, mitotic midbody, and centromeres. Smurf2 depletion or the expression of a catalytically inactive Smurf2 results in misaligned and lagging chromosomes, premature anaphase onset, and defective cytokinesis. Smurf2 inactivation prevents nocodazole-treated cells from accumulating cyclin B and securin and prometaphase arrest. The silencing of Cdc20 in Smurf2-depleted cells restores mitotic accumulation of cyclin B and securin. Smurf2 depletion results in enhanced polyubiquitination and degradation of Mad2, a critical checkpoint effector. Mad2 is mislocalized in Smurf2-depleted cells, suggesting that Smurf2 regulates the localization and stability of Mad2. These data indicate that Smurf2 is a novel mitotic regulator.

scholarly journals The roles of Fzy/Cdc20 and Fzr/Cdh1 in regulating the destruction of cyclin B in space and time

2002 ◽  
Vol 157 (7) ◽  
pp. 1139-1149 ◽  
Author(s):  
Jordan W. Raff ◽  
Kim Jeffers ◽  
Jun-yong Huang
Keyword(s):  
Cell Cycle ◽  
Triple Point ◽  
Mitotic Spindle ◽  
Cyclin B ◽  
Second Phase ◽  
Point Mutant ◽  
Two Phases ◽  
Drosophila Cells

In Drosophila cells cyclin B is normally degraded in two phases: (a) destruction of the spindle-associated cyclin B initiates at centrosomes and spreads to the spindle equator; and (b) any remaining cytoplasmic cyclin B is degraded slightly later in mitosis. We show that the APC/C regulators Fizzy (Fzy)/Cdc20 and Fzy-related (Fzr)/Cdh1 bind to microtubules in vitro and associate with spindles in vivo. Fzy/Cdc20 is concentrated at kinetochores and centrosomes early in mitosis, whereas Fzr/Cdh1 is concentrated at centrosomes throughout the cell cycle. In syncytial embryos, only Fzy/Cdc20 is present, and only the spindle-associated cyclin B is degraded at the end of mitosis. A destruction box–mutated form of cyclin B (cyclin B triple-point mutant [CBTPM]–GFP) that cannot be targeted for destruction by Fzy/Cdc20, is no longer degraded on spindles in syncytial embryos. However, CBTPM–GFP can be targeted for destruction by Fzr/Cdh1. In cellularized embryos, which normally express Fzr/Cdh1, CBTPM–GFP is degraded throughout the cell but with slowed kinetics. These findings suggest that Fzy/Cdc20 is responsible for catalyzing the first phase of cyclin B destruction that occurs on the mitotic spindle, whereas Fzr/Cdh1 is responsible for catalyzing the second phase of cyclin B destruction that occurs throughout the cell. These observations have important implications for the mechanisms of the spindle checkpoint.

scholarly journals Protein Phosphatase 2A Stabilizes Human Securin, Whose Phosphorylated Forms Are Degraded via the SCF Ubiquitin Ligase

2006 ◽  
Vol 26 (11) ◽  
pp. 4017-4027 ◽  
Author(s):  
Ana M. Gil-Bernabé ◽  
Francisco Romero ◽  
M. Cristina Limón-Mortés ◽  
María Tortolero
Keyword(s):  
Ubiquitin Ligase ◽  
Protein Phosphatase ◽  
Protein Phosphatase 2A ◽  
Anaphase Promoting Complex ◽  
Anaphase Onset ◽  
Chromatid Segregation ◽  
Phosphatase 2A ◽  
F Box Protein

ABSTRACT Sister chromatid segregation is triggered at the metaphase-to-anaphase transition by the activation of the protease separase. For most of the cell cycle, separase activity is kept in check by its association with the inhibitory chaperone securin. Activation of separase occurs at anaphase onset, when securin is targeted for destruction by the anaphase-promoting complex or cyclosome E3 ubiquitin protein ligase. This results in the release of the cohesins from chromosomes, which in turn allows the segregation of sister chromatids to opposite spindle poles. Here we show that human securin (hSecurin) forms a complex with enzymatically active protein phosphatase 2A (PP2A) and that it is a substrate of the phosphatase, both in vitro and in vivo. Treatment of cells with okadaic acid, a potent inhibitor of PP2A, results in various hyperphosphorylated forms of hSecurin which are extremely unstable, due to the action of the Skp1/Cul1/F-box protein complex ubiquitin ligase. We propose that PP2A regulates hSecurin levels by counteracting its phosphorylation, which promotes its degradation. Misregulation of this process may lead to the formation of tumors, in which overproduction of hSecurin is often observed.

scholarly journals Rapid microtubule-independent dynamics of Cdc20 at kinetochores and centrosomes in mammalian cells

2002 ◽  
Vol 158 (5) ◽  
pp. 841-847 ◽  
Author(s):  
Marko J. Kallio ◽  
Victoria A. Beardmore ◽  
Jasminder Weinstein ◽  
Gary J. Gorbsky
Keyword(s):  
Mitotic Spindle ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Anaphase Promoting Complex ◽  
Late Prophase ◽  
Checkpoint Signaling ◽  
Spindle Poles ◽  
Anaphase Onset ◽  
Late Telophase ◽  
Green Fluorescent

Cdc20 is a substrate adaptor and activator of the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase whose activity is required for anaphase onset and exit from mitosis. A green fluorescent protein derivative, Cdc20–GFP, bound to centrosomes throughout the cell cycle and to kinetochores from late prophase to late telophase. We mapped distinct domains of Cdc20 that are required for association with kinetochores and centrosomes. FRAP measurements revealed extremely rapid dynamics at the kinetochores (t1/2 = 5.1 s) and spindle poles (t1/2 = 4.7 s). This rapid turnover is independent of microtubules. Rapid transit of Cdc20 through kinetochores may ensure that spindle checkpoint signaling at unattached/relaxed kinetochores can continuously inhibit APC/CCdc20 targeting of anaphase inhibitors (securins) throughout the cell until all the chromosomes are properly attached to the mitotic spindle.

scholarly journals Checkpoint Protein BubR1 Acts Synergistically with Mad2 to Inhibit Anaphase-promoting Complex

2002 ◽  
Vol 13 (3) ◽  
pp. 755-766 ◽  
Author(s):  
Guowei Fang
Keyword(s):  
Ubiquitin Ligase ◽  
Mitotic Spindle ◽  
Spindle Assembly Checkpoint ◽  
Kinase Activity ◽  
Metaphase Plate ◽  
Anaphase Promoting Complex ◽  
Checkpoint Control ◽  
Sister Chromatids ◽  
Direct Binding ◽  
And Function

The spindle assembly checkpoint monitors the attachment of kinetochores to the mitotic spindle and the tension exerted on kinetochores by microtubules and delays the onset of anaphase until all the chromosomes are aligned at the metaphase plate. The target of the checkpoint control is the anaphase-promoting complex (APC)/cyclosome, a ubiquitin ligase whose activation by Cdc20 is required for separation of sister chromatids. In response to activation of the checkpoint, Mad2 binds to and inhibits Cdc20-APC. I show herein that in checkpoint-arrested cells, human Cdc20 forms two separate, inactive complexes, a lower affinity complex with Mad2 and a higher affinity complex with BubR1. Purified BubR1 binds to recombinant Cdc20 and this interaction is direct. Binding of BubR1 to Cdc20 inhibits activation of APC and this inhibition is independent of its kinase activity. Quantitative analysis indicates that BubR1 is 12-fold more potent than Mad2 as an inhibitor of Cdc20. Although at high protein concentrations BubR1 and Mad2 each is sufficient to inhibit Cdc20, BubR1 and Mad2 mutually promote each other's binding to Cdc20 and function synergistically at physiological concentrations to quantitatively inhibit Cdc20-APC. Thus, BubR1 and Mad2 act cooperatively to prevent premature separation of sister chromatids by directly inhibiting APC.

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