A Hec of a microtubule attachment.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
P Todd Stukenberg
Publications and source records attributed to P Todd Stukenberg.
Aurora-A kinase and inhibitor-2 regulate the cyclin threshold for mitotic entry in Xenopus early embryonic cell cycles.
The abrupt activation of CDK1 during mitotic entry requires suppression of CDK activity until a threshold concentration of cyclin B is synthesized, triggering the activation of a large pool of CDK. The cellular mechanisms that define the concentration of cyclin B at which the threshold occurs are unknown. Here we demonstrate that this threshold is regulated by Aurora-A kinase and phosphatase Inhibitor-2. In Xenopus CSF extracts that actively translate cyclin B1, immunodepletion of either endogenous xInhibitor-2 or endogenous xAurora-A caused delayed mitotic entry and normal timing was restored by addition of the respective recombinant proteins. Aurora-A depleted extracts also could be rescued by the addition of full-length xInhibitor-2, but not an xInhibitor-2 truncated of its PP1 binding motif. This demonstrates that inhibition of PP1 was required to compensate for the absence of Aurora-A. To test the hypothesis that the delays in mitotic entry in CSF extracts were due to increases in cyclin B thresholds, we employed interphase extracts, which are driven into mitosis by the addition of recombinant cyclin B in a nonlinear (threshold) dose-response. Neutralization of endogenous xInhibitor-2 or xAurora with antibodies increased the cyclin B threshold concentration. Alternatively, the addition of exogenous Aurora-A or Inhibitor-2 lowered the concentration of cyclin B that triggered CDK activation. Because the cyclin B threshold could be raised or lowered by changing the amount of either Aurora-A or Inhibitor-2, the results demonstrate these regulatory proteins are involved in a signaling loop required to create the switching behavior characteristic of mitotic entry.
Animals↗
Aurora B is enriched at merotelic attachment sites, where it regulates MCAK.
Kinetochores often form merotelic attachments, in which a single kinetochore is attached to microtubules from both spindle poles. These attachments can result in improper chromosome segregation and are a significant source of aneuploidy, a hallmark of cancer. Aurora B kinase and the kinesin-13 microtubule depolymerase mitotic-centromere-associated kinesin (MCAK) are required to release improper microtubule attachments. Aurora B regulates MCAK's activity and localization. We set out to understand why MCAK and Aurora B are more abundant at some metaphase-aligned centromeres but are present at low amounts on most others. We found that members of the Aurora B complex are specifically enriched at merotelic attachment sites. We also found that Aurora B does not require its activity to become enriched at these sites; however, inhibition of Aurora B activity causes a significant increase in the number of merotelic attachments per cell. Aurora B activity enriches MCAK at merotelic attachments and phosphorylates MCAK on residues that regulate its microtubule depolymerase activity. These data demonstrate that proteins that resolve the defect are specifically localized to merotelic attachments, where their enzymatic activities are regulated.
Animals↗
The reversibility of mitotic exit in vertebrate cells.
A guiding hypothesis for cell-cycle regulation asserts that regulated proteolysis constrains the directionality of certain cell-cycle transitions. Here we test this hypothesis for mitotic exit, which is regulated by degradation of the cyclin-dependent kinase 1 (Cdk1) activator, cyclin B. Application of chemical Cdk1 inhibitors to cells in mitosis induces cytokinesis and other normal aspects of mitotic exit, including cyclin B degradation. However, chromatid segregation fails, resulting in entrapment of chromatin in the midbody. If cyclin B degradation is blocked with a proteasome inhibitor or by expression of non-degradable cyclin B, Cdk inhibitors will nonetheless induce mitotic exit and cytokinesis. However, if after mitotic exit, the Cdk1 inhibitor is washed free from cells in which cyclin B degradation is blocked, the cells can revert back to M phase. This reversal is characterized by chromosome recondensation, nuclear envelope breakdown, assembly of microtubules into a mitotic spindle, and in most cases, dissolution of the midbody, reopening of the cleavage furrow, and realignment of chromosomes at the metaphase plate. These findings demonstrate that proteasome-dependent degradation of cyclin B provides directionality for the M phase to G1 transition.
Animals↗
INCENP and Aurora B promote meiotic sister chromatid cohesion through localization of the Shugoshin MEI-S332 in Drosophila.
The chromosomal passenger complex protein INCENP is required in mitosis for chromosome condensation, spindle attachment and function, and cytokinesis. Here, we show that INCENP has an essential function in the specialized behavior of centromeres in meiosis. Mutations affecting Drosophila incenp profoundly affect chromosome segregation in both meiosis I and II, due, at least in part, to premature sister chromatid separation in meiosis I. INCENP binds to the cohesion protector protein MEI-S332, which is also an excellent in vitro substrate for Aurora B kinase. A MEI-S332 mutant that is only poorly phosphorylated by Aurora B is defective in localization to centromeres. These results implicate the chromosomal passenger complex in directly regulating MEI-S332 localization and, therefore, the control of sister chromatid cohesion in meiosis.
Animals↗
Measuring the stoichiometry and physical interactions between components elucidates the architecture of the vertebrate kinetochore.
Vertebrate kinetochores contain over 50 different proteins organized into three distinct regions: the inner plate, outer plate, and fibrous corona. The present study characterizes numerous precursors of kinetochore assembly in a system free of centromeric chromatin, Xenopus extracts. Hydrodynamic analysis suggests there are a minimum of two monomeric proteins and six pre-assembled complexes that accumulate on centromeres to form the kinetochore. The inner and outer kinetochore assemble from at least two distinct kinetochore complexes containing the proteins Mis12, Zwint, and Ndc80, all of which interact by immunoprecipitation. There is also a network of interactions between the fibrous corona proteins that is dissociated by microtubules. We quantify the number of molecules of specific proteins assembled into a single kinetochore. There are between 800 and 1200 molecules of the measured inner and outer kinetochore proteins, demonstrating that the components in these regions are in similar stoichiometry. In contrast, the measured fibrous corona proteins are present at 250-300 molecules per kinetochore. Zwint, but not Mis12, requires the Ndc80 complex for assembly into the kinetochore. Further, Ndc80 requires Zwint for assembly, indicating a co-dependency for these two proteins. Our data provide a model for the structural architecture and assembly pathway of the vertebrate kinetochore.
Animals↗
Polo-like kinase 1 creates the tension-sensing 3F3/2 phosphoepitope and modulates the association of spindle-checkpoint proteins at kinetochores.
BACKGROUND: In mitosis, a mechanochemical system recognizes tension that is generated by bipolar microtubule attachment to sister kinetochores. This is translated into multiple outputs including the stabilization of microtubule attachments, changes in kinetochore protein dynamics, and the silencing of the spindle checkpoint. How kinetochores sense tension and translate this into various signals represent critical unanswered questions. The kinetochores of chromosomes not under tension are specifically phosphorylated at an epitope recognized by the 3F3/2 monoclonal antibody. Determining the kinase that generates the 3F3/2 phosphoepitope at kinetochores should reveal an important component of this system that regulates mitotic progression. RESULTS: We demonstrate that Polo-like kinase 1 (Plk1) creates the 3F3/2 phosphoepitope on mitotic kinetochores. In a permeabilized in vitro cell system, the depletion of Xenopus Plk1 from M phase extract leads to the loss of 3F3/2 kinase activity. Purified recombinant Plk1 is sufficient to generate the 3F3/2 phosphoepitope in this system. Using siRNA, we show that the reduction of Plk1 protein levels significantly diminishes 3F3/2 phosphoepitope expression at kinetochores. The consensus phosphorylation sites of Plk1 show strong similarity to the 3F3/2 phosphoepitope sequence determined by phosphopeptide mapping. The inhibition of Plk1 by siRNA alters the normal kinetochore association of Mad2, Cenp-E, Hec1/Ndc80, Spc24, and Cdc20 and induces a spindle-checkpoint-mediated mitotic arrest. CONCLUSIONS: Plk1 generates the 3F3/2 phosphoepitope at kinetochores that are not under tension and contributes to the normal kinetochore association of several key proteins important in checkpoint signaling. Mechanical tension regulates Plk1 accumulation at kinetochores and possibly its kinase activity.
Amino Acid Motifs↗
Mechanism of Aurora B activation by INCENP and inhibition by hesperadin.
Aurora family serine/threonine kinases control mitotic progression, and their deregulation is implicated in tumorigenesis. Aurora A and Aurora B, the best-characterized members of mammalian Aurora kinases, are approximately 60% identical but bind to unrelated activating subunits. The structure of the complex of Aurora A with the TPX2 activator has been reported previously. Here, we report the crystal structure of Aurora B in complex with the IN-box segment of the inner centromere protein (INCENP) activator and with the small molecule inhibitor Hesperadin. The Aurora B:INCENP complex is remarkably different from the Aurora A:TPX2 complex. INCENP forms a crown around the small lobe of Aurora B and induces the active conformation of the T loop allosterically. The structure represents an intermediate state of activation of Aurora B in which the Aurora B C-terminal segment stabilizes an open conformation of the catalytic cleft, and a critical ion pair in the kinase active site is impaired. Phosphorylation of two serines in the carboxyl terminus of INCENP generates the fully active kinase.
Amino Acid Sequence↗
Two complexes of spindle checkpoint proteins containing Cdc20 and Mad2 assemble during mitosis independently of the kinetochore in Saccharomyces cerevisiae.
Favored models of spindle checkpoint signaling propose that two inhibitory complexes (Mad2-Cdc20 and Mad2-Mad3-Bub3-Cdc20) must be assembled at kinetochores in order to inhibit mitosis. We have directly tested this model in the budding yeast Saccharomyces cerevisiae. The proteins Mad2, Mad3, Bub3, Cdc20, and Cdc27 in yeast were quantified, and there are sufficient amounts to form stoichiometric inhibitors of Cdc20 and the anaphase-promoting complex. Mad2 is present in two separate complexes in cells arrested in mitosis with nocodazole. There is a small amount of Mad2-Mad3-Bub3-Cdc20 and a much larger amount of a complex that contains Mad2-Cdc20. We use conditional mutants to show that both Mad2 and Mad3 are essential for establishment and maintenance of the spindle checkpoint. Both spindle checkpoint complexes containing Mad2 form in mitosis, not in response to checkpoint activation. The kinetochore is not required to form either complex. We propose that the conversion of Mad1-Mad2 to Cdc20-Mad2, a key step in generating inhibitory checkpoint complexes, is limited to mitosis by the availability of Cdc20 and is kinetochore independent.
Cdc20 Proteins↗
Purification of the Ndc80 kinetochore subcomplex from Xenopus eggs.
The identification of protein binding partners often facilitates understanding of protein complex function. However, identifying binding partners has proven difficult because proteins are often bound to insoluble structures or are only present during certain stages of the cell cycle. Fortunately, Xenopus eggs stockpile many proteins, which are typically insoluble, as soluble subcomplexes to facilitate rapid early embryonic divisions. We exploited this by developing a purification scheme using Xenopus egg extracts to isolate Coomassie-stainable amounts of the xNdc80 kinetochore complex. In this scheme Xenopus eggs are directly made into a mitotic high-speed supernatant and then flowed over three chromatographic columns: heparin, Mono-Q, and Superose 6 gel filtration columns. A final immunoprecipitation is then performed from the peak Superose 6 column to yield Ndc80 complex purified to homogeneity. With minor modification and manipulation of Xenopus egg extracts, this protocol can easily be adapted for purification of other protein complexes.
Animals↗
Triggering p53 after cytokinesis failure.
Cells that fail to divide during cytokinesis often arrest in the next G1 phase by a mysterious mechanism that depends upon p53. What triggers this arrest is unclear. New studies, including a report in this issue (Uetake and Sluder, 2004) suggest that this arrest does not occur because cells are polyploid, are binucleate, have multiple centrosome, or have failed cytokinesis, making this phenomenon even more puzzling.
Animals↗
Activation of Aurora-A kinase by protein phosphatase inhibitor-2, a bifunctional signaling protein.
Aurora-A kinase is necessary for centrosome maturation, for assembly and maintenance of a bipolar spindle, and for proper chromosome segregation during cell division. Aurora-A is an oncogene that is overexpressed in multiple human cancers. Regulation of kinase activity apparently depends on phosphorylation of Thr-288 in the T-loop. In addition, interactions with targeting protein for Xenopus kinesin-like protein 2 (TPX2) allosterically activate Aurora-A. The Thr-288 phosphorylation is reversed by type-1 protein phosphatase (PP1). Mutations in the yeast Aurora, Ipl1, are suppressed by overexpression of Glc8, the yeast homolog of phosphatase inhibitor-2 (I-2). In this study, we show that human I-2 directly and specifically stimulated recombinant human Aurora-A activity in vitro. The I-2 increase in kinase activity was not simply due to inhibition of PP1 because it was not mimicked by other phosphatase inhibitors. Furthermore, activation of Aurora-A was unaffected by deletion of the I-2 N-terminal PP1 binding motif but was eliminated by deletion of the I-2 C-terminal domain. Aurora-A and I-2 were recovered together from mitotic HeLa cells. Kinase activation by I-2 and TPX2 was not additive and occurred without a corresponding increase in T-loop phosphorylation. These results suggest that both I-2 and TPX2 function as allosteric activators of Aurora-A. This implies that I-2 is a bifunctional signaling protein with separate domains to inhibit PP1 and directly stimulate Aurora-A kinase.
Animals↗
A signalling pathway controlling c-Myc degradation that impacts oncogenic transformation of human cells.
The stability of c-Myc is regulated by multiple Ras effector pathways. Phosphorylation at Ser 62 stabilizes c-Myc, whereas subsequent phosphorylation at Thr 58 is required for its degradation. Here we show that Ser 62 is dephosphorylated by protein phosphatase 2A (PP2A) before ubiquitination of c-Myc, and that PP2A activity is regulated by the Pin1 prolyl isomerase. Furthermore, the absence of Pin1 or inhibition of PP2A stabilizes c-Myc. A stable c-Myc(T58A) mutant that cannot bind Pin1 or be dephosphorylated by PP2A replaces SV40 small T antigen in human cell transformation and tumorigenesis assays. Therefore, small T antigen, which inactivates PP2A, exerts its oncogenic potential by preventing dephosphorylation of c-Myc, resulting in c-Myc stabilization. Thus, Ras-dependent signalling cascades ensure transient and self-limiting accumulation of c-Myc, disruption of which contributes to human cell oncogenesis.
Amino Acid Sequence↗
Aurora B phosphorylates centromeric MCAK and regulates its localization and microtubule depolymerization activity.
BACKGROUND: Sister kinetochores must bind microtubules in a bipolar fashion to equally segregate chromosomes during mitosis. The molecular mechanisms underlying this process remain unclear. Aurora B likely promotes chromosome biorientation by regulating kinetochore-microtubule attachments. MCAK (mitotic centromere-associated kinesin) is a Kin I kinesin that can depolymerize microtubules. These two proteins both localize to mitotic centromeres and have overlapping mitotic functions, including regulation of microtubule dynamics, proper chromosome congression, and correction of improper kinetochore-microtubule attachments. RESULTS: We show that Aurora B phosphorylates and regulates MCAK both in vitro and in vivo. Specifically, we mapped six Aurora B phosphorylation sites on MCAK in both the centromere-targeting domain and the neck region. Aurora B activity was required to localize MCAK to centromeres, but not to spindle poles. Aurora B phosphorylation of serine 196 in the neck region of MCAK inhibited its microtubule depolymerization activity. We found that this key site was phosphorylated at centromeres and anaphase spindle midzones in vivo. However, within the inner centromere there were pockets of both phosphorylated and unphosphorylated MCAK protein, suggesting that phosphate turnover is crucial in the regulation of MCAK activity. Addition of alpha-p-S196 antibodies to Xenopus egg extracts or injection of alpha-p-S196 antibodies into cells caused defects in chromosome positioning and/or segregation. CONCLUSIONS: We have established a direct link between the microtubule depolymerase MCAK and Aurora B kinase. Our data suggest that Aurora B both positively and negatively regulates MCAK during mitosis. We propose that Aurora B biorients chromosomes by directing MCAK to depolymerize incorrectly oriented kinetochore microtubules.
Animals↗
The vertebrate Ndc80 complex contains Spc24 and Spc25 homologs, which are required to establish and maintain kinetochore-microtubule attachment.
How kinetochores bind to microtubules and move on the mitotic spindle remain unanswered questions. Multiple systems have implicated the Ndc80/Hec1 (Ndc80) kinetochore complex in kinetochore-microtubule interaction and spindle checkpoint activity. In budding yeast, Ndc80 copurifies with three additional interacting proteins: Nuf2, Spc24, and Spc25. Although functional vertebrate homologs of Ndc80 and Nuf2 exist, extensive sequence similarity searches have not uncovered homologs of Spc24 and Spc25. We have purified the xNdc80 complex to homogeneity from Xenopus egg extracts and identified two novel interacting proteins. Although the sequences have greatly diverged, we have concluded that these are the functional homologs of the yeast Spc24 and Spc25 proteins based on limited sequence similarity, common coiled-coil domains, kinetochore localization, similar phenotypes, and copurification with xNdc80 and xNuf2. Using both RNAi and antibody injection experiments, we have extended previous characterization of the complex and found that Spc24 and Spc25 are required not only to establish, but also to maintain kinetochore-microtubule attachments and metaphase alignment. In addition, we show that Spc24 and Spc25 are required for chromosomal movement to the spindle poles in anaphase.
Anaphase↗
Analyzing the spindle checkpoint in yeast and frogs.
The spindle checkpoint is an evolutionarily conserved regulatory mechanism that ensures correct segregation of chromosomes at mitosis and meiosis. The kinetochore plays an integral role in spindle checkpoint signaling by integrating chromosome attachment to the spindle with cell cycle progression. A single kinetochore can inhibit cell cycle progression in the absence of proper spindle attachment or tension from bipolar orientation. Recent advances have shed light on how the kinetochore measures these situations, transduces a signal, and inhibits the entry into anaphase.
Anaphase↗
Mitosis: long-range signals guide microtubules.
The formation of microtubule-derived structures is thought to involve nucleation in all directions and stabilization of microtubules that contact relevant targets. A recent study indicates, during mitosis, there may be a third step, in which microtubule growth is directed over long distances without physical contact between the target and microtubules.
Microtubules↗
The highly conserved Ndc80 complex is required for kinetochore assembly, chromosome congression, and spindle checkpoint activity.
We show that the Xenopus homologs of Ndc80/Tid3/HEC1 (xNdc80) and Nuf2/MPP1/Him-10 (xNuf2) proteins physically interact in a 190-kD complex that associates with the outer kinetochore from prometaphase through anaphase. Injecting function-blocking antibodies to either xNdc80 or xNuf2 into XTC cells caused premature exit from mitosis without detectable chromosome congression or anaphase movements. Injected cells did not arrest in response to microtubule drugs, showing that the complex is required for the spindle checkpoint. Kinetochores assembled in Xenopus extracts after immunodepletion of the complex did not contain xRod, xZw10, xP150 glued (Dynactin), xMad1, xMad2, xBub1, and xBub3, demonstrating that the xNdc80 complex is required for functional kinetochore assembly. In contrast, function-blocking antibodies did not affect the localization of other kinetochore proteins when added to extracts containing previously assembled kinetochores. These extracts with intact kinetochores were deficient in checkpoint signaling, suggesting that the Ndc80 complex participates in the spindle checkpoint. We also demonstrate that the spindle checkpoint can arrest budding yeast cells lacking Ndc80 or Nuf2, whereas yeast lacking both proteins fail to arrest in mitosis. Systematic deletion of yeast kinetochore genes suggests that the Ndc80 complex has a unique role in spindle checkpoint signaling. We propose that the Ndc80 complex has conserved roles in kinetochore assembly, chromosome congression, and spindle checkpoint signaling.
Anaphase↗