Budding Yeast Swe1 Is Involved in the Control of Mitotic Spindle Elongation and Is Regulated by Cdc14 Phosphatase during Mitosis

Cyclin-dependent kinase (Cdk1) activity is required for mitotic entry, and this event is restrained by an inhibitory phosphorylation of the catalytic subunit Cdc28 on a conserved tyrosine (Tyr¹⁹). This modification is brought about by the protein kinase Swe1 that inhibits Cdk1 activation thus blocking mitotic entry. Swe1 levels are regulated during the cell cycle, and they decrease during G₂/M concomitantly to Cdk1 activation, which drives entry into mitosis. However, after mitotic entry, a pool of Swe1 persists, and we collected evidence that it is involved in controlling mitotic spindle elongation. We also describe that the protein phosphatase Cdc14 is implicated in Swe1 regulation; in fact, we observed that Swe1 dephosphorylation in vivo depends on Cdc14 that, in turn, is able to control its subcellular localization. In addition we show that the lack of Swe1 causes premature mitotic spindle elongation and that high levels of Swe1 block mitotic spindle elongation, indicating that Swe1 inhibits this process. Importantly, these effects are not dependent upon the role of in Cdk1 inhibition. These data fit into a model in which Cdc14 binds and inhibits Swe1 to allow timely mitotic spindle elongation.     

Rac1-dependent recruitment of PAK2 to G2 phase centrosomes and their roles in the regulation of mitotic entry

During mitotic entry, the centrosomes provide a scaffold for initial activation of the CyclinB/Cdk1 complex, the mitotic kinase Aurora A, and the Aurora A-activating kinase p21-activated kinase (PAK). The activation of PAK at the centrosomes is yet regarded to happen independently of the Rho-GTPases Rac/Cdc42. In this study, Rac1 (but not RhoA or Cdc42) is presented to associate with the centrosomes from early G₂ phase until prometaphase in a cell cycle-dependent fashion, as evidenced by western blot analysis of prepared centrosomes and by immunolabeling. PAK associates with the G₂/M-phase centrosomes in a Rac1-dependent fashion. Furthermore, specific inhibition of Rac1 by C. difficile toxinB-catalyzed glucosylation or by knockout results in inhibited activation of PAK1/2, Aurora A, and the CyclinB/Cdk1 complex in late G₂ phase/prophase and delayed mitotic entry. Inhibition of PAK activation at late G₂-phase centrosomes caused by Rac1 inactivation coincides with impeded activation of Aurora A and the CyclinB/Cdk1 complex and delayed mitotic entry.     

A functional role for p38 MAPK in modulating mitotic transit in the absence of stress

Although p38 MAPK is known to be activated in response to various environmental stresses and to have inhibitory roles in cell proliferation and tumor progression, its role in cell cycle progression in the absence of stress is unknown in most cell types. In the case of G(2)/M cell cycle control, p38 activation has been shown to trigger a rapid G(2)/M cell cycle checkpoint after DNA damage stress and a spindle checkpoint after microtubule disruption. In the course of our studies, we observed that p38 became actively phosphorylated, and its kinase activity increased transiently during G(2)/M cell cycle transition. Using an immunocytochemistry approach, the active form of p38 was found at the centrosome from late G(2) throughout mitosis, which suggests functional relevance for active p38 protein during mitotic entry. A closer examination reveals that p38 inhibition by pharmacologic inhibitors significantly accelerated the timing of mitotic entry. In addition, long term exposure of the inhibitor enhanced Cdc2 activity. These results indicate that p38 activity during G(2)/M may be involved in a mechanism for fine tuning the initiation of mitosis and perhaps transit of mitosis. Consistent with our previous findings, Cdc25B was phosphorylated on serine 309 at the centrosome during G(2)/M when p38 was active at this site; Cdc25B phosphorylation inhibits Cdc25B activity, and this phosphorylation was found to be p38-dependent. Taken together, our findings suggest that p38 regulates the timing of mitotic entry via modulation of Cdc25B activity under normal nonstress conditions.     

Revised genetic requirements for the decatenation G2 checkpoint: The role of ATM

The decatenation G2 checkpoint is proposed to delay cellular progression from G2 into mitosis when intertwined daughter chromatids are insufficiently decatenated. Previous studies indicated that the ATM- and Rad3-related (ATR) checkpoint kinase, but not the ataxia telangiectasia-mutated (ATM) kinase, was required for decatenation G2 checkpoint function. Here, we show that the method used to quantify decatenation G2 checkpoint function can influence the identification of genetic requirements for the checkpoint. Normal human diploid fibroblast (NHDF) lines responded to the topoisomerase II (topo II) catalytic inhibitor ICRF-193 with a stringent G2 arrest and a reduction in the mitotic index. While siRNA-mediated depletion of ATR and CHEK1 increased the mitotic index in ICRF-193 treated NHDF lines, depletion of these proteins did not affect the mitotic entry rate, indicating that the decatenation G2 checkpoint was functional. These results suggest that ATR and CHEK1 are not required for the decatenation G2 checkpoint, but may influence mitotic exit after inhibition of topo II. A re-evaluation of ataxia telangiectasia (AT) cell lines using the mitotic entry assay indicated that ATM was required for the decatenation G2 checkpoint. Three NHDF cell lines responded to ICRF-193 with a mean 98% inhibition of the mitotic entry rate. Examination of the mitotic entry rates in AT fibroblasts upon treatment with ICRF-193 revealed a significantly attenuated decatenation G2 checkpoint response, with a mean 59% inhibition of the mitotic entry rate. In addition, a normal lymphoblastoid line exhibited a 95% inhibition of the mitotic entry rate after incubation with ICRF-193, whereas two AT lymphoblastoid lines displayed only 36% and 20% inhibition of the mitotic entry rate. Stable depletion of ATM in normal human fibroblasts with short hairpin RNA also attenuated decatenation G2 checkpoint function by an average of 40%. Western immunoblot analysis demonstrated that treatment with ICRF-193 induced ATM autophosphorylation and ATM-dependent phosphorylation of Ser15-p53 and Thr68 in Chk2, but no appreciable phosphorylation of Ser139-H2AX or Ser345-Chk1. The results suggest that inhibition of topo II induces ATM to phosphorylate selected targets that contribute to a G2 arrest independently of DNA damage.     

Analysis of the mitotic exit control system using locked levels of stable mitotic cyclin

Cyclin‐dependent kinase (Cdk) both promotes mitotic entry (spindle assembly and anaphase) and inhibits mitotic exit (spindle disassembly and cytokinesis), leading to an elegant quantitative hypothesis that a single cyclin oscillation can function as a ratchet to order these events. This ratchet is at the core of a published ODE model for the yeast cell cycle. However, the ratchet model requires appropriate cyclin dose–response thresholds. Here, we test the inhibition of mitotic exit in budding yeast using graded levels of stable mitotic cyclin (Clb2). In opposition to the ratchet model, stable levels of Clb2 introduced dose‐dependent delays, rather than hard thresholds, that varied by mitotic exit event. The ensuing cell cycle was highly abnormal, suggesting a novel reason for cyclin degradation. Cdc14 phosphatase antagonizes Clb2–Cdk, and Cdc14 is released from inhibitory nucleolar sequestration independently of stable Clb2. Thus, Cdc14/Clb2 balance may be the appropriate variable for mitotic regulation. Although our results are inconsistent with the aforementioned ODE model, revision of the model to allow Cdc14/Clb2 balance to control mitotic exit corrects these discrepancies, providing theoretical support for our conclusions.     

Inhibition of cdc2 activation by INH/PP2A.

INH, a type 2A protein phosphatase (PP2A), negatively regulates entry into M phase and the cyclin B-dependent activation of cdc2 in Xenopus extracts. INH appears to be central to the mechanism of the trigger for mitotic initiation, as it prevents the premature activation of cdc2. We first show that INH is a conventional form of PP2A with a B alpha regulatory subunit. We next explore the mechanism by which it inhibits cdc2 activation by examining the effect of purified PP2A on the reaction pathways controlling cdc2 activity. Our results suggest that although PP2A inhibits the switch in tyrosine kinase and tyrosine phosphatase activities accompanying mitosis, this switch is a consequence of the inhibition of some other rate-limiting event. In the preactivation phase, PP2A inhibits the pathway leading to T161 phosphorylation, suggesting that this activity may be one of the rate-limiting events for transition. However, our results also suggest that the accumulation of active cdc2/cyclin complexes during the lag is only one of the events required for triggering entry into mitosis.     

除草剂普杀特（Imazethapyr）对玉米根尖蛋白质和DNA合成的影响

除草剂普杀特（Ｉｍａｚｅｔｈａｐｙｒ）对玉米根尖蛋白质和ＤＮＡ合成的影响刘支前（北京农业大学农业应用化学系,北京１０００９４）关键词：普杀特,玉米,蛋白质,ＤＮＡ８０年代美国氰胺公司开发的咪唑淋酮类除草剂,由于其高效低毒、杀草谱广,又因对某些农作物具...     

Cyclin B Translation Depends on mTOR Activity after Fertilization in Sea Urchin Embryos

The cyclin B/CDK1 complex is a key regulator of mitotic entry. Using PP242, a specific ATP-competitive inhibitor of mTOR kinase, we provide evidence that the mTOR signalling pathway controls cyclin B mRNA translation following fertilization in Sphaerechinus granularis and Paracentrotus lividus. We show that PP242 inhibits the degradation of the cap-dependent translation repressor 4E-BP (eukaryotic initiation factor 4E-Binding Protein). PP242 inhibits global protein synthesis, delays cyclin B accumulation, cyclin B/CDK1 complex activation and consequently entry into the mitotic phase of the cell cycle triggered by fertilization. PP242 inhibits cyclin B mRNA recruitment into active polysomes triggered by fertilization. An amount of cyclin B mRNA present in active polysomes appears to be insensitive to PP242 treatment. Taken together, our results suggest that, following sea urchin egg fertilization, cyclin B mRNA translation is controlled by two independent mechanisms: a PP242-sensitive and an additional PP242-insentitive mechanism.     

Greatwall maintains mitosis through regulation of PP2A

Greatwall (GW) is a new kinase that has an important function in the activation and the maintenance of cyclin B–Cdc2 activity. Although the mechanism by which it induces this effect is unknown, it has been suggested that GW could maintain cyclin B–Cdc2 activity by regulating its activation loop. Using Xenopus egg extracts, we show that GW depletion promotes mitotic exit, even in the presence of a high cyclin B–Cdc2 activity by inducing dephosphorylation of mitotic substrates. These results indicate that GW does not maintain the mitotic state by regulating the cyclin B–Cdc2 activation loop but by regulating a phosphatase. This phosphatase is PP2A; we show that (1) PP2A binds GW, (2) the inhibition or the specific depletion of this phosphatase from mitotic extracts rescues the phenotype induced by GW inactivation and (3) the PP2A‐dependent dephosphorylation of cyclin B–Cdc2 substrates is increased in GW‐depleted Xenopus egg extracts. These results suggest that mitotic entry and maintenance is not only mediated by the activation of cyclin B–Cdc2 but also by the regulation of PP2A by GW.     

Transient inhibition of L929 cell mitosis and locomotion by argon ion laser irradiation

Summary Argon ion laser irradiation of L929 cells transiently inhibits both entry into and passage through mitosis without affecting clonogenic survival. Anaphase mitotic figures virtually disappear from irradiated cell monolayers although prophase + metaphase mitotic figures can still be identified. The total number of mitotic figures does not change significantly and time-lapse video recording shows that cells do not enter mitosis following irradiation. This effect is dependent on light dose within the 900–2700 J/cm² range and persists for 10–48 h depending on the initial light exposure. Inhibition of cell locomotion and subsequent recovery were observed to occur over a similar time course. The possible contribution of these phenomena must be considered whenever biological systems are exposed to argon ion laser irradiation.     

Coupling of Cdc20 inhibition and activation by BubR1

Tight regulation of the APC/C-Cdc20 ubiquitin ligase that targets cyclin B1 for degradation is important for mitotic fidelity. The spindle assembly checkpoint (SAC) inhibits Cdc20 through the mitotic checkpoint complex (MCC). In addition, phosphorylation of Cdc20 by cyclin B1–Cdk1 independently inhibits APC/C–Cdc20 activation. This creates a conundrum for how Cdc20 is activated before cyclin B1 degradation. Here, we show that the MCC component BubR1 harbors both Cdc20 inhibition and activation activities, allowing for cross-talk between the two Cdc20 inhibition pathways. Specifically, BubR1 acts as a substrate specifier for PP2A-B56 to enable efficient Cdc20 dephosphorylation in the MCC. A mutant Cdc20 mimicking the dephosphorylated state escapes a mitotic checkpoint arrest, arguing that restricting Cdc20 dephosphorylation to the MCC is important. Collectively, our work reveals how Cdc20 can be dephosphorylated in the presence of cyclin B1-Cdk1 activity without causing premature anaphase onset.     

Stathmin and microtubules regulate mitotic entry in HeLa cells by controlling activation of both Aurora kinase A and Plk1

Depletion of stathmin, a microtubule (MT) destabilizer, delays mitotic entry by ∼4 h in HeLa cells. Stathmin depletion reduced the activity of CDC25 and its upstream activators, Aurora A and Plk1. Chemical inhibition of both Aurora A and Plk1 was sufficient to delay mitotic entry by 4 h, while inhibiting either kinase alone did not cause a delay. Aurora A and Plk1 are likely regulated downstream of stathmin, because the combination of stathmin knockdown and inhibition of Aurora A and Plk1 was not additive and again delayed mitotic entry by 4 h. Aurora A localization to the centrosome required MTs, while stathmin depletion spread its localization beyond that of γ-tubulin, indicating an MT-dependent regulation of Aurora A activation. Plk1 was inhibited by excess stathmin, detected in in vitro assays and cells overexpressing stathmin–cyan fluorescent protein. Recruitment of Plk1 to the centrosome was delayed in stathmin-depleted cells, independent of MTs. It has been shown that depolymerizing MTs with nocodazole abrogates the stathmin-depletion induced cell cycle delay; in this study, depolymerization with nocodazole restored Plk1 activity to near normal levels, demonstrating that MTs also contribute to Plk1 activation. These data demonstrate that stathmin regulates mitotic entry, partially via MTs, to control localization and activation of both Aurora A and Plk1.     

Regulated activity of PP2A–B55δ is crucial for controlling entry into and exit from mitosis in Xenopus egg extracts

Entry into mitosis depends on the activity of cyclin‐dependent kinases (CDKs). Conversely, exit from mitosis occurs when mitotic cyclins are degraded, thereby extinguishing CDK activity. Exit from mitosis must also require mitotic phosphoproteins to revert to their interphase hypophosphorylated forms, but there is a controversy about which phosphatase(s) is/are responsible for dephosphorylating the CDK substrates. We find that PP2A associated with a B55δ subunit is relatively specific for a model mitotic CDK substrate in Xenopus egg extracts. The phosphatase activity measured by this substrate is regulated during the cell cycle—high in interphase and suppressed during mitosis. Depletion of PP2A–B55δ (in interphase) from ‘cycling’ frog egg extracts accelerated their entry into mitosis and kept them indefinitely in mitosis. When PP2A–B55δ was depleted from mitotic extracts, however, exit from mitosis was hardly delayed, showing that other phosphatase(s) are also required for mitotic exit. Increasing the concentration of PP2A–B55δ in extracts by adding recombinant enzyme inhibited the entry into mitosis. This form of PP2A seems to be a key regulator of entry into and exit from mitosis.     

Apparent noncompetitive inhibition of choline transport in erythrocytes by inhibitors bound at the substrate site

Summary According to the conventional carrier model, an inhibitor bound at the substrate transfer site inhibits competitively when on the same side of the membrane as the substrate, but noncompetitively when on the opposite side. This prediction was tested with the nonpenetrating choline analog dimethyl-n-pentyl (2-hydroxyethyl) ammonium ion. In zerotrans entry and infinitetrans entry experiments, where the labeled substrate and the inhibitor occupy the same compartment, the inhibition was competitive, but in zerotrans exit it was noncompetitive, in accord with the model. Similar behavior was seen with dimethyl-n-decyl (2-hydroxyethyl) ammonium ion. With this property of the choline transport system established, it becomes possible to estimate the relative affinity inside and outside of inhibitors present on both sides of the membrane. The tertiary amine, dibutylaminoethanol, which enters the cell by simple diffusion, is such an inhibitor. Here the inhibition kinetics were the reverse of those for nonpenetrating inhibitors; zerotrans and infinitetrans exit was inhibited competitively, and zerotrans entry noncompetitively. It follows that dibutylaminoethanol binds predominantly to the inner carrier form.     

Genetic depletion of Polo‐like kinase 1 leads to embryonic lethality due to mitotic aberrancies

Polo‐like kinase 1 (PLK1) is a serine/threonine kinase that plays multiple and essential roles during the cell division cycle. Its inhibition in cultured cells leads to severe mitotic aberrancies and cell death. Whereas previous reports suggested that Plk1 depletion in mice leads to a non‐mitotic arrest in early embryos, we show here that the bi‐allelic Plk1 depletion in mice certainly results in embryonic lethality due to extensive mitotic aberrations at the morula stage, including multi‐ and mono‐polar spindles, impaired chromosome segregation and cytokinesis failure. In addition, the conditional depletion of Plk1 during mid‐gestation leads also to severe mitotic aberrancies. Our data also confirms that Plk1 is completely dispensable for mitotic entry in vivo. On the other hand, Plk1 haploinsufficient mice are viable, and Plk1‐heterozygous fibroblasts do not harbor any cell cycle alterations. Plk1 is overexpressed in many human tumors, suggesting a therapeutic benefit of inhibiting Plk1, and specific small‐molecule inhibitors for this kinase are now being evaluated in clinical trials. Therefore, the different Plk1 mouse models here presented are a valuable tool to reexamine the relevance of the mitotic kinase Plk1 during mammalian development and animal physiology.     

Cell cycle checkpoint regulators reach a zillion

Entry into mitosis is regulated by a checkpoint at the boundary between the G₂ and M phases of the cell cycle (G₂/M). In many organisms, this checkpoint surveys DNA damage and cell size and is controlled by both the activation of mitotic cyclin-dependent kinases (Cdks) and the inhibition of an opposing phosphatase, protein phosphatase 2A (PP2A). Misregulation of mitotic entry can often lead to oncogenesis or cell death. Recent research has focused on discovering the signaling pathways that feed into the core checkpoint control mechanisms dependent on Cdk and PP2A. Herein, we review the conserved mechanisms of the G₂/M transition, including recently discovered upstream signaling pathways that link cell growth and DNA replication to cell cycle progression. Critical consideration of the human, frog and yeast models of mitotic entry frame unresolved and emerging questions in this field, providing a prediction of signaling molecules and pathways yet to be discovered.     

Alzheimer Aβ disrupts the mitotic spindle and directly inhibits mitotic microtubule motors

Chromosome mis-segregation and aneuploidy are greatly induced in Alzheimer’s disease and models thereof by mutant forms of the APP and PS proteins and by their product, the Ab peptide. Here we employ human somatic cells and Xenopus egg extracts to show that Aβ impairs the assembly and maintenance of the mitotic spindle. Mechanistically, these defects result from Aβ’s inhibition of mitotic motor kinesins, including Eg5, KIF4A and MCAK. In vitro studies show that oligomeric Aβ directly inhibits recombinant MCAK by a noncompetitive mechanism. In contrast, inhibition of Eg5 and KIF4A is competitive with respect to both ATP and microtubules, indicating that Aβ interferes with their interactions with the microtubules of the mitotic spindle. Consistently, increased levels of polymerized microtubules or of the microtubule stabilizing protein Tau significantly decrease the inhibitory effect of Aβ on Eg5 and KIF4A. Together, these results indicate that by disrupting the interaction between specific kinesins and microtubules and by exerting a direct inhibitory effect on the motor activity, excess Ab deregulates the mechanical forces that govern the spindle and thereby leads to the generation of defective mitotic structures. The resulting defect in neurogenesis can account for the over 30% aneuploid/hyperploid, degeneration-prone neurons observed in Alzheimer disease brain. The finding of mitotic motors including Eg5 in mature post-mitotic neurons implies that their inhibition by Ab may also disrupt neuronal function and plasticity.     

Hydrogen peroxide inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells

Hydrogen peroxide (H(2)O(2)) induces a number of events, which are also induced by mitogens. Since the progression through the G1 phase of the cell cycle is dependent on mitogen stimulation, we were interested to study the effect of H(2)O(2) on the cell cycle progression. This study demonstrates that H(2)O(2) inhibits DNA synthesis in a dose-dependent manner when given to cells in mitosis or at different points in the G1 phase. Interestingly, mitotic cells treated immediately after synchronization are significantly more sensitive to H(2)O(2) than cells treated in the G1, and this is due to the inhibition of the cell spreading after mitosis by H(2)O(2). H(2)O(2) reversibly inhibits focal adhesion activation and stress fiber formation of mitotic cells, but not those of G1 cells. The phosphorylation of MAPK is also reversibly inhibited in both mitotic and G1 cells. Taken together, H(2)O(2) is probably responsible for the inhibition of the expression of cyclin D1 and cyclin A observed in cells in both phases. In conclusion, H(2)O(2) inhibits cell cycle progression by inhibition of the spreading of mitotic CHO cells. This may play a role in pathological processes in which H(2)O(2) is generated.     

Plk1 Inhibition Causes Post-Mitotic DNA Damage and Senescence in a Range of Human Tumor Cell Lines

Plk1 is a checkpoint protein whose role spans all of mitosis and includes DNA repair, and is highly conserved in eukaryotes from yeast to man. Consistent with this wide array of functions for Plk1, the cellular consequences of Plk1 disruption are diverse, spanning delays in mitotic entry, mitotic spindle abnormalities, and transient mitotic arrest leading to mitotic slippage and failures in cytokinesis. In this work, we present the in vitro and in vivo consequences of Plk1 inhibition in cancer cells using potent, selective small-molecule Plk1 inhibitors and Plk1 genetic knock-down approaches. We demonstrate for the first time that cellular senescence is the predominant outcome of Plk1 inhibition in some cancer cell lines, whereas in other cancer cell lines the dominant outcome appears to be apoptosis, as has been reported in the literature. We also demonstrate strong induction of DNA double-strand breaks in all six lines examined (as assayed by γH2AX), which occurs either during mitotic arrest or mitotic-exit, and may be linked to the downstream induction of senescence. Taken together, our findings expand the view of Plk1 inhibition, demonstrating the occurrence of a non-apoptotic outcome in some settings. Our findings are also consistent with the possibility that mitotic arrest observed as a result of Plk1 inhibition is at least partially due to the presence of unrepaired double-strand breaks in mitosis. These novel findings may lead to alternative strategies for the development of novel therapeutic agents targeting Plk1, in the selection of biomarkers, patient populations, combination partners and dosing regimens.     

Mitotic entry: The interplay between Cdk1, Plk1 and Bora

Polo-like kinase 1 (Plk1) is an important mitotic kinase that is crucial for entry into mitosis after recovery from DNA damage-induced cell cycle arrest. Plk1 activation is promoted by the conserved protein Bora (SPAT-1 in C. elegans), which stimulates the phosphorylation of a conserved residue in the activation loop by the Aurora A kinase. In a recent article published in Cell Reports, we show that the master mitotic kinase Cdk1 contributes to Plk1 activation through SPAT-1/Bora phosphorylation. We identified 3 conserved Sp/Tp residues that are located in the N-terminal, most conserved part, of SPAT-1/Bora. Phosphorylation of these sites by Cdk1 is essential for Plk1 function in mitotic entry in C. elegans embryos and during DNA damage checkpoint recovery in mammalian cells. Here, using an untargeted Förster Resonance Energy Transfer (FRET) biosensor to monitor Plk1 activation, we provide additional experimental evidence supporting the importance of these phosphorylation sites for Plk1 activation and subsequent mitotic entry after DNA damage. We also briefly discuss the mechanism of Plk1 activation and the potential role of Bora phosphorylation by Cdk1 in this process. As Plk1 is overexpressed in cancer cells and this correlates with poor prognosis, understanding how Bora contributes to Plk1 activation is paramount for the development of innovative therapeutical approaches.