The wis1 protein kinase is a dosage-dependent regulator of mitosis in Schizosaccharomyces pombe.

The wis1+ gene encodes a newly identified mitotic control element in Schizosaccharomyces pombe. It was isolated by virtue of its interaction with the mitotic control genes cdc25, wee1 and win1. The wis1+ gene potentially encodes a 66 kDa protein with homology to the serine/threonine family of protein kinases. wis1+ plays an important role in the regulation of entry into mitosis, as it shares with cdc25+ and nim1+/cdr1+ the property of inducing mitosis in a dosage-dependent manner. Increased levels of wis1+ expression cause mitotic initiation to occur at a reduced cell size. Loss of wis1+ function does not prevent vegetative growth and division, though wis1- cells show an elongated morphology, indicating that their entry into mitosis and cell division is delayed relative to wild type cells. wis1- cells undergo a rapid reduction of viability upon entry into stationary phase, suggesting a role for wis1+ in the integration of nutritional sensing with the control over entry into mitosis.     

B23/nucleophosmin serine 4 phosphorylation mediates mitotic functions of polo-like kinase 1

Phosphoprotein profiling by Kinetworks trade mark analysis of M-phase-arrested HeLa cells by nocodazole treatment revealed that a novel mitosis-specific phosphorylation event occurred in the nucleolar protein B23/nucleophosmin at a conserved Ser-4 residue. Consistent with the resemblance of the Ser-4 phosphorylation site to the Polo-like kinase 1 (Plk1) consensus recognition sequence, inhibition of Plk1 by a kinase-defective mutation (K82M) abrogated B23 Ser-4 phosphorylation, whereas activation of Plk1 by a constitutively active mutation (T210D) enhanced its phosphorylation following in vivo transfection and in vitro phosphorylation assays. Depletion of endogenous Plk1 by RNA interference abolished B23 Ser-4 phosphorylation. The physical interaction of Plk1 and B23 was further demonstrated by their co-immunoprecipitation and glutathione S-transferase fusion protein pull-down assays. Interference of Ser-4 phosphorylation of B23 induced multiple mitotic defects in HeLa cells, including aberrant numbers of centrosomes, elongation and fragmentation of nuclei, and incomplete cytokinesis. The phenotypes of B23 mutants are reminiscent of a subset of those described previously in Plk1 mutants. Our findings provide insights into the biochemical mechanism underlying the role of Plk1 in mitosis regulation through the identification of Ser-4 in B23 as a major physiological substrate of Plk1.     

Overexpression of the nucleolar protein nucleophosmin/B23 in collagen lattice-cultured fibroblasts: Potential role in the control of protein synthesis

Fibroblasts cultivated in tridimensional collagen lattices exhibit a downregulation of protein synthesis, related to decreased ribosomal RNA (rRNA) content and half life, when compared to monolayer cultivated cells. The involvement in this process of nucleophosmin/B23, a nucleolar phosphoprotein with ribonuclease properties, was checked. We compared production of nucleophosmin/B23 in monolayer and collagen lattice cultured fibroblasts. A significant increase of nucleophosmin/B23 mRNA levels was noticed in lattice-cultured fibroblasts vs monolayers (+154%, p < 0.05). A concomitant enhancement of nucleolar nucleophosmin/B23 content was found (+112%, p < 0.001). Simultaneously, ribonuclease activity contained in nucleolar extracts from collagen lattice-cultured fibroblasts was significantly increased (+54%, p < 0.01). These data demonstrate that extracellular collagen matrix induces the overexpression of nucleophosmin/B23, and suggest that the regulation of protein syntheses in collagen lattice cultures may be explained, at least partly, by an increased degradation of neosynthesized rRNAs dependent on nucleophosmin.     

Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells

In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.     

Dual phosphorylation controls Cdc25 phosphatases and mitotic entry

Negative regulation of the Cdc25C protein phosphatase by phosphorylation on Ser 216, the 14-3-3-binding site, is an important regulatory mechanism used by cells to block mitotic entry under normal conditions and after DNA damage. During mitosis, Cdc25C is not phosphorylated on Ser 216 and ionizing radiation (IR) does not induce either phosphorylation of Ser 216, or binding to 14-3-3. Here, we show that Cdc25C is phosphorylated on Ser 214 during mitosis, which in turn prevents phosphorylation of Ser 216. Mutation of Ser 214 to Ala reconstitutes Ser 216 phosphorylation and 14-3-3 binding during mitosis. Introduction of exogenous Cdc25C(S214A) into HeLa cells depleted of endogenous Cdc25C results in a substantial delay to mitotic entry. This effect was fully reversed in a S214A/S216A double-mutant, implying that the inhibitory effect of S214A mutant was entirely dependent on Ser 216 phosphorylation. A similar regulatory mechanism may also apply to another mitotic phosphatase, Cdc25B, as well as mitotic phosphatases of other species, including Xenopus laevis. We propose that this pathway ensures that Cdc2 remains active once mitosis is initiated and is a key control mechanism for maintaining the proper order of cell-cycle transitions.     

Characterization and cellular localization of nucleophosmin/B23 in HeLa cells treated with selected cytotoxic agents (studies of B23-translocation mechanism).

Previous studies indicated that nucleophosmin/B23, an abundant nucleolar phosphoprotein, accumulated in the nucleoplasm (B23-translocation) of cells after exposure to selected cytotoxic drugs. Attempts were made to understand the B23-translocation mechanism. This paper reports that: (1) B23-translocation is a reversible process. Upon removal of camptothecin, which induced B23-translocation in HeLa cells, nucleophosmin/B23 relocalized into nucleoli within 2 h. Relocation occurs in the presence of cycloheximide which inhibits new protein synthesis. There is no reduction or degradation of nucleophosmin/B23 detected during drug treatments. Nucleophosmin/B23 has a half-life of 18-20 h. Taken together, these results indicate that B23-translocation is a reversible process. Drug treatment causes redistribution of nucleophosmin/B23 in nucleoplasm. (2) Inhibition of RNA synthesis does not cause the B23-translocation. Over 80% of RNA synthesis was inhibited in HeLa cells by treatment with actinomycin D, camptothecin, and methotrexate. While actinomycin D and camptothecin cause B23-translocation in all cells, 40% of methotrexate-treated cells remain untranslocated. (3) There is no significant change of phosphorylation in nucleophosmin/B23 during drug treatment. An identical oligomeric cross-linkage pattern was obtained in drug-treated cells. (4) HeLa cells treated with B23-translocation effective drugs have small and round nucleoli while control cells have large and irregular-shaped nucleoli.     

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.     

Chk1-Dependent Regulation of Cdc25B Functions to Coordinate Mitotic Events

The coordination of mitotic spindle formation and chromatin condensation is an essential prerequisite for successful mitosis. Both events are thought to be initiated by cyclin B/Cdk1, whose initial activation occurs in late prophase at the centrosomes. Recently, we have shown that Chk1 localizes to interphase centrosomes and thereby negatively regulates entry into mitosis by preventing premature activation of cyclin B/Cdk1. Here, we demonstrate that inhibition of Chk1 kinase induces mitotic entry with regular spindle assembly but aberrant and mislocalized chromatin. This effect, which we have termed the ‘paraspindle’ phenotype, was reverted by downregulation of Cdc25B phosphatase using siRNA, which restored normal mitosis with regular chromatin. Analogous to Chk1 inhibition, the ‘paraspindle’ phenotype was induced by overexpression of Cdc25B but not Cdc25A. Our results suggest that Chk1 functions to coordinate mitotic events through regulation of Cdc25B.     

Effect of roscovitine,a selective cyclin B-dependent kinase 1 inhibitor,on assembly of the nucleolus in mitosis

It is well known that at the beginning of mitosis the nucleolus disassembles but then reassembles at the end of mitosis. However, the mechanisms of these processes are still unclear. In the present work, we show for the first time that selective inhibition of cyclin B-dependent kinase 1 (CDK1) by roscovitine induces premature assembly of the nucleolus in mammalian cells in metaphase. Treatment of metaphase cells with roscovitine induces formation of structures in their cytoplasm that contain major proteins of the mature nucleolus participating in rRNA processing, such as B23/nucleophosmin, C23/nucleolin, fibrillarin, Nop52, as well as partially processed (immature) 46-45S pre-rRNA. This effect is reproducible in cells of various types; this indicates that general mechanisms regulate early stages of the nucleolus reassembly with CDK1 participation in mammalian cells. Based on our and literature data, we suggest that inactivation of the CDK1-cyclin B complex at the end of mitosis results in dephosphorylation of B23/nucleophosmin and C23/nucleolin; this facilitates their interaction with pre-rRNA and leads to formation of insoluble supramolecular complexes--nucleolus-derived foci.     

Inhibiting Crm1 causes the formation of excess acentriolar spindle poles containing NuMA and B23, but does not affect centrosome numbers

Background. B23/nucleophosmin is present on spindle poles at metaphase. Migration of B23 to the poles is under the control of exportin Crm1. B23 at the centrosome plays a role in the control centrosome duplication. Results. h‐Tert‐RPE1 cells blocked in prometaphase with low doses of Nocodazol showed a progression to mitosis if Crm1 exportin was inhibited. Under these conditions, the formation of accessory poles containing γ‐tubulin, NuMA (nuclear‐mitotic‐apparatus) and B23 was induced at metaphase. No effect on centrosome number was observed. In quiescent h‐Tert‐RPE1 cells, when Crm1 was active, B23 was not detected at the centrosome as well as B23‐mutants reported to block centrosome duplication. In addition, the modification of B23 nucleo‐cytoplasmic shuttling showed no effect on centrosome duplication. Conclusion. Inhibition of Crm1 in early metaphase favours the formation of supplementary acentriolar spindle poles. B23 and NuMA are present at these poles that ultimately focus around the centrosome. Inhibition of Crm1 at metaphase has no effect on the control of centrosome numbers.     

Effect of serine/threonine protein kinases and protein phosphatases inhibitors on mitosis progression in a synchronized tobacco BY-2 culture

In order to investigate the role of various serine/threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells, the influence of cyclin(olomoucine) and Ca²⁺/calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine), and a protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin-dependent protein kinases and protein kinase C caused a prophase delay, reduced the mitotic index, and displaced the mitotic peak as compared with control cells. Inhibition of Ca²⁺/calmodulin-dependent protein kinases enhanced the cells entry into prophase and delayed their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances synchronized BY-2 cells entering into all phases of mitosis.     

The role of stathmin in the regulation of the cell cycle

Stathmin is the founding member of a family of proteins that play critically important roles in the regulation of the microtubule cytoskeleton. Stathmin regulates microtubule dynamics by promoting depolymerization of microtubules and/or preventing polymerization of tubulin heterodimers. Upon entry into mitosis, microtubules polymerize to form the mitotic spindle, a cellular structure that is essential for accurate chromosome segregation and cell division. The microtubule-depolymerizing activity of stathmin is switched off at the onset of mitosis by phosphorylation to allow microtubule polymerization and assembly of the mitotic spindle. Phosphorylated stathmin has to be reactivated by dephosphorylation before cells exit mitosis and enter a new interphase. Interfering with stathmin function by forced expression or inhibition of expression results in reduced cellular proliferation and accumulation of cells in the G2/M phases of the cell cycle. Forced expression of stathmin leads to abnormalities in or a total lack of mitotic spindle assembly and arrest of cells in the early stages of mitosis. On the other hand, inhibition of stathmin expression leads to accumulation of cells in the G2/M phases and is associated with severe mitotic spindle abnormalities and difficulty in the exit from mitosis. Thus, stathmin is critically important not only for the formation of a normal mitotic spindle upon entry into mitosis but also for the regulation of the function of the mitotic spindle in the later stages of mitosis and for the timely exit from mitosis. In this review, we summarize the early studies that led to the identification of the important mitotic function of stathmin and discuss the present understanding of its role in the regulation of microtubules dynamics during cell-cycle progression. We also describe briefly other less mature avenues of investigation which suggest that stathmin may participate in other important biological functions and speculate about the future directions that research in this rapidly developing field may take.     

Aurora-B regulates RNA methyltransferase NSUN2

Disassembly of the nucleolus during mitosis is driven by phosphorylation of nucleolar proteins. RNA processing stops until completion of nucleolar reformation in G(1) phase. Here, we describe the RNA methyltransferase NSUN2, a novel substrate of Aurora-B that contains an NOL1/NOP2/sun domain. NSUN2 was concentrated in the nucleolus during interphase and was distributed in the perichromosome and cytoplasm during mitosis. Aurora-B phosphorylated NSUN2 at Ser139. Nucleolar proteins NPM1/nucleophosmin/B23 and nucleolin/C23 were associated with NSUN2 during interphase. In mitotic cells, association between NPM1 and NSUN2 was inhibited, but NSUN2-S139A was constitutively associated with NPM1. The Aurora inhibitor Hesperadin induced association of NSUN2 with NPM1 even in mitosis, despite the silver staining nucleolar organizer region disassembly. In vitro methylation experiments revealed that the Aurora-B-phosphorylation and the phosphorylation-mimic mutation (S139E) suppressed methyltransferase activities of NSUN2. These results indicate that Aurora-B participates to regulate the assembly of nucleolar RNA-processing machinery and the RNA methyltransferase activity of NSUN2 via phosphorylation at Ser139 during mitosis.     

A topoisomerase II-dependent checkpoint in G2-phase plant cells can be bypassed by ectopic expression of mitotic cyclin B2

DNA topoisomerase II is required for mitotic chromosome condensation and segregation. Here we characterize the effects of inhibiting DNA topoisomerase II activity in plant cells using the non-DNA damaging topoisomerase II inhibitor ICRF-193. We report that ICRF-193 abrogated chromosome condensation in cultured alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.) mitoses and led to bridged chromosomes at anaphase. Moreover, ICRF-193 treatment delayed entry into mitosis, increasing the frequency of cells having a pre-prophase band of microtubules, a marker of late G2 and prophase, and delaying the activation of cyclin-dependent kinase. These data suggest the existence of a late G2 checkpoint in plant cells that is activated in the absence of topoisomerase II activity. To determine whether the checkpoint-induced delay was a result of reduced cyclindependent kinase activity, mitotic cyclin B2 was ectopically expressed. Cyclin B2 bypassed the ICRF-193-induced delay before mitosis, and correspondingly, reduced the frequency of interphase cells with a pre-prophase band. These data provide evidence that plant cells possess a topoisomerase II-dependent G2 cell cycle checkpoint that transiently inhibits mitotic CDK activation and entry into mitosis, and that is overridden by raising the level of CDK activity through the ectopic expression of a plant mitotic cyclin.     

APOLLON Protein Promotes Early Mitotic CYCLIN A Degradation Independent of the Spindle Assembly Checkpoint

In the mammalian cell cycle, both CYCLIN A and CYCLIN B are required for entry into mitosis, and their elimination is also essential to complete the process. During mitosis, CYCLIN A and CYCLIN B are ubiquitylated by the anaphase-promoting complex/cyclosome (APC/C) and then subjected to proteasomal degradation. However, CYCLIN A, but not CYCLIN B, begins to be degraded in the prometaphase when APC/C is inactivated by the spindle assembly checkpoint (SAC). Here, we show that APOLLON (also known as BRUCE or BIRC6) plays a role in SAC-independent degradation of CYCLIN A in early mitosis. APPOLON interacts with CYCLIN A that is not associated with cyclin-dependent kinases. APPOLON also interacts with APC/C, and it facilitates CYCLIN A ubiquitylation. In APPOLON-deficient cells, mitotic degradation of CYCLIN A is delayed, and the total, but not the cyclin-dependent kinase-bound, CYCLIN A level was increased. We propose APPOLON to be a novel regulator of mitotic CYCLIN A degradation independent of SAC.     

Tome-1, a trigger of mitotic entry,is degraded during G1 via the APC

Entry into mitosis requires the activation of cdk1/cyclin B, while mitotic exit is achieved when the same kinase activity decreases, as cyclin B is degraded. Cyclin B proteolysis is mediated by the anaphase promoting complex, or APC, an E3 ligase that is active at anaphase in mitosis through G1. We have identified a G1 substrate of the APC that we have termed Tome-1, for trigger of mitotic entry. Tome-1 is a cytosolic protein required for proper activation of cdk1/cyclin B and mitotic entry. Tome-1 associates with Skp-1 and is required for degradation of the cdk1 inhibitory tyrosine kinase wee1; Tome-1 therefore appears to be acting as part of an SCF-type E3 for wee1. Degradation of Tome-1 during G1 allows for wee 1 accumulation during interphase, thereby providing a critical link between the APC and SCF pathways in regulation of cdk1/cyclin B activity and thus mitotic entry and exit.     

A Topoisomerase II-Dependent Checkpoint in G2-Phase Plant Cells Can Be Bypassed by Ectopic Expression of Mitotic Cyclin B2

DNA topoisomerase II is required for mitotic chromosome condensation and segregation. Here we characterize the effects of inhibiting DNA topoisomerase II activity in plant cells using the non-DNA damaging topoisomerase II inhibitor ICRF-193. We report that ICRF-193 abrogated chromosome condensation in cultured alfalfa (Medicago sativa L.) and tobacco (Nicotiana tabaccum L.) mitoses and led to bridged chromosomes at anaphase. Moreover, ICRF-193 treatment delayed entry into mitosis, increasing the frequency of cells having a pre-prophase band of microtubules, a marker of late G2 and prophase, and delaying the activation of cyclin-dependent kinase. These data suggest the existence of a late G2 checkpoint in plant cells that is activated in the absence of topoisomerase II activity. To determine whether the checkpoint-induced delay was a result of reduced cyclin-dependent kinase activity, mitotic cyclin B2 was ectopically expressed. Cyclin B2 bypassed the ICRF-193-induced delay before mitosis, and correspondingly, reduced the frequency of interphase cells with a pre-prophase band. These data provide evidence that plant cells possess a topoisomerase II-dependent G2 cell cycle checkpoint that transiently inhibits mitotic CDK activation and entry into mitosis, and that is overridden by raising the level of CDK activity through the ectopic expression of a plant mitotic cyclin.

Key Words:

Plant cyclin B2, Topoisomerase II, ICRF-193, G2 checkpoint, Microtubules