Human Cdc14B promotes progression through mitosis by dephosphorylating Cdc25 and regulating Cdk1/cyclin B activity

Entry into and progression through mitosis depends on phosphorylation and dephosphorylation of key substrates. In yeast, the nucleolar phosphatase Cdc14 is pivotal for exit from mitosis counteracting Cdk1-dependent phosphorylations. Whether hCdc14B, the human homolog of yeast Cdc14, plays a similar function in mitosis is not yet known. Here we show that hCdc14B serves a critical role in regulating progression through mitosis, which is distinct from hCdc14A. Unscheduled overexpression of hCdc14B delays activation of two master regulators of mitosis, Cdc25 and Cdk1, and slows down entry into mitosis. Depletion of hCdc14B by RNAi prevents timely inactivation of Cdk1/cyclin B and dephosphorylation of Cdc25, leading to severe mitotic defects, such as delay of metaphase/anaphase transition, lagging chromosomes, multipolar spindles and binucleation. The results demonstrate that hCdc14B-dependent modulation of Cdc25 phosphatase and Cdk1/cyclin B activity is tightly linked to correct chromosome segregation and bipolar spindle formation, processes that are required for proper progression through mitosis and maintenance of genomic stability.     

Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1-Cdk1 at the centrosome

Cdc25 phosphatases are essential for the activation of mitotic cyclin-Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear. Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis. We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1-Cdk1 and cyclin A-Cdk2 complexes and a delayed accumulation of cyclin B1 protein. Further, three-dimensional time-lapse microscopy and quantification of Cdk1 phosphorylation versus cyclin B1 levels in individual cells revealed that Cdc25A and -B exert specific functions in the initiation of mitosis: Cdc25A may play a role in chromatin condensation, whereas Cdc25B specifically activates cyclin B1-Cdk1 on centrosomes.     

WARTS tumor suppressor is phosphorylated by Cdc2/cyclin B at spindle poles during mitosis

Identification of physiological substrates for Cdc2/cyclin B is crucial for understanding the functional link between mitotic events and Cdc2/cyclin B activation. A human homologue of the Drosophila warts tumor suppressor, termed WARTS, is a serine/threonine kinase and a dynamic component of the mitotic apparatus. We have found that Cdc2/cyclin B forms a complex with a fraction of WARTS in the centrosome and phosphorylates the Ser613 site of WARTS during mitosis. Immunocytochemical analysis has shown that the S613-phosphorylated WARTS appears in the spindle poles at prometaphase and disappears at telophase. Our findings suggest that Cdc/cyclin B regulates functions of WARTS on the mitotic apparatus.     

Purine synthesis de novo and salvage in hypoxanthine phosphoribosyltransferase-deficient mice

Extreme degrees of hypoxanthine phosphoribosyltransferase (HPRT) deficiency in man are associated with gross sex-linked neurological dysfunction, gout and urinary stones (the Lesch-Nyhan or 'complete HPRT-deficiency' syndrome). The less severe degrees of enzyme deficiency (sex-linked recessive gout and/or urolithiasis or the 'partial HPRT-deficiency' syndrome) may be associated with minor neurological manifestations. Whole body purine synthesis de novo is accelerated in both these groups of patients. A strain of mice with an experimentally produced mutation at the HPRT locus showed some residual 'apparent HPRT activity' in brain, liver, testicular, splenic, kidney and ovarian tissues but not in erythrocyte haemolysates. The mutation removes exons 1 and 2 of the coding region of the gene together with the promotor and about 10 kb of upstream sequence from the gene. It is therefore possible that the observed 'apparent HPRT activity' in these mice is due to the operation of an alternative metabolic pathway. Purine synthesis de novo was markedly accelerated in their brain, testicular, splenic and kidney tissues. It was not accelerated in the liver tissue of male mice hemizygous for the mutation and the degree of acceleration in the female homozygotes only just reached statistical significance at the p = 0.02 level. This observation casts doubt on the importance of modulations in the rate of hepatic purine synthesis de novo as a mechanism for maintaining a steady supply of purines for translocation to other organs.     

Bub1 and the multilayered inhibition of Cdc20-APC/C in mitosis

To ensure the accuracy of chromosome segregation in mitosis, the spindle checkpoint blocks the activity of the anaphase-promoting complex APC/C until all chromosomes are properly bi-orientated on the metaphase spindle. How the checkpoint machinery actually inhibits the APC/C is still unclear. A new paper by Tang and coworkers helps further our understanding of this complex and fundamental process.     

A cyclin B homolog in S. cerevisiae: chronic activation of the Cdc28 protein kinase by cyclin prevents exit from mitosis.

A cyclin B homolog was identified in Saccharomyces cerevisiae using degenerate oligonucleotides and the polymerase chain reaction. The protein, designated Scb1, has a high degree of similarity with B-type cyclins from organisms ranging from fission yeast to human. Levels of SCB1 mRNA and protein were found to be periodic through the cell cycle, with maximum accumulation late, most likely in the G2 interval. Deletion of the gene was found not to be lethal, and subsequently other B-type cyclins have been found in yeast functionally redundant with Scb1. A mutant allele of SCB1 that removes an amino-terminal fragment of the encoded protein thought to be required for efficient degradation during mitosis confers a mitotic arrest phenotype. This arrest can be reversed by inactivation of the Cdc28 protein kinase, suggesting that cyclin-mediated arrest results from persistent protein kinase activation.     

Cyclin destruction in mitosis: a crucial task of Cdc20

Proteolytic destruction of cyclins is a fundamental process for cell division. At the end of mitosis, degradation of mitotic cyclins results in the inactivation of cyclin-dependent kinases. Cyclin proteolysis is triggered by the anaphase-promoting complex/cyclosome (APC/C), a multi-subunit complex which contains ubiquitin ligase activity. Recent data in yeast demonstrated that a partial degradation of the mitotic cyclin Clb2, mediated by APC/C and its activator protein Cdc20, is essential and sufficient for the mitotic exit. Remarkably, a complete inactivation of cyclin-dependent kinases seems to be not essential. This review discusses recent novel insights into cyclin destruction and its implications for the mitotic exit.     

Phosphorylation-dependent binding of cyclin B1 to a Cdc6-like domain of human separase

Sister chromatids are held together by the ring-shaped cohesin complex, which likely entraps both DNA-double strands in its middle. This tie is resolved in anaphase when separase, a giant protease, becomes active and cleaves the kleisin subunit of cohesin. Premature activation of separase and, hence, chromosome missegregation are prevented by at least two inhibitory mechanisms. Although securin has long been appreciated as a direct inhibitor of separase, surprisingly its loss has basically no phenotype in mammals. Phosphorylation-dependent binding of Cdk1 constitutes an alternative way to inhibit vertebrate separase. Its importance is illustrated by the premature loss of cohesion when Cdk1-resistant separase is expressed in mammalian cells without or with limiting amounts of securin. Here, we demonstrate that crucial inhibitory phosphorylations occur within a region of human separase that is also shown to make direct contact with the cyclin B1 subunit of Cdk1. This region exhibits a weak homology to Saccharomyces cerevisiae Cdc6 of similar Cdk1 binding behavior, thereby establishing phosphoserine/threonine-mediated binding of partners as a conserved characteristic of B-type cyclins. In contrast to the Cdc6-like domain, the previously identified serine 1126 phosphorylation is fully dispensable for Cdk1 binding to separase fragments. This suggests that despite its in vivo relevance, it promotes complex formation indirectly, possibly by inducing a conformational change in full-length separase.     

Dose-dependent effects of stable cyclin B1 on progression through mitosis in human cells

The disassembly of the mitotic spindle and exit from mitosis require the inactivation of Cdk1. Here, we show that expression of nondegradable cyclinB1 causes dose-dependent mitotic arrest phenotypes. By monitoring chromosomes in living cells, we determined that pronounced overexpression of stable cyclinB1 entailed metaphase arrest without detectable sister chromatid separation, while moderate overexpression arrested cells in a pseudometaphase state, in which separated sister chromatids were kept at the cellular equator by a bipolar 'metaphase-like' spindle. Chromosomes that left the pseudometaphase plate became pulled back and individual kinetochores were found to be merotelically attached to both spindle poles in stable cyclinB1 arrested cells. Inactivation of the chromokinesin hKid, by RNAi or antibody microinjection, prevented the formation of stable bipolar spindles and the 'metaphase-like' alignment of chromosomes in cells expressing stable cyclinB1. These experiments show that cyclinB1 is able to maintain a bipolar spindle even after sister chromatids had become separated and suggest an important role of hKid in this process. Cells expressing low levels of nondegradable cyclinB1 progressed further in mitosis and arrested in telophase.     

Redundant pathways for Cdc2 activation in Xenopus oocyte: either cyclin B or Mos synthesis

Xenopus oocytes are arrested in meiotic prophase I. Progesterone induces the resumption of meiotic maturation, which requires continuous protein synthesis to bring about Cdc2 activation. The identification of the newly synthesized proteins has long been a goal. Two plausible candidates have received extensive study. The synthesis of cyclin B and of c-Mos, a kinase that activates the mitogen-activated protein kinase pathway in oocytes, is clearly upregulated by translational control in response to progesterone. Recent studies suggest that ablation of either c-Mos or cyclin B synthesis by antisense oligonucleotides does not block meiotic maturation. Here, however, we show that when both pathways are simultaneously inhibited, progesterone no longer triggers maturation; adding back either c-Mos or cyclin B restores meiotic maturation. We conclude that the specific synthesis of either B-type cyclins or c-Mos, induced by progesterone, is required to induce meiotic maturation. The two pathways seem to be functionally redundant.     

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

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.     

不同温度下酿酒酵母细胞分裂周期蛋白Cdc5在有丝分裂中的分子动力学研究

[目的] 探究不同温度下酿酒酵母细胞分裂周期蛋白Cdc5蛋白在有丝分裂中的分子动力学变化。[方法] 本研究以酿酒酵母（Saccharomyces cerevisiae）为材料,采用活细胞成像的方法,探究Cdc5蛋白在不同温度下在酿酒酵母有丝分裂过程中的精细分子动力学变化;通过测量OD₅₉₅绘制生长曲线图,看其宏观的分裂情况是否与微观下Cdc5蛋白的分子动力学变化一致;利用流式细胞术检测细胞的细胞周期变化的情况。[结果] 在胞质分裂时,Cdc5蛋白从母细胞进入子细胞,并在芽颈处发生聚集。25℃条件下细胞中Cdc5蛋白在芽颈处的聚集时间长,37℃条件下Cdc5蛋白在芽颈处聚集时间短,两者间存在显著差异;但两个温度下,细胞中Cdc5蛋白的表达量没有显著性差异。同时,温度也会影响Cdc5蛋白在降解过程中的动力学行为,包括Cdc5蛋白在母细胞与子细胞中荧光强度峰值出现的次数和时间。生长曲线结果显示,酿酒酵母单一细胞分裂周期的变化影响了其宏观的细胞生长,且酵母分裂速度越快,子细胞长宽比越小;细胞周期结果表明,37℃下Cdc5蛋白的动力学变化与酿酒酵母细胞周期变化一致,酿酒酵母细胞周期从G₀/G₁期进入S期,亦加速了酿酒酵母的分裂。[结论] 本研究首次探究了不同温度下酿酒酵母有丝分裂中Cdc5蛋白的精细分子动力学及对应的酵母的宏观生长情况,结果表明温度会对Cdc5蛋白的动力学产生影响,且其精细分子动力学与酿酒酵母的分裂速度成正相关,该结果为进一步研究其在细胞有丝分裂中的功能提供了前期研究基础。     

Regulation of de novo phosphatidylinositol synthesis

Mechanisms that function to regulate the rate of de novo phosphatidylinositol (PtdIns) synthesis in mammalian cells have not been elucidated. In this study, we characterize the effect of phorbol ester treatment on de novo PtdIns synthesis in C3A human hepatoma cells. Incubation of cells with 12-O-tetradecanoyl phorbol 13-acetate (TPA) initially (1-6 h) results in a decrease in precursor incorporation into PtdIns; however, at later times (18-24 h), a marked increase is observed. TPA-induced glucose uptake from the medium is not required for observation of the stimulation of PtdIns synthesis, because the effect is apparent in glucose-free medium. Inhibition of the activation of arachidonic acid substantially blocks the synthesis of PtdIns but has no effect on the synthesis of phosphatidylcholine (PtdCho). Increasing the concentration of cellular phosphatidic acid by blocking its conversion to diacylglycerol, on the other hand, enhances the synthesis of PtdIns and inhibits the synthesis of PtdCho. The TPA-induced stimulation of PtdIns synthesis is not the result of the concomitant TPA-induced G1 arrest, because G1 arrest induced by mevastatin has no effect on PtdIns synthesis. Inhibition of protein kinase C activity blocks the stimulatory action of TPA on de novo synthesis of PtdIns but has no effect on TPA-induced inhibition. Potential sites of enzymatic regulation are discussed.     

A novel role for Cdk1/cyclin B in regulating B-raf activation at mitosis

MAPK activity is important during mitosis for spindle assembly and maintenance of the spindle checkpoint arrest. We previously identified B-Raf as a critical activator of the MAPK cascade during mitosis in Xenopus egg extracts and showed that B-Raf activation is regulated in an M-phase-dependent manner. The mechanism that mediates B-Raf activation at mitosis has not been elucidated. Interestingly, activation of 95-kDa B-Raf at mitosis does not require phosphorylation of Thr-599 and Ser-602 residues (Thr-633 and Ser-636 in Xenopus B-Raf), previously shown to be essential for B-Raf activation by Ras. Instead, we provide evidence for Cdk1/cyclin B in mediating mitotic activation of B-Raf. In particular, Cdk1/cyclin B complexes associate with B-Raf at mitosis in Xenopus egg extracts and contribute to its phosphorylation. Mutagenesis and in vitro kinase assays demonstrated that Cdk1/cyclin B directly phosphorylates B-Raf at Serine-144, which is part of a conserved Cdk1 preferential consensus site (S(144)PQK). Importantly, phosphorylation of Ser-144 is absolutely required for mitotic activation of B-Raf and subsequent activation of the MAPK cascade. However, substitution of a phospho-mimicking amino acid at Ser-144 failed to produce a constitutive active B-Raf indicating that, in addition of Ser-144 phosphorylation, other regulatory events may be needed to activate B-Raf at mitosis. Taken together, our data reveal a novel cell cycle mechanism for activating the B-Raf/MEK/MAPK cascade.     

Centrosomal and cytoplasmic Cdc2/cyclin B1 activation precedes nuclear mitotic events

The activation of cdc2/cyclin B is the trigger for entry into mitosis. The mechanism of cdc2/cyclin B activation is complex, but the final step is the dephosphorylation of the Thr14 and Tyr15 residues on the cdc2 subunit, catalyzed by a member of the Cdc25 family of phosphatases. Cdc2/cyclin B1 accumulates at the centrosome in late G2 phase and has been implicated in the conversion of the centrosome from an interphase to a mitotic microtubule organizing center. Here we demonstrate biochemically that cdc2/cyclin B1 accumulates at the centrosome in late G2 as the inactive, phosphotyrosine 15 form and that the centrosomal cdc2/cyclin B1 can be activated in vitro by recombinant cdc25B. We provide evidence that a portion of the cdc2/cyclin B1 translocated into the nucleus in prophase is the inactive tyrosine-15-phosphorylated form. At this time the centrosomal and cytoplasmic cdc2/cyclin B1 is already active. This provides evidence that the activation of cdc2/cyclin B1 is initiated in the cytoplasm and that full activation of the translocated pool occurs in the nucleus.     

Distinct sequence elements of cyclin B1 promote localization to chromatin, centrosomes, and kinetochores during mitosis

The mitotic cyclins promote cell division by binding and activating cyclin-dependent kinases (CDKs). Each cyclin has a unique pattern of subcellular localization that plays a vital role in regulating cell division. During mitosis, cyclin B1 is known to localize to centrosomes, microtubules, and chromatin. To determine the mechanisms of cyclin B1 localization in M phase, we imaged full-length and mutant versions of human cyclin B1-enhanced green fluorescent protein in live cells by using spinning disk confocal microscopy. In addition to centrosome, microtubule, and chromatin localization, we found that cyclin B1 also localizes to unattached kinetochores after nuclear envelope breakdown. Kinetochore recruitment of cyclin B1 required the kinetochore proteins Hec1 and Mad2, and it was stimulated by microtubule destabilization. Mutagenesis studies revealed that cyclin B1 is recruited to kinetochores through both CDK1-dependent and -independent mechanisms. In contrast, localization of cyclin B1 to chromatin and centrosomes is independent of CDK1 binding. The N-terminal domain of cyclin B1 is necessary and sufficient for chromatin association, whereas centrosome recruitment relies on sequences within the cyclin box. Our data support a role for cyclin B1 function at unattached kinetochores, and they demonstrate that separable and distinct sequence elements target cyclin B1 to kinetochores, chromatin, and centrosomes during mitosis.     

Cdc25B and Cdc25C differ markedly in their properties as initiators of mitosis.

We have used time-lapse fluorescence microscopy to study the properties of the Cdc25B and Cdc25C phosphatases that have both been implicated as initiators of mitosis in human cells. To differentiate between the functions of the two proteins, we have microinjected expression constructs encoding Cdc25B or Cdc25C or their GFP-chimeras into synchronized tissue culture cells. This assay allows us to express the proteins at defined points in the cell cycle. We have followed the microinjected cells by time-lapse microscopy, in the presence or absence of DNA synthesis inhibitors, and assayed whether they enter mitosis prematurely or at the correct time. We find that overexpressing Cdc25B alone rapidly causes S phase and G2 phase cells to enter mitosis, whether or not DNA replication is complete, whereas overexpressing Cdc25C does not cause premature mitosis. Overexpressing Cdc25C together with cyclin B1 does shorten the G2 phase and can override the unreplicated DNA checkpoint, but much less efficiently than overexpressing Cdc25B. These results suggest that Cdc25B and Cdc25C do not respond identically to the same cell cycle checkpoints. This difference may be related to the differential localization of the proteins; Cdc25C is nuclear throughout interphase, whereas Cdc25B is nuclear in the G1 phase and cytoplasmic in the S and G2 phases. We have found that the change in subcellular localization of Cdc25B is due to nuclear export and that this is dependent on cyclin B1. Our data suggest that although both Cdc25B and Cdc25C can promote mitosis, they are likely to have distinct roles in the controlling the initiation of mitosis.