Bub1 regulates chromosome segregation in a kinetochore-independent manner

The kinetochore-bound protein kinase Bub1 performs two crucial functions during mitosis: it is essential for spindle checkpoint signaling and for correct chromosome alignment. Interestingly, Bub1 mutations are found in cancer tissues and cancer cell lines. Using an isogenic RNA interference complementation system in transformed HeLa cells and untransformed RPE1 cells, we investigate the effect of structural Bub1 mutants on chromosome segregation. We demonstrate that Bub1 regulates mitosis through the same mechanisms in both cell lines, suggesting a common regulatory network. Surprisingly, Bub1 can regulate chromosome segregation in a kinetochore-independent manner, albeit at lower efficiency. Its kinase activity is crucial for chromosome alignment but plays only a minor role in spindle checkpoint signaling. We also identify a novel conserved motif within Bub1 (amino acids 458–476) that is essential for spindle checkpoint signaling but does not regulate chromosome alignment, and we show that several cancer-related Bub1 mutants impair chromosome segregation, suggesting a possible link to tumorigenesis.     

The Aurora/Ipl1p kinase family: regulators of chromosome segregation and cytokinesis.

Members of the Aurora/Ipl1p family of mitotically regulated serine/threonine kinases are emerging as key regulators of chromosome segregation and cytokinesis. Proper chromosome segregation and cytokinesis ensure that each daughter cell receives the full complement of genetic material. Defects in these processes can lead to aneuploidy and the propagation of genetic abnormalities. This review discusses the Aurora/Ipl1p kinases in terms of their protein structure and proposed function in mitotic cells and also the potential role of aurora2 in human cancer.     

Incenp and an aurora-like kinase form a complex essential for chromosome segregation and efficient completion of cytokinesis

BACKGROUND: In animal cells, cytokinesis begins shortly after the sister chromatids move to the spindle poles. The inner centromere protein (Incenp)has been implicated in both chromosome segregation and cytokinesis, but it is not known exactly how it mediates these two distinct processes. RESULTS: We identified two Caenorhabditis elegans proteins, ICP-1 and ICP-2, with significant homology in their carboxyl termini to the corresponding region of vertebrate Incenp. Embryos depleted of ICP-1 by RNA-mediated interference had defects in both chromosome segregation and cytokinesis. Depletion of the Aurora-like kinase AIR-2 resulted in a similar phenotype. The carboxy-terminal region of Incenp is also homologous to that in Sli15p, a budding yeast protein that functions with the yeast Aurora kinase Ipl1p. ICP-1 bound C. elegans AIR-2 in vitro, and the corresponding mammalian orthologs Incenp and AIRK2 could be co-immunoprecipitated from cell extracts. A significant fraction of embryos depleted of ICP-1 and AIR-2 completed one cell division over the course of several cell cycles. ICP-1 promoted the stable localization of ZEN-4 (also known as CeMKLP1), a kinesin-like protein required for central spindle assembly. CONCLUSIONS: ICP-1 and AIR-2 are part of a complex that is essential for chromosome segregation and for efficient completion of cytokinesis. We propose that this complex acts by promoting dissolution of sister chromatid cohesion and the assembly of the central spindle.     

Phosphatase inhibitor-2 balances protein phosphatase 1 and aurora B kinase for chromosome segregation and cytokinesis in human retinal epithelial cells

Mitosis in Saccharomyces cerevisiae depends on IPL1 kinase, which genetically interacts with GLC8. The metazoan homologue of GLC8 is inhibitor-2 (I-2), but its function is not understood. We found endogenous and ectopic I-2 localized to the spindle, midzone, and midbody of mitotic human epithelial ARPE-19 cells. Knockdown of I-2 by RNA interference produced multinucleated cells, with supernumerary centrosomes, multipolar spindles and lagging chromosomes during anaphase. These defects did not involve changes in levels of protein phosphatase-1 (PP1), and the multinuclear phenotype was rescued by overexpression of I-2. Appearance of multiple nuclei and supernumerary centrosomes required progression through the cell cycle and I-2 knockdown cells failed cytokinesis, as observed by time-lapse microscopy. Inhibition of Aurora B by hesperadin produced multinucleated cells and reduced H3S10 phosphorylation. I-2 knockdown enhanced this latter effect. Partial knockdown of PP1Cα prevented multiple nuclei caused by either knockdown of I-2 or treatment with hesperadin. Expression of enhanced green fluorescent protein-I-2 or hemagglutinin-I-2 made cells resistant to hesperadin. We propose that I-2 acts to enhance Aurora B by inhibiting specific PP1 holoenzymes that dephosphorylate Aurora B substrates necessary for chromosome segregation and cytokinesis. Conserved together throughout eukaryotic evolution, I-2, PP1 and Aurora B function interdependently during mitosis.     

Role of chromosomal passenger complex in chromosome segregation and cytokinesis

Chromosomal passenger proteins associate with chromosomes early in mitosis and transfer to the spindle at ana/telophase. Recent results show that aurora B/AIM-1 (aurora and Ipl1-like midbody-associated protein kinase), which is responsible for mitotic histone H3 phosphorylation, INCENP (Inner Centromere protein) and Survivin/BIR are in a macromolecular complex as novel chromosomal passenger proteins. Aurora B/AIM-1 can bind to Survivin and the C-terminal region of INCENP, respectively, and colocalizes with both proteins to the centromeres, midzone and midbody. Disruption of either aurora B/AIM-1 or INCENP function leads to sever defects in chromosome segregation and cytokinesis. Moreover, the formation of the central spindle through anaphase to cytokinesis is also disrupted severely. These data suggest that chromosomal passenger complex is required for proper chromosome segregation by phosphorylating histone H3, and cytokinesis by ensuring the correct assembly of the midzone and midbody microtubule. Chromosomal passenger protein complex may couple chromosome segregation with cytokinesis.     

The Schizosaccharomyces pombe aurora-related kinase Ark1 interacts with the inner centromere protein Pic1 and mediates chromosome segregation and cytokinesis

The chromosomal passenger proteins aurora-B, survivin, and inner centromere protein (INCENP) have been implicated in coordinating chromosome segregation with cell division. This work describes the interplay between aurora, survivin, and INCENP orthologs in the fission yeast Schizosaccharomyces pombe and defines their roles in regulating chromosome segregation and cytokinesis. We describe the cloning and characterization of the aurora-related kinase gene ark1(+), demonstrating that it is an essential gene required for sister chromatid segregation. Cells lacking Ark1p exhibit the cut phenotype, DNA fragmentation, and other defects in chromosome segregation. Overexpression of a kinase-defective version of Ark1, Ark1-K147R, inhibits cytokinesis, with cells exhibiting an elongated, multiseptate phenotype. Ark1p interacts physically and/or genetically with the survivin and INCENP orthologs Bir1p and Pic1p. We identified Pic1p in a two-hybrid screen for Ark1-K147R interacting partners and went on to map domains in both proteins that mediate their binding. Pic1p residues 925-972 are necessary and sufficient for Ark1p binding, which occurs through the kinase domain. As with Ark1-K147R, overexpression of Ark1p-binding fragments of Pic1p leads to multiseptate phenotypes. We also provide evidence that the dominant-negative effect of Ark1-K147R requires Pic1p binding, indicating that the formation of Ark1p-Pic1p complexes is required for the execution of cytokinesis.     

Inactivation of Cdk1/Cyclin B in metaphase-arrested mouse FT210 cells induces exit from mitosis without chromosome segregation or cytokinesis and allows passage through another cell cycle

It is well known that inactivation of Cdk1/Cyclin B is required for cells to exit mitosis. The work reported here tests the hypothesis that Cdk1/Cyclin B inactivation is not only necessary but also sufficient to induce mitotic exit and reestablishment of the interphase state. This hypothesis predicts that inactivation of Cdk1 in metaphase-arrested cells will induce the M to G1-phase transition. It is shown that when mouse FT210 cells (in which Cdk1 is temperature-sensitive) are arrested in metaphase and then shifted to their non-permissive temperature, they rapidly exit mitosis as evidenced by reassembly of interphase nuclei, decondensation of chromosomes, and dephosphorylation of histones H1 and H3. The resulting interphase cells are functionally normal as judged by their ability to progress through another cell cycle. However, they have double the normal number of chromosomes because they previously bypassed anaphase, chromosome segregation, and cytokinesis. These results, taken together with other observations in the literature, strongly suggest that in mammalian cells, inactivation of Cdk1/cyclin B is the trigger for mitotic exit and reestablishment of the interphase state.     

sepB: an Aspergillus nidulans gene involved in chromosome segregation and the initiation of cytokinesis.

In Aspergillus nidulans conidia, cytokinesis (septation) is delayed until three rounds of nuclear division have been completed. This has permitted the identification of essential genes that are involved in the coordination of cytokinesis with nuclear division. Conditional mutations in the sepB gene block septation but allow germinating spores to complete the first three rounds of nuclear division at restrictive temperature. sepB3 mutants demonstrate transient delays in M-phase, accumulate aneuploid nuclei and show defects in chromosome segregation. Molecular analysis of the sepB gene reveals that it is essential and possesses limited similarity to the CTF4 gene of Saccharomyces cerevisiae. Using temperature-shift analysis we show that sepB is required after the first nuclear division but before the onset of cytokinesis. A failure to execute the sepB function results in a block to nuclear division and leads to cell death at a time when wild-type cells would be undergoing cytokinesis. Finally, we demonstrate that sepB is also required for the uninucleate cell divisions of developing conidiophores. Our results suggest that sepB3 mutants accumulate specific nuclear defects that do not arrest mitosis, but block the initiation of septum formation. Thus, proper chromosome segregation and a functional sepB gene are required to initiate cytokinesis.     

Conditional mutations in gamma-tubulin reveal its involvement in chromosome segregation and cytokinesis

gamma-Tubulin is a conserved essential protein required for assembly and function of the mitotic spindle in humans and yeast. For example, human gamma-tubulin can replace the gamma-tubulin gene in Schizosaccharomyces pombe. To understand the structural/functional domains of gamma-tubulin, we performed a systematic alanine-scanning mutagenesis of human gamma-tubulin (TUBG1) and studied phenotypes of each mutant allele in S. pombe. Our screen, both in the presence and absence of the endogenous S. pombe gamma-tubulin, resulted in 11 lethal mutations and 12 cold-sensitive mutations. Based on structural mapping onto a homology model of human gamma-tubulin generated by free energy minimization, all deleterious mutations are found in residues predicted to be located on the surface, some in positions to interact with alpha- and/or beta-tubulins in the microtubule lattice. As expected, one class of tubg1 mutations has either an abnormal assembly or loss of the mitotic spindle. Surprisingly, a subset of mutants with abnormal spindles does not arrest in M phase but proceeds through anaphase followed by abnormal cytokinesis. These studies reveal that in addition to its previously appreciated role in spindle microtubule nucleation, gamma-tubulin is involved in the coordination of postmetaphase events, anaphase, and cytokinesis.     

Cdc37 is essential for chromosome segregation and cytokinesis in higher eukaryotes

Cdc37 has been shown to be required for the activity and stability of protein kinases that regulate different stages of cell cycle progression. However, little is known so far regarding interactions of Cdc37 with kinases that play a role in cell division. Here we show that the loss of function of Cdc37 in Drosophila leads to defects in mitosis and male meiosis, and that these phenotypes closely resemble those brought about by the inactivation of Aurora B. We provide evidence that Aurora B interacts with and requires the Cdc37/Hsp90 complex for its stability. We conclude that the Cdc37/Hsp90 complex modulates the function of Aurora B and that most of the phenotypes brought about by the loss of Cdc37 function can be explained by the inactivation of this kinase. These observations substantiate the role of Cdc37 as an upstream regulatory element of key cell cycle kinases.     

The nuclear protein p34SEI-1 regulates the kinase activity of cyclin-dependent kinase 4 in a concentration-dependent manner

Previous studies have shown that p34(SEI-1), also known as TRIP-Br1, is involved in cell cycle regulations by interacting with a number of important proteins including CDK4. However, the detailed mechanism and structural basis of the interaction remains to be determined. We report the use of in vitro studies to address these problems. First, it was shown that p34(SEI-1) binds to CDK4 directly, and the binding does not compete directly with p16. In the presence of p16, a quaternary complex is formed between p34(SEI-1), CDK4, cyclin D2, and p16. Second, it was found that p34(SEI-1) activates the kinase activity of CDK4 at lower concentrations (reaching the maximum at 500 nM) but inhibits the same activity at higher concentrations, implying that p34(SEI-1)-mediated CDK4 activation is dose-dependent. Again, the effects of p34(SEI-1) and p16 are independent of each other. Third, it was shown that p34(SEI-1) possesses a LexA-mediated transactivation activity. Finally, a set of truncation mutants were used to dissect the structural elements responsible for the different functions of p34(SEI-1). The results indicate that the fragment 30-160 can bind, activate, and inhibit CDK4; the fragment 30-132 can bind and activate but does not inhibit CDK4, while the fragment 30-88 cannot bind, activate, or inhibit but retains the LexA-mediated transactivation activity.     

Inhibition of c-Jun N-terminal kinase 2 expression suppresses growth and induces apoptosis of human tumor cells in a p53-dependent manner

c-Jun N-terminal kinase (JNK) plays a critical role in coordinating the cellular response to stress and has been implicated in regulating cell growth and transformation. To investigate the growth-regulatory functions of JNK1 and JNK2, we used specific antisense oligonucleotides (AS) to inhibit their expression. A survey of several human tumor cell lines revealed that JNKAS treatment markedly inhibited the growth of cells with mutant p53 status but not that of cells with normal p53 function. To further examine the influence of p53 on cell sensitivity to JNKAS treatment, we compared the responsiveness of RKO, MCF-7, and HCT116 cells with normal p53 function to that of RKO E6, MCF-7 E6, and HCT116 p53(-/-), which were rendered p53 deficient by different methods. Inhibition of JNK2 (and to a lesser extent JNK1) expression dramatically reduced the growth of p53-deficient cells but not that of their normal counterparts. JNK2AS-induced growth inhibition was correlated with significant apoptosis. JNK2AS treatment induced the expression of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1) in parental MCF-7, RKO, and HCT116 cells but not in the p53-deficient derivatives. That p21(Cip1/Waf1) expression contributes to the survival of JNK2AS-treated cells was supported by additional experiments demonstrating that p21(Cip1/Waf1) deficiency in HCT116 cells also results in heightened sensitivity to JNKAS treatment. Our results indicate that perturbation of JNK2 expression adversely affects the growth of otherwise nonstressed cells. p53 and its downstream effector p21(Cip1/Waf1) are important in counteracting these detrimental effects and promoting cell survival.     

Inhibition of casein kinase 1-epsilon induces cancer-cell-selective,PERIOD2-dependent growth arrest

Background  

Kinases are under extensive investigation as targets for drug development. Discovering novel kinases whose inhibition induces cancer-cell-selective lethality would be of value. Recent advances in RNA interference have enabled the realization of this goal.     

Inhibition of neddylation induces mitotic defects and alters MKLP1 accumulation at the midbody during cytokinesis

The cullin-RING E3 ubiquitin ligases (CRLs) play crucial roles in modulating the stability of proteins in the cell and are, in turn, regulated by post-translational modification by the ubiquitin-like (Ubl) protein NEDD8. This process, termed neddylation, is reversible through the action of the COP9 signalosome (CSN); a multi-subunit metalloprotease conserved among eukaryotes that plays direct or indirect roles in DNA repair, cell signaling and cell cycle regulation in part through modulating the activity of the CRLs. Previously, inhibition of CRL neddylation by MLN4924, a small molecule inhibitor of the NEDD8-activating enzyme 1 (NAE1), was shown to induce interphase cell cycle arrest and cell death. Using fixed and living cell microscopy, we re-evaluated the cell cycle effects of inhibition of neddylation by MLN4924 in both asynchronous and mitotic cell populations. Consistent with previous studies, treatment of asynchronous cells with MLN4924 increased CDT1 expression levels, induced G2 arrest and increased nuclear size. However, in synchronized cells treated in mitosis, mitotic defects were observed including lagging chromosomes and binucleated daughter cells. Consistent with neddylation and deneddylation playing a role in cytokinesis, NEDD8, as well as subunits of the CSN, could be localized at the midbody and cleavage furrow. Finally, treatment of mitotic cells with MLN4924 induced the premature accumulation of MKLP1 at the cleavage furrow, a key regulator of cytokinesis, which was concomitant with increased abscission delay and failure. Thus, these studies uncover an uncharacterized mitotic effect of MLN4924 on MKLP1 accumulation at the midbody and support a role for neddylation during cytokinesis.

Abbreviations: CSN, COP9 Signalosome; MKLP1, mitotic kinesin-like protein 1; NEDD8, Neural precursor cell Expressed, Developmentally Down-regulated 8.     

Extracellular alpha-synuclein induces calpain-dependent overactivation of cyclin-dependent kinase 5 in vitro

Extracellular alpha-synuclein (ASN) could be involved in the pathomechanism of Parkinson’s disease (PD) via disturbances of calcium homeostasis, activation of nitric oxide synthase and oxidative/nitrosative stress. In this study we analyzed the role of cyclin-dependent kinase 5 (Cdk5) in the molecular mechanism(s) of ASN toxicity.     

The BRCT domains of ECT2 have distinct functions during cytokinesis

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Centrin1 is required for organelle segregation and cytokinesis in Trypanosoma brucei

Centrin is a calcium-binding centrosome/basal body-associated protein involved in duplication and segregation of these organelles in eukaryotes. We had shown that disruption of one of the centrin genes (centrin1) in Leishmania amastigotes resulted in failure of both basal body duplication and cytokinesis. Here, we undertook to define the role of centrin1 (TbCen1) in the duplication and segregation of basal body and its associated organelles kinetoplast and Golgi, as well as its role in cytokinesis of the procyclic form of Trypanosoma brucei by depleting its protein using RNA inhibition methodology. TbCen1-depleted cells showed significant reduction in growth compared with control cells. Morphological analysis of these cells showed they were large and pleomorphic with multiple detached flagella. Both immunofluorescence assays using organelle-specific antibodies and electron microscopic analysis showed that TbCen1-deficient cells contained multiple basal bodies, kinetoplasts, Golgi, and nuclei. These multiple organelles were, however, closely clustered together, indicating duplication without segregation in the absence of centrin. This failure in organelle segregation may be the likely cause of inhibition of cytokinesis, suggesting for the first time a new and unique role for centrin in the segregation of organelles without affecting their multiplication in the procyclic form of T. brucei.