Analysis of Polo-like kinase Cdc5 in the meiosis recombination checkpoint

In meiosis, accumulation of recombination intermediates or defects in chromosome synapsis trigger checkpoint-mediated arrest in prophase I. Such 'checkpoints' are important surveillance mechanisms that ensure temporal dependence of cell cycle events. The budding yeast Polo-like kinase, Cdc5, has been identified as a key regulator of the meiosis I chromosome segregation pattern. Here we have analysed the role of Cdc5 in the recombination checkpoint and observed that Polo-like kinase is not required for checkpoint activation in yeast meiosis. Surprisingly, depletion of CDC5 in the Drad17 checkpoint-defective background resulted in nuclear fragmentation to levels even higher than that observed inDdmc1 Drad17 cells that bypass the checkpoint arrest despite accumulating DNA double-strand breaks. The spindle morphology of Cdc5-depleted cells included short, thick metaphase I spindles in mononucleate cells and disassembled spindles in binucleate and tetranucleate cells, although this phenotype does not appear to be the cause of the nuclear fragmentation. An exaggeration of chromosome synapsis defects occurred in Cdc5-depleted Drad17 cells and may contribute to the nuclear fragmentation phenotype. The analysis also uncovered a role for Cdc5 in maintaining spindle integrity in Ddmc1 Drad17 cells. Further analysis confirmed that adaptation to DNA damage does occur in meiosis and that CDC5 is required for this process. The cdc5-ad mutation that renders cells unable to adapt to DNA damage in mitosis did not affect checkpoint adaptation in meiosis, indicating that the mechanisms of checkpoint adaptation in mitosis and meiosis are not fully conserved.     

The role of Cdc25 phosphatases in cell cycle checkpoints

Summary The major driving forces in the eukaryotic cell cycle are the cyclin-dependent kinases (Cdk). Cdks can be activated through dephosphorylation of inhibitory phosphorylations catalyzed by the Cdc25 phosphatase family. In higher-eukaryotic cells, there exist three Cdc25 family members, Cdc25A, Cdc25B, and Cdc25C. While Cdc25A plays a major role at the G₁-to-S phase transition, Cdc25B and C are required for entry into mitosis. The regulation of Cdc25C is crucial for the operation of the DNA-damage checkpoint. Two protein kinases, Chk1 and Cds1, can be activated in response to DNA damage or in the presence of unreplicated DNA. Chk1 and Cds1 may phosphorylate Cdc25C to prevent entry into mitosis through inhibition of Cdc2 (Cdk1) dephosphorylation.     

Normal cell cycle and checkpoint responses in mice and cells lacking Cdc25B and Cdc25C protein phosphatases

The Cdc25 family of protein phosphatases positively regulates cell division by activating cyclin-dependent protein kinases (CDKs). In humans and rodents, there are three Cdc25 family members--denoted Cdc25A, Cdc25B, and Cdc25C--that can be distinguished based on their subcellular compartmentalizations, their abundances and/or activities throughout the cell cycle, the CDKs that they target for activation, and whether they are overexpressed in human cancers. In addition, murine forms of Cdc25 exhibit distinct patterns of expression throughout development and in adult tissues. These properties suggest that individual Cdc25 family members contribute distinct biological functions in embryonic and adult cell cycles of mammals. Interestingly, mice with Cdc25C disrupted are healthy, and cells derived from these mice exhibit normal cell cycles and checkpoint responses. Cdc25B-/- mice are also generally normal (although females are sterile), and cells derived from Cdc25B-/- mice have normal cell cycles. Here we report that mice lacking both Cdc25B and Cdc25C are obtained at the expected Mendelian ratios, indicating that Cdc25B and Cdc25C are not required for mouse development or mitotic entry. Furthermore, cell cycles, DNA damage responses, and Cdc25A regulation are normal in cells lacking Cdc25B and Cdc25C. These findings indicate that Cdc25A, or possibly other phosphatases, is able to functionally compensate for the loss of Cdc25B and Cdc25C in mice.     

TOUSLED kinase activity oscillates during the cell cycle and interacts with chromatin regulators

The TOUSLED (TSL)-like nuclear protein kinase family is highly conserved in plants and animals. tsl loss of function mutations cause pleiotropic defects in both leaf and flower development, and growth and initiation of floral organ primordia is abnormal, suggesting that basic cellular processes are affected. TSL is more highly expressed in exponentially growing Arabidopsis culture cells than in stationary, nondividing cells. While its expression remains constant throughout the cell cycle in dividing cells, TSL kinase activity is higher in enriched late G2/M-phase and G1-phase populations of Arabidopsis suspension culture cells compared to those in S-phase. tsl mutants also display an aberrant pattern and increased expression levels of the mitotic cyclin gene CycB1;1, suggesting that TSL represses CycB1;1 expression at certain times during development or that cells are delayed in mitosis. TSL interacts with and phosphorylates one of two Arabidopsis homologs of the nucleosome assembly/silencing protein Asf1 and histone H3, as in humans, and a novel plant SANT/myb-domain protein, TKI1, suggesting that TSL plays a role in chromatin metabolism.     

Polo-like kinase 3 is Golgi localized and involved in regulating Golgi fragmentation during the cell cycle

The Golgi apparatus undergoes extensive fragmentation during mitosis in animal cells. Protein kinases play a critical role during fragmentation of the Golgi apparatus. We reported here that Polo-like kinase 3 (Plk3) may be an important mediator during Golgi breakdown. Specifically, Plk3 was concentrated at the Golgi apparatus in HeLa and A549 cells during interphase. At the onset of mitosis, Plk3 signals disintegrated and redistributed in a manner similar to those of Golgi stacks. Nocodazole activated Plk3 kinase activity, correlating with redistribution of Plk3 signals and Golgi fragmentation. In addition, treatment with brefeldin A (BFA), a Golgi-specific poison, also resulted in disappearance of concentrated Plk3 signals. Plk3 interacted with giantin, a Golgi-specific protein. Expression of Plk3, but not the kinase-defective Plk3 (Plk3(K52R)), resulted in significant Golgi breakdown. Given its role in cell cycle progression, Plk3 may be a protein kinase involved in regulation of Golgi fragmentation during the cell cycle.     

Genetic dissection of mammalian Cdc7 kinase: cell cycle and developmental roles

Cdc7, originally discovered by Hartwell as a budding yeast mutant that arrests immediately before the onset of S phase, is conserved through evolution and plays essential roles in initiation of mitotic DNA replication. Inducible inactivation of Cdc7 in mouse embryonic stem cells leads to rapid cessation of DNA synthesis and the subsequent activation of checkpoint responses, resulting in p53 activation and eventually p53-mediated apoptosis. This indicates a requirement of Cdc7 kinase for ongoing replication of mammalian genomes, and loss of Cdc7 kinase presumably generates arrested replication fork signals. Cdc7-/- mice or embryonic fibroblast cells (MEFs) expressing a low level of transgene-encoded Cdc7 protein are viable but exhibit reduced body size with impaired germ cell development and decreased cell proliferation. Interestingly, these phenotypes are largely corrected by the presence of an additional copy of the transgene, resulting in increased level of Cdc7 expression. This indicates the requirement of a critical level of Cdc7 for normal cell proliferation and development of specific organs. These results from mammals will be discussed in conjunction with the pleiotropic effects of Cdc7 mutation observed in yeasts.     

Cdc25A is a novel phosphatase functioning early in the cell cycle.

The cdc25+ tyrosine phosphatase is a key mitotic inducer of the fission yeast Schizosaccharomyces pombe, controlling the timing of the initiation of mitosis. Mammals contain at least three cdc25+ homologues called cdc25A, cdc25B and cdc25C. In this study we investigate the biological function of cdc25A. Although very potent in rescuing the S.pombe cdc25 mutant, cdc25A is less structurally related to the S.pombe enzyme. Northern and Western blotting detection reveals that unlike cdc25B, cdc25C and cdc2, cdc25A is predominantly expressed in late G1. Moreover, immunodepletion of cdc25A in rat cells by microinjection of a specific antibody effectively blocks their cell cycle progression from G1 into the S phase, as determined by laser scanning single cell cytometry. These results indicate that cdc25A is not a mitotic regulator but a novel phosphatase that plays a crucial role in the start of the cell cycle. In view of its strong ability to activate cdc2 kinase and its specific expression in late G1, cdc2-related kinases functioning early in the cell cycle may be targets for this phosphatase.     

Dbf4 regulates the Cdc5 Polo-like kinase through a distinct non-canonical binding interaction

Cdc7-Dbf4 is a conserved, two-subunit kinase required for initiating eukaryotic DNA replication. Recent studies have shown that Cdc7-Dbf4 also regulates the mitotic exit network (MEN) and monopolar homolog orientation in meiosis I (Matos, J., Lipp, J. J., Bogdanova, A., Guillot, S., Okaz, E., Junqueira, M., Shevchenko, A., and Zachariae, W. (2008) Cell 135, 662-678 and Miller, C. T., Gabrielse, C., Chen, Y. C., and Weinreich, M. (2009) PLoS Genet. 5, e1000498). Both activities likely involve a Cdc7-Dbf4 interaction with Cdc5, the single Polo-like kinase in budding yeast. We previously showed that Dbf4 binds the Cdc5 polo-box domain (PBD) via an ～40-residue N-terminal sequence, which lacks a PBD consensus binding site (S(pS/pT)(P/X)), and that Dbf4 inhibits Cdc5 function during mitosis. Here we identify a non-consensus PBD binding site within Dbf4 and demonstrate that the PBD-Dbf4 interaction occurs via a distinct PBD surface from that used to bind phosphoproteins. Genetic and biochemical analysis of multiple dbf4 mutants indicate that Dbf4 inhibits Cdc5 function through direct binding. Surprisingly, mutation of invariant Cdc5 residues required for binding phosphorylated substrates has little effect on yeast viability or growth rate. Instead, cdc5 mutants defective for binding phosphoproteins exhibit enhanced resistance to microtubule disruption and an increased rate of spindle elongation. This study, therefore, details the molecular nature of a new type of PBD binding and reveals that Cdc5 targeting to phosphorylated substrates likely regulates spindle dynamics.     

Arrest of cell cycle by Amida which is phosphorylated by Cdc2 kinase

Amida was first isolated from a rat hippocampal cDNA library as an Arc-associated protein. Although previous studies have shown that Amida mRNA is predominantly expressed and developmentally regulated in rat testis and overexpression induces apoptosis, the function of Amida remains unclear. In this study, we found that overexpression of Amida inhibited cell growth. Flow cytometry analysis showed that Amida caused cell cycle inhibition in the S-phase and blocked cell cycle from entry into mitosis. Attempting to elucidate Amida effect on the cell cycle, we found that Amida was interacted with Cdc2 in mitosis and Amida's overexpression resulted in a decrease in Cdc2 kinase activity. In addition, Amida showed DNA-binding ability with DNA-affinity column chromatography. A region (aa, 76–189) between the two nuclear localization signals was found to be responsible for cell growth inhibition and DNA-binding activity, implying that DNA-binding activity may be necessary for Amida to repress cell cycle. Moreover, Amida was phosphorylated by Cdc2 kinase in vitro and Ser-180 of Amida was identified as the phosphorylation site. Furthermore, AmidaS180G (eliminate phosphorylation of Ser-180) showed stronger DNA-binding activity. Taken together, the data suggest that Amida may play an important role in cell cycle and may be partly regulated by Cdc2 kinase.     

Polo-like kinase Cdc5 promotes chiasmata formation and cosegregation of sister centromeres at meiosis I

During meiosis, two rounds of chromosome segregation occur after a single round of DNA replication, producing haploid progeny from diploid progenitors. Three innovations in chromosome behaviour during meiosis I accomplish this unique division. First, crossovers between maternal and paternal sister chromatids (detected cytologically as chiasmata) bind replicated maternal and paternal chromosomes together. Second, sister kinetochores attach to microtubules from the same pole (mono-polar orientation), causing maternal and paternal centromere pairs (and not sister chromatids) to be separated. Third, sister chromatid cohesion near centromeres is preserved at anaphase I when cohesion along chromosome arms is destroyed. The finding that destruction of mitotic cohesion is regulated by Polo-like kinases prompted us to investigate the meiotic role of the yeast Polo-like kinase Cdc5. We show here that cells lacking Cdc5 synapse homologues and initiate recombination normally, but fail to efficiently resolve recombination intermediates as crossovers. They also fail to properly localize the Lrs4 (ref. 3) and Mam1 (ref. 4) monopolin proteins, resulting in bipolar orientation of sister kinetochores. Cdc5 is thus required both for the formation of chiasmata and for cosegregation of sister centromeres at meiosis I.     

The cyclin-dependent kinase Cdc28p regulates distinct modes of Cdc6p proteolysis during the budding yeast cell cycle

BACKGROUND: Cdc28p, the major cyclin-dependent kinase in budding yeast, prevents re-replication within each cell cycle by preventing the reassembly of Cdc6p-dependent pre-replicative complexes (pre-RCs) once origins have fired. Cdc6p is a rapidly degraded protein that must be synthesised in each cell cycle and is present only during the G1 phase. RESULTS: We found that, at different times in the cell cycle, there are distinct modes of Cdc6p proteolysis. Before Start, Cdc6p proteolysis did not require either the anaphase-promoting complex (APC/C) or the SCF complex, which mediate the major cell cycle regulated ubiquitination pathways, nor did it require Cdc28p activity or any of the potential Cdc28p phosphorylation sites in Cdc6p. In fact, the activation of B cyclin (Clb)-Cdc28p kinase inactivated this pathway of Cdc6p degradation later in the cell cycle. Activation of the G1 cyclins (Clns) caused Cdc6p degradation to become extremely rapid. This degradation required the SCF(CDC4) and Cdc28p consensus sites in Cdc6p, but did not require Clb5 and Clb6. Later in the cell cycle, SCF(CDC4)-dependent Cdc6p proteolysis remained active but became less rapid. CONCLUSIONS: Levels of Cdc6p are regulated in several ways by the Cdc28p cyclin-dependent kinase. The Cln-dependent elimination of Cdc6p, which does not require the S-phase-promoting cyclins Clb5 and Clb6, suggests that the ability to assemble pre-RCs is lost before, not concomitant with, origin firing.     

The cell cycle and development: redundant roles of cell cycle regulators

The existence of families of cell cycle regulators reflects the need by a developing organism to precisely control proliferation of its cells and also suggests that family members may play redundant roles. Recent advances have shown redundancy to be a theme in development.     

Polo-like kinases: positive regulators of cell division from start to finish

Present in organisms ranging from yeast to man, homologues of the Drosophila Polo kinase control multiple stages of cell division. At the onset of mitosis, Polo-like kinases (Plks) function in centrosome maturation and bipolar spindle formation, and they contribute to the activation of cyclin-dependent kinase (Cdk)1—cyclin B. Subsequently, they are required for the inactivation of Cdk1 and exit from mitosis. In the absence of Plk function, mitotic cyclins fail to be destroyed, indicating that Plks are important regulators of the anaphase-promoting complex/cyclosome (APC/C), a key component of the ubiquitin-dependent proteolytic degradation pathway. Finally, recent evidence implicates Plks in the temporal and spatial coordination of cytokinesis.     

A casein kinase 1 and PAR proteins regulate asymmetry of a PIP(2) synthesis enzyme for asymmetric spindle positioning

Spindle positioning is an essential feature of asymmetric cell division. The conserved PAR proteins together with heterotrimeric G proteins control spindle positioning in animal cells, but how these are linked is not known. In C. elegans, PAR protein activity leads to asymmetric spindle placement through cortical asymmetry of Galpha regulators GPR-1/2. Here, we establish that the casein kinase 1 gamma CSNK-1 and a PIP(2) synthesis enzyme (PPK-1) transduce PAR polarity to asymmetric Galpha regulation. PPK-1 is posteriorly enriched in the one-celled embryo through PAR and CSNK-1 activities. Loss of CSNK-1 causes uniformly high PPK-1 levels, high symmetric cortical levels of GPR-1/2 and LIN-5, and increased spindle pulling forces. In contrast, knockdown of ppk-1 leads to low GPR-1/2 levels and decreased spindle forces. Furthermore, loss of CSNK-1 leads to increased levels of PIP(2). We propose that asymmetric generation of PIP(2) by PPK-1 directs the posterior enrichment of GPR-1/2 and LIN-5, leading to posterior spindle displacement.     

RNA interference pinpoints regulators of cell size and the cell cycle

Cell-based genome-wide RNA interference screens are being used to address an increasingly broad spectrum of biological questions. In one recent screen, Drosophila cell cultures treated with double-stranded RNA were analyzed by flow cytometry, providing a wealth of new information and identifying 488 regulators of the cell cycle, cell size, and cell death.     

Ability of human CDC25B phosphatase splice variants to replace the function of the fission yeast Cdc25 cell cycle regulator

CDC25 phosphatases are essential and evolutionary-conserved actors of the eukaryotic cell cycle control. To examine and compare the properties of three splicing variants of human CDC25B, recombinant fission yeast strains expressing the human proteins in place of the endogenous Cdc25 were generated and characterized. We report, that the three CDC25B variants: (i) efficiently replace the yeast counterpart in vegetative growth, (ii) partly restore the gamma and UV radiation DNA damage-activated checkpoint, (iii) fail to restore the DNA replication checkpoint activated by hydroxyurea. Although these yeast strains do not reveal the specific functions of the human CDC25B variants, they should provide useful screening tools for the identification of new cell cycle regulators and pharmacological inhibitors of CDC25 phosphatase.     

HSP25 inhibits protein kinase C delta-mediated cell death through direct interaction

Heat shock protein 25 (HSP25) interferes negatively with apoptosis through several pathways that involve its direct interaction with cytochrome c or Akt. Here we show that HSP25 inhibits protein kinase C (PKC) delta-mediated cell death through direct interaction. HSP25 binds to kinase-active PKCdelta to inhibit its kinase activity and translocation to the membrane, which results in reduced cell death. Deletion constructs of HSP25 and PKCdelta identified amino acids 90-103 of HSP25 and the C-terminal V5 region of PKCdelta as binding sites. In addition, the interaction between HSP25 and PKCdelta induced HSP25 phosphorylation at Ser-15 and Ser-86, and these phosphorylations permitted HSP25 release from PKCdelta. Based on these observations, we propose that after PKCdelta activation, HSP25 binds to the exposed V5 region of PKCdelta. This novel function of HSP25 accounts for its cytoprotective properties via the inhibition of PKCdelta and the enhancement of HSP25 phosphorylation.     

Screening of cell cycle fusion proteins to identify kinase signaling networks

Kinase signaling networks are well-established mediators of cell cycle transitions. However, how kinases interact with the ubiquitin proteasome system (UPS) to elicit protein turnover is not fully understood. We sought a means of identifying kinase-substrate interactions to better understand signaling pathways controlling protein degradation. Our prior studies used a luciferase fusion protein to uncover kinase networks controlling protein turnover. In this study, we utilized a similar approach to identify pathways controlling the cell cycle protein p27^Kip1. We generated a p27^Kip1-luciferase fusion and expressed it in cells incubated with compounds from a library of pharmacologically active compounds. We then compared the relative effects of the compounds on p27^Kip1-luciferase fusion stabilization. This was combined with in silico kinome profiling to identify potential kinases inhibited by each compound. This approach effectively uncovered known kinases regulating p27^Kip1 turnover. Collectively, our studies suggest that this parallel screening approach is robust and can be applied to fully understand kinase-ubiquitin pathway interactions.