Dissertations / Theses on the topic 'Polo kinases/Cdc5'

In budding yeast, progression through anaphase and exit from mitosis are controlled by the conserved protein phosphatase Cdc14. The activity of Cdc14 is regulated in space and time by changes in its subcellular localization. For most of the cell cycle up to metaphase, the phosphatase is sequestered in the nucleolus, by binding to a competitive inhibitor called Cfi1 (also known as Net1) (Shou et al., 1999; Straight et al., 1999; Visintin et al., 1999). During anaphase, Cdc14 is released from its inhibitor by the sequential activation of two signaling cascades, the Cdc Fourteen Early Anaphase Release (FEAR) network and the Mitotic Exit Network (MEN). Once released Cdc14 spreads throughout the nucleus and the cytoplasm, where it reaches its targets and promotes progression through and exit from mitosis (Pereira et al., 2002; Shou et al., 1999; Stegmeier et al., 2002; Visintin et al., 1999; Yoshida et al., 2002). Several in vivo and in vitro observations suggest that phosphorylation of Cdc14 and/or Cfi1 is responsible for the dissociation of Cdc14 from its inhibitor. Three kinases have been implicated in the process: the polo-like kinase Cdc5, the Clb2-Cdk complex and the MEN kinase Dbf2 (Azzam et al., 2004; Geymonat et al., 2003; Hu and Elledge, 2002; Hu et al., 2001; Mohl et al., 2009; Pereira et al., 2002; Queralt et al., 2006; Stegmeier et al., 2002; Visintin et al., 2003; Yoshida and Toh-e, 2002). The aim of my project was to assess the contribution of the above-mentioned kinases and to identify the molecular mechanisms by which these kinases mediate the release of Cdc14 from its inhibitor. By modulating the kinases of interest alone or in mutual combination we found that Cdc14 is released from the nucleolus by the combined activity of two kinases, Cdc5 always and either Clb-Cdks or Dbf2. Once active, Cdc14 triggers a negative feedback loop that, in the presence of stable levels of mitotic cyclins, generates periodic cycles of Cdc14 release and sequestration. Similar phenotypes have been described for yeast bud formation and centrosome duplication. A common theme emerges where events that must happen only once per cycle, although intrinsically capable of oscillations, are limited to one occurrence by their coupling with the cyclin-Cdk cell cycle engine.

The integrity of genomic DNA is continuously jeopardized through of environmental stresses such as UV light, ionizing radiations and various chemicals in addition to cellular byproducts such as reactive oxygen species. Furthermore, structural or chemical hindrances also affect the basic cellular processes (replication, transcription and translation) compromising genome stability. All the eukaryotic cells have thus evolved mechanisms to detect such genomic lesions and activate a surveillance mechanisms termed as checkpoint activation to arrest cell cycle, which in term provide time to repair the lesion using a suitable pathway to maintain genome stability. The resumption of cell cycle after the repair is also an important and finely regulated mechanisms. Indeed, resumption of cell cycle in case of faulty/un-repaired damage compromises genome integrity and may lead to cancer. In this thesis, I studied the role of Polo-kinase Cdc5 and DNA repair scaffold complex-Slx-Rtt107, specifically in response to one of the most deleterious lesion, DNA double strand break (DSB) in budding yeast Saccharomyces cerevisiae. The human counterpart Polo-like kinase 1 is overexpressed in many cancers, while Slx4/FANCP is one of the proteins involved in Fanconi anemia repair pathway. In first part, we characterized the role of phosphorylation of Threonine 238 in the activation loop of the Cdc5 kinase domain in unperturbed cell cycle and in response to repairable and unrepairable DSB. Using alanine/ aspartic acid mutagenesis and genetic approaches we delineated the requirement of T238 phosphorylation of Cdc5. Interestingly, we discovered that absence of T238 phosphorylation of Cdc5, even though doesn’t affect the normal cell cycle, affects kinase activity and leads to defect in checkpoint adaptation and recovery after one DSB. Importantly, we also found that cdc5-T238A cells also have altered genome stability, assessed by using multiple genetic approaches. In second part, we characterized the role of Slx4-Rtt107 complex in modulating the level of checkpoint signalling and initial processing of DSB. Indeed in the absence of functional Slx4-Rtt107 complex, we found slower processing of DSB and hyper-activated checkpoint signalling which is due to increased binding of checkpoint adaptor protein Rad9 at the lesion. Importantly, this hyper-activated checkpoint has consequent effect on cell cycle resumption and proliferation in response to various DNA damaging agents.

Proper chromosome segregation requires an orderly sequence of events, whereby spindle elongation follows the dissolution of sister chromatid linkages. Chromosome segregation starts at the onset of anaphase when the separase triggers the cleavage of cohesin, a protein complex that holds sister chromatids together. Next, chromatids are segregated into the daughter cells by the pulling force of the mitotic spindle. The mitotic spindle is a sophisticated and complex machinery built of microtubules, microtubule associated proteins and motor proteins. Despite the fundamental role of the mitotic spindle, the molecular mechanisms underlying its regulation remain elusive. Proper spindle function requires that microtubule dynamics are stabilized at anaphase. This change in microtubule dynamics is perceived as dictated by a shift in the balance of kinase and phosphatase activities in favor of the phosphatases. The finding that cells simultaneously lacking the polo-like kinase Cdc5 and the phosphatase Cdc14 cannot progress through anaphase albeit having cleaved cohesin due to defects in spindle elongation, challenges the view of mitotic exit as a time for protein dephosphorylation. The aim of my work is to understand the molecular mechanism by which the two proteins contribute to anaphase spindle elongation, with a particular focus on the role of Cdc5. We identified the kinesin 5 motor protein Cin8 as a key target of the “Cdc14-Cdc5” spindle elongation pathway. We show that besides being dephosphorylated by Cdc14, Cin8 is also phosphorylated by Cdc5 on residues S409 and S441, and that this phosphorylation is crucial for the function of the kinesin in anaphase spindle elongation, likely because of the impact it has on the interaction between Cin8 and microtubules. Since these residues, S409 and S441, are located within a highly conserved stretch of amino acids, it will be interesting to test whether this regulation is conserved in other vertebrates as well. The finding that Cin8 is simultaneously a substrate of a kinase and a phosphatase sheds light on the complexity of mitotic exit regulation and is in complete agreement with recent data showing that approximately equal numbers of phosphosites are phosphorylated and dephosphorylated during mitotic progression and exit. Since it appears that phosphorylation and dephosphorylation events are equally important to the point that kinases and phosphatases cooperate to regulate the same substrates, the view of mitotic exit as the realm of phosphatases is dismantled and the continuous need for single molecule studies in addition to global analyses investigation is put forward.

During mitosis the newly replicated genetic material, organized in sister chromatids, is equally subdivided into the daughter cells through a fine-regulated process called chromosome segregation. Sister chromatids are held together and identified as sisters by cohesin. At the metaphase-to-anaphase transition, when all chromatids are correctly attached to the spindle, cohesin is cleaved and chromosome segregation initiates. Beside cohesin, all linkages between sister chromatids need to be removed to allow for their complete separation. Additional linkages include DNA linkages (or sister chromatid intertwines, SCIs), such as recombination intermediates and DNA catenanes. In Saccharomyces cerevisiae a mutant that lacks the activities of the Polo-like kinase Cdc5 and the phosphatase Cdc14, two major mitotic regulators, has been identified that proved to be particularly suitable for studying SCIs that persist in mitosis. The cdc5 cdc14 double mutant arrests with short and stable mitotic spindles and unseparated nuclei, despite having cleaved cohesin. In addition to having a spindle elongation defect, these cells are also impaired in the resolution of cohesin-independent linkages between chromatids. We found that these linkages mostly consist of DNA catenanes, that persist in cdc5 cdc14 cells at their terminal arrest and that are sufficient to counteract spindle elongation. Our results suggest that Cdc5 is required for their resolution. This finding, together with the knowledge that Cdc5 promotes Cdc14 activation and that both proteins are essential for spindle elongation and mitotic exit, allows us to speculate that they coordinate different aspects of chromosome segregation to guarantee genome integrity throughout mitosis.

Meiotic recombination is initiated by self-inflicted DNA breaks and primarily involves homologous chromosomes, whereas mitotic recombination involves sister chromatids. Whilst the mitotic recombinase Rad51 exists during meiosis, its activity is suppressed in favour of the meiosis-specific recombinase, Dmc1, thus establishing a meiosis-specific mode of homologous recombination (HR). A key contributor to the suppression of Rad51 activity is the synaptonemal complex (SC), a meiosis-specific chromosomal structure that adheres homologous chromosomes along their entire lengths. Here, in budding yeast, we show that two major cell cycle kinases, Dbf4-dependent Cdc7 kinase (DDK) and Polo-kinase (Cdc5), collaborate to link the mode change of HR to the meiotic cell cycle by. This regulation of HR is through the SC. During prophase I, DDK is shown to maintain SC integrity and thus inhibition of Rad51. Cdc5, which is produced during the prophase I/metaphase I transition, interacts with DDK to cooperatively destroy the SC and remove Rad51 inhibition. By enhancing the interaction between DDK and Cdc5 or depleting DDK at late prophase I, meiotic DNA breaks are repaired even in the absence of Dmc1 by utilising Rad51. We propose that the interplay between DDK and Polo-kinase reactivates mitotic HR mechanisms to ensure complete repair of DNA breaks before meiotic chromosomem segregation.

During mitosis, the newly duplicated genetic material, organized in pairs of sister chromatids, is distributed between the daughter cells by the spindle machinery, in a process called chromosome segregation. Errors in this process can compromise genome integrity. Faithful chromosome segregation requires the removal of all sorts of cohesion between sister chromatids. Although the main contributors of sister chromatid cohesion are cohesin complexes, which are cleaved at anaphase onset, another source of cohesion is represented by DNA linkages, also called Sister Chromatid Intertwines (SCIs). These linkages comprise unreplicated segments, recombination intermediates, and double-stranded catenanes. If not properly removed, SCIs can break during cell division causing DNA damage and jeopardizing genome stability. Although most DNA linkages are removed before mitosis, their complete resolution only occurs concomitantly with chromosome segregation, in a process whose regulation is still poorly understood. In this thesis, to investigate the mechanisms of SCI resolution during mitosis, we exploited the unique phenotype of S. cerevisiae cells lacking the activities of the polo-like kinase Cdc5 and the Cdk-counteracting phosphatase Cdc14. These cells arrest after cohesin cleavage, with short bipolar spindles and undivided nuclei, because impaired in spindle elongation. Evidence suggests that cdc5 cdc14 cells are also impaired in sister chromatid separation, due to the presence of unresolved SCIs, and previous work in our laboratory revealed that these linkages mainly consist of DNA catenanes. Here, we found that both Cdc14 and Cdc5 contribute to the resolution of DNA linkages, with different functions. Cdc14 is mainly involved in nucleolar segregation and processing of recombination intermediates, while Cdc5 seems to act through a more generalized mechanism and promote the removal of DNA catenanes. At the molecular level, Cdc14 acts through its known substrate Yen1. On the other hand, we found that Cdc5 controls post-translational modification of the decatenating enzyme Top2 during mitosis, particularly conjugation with small ubiquitin-like modifier (SUMO) and, possibly, also phosphorylation. The polo-like kinase is known to inactivate the SUMO protease Ulp2 in metaphase, thus increasing SUMOylation of Ulp2 substrates, like Top2. Since the decatenation defect of cdc5 cells correlates with a dysregulation of the SUMO pathway and this pathway is known to regulate sister chromatid cohesion, we speculate that the hyperactivation of Ulp2 may be the reason behind the sister chromatid separation defect of cdc5 cells. Taken together, our findings integrate the current knowledge of the mechanisms of sister chromatid separation and allow us to propose a model that foresees Cdc5 and Cdc14 coordinating cohesin cleavage and spindle elongation with the removal of DNA intertwines.

Checkpoints are surveillance mechanisms that constitute a barrier to oncogenesis by preserving genome integrity. Loss of checkpoint function is an early event in tumorigenesis. Polo kinases (Plks) are fundamental regulators of cell cycle progression in all eukaryotes and are frequently overexpressed in tumors. Through their polo box domain, Plks target multiple substrates previously phosphorylated by CDKs and MAPKs. In response to DNA damage, Plks are temporally inhibited in order to maintain the checkpoint-dependent cell cycle block while their activity is required to silence the checkpoint response and resume cell cycle progression. Here, we report that, in budding yeast, overproduction of the Cdc5 polo kinase overrides the checkpoint signaling induced by double strand DNA breaks (DSBs), preventing the phosphorylation of several Mec1/ATR targets, including Ddc2/ATRIP, the checkpoint mediator Rad9, and the transducer kinase Rad53/CHK2. We also show that high levels of Cdc5 slow down DSB processing in a Rad9-dependent manner, but do not prevent the binding of checkpoint factors to a single DSB. Finally, we provide evidence that Sae2, the functional ortholog of human CtIP, which regulates DSB processing and inhibits checkpoint signaling, is regulated by Cdc5. We propose that Cdc5 interferes with the checkpoint response to DSBs acting at multiple levels in the signal transduction pathway and at an early step required to resect DSB ends.

Lors de la division cellulaire, une cellule mère doit dupliquer (interphase) puis ségréger son matériel génétique de façon égale entre les deux cellules filles (mitose). Entre ces deux étapes, la cellule subit une réorganisation drastique gouvernée par l’acteur majeur Cdk1-Cycline B, conduisant à l’entrée en mitose. L’activation de cette kinase est régulée par une boucle d’auto-amplification où les premières molécules de Cdk1-Cycline B stimulent l’activation des suivantes. Il a été montré que la kinase Plk1 initie cette boucle d’auto-amplification en stimulant les activateurs et en réprimant les inhibiteurs de Cdk1-Cycline B en amont. Pour que cette kinase soit totalement active, elle doit être elle-même activée par Aurora A, en présence de son co-activateur Bora. Il est crucial de comprendre comment tous ces acteurs se coordonnent dans l’espace et dans le temps pour déclencher l’entrée en mitose car un dérèglement pourrait amener à une ségrégation de l’ADN anarchique, conduisant à la formation de tumeurs et l’apparition de cancers. Au cours de ma thèse, j’ai tout d’abord contribué à la mise en évidence d’un mécanisme conservé d’activation de Plk1 dans les cellules humaines et chez C. elegans (PLK-1), impliquant le co-activateur Bora ou SPAT-1 chez C. elegans. Nous avons montré que la phosphorylation de SPAT-1 par Cdk1-Cycline B induit son interaction avec PLK-1, ce qui promeut la phosphorylation de PLK-1 par Aurora A et donc son activation in vitro. Ce mécanisme phospho-dépendant de SPAT-1 est important in vivo pour contrôler dans le temps l’entrée en mitose. De plus, l’activation de Plk1 in vitro avec les protéines humaines suggèrent fortement une conservation du mécanisme. Nous avons ensuite montré que la phosphorylation de Bora et de SPAT-1 par Cdk1 sur les résidus S41, S112, S137 et S119, S190, T229 respectivement, est nécessaire à leur interaction avec Plk1/PLK-1, déclenchant ensuite l’activation de Plk1/PLK-1 et l’entrée en mitose. Ces résultats démontrent que Bora/SPAT-1 phosphorylée fait partie de la boucle d’auto-amplification de Cdk1-Cycline B via l’activation de Plk1, permettant à terme d’activer de façon irréversible les acteurs de l’entrée en mitose. Par la suite, je me suis focalisée sur le rôle de PLK-1 dans la rupture de l’enveloppe nucléaire en utilisant l’embryon de C. elegans comme système modèle. Après avoir démontré que PLK-1 est cruciale pour la rupture de l’enveloppe nucléaire dans les embryons, j’ai observé une localisation de PLK-1 à l’enveloppe nucléaire avant sa rupture et j’ai identifié un complexe de nucléoporines impliqué dans ce processus. En effet, NPP-1, NPP-4 et NPP-11 dont la fonction est de réguler le transport nucléo-cytoplasmique, ont également un second rôle dans le recrutement de PLK-1 aux pores nucléaires. PLK-1 interagit avec ses substrats phosphorylés par deux types de mécanismes d’amorçage Plk1-dépendant et indépendant, impliquant une autre kinase en amont comme Cdk1-Cycline B par exemple. J’ai montré que le recrutement de PLK-1 aux pores dépend des deux mécanismes, nécessitant donc une coordination entre Cdk1-Cycline B et PLK-1. Une fois que PLK-1 est au centre du pore nucléaire, elle peut alors probablement phosphoryler de nombreuses nucléoporines et participer au désassemblage des pores, conduisant à la rupture de l’enveloppe nucléaire
During cell division, a mother cell duplicates (interphase) and then segregate its genetic material equally between the two daughter cells (mitosis). Between these two stages, the cell undergoes a drastic reorganization governed by the major actor Cdk1-Cyclin B, leading to mitotic entry. The activation of this kinase is regulated by an auto-amplification loop where the first molecules of Cdk1-Cyclin B stimulate activation of the following. Plk1 kinase has been shown to initiate this self-amplification loop by stimulating activators and repressing upstream Cdk1-Cyclin B inhibitors. For this kinase to be fully active, it must itself be activated by Aurora A, in the presence of its coactivator Bora. It is crucial to understand how all these actors coordinate in space and time to trigger mitotic entry because a disruption could lead to a segregation of anarchic DNA, leading to the formation of tumors and the appearance of cancers. During my thesis, I first contributed to demonstrate a conserved mechanism of Plk1 activation in human cells and in C. elegans (PLK-1), involving the coactivator Bora or SPAT-1 in C. elegans. We have shown that the phosphorylation of SPAT-1 by Cdk1-Cyclin B induces its interaction with PLK-1, which promotes the phosphorylation of PLK-1 by Aurora A and thus its activation in vitro. This phosphory-dependent mechanism of SPAT-1 is important in vivo for controlling the entry into mitosis over time. In addition, activation of Plk1 in vitro with human proteins strongly suggests conservation of the mechanism. We then showed that the phosphorylation of Bora and SPAT-1 by Cdk1 on residues S41, S112, S137 and S119, S190, T229 respectively, is necessary for their interaction with Plk1 / PLK-1, then triggering the activation of Plk1 / PLK-1 and mitotic entry. These results demonstrate that phosphorylated Bora / SPAT-1 is part of the self-amplification loop of Cdk1-Cyclin B via the activation of Plk1, ultimately enabling irreversible activation of the actors of mitotic entry. Subsequently, I focused on the role of PLK-1 in nuclear envelope breakdown using the C. elegans early embryo as a model system. After demonstrating that PLK-1 is crucial for the nuclear envelope breakdown in embryos, I observed a localization of PLK-1 to the nuclear envelope before its rupture and I identified a nucleoporin complex involved in this process. Indeed, NPP-1, NPP-4 and NPP-11 whose function is to regulate nucleo-cytoplasmic transport, also have a second role in the recruitment of PLK-1 to nuclear pores. PLK-1 interacts with its phosphorylated substrates by two types of Plk1-dependent and independent priming mechanisms, involving another upstream kinase such as Cdk1-Cyclin B for example. I have shown that the recruitment of PLK-1 to the pores depends on both mechanisms, thus requiring coordination between Cdk1-Cyclin B and PLK-1. Once PLK-1 is at the center of the nuclear pore, it can probably phosphorylate many nucleoporins and participate in the disassembly of pores, leading to tnuclear envelope breakdown

During development, the precise regulation of the processes of proliferation, migration, and differentiation is required to establish proper organ structure and function and to prevent the deregulation that can lead to disease, such as cancer. Improved understanding of the signals that regulate these processes is therefore necessary to both gain insight into the mechanisms by which organ development proceeds and to identify strategies for treating the consequences of deregulation of these processes. In the cerebellum, some of the factors that regulate these processes have been identified but remain incompletely understood. Our studies have focused on the signals that regulate the migration of cerebellar granule neuron progenitors (GNPs) and the contribution of the SDF-1/CXCR4 signaling axis to postnatal cerebellar development. Using conditional knockout mice to delete CXCR4 specifically in GNPs, we show that loss of CXCR4 results in premature migration of a subset of GNPs throughout postnatal development that are capable of proliferation and survival outside of their normal mitogenic niche. Loss of CXCR4 also causes a reduction in the activity of the Sonic hedgehog (SHH) pathway (the primary mitogen for GNPs) but does not affect GNP proliferation, differentiation, or capacity for tumor formation. Our data suggest that while other factors likely contribute, SDF-1/CXCR4 signaling is necessary for proper migration of GNPs throughout cerebellar development.

In addition to understanding the signals that regulate normal development, the identification of vulnerabilities of established tumors is also necessary to improve cancer treatment. One strategy to improve treatment involves targeting the cells that are critical for maintaining tumor growth, known as tumor-propagating cells (TPCs). In the context of the cerebellar tumor medulloblastoma (MB), we have previously identified a population of TPCs in tumors from patched mutant mice that express the cell surface carbohydrate antigen CD15/SSEA-1. Here, we employed multiple approaches in an effort to target these cells, including a biochemical approach to identify molecules that carry the CD15 carbohydrate epitope as well as an immunotoxin approach to specifically target CD15-expressing cells. Unfortunately, these strategies were ultimately unsuccessful, but an alternative approach that recognized a vulnerability of CD15+ cells was identified. We show that CD15+ cells express elevated levels of genes associated with the G2/M phases of the cell cycle, progress more rapidly through the cell cycle than CD15- cells, and contain an increased proportion of cells in G2/M. Exposure of tumor cells to inhibitors of Aurora and Polo-like kinases, key regulators of G2/M, induces cell cycle arrest, apoptosis and enhanced sensitivity to conventional chemotherapy, and treatment of tumor-bearing mice with these agents significantly inhibits tumor progression. Importantly, cells from human patient-derived MB xenografts are also sensitive to Aurora and Polo-like kinase inhibitors. Our findings suggest that targeting G2/M regulators may represent a novel approach for the treatment of human MB.

Dissertation