Genetic enhancers of partial PLK1 inhibition reveal hypersensitivity to kinetochore perturbations.

Polo-like kinase 1 (PLK1) is a serine/threonine kinase required for mitosis and cytokinesis. As cancer cells are often hypersensitive to partial PLK1 inactivation, chemical inhibitors of PLK1 have been developed and tested in clinical trials. However, these small molecule inhibitors alone are not completely effective. PLK1 promotes numerous molecular and cellular events in the cell division cycle and it is unclear which of these events most crucially depend on PLK1 activity. We used a CRISPR-based genome-wide screening strategy to identify genes whose inactivation enhances cell proliferation defects upon partial chemical inhibition of PLK1. Genes identified encode proteins that are functionally linked to PLK1 in multiple ways, most notably factors that promote centromere and kinetochore function. Loss of the kinesin KIF18A or the outer kinetochore protein SKA1 in PLK1-compromised cells resulted in mitotic defects, activation of the spindle assembly checkpoint and nuclear reassembly defects. We also show that PLK1-dependent CENP-A loading at centromeres is extremely sensitive to partial PLK1 inhibition. Our results suggest that partial inhibition of PLK1 compromises the integrity and function of the centromere/kinetochore complex, rendering cells hypersensitive to different kinetochore perturbations. We propose that KIF18A is a promising target for combinatorial therapies with PLK1 inhibitors.

The spindle assembly checkpoint and the spatial activation of Polo kinase determine the duration of cell division and prevent tumor formation.

The maintenance of a restricted pool of asymmetrically dividing stem cells is essential for tissue homeostasis. This process requires the control of mitotic progression that ensures the accurate chromosome segregation. In addition, this event is coupled to the asymmetric distribution of cell fate determinants in order to prevent stem cell amplification. How this coupling is regulated remains poorly described. Here, using asymmetrically dividing Drosophila neural stem cells (NSCs), we show that Polo kinase activity levels determine timely Cyclin B degradation and mitotic progression independent of the spindle assembly checkpoint (SAC). This event is mediated by the direct phosphorylation of Polo kinase by Aurora A at spindle poles and Aurora B kinases at centromeres. Furthermore, we show that Aurora A-dependent activation of Polo is the major event that promotes NSC polarization and together with the SAC prevents brain tumor growth. Altogether, our results show that an Aurora/Polo kinase module couples NSC mitotic progression and polarization for tissue homeostasis.

Spatiotemporal coordination of Greatwall-Endos-PP2A promotes mitotic progression.

Mitotic entry involves inhibition of protein phosphatase 2A bound to its B55/Tws regulatory subunit (PP2A-B55/Tws), which dephosphorylates substrates of mitotic kinases. This inhibition is induced when Greatwall phosphorylates Endos, turning it into an inhibitor of PP2A-Tws. How this mechanism operates spatiotemporally in the cell is incompletely understood. We previously reported that the nuclear export of Greatwall in prophase promotes mitotic progression. Here, we examine the importance of the localized activities of PP2A-Tws and Endos for mitotic regulation. We find that Tws shuttles through the nucleus via a conserved nuclear localization signal (NLS), but expression of Tws in the cytoplasm and not in the nucleus rescues the development of tws mutants. Moreover, we show that Endos must be in the cytoplasm before nuclear envelope breakdown (NEBD) to be efficiently phosphorylated by Greatwall and to bind and inhibit PP2A-Tws. Disrupting the cytoplasmic function of Endos before NEBD results in subsequent mitotic defects. Evidence suggests that this spatiotemporal regulation is conserved in humans.

A unified view of spatio-temporal control of mitotic entry: Polo kinase as the key.

The Polo kinase is an essential regulator of cell division. Its ability to regulate multiple events at distinct subcellular locations and times during mitosis is remarkable. In the last few years, a much clearer mechanistic understanding of the functions and regulation of Polo in cell division has emerged. In this regard, the importance of coupling changes in activity with changes in localization is striking, both for Polo itself and for its upstream regulators. This review brings together several new pieces of the puzzle that are gradually revealing how Polo is regulated, in space and time, to enable its functions in the early stages of mitosis in animal cells. As a result, a unified view of how mitotic entry is spatio-temporally regulated is emerging.

Psychosocial Features of Neurofibromatosis Type 1 in Children and Adolescents.

Neurofibromatosis type 1 (NF1) is a common neurologic condition associated with a wide variety of developmental deficits that have an important impact on children and adolescents. OBJECTIVE: This article aims to document the psychosocial features of NF1 and to report the interventions described to address the needs of pediatric patients with NF1. METHODS: A literature review was conducted concerning the social life, mental health, and quality of life (QOL) of children and adolescents with NF1 as well as the psychosocial interventions addressed to this population. RESULTS: Compared to unaffected children and adolescents of the general population, pediatric patients with NF1 have an increased risk of having social difficulties, mental health disorders, behavioral and emotional problems, as well as diminished QOL. Only 3 articles describe interventions within the NF1 population to address these difficulties. CONCLUSION: There is a need to develop and assess psychosocial interventions for patients with NF1.

Several inhibitors of the Plk1 Polo-Box Domain turn out to be non-specific protein alkylators.

For almost a decade, there has been much interest in the development of chemical inhibitors of Polo-like kinase 1 (Plk1) protein interactions. Plk1 is a master regulator of the cell division cycle that controls numerous substrates. It is a promising target for cancer drug development. Inhibitors of the kinase domain of Plk1 had some success in clinical trials. However, they are not perfectly selective. In principle, Plk1 can also be inhibited by interfering with its protein interaction domain, the Polo-Box Domain (PBD). Selective chemical inhibitors of the PBD would constitute tools to probe for PBD-dependent functions of Plk1 and could be advantageous in cancer therapy. The discovery of Poloxin and thymoquinone as PBD inhibitors indicated that small, cell-permeable chemical inhibitors could be identified. Other efforts followed, including ours, reporting additional molecules capable of blocking the PBD. It is now clear that, unfortunately, most of these compounds are non-specific protein alkylators (defined here as groups covalently added via a carbon) that have little or no potential for the development of real Plk1 PBD-specific drugs. This situation should be minded by biologists potentially interested in using these compounds to study Plk1. Further efforts are needed to develop selective, cell-permeable PBD inhibitors.

Identification of Polo-like kinase 1 interaction inhibitors using a novel cell-based assay.

Polo-like kinase 1 (Plk1) plays several roles in cell division and it is a recognized cancer drug target. Plk1 levels are elevated in cancer and several types of cancer cells are hypersensitive to Plk1 inhibition. Small molecule inhibitors of the kinase domain (KD) of Plk1 have been developed. Their selectivity is limited, which likely contributes to their toxicity. Polo-like kinases are characterized by a Polo-Box Domain (PBD), which mediates interactions with phosphorylation substrates or regulators. Inhibition of the PBD could allow better selectivity or result in different effects than inhibition of the KD. In vitro screens have been used to identify PBD inhibitors with mixed results. We developed the first cell-based assay to screen for PBD inhibitors, using Bioluminescence Resonance Energy Transfer (BRET). We screened through 112 983 compounds and characterized hits in secondary biochemical and biological assays. Subsequent Structure-Activity Relationship (SAR) analysis on our most promising hit revealed that it requires an alkylating function for its activity. In addition, we show that the previously reported PBD inhibitors thymoquinone and Poloxin are also alkylating agents. Our cell-based assay is a promising tool for the identification of new PBD inhibitors with more drug-like profiles using larger and more diverse chemical libraries.

The Greatwall-PP2A axis in cell cycle control.

Cell cycle progression is largely controlled by reversible protein phosphorylation mediated by cyclically activated kinases and phosphatases. It has long been known that cyclin B-Cdk1 activation triggers mitotic entry, and the enzymatic network controlling its activation and inactivation has been well characterized. Much more recently protein phosphatase 2A (PP2A) together with its B55 regulatory subunit has been recognized as the major activity dephosphorylating Cdk1 targets. Moreover, PP2A-B55 activity is high in late M phase and interphase, but low at mitotic entry. A series of discoveries in the fly and frog model systems have uncovered the molecular mechanism mediating this regulation. The Greatwall (Gwl) kinase activates endosulfines, which become specific inhibitors of PP2A-B55. Cdk1-dependent activation of Gwl at mitotic entry leads to PP2A-B55 downregulation, which synergizes with Cdk1 activation to promote the phosphorylated states of several mitotic substrates. Much less is known on the mechanisms inactivating Gwl and endosulfines at mitotic exit. Recent reports show the importance of spatiotemporal regulation of Gwl, endosulfines, and PP2A-B55 for cell cycle progression. The various systems and cell types differ in their dependence on the Gwl-PP2A axis for cell cycle progression. Moreover, this pathway also regulates gene expression in yeast, and this function could be conserved in metazoans.

Polo-like kinase-activating kinases: Aurora A, Aurora B and what else?

The events of cell division are regulated by a complex interplay between kinases and phosphatases. Cyclin-dependent kinases (Cdks), polo-like kinases (Plks) and Aurora kinases play central roles in this process. Polo kinase (Plk1 in humans) regulates a wide range of events in mitosis and cytokinesis. To ensure the accuracy of these processes, polo activity itself is subject to complex regulation. Phosphorylation of polo in its T loop (or activation loop) increases its kinase activity several-fold. It has been shown that Aurora A kinase, with its co-factor Bora, activates Plk1 in G(2), and that this is essential for recovery from cell cycle arrest induced by DNA damage. In a recent article published in PLoS Biology, we report that Drosophila polo is activated by Aurora B kinase at centromeres, and that this is crucial for polo function in regulating chromosome dynamics in prometaphase. Our results suggest that this regulatory pathway is conserved in humans. Here, we propose a model for the collaboration between Aurora B and polo in the regulation of kinetochore attachment to microtubules in early mitosis. Moreover, we suggest that Aurora B could also function to activate Polo/Plk1 in cytokinesis. Finally, we discuss recent findings and open questions regarding the activation of polo and polo-like kinases by different kinases in mitosis, cytokinesis and other processes.

Polo-like kinases: conservation and divergence in their functions and regulation.

Polo-like kinases (Plks) are potent regulators of M phase that are conserved from yeasts to humans. Their roles in mitotic entry, spindle pole functions and cytokinesis are broadly conserved despite physical and molecular differences in these processes in disparate organisms. Plks are characterized by their Polo-box domain, which mediates protein interactions. They are additionally controlled by phosphorylation, proteolysis and transcription, depending on the biological context. Plks are now recognized to link cell division to developmental processes and to function in differentiated cells. A comparison of Plk function and regulation between organisms offers insight into the rich variations of cell division.

A bitter PP1 fights the sweet polo.

In a recent paper in Developmental Cell, Yamashiro et al. (2008) report that the PP1 regulatory subunit MYPT1 interacts with PLK1 and antagonizes essential mitotic functions of PLK1, at least in part by promoting the dephosphorylation of PLK1 at Thr210.

Yeast Polo-like kinase substrates are nailed with the right tools.

A platform has been built combining chemical genetics and bioinformatics to screen the proteome for physiological substrates of the Polo-like kinase in budding yeast. A novel role for this kinase in regulating the mitotic spindle is revealed.

Targeted proteomic study of the cyclin-Cdk module.

The cell division cycle of the yeast S. cerevisiae is driven by one Cdk (cyclin-dependent kinase), which becomes active when bound to one of nine cyclin subunits. Elucidation of Cdk substrates and other Cdk-associated proteins is essential for a full understanding of the cell cycle. Here, we report the results of a targeted proteomics study using affinity purification coupled to mass spectrometry. Our study identified numerous proteins in association with particular cyclin-Cdk complexes. These included phosphorylation substrates, ubiquitination-degradation proteins, adaptors, and inhibitors. Some associations were previously known, and for others, we confirmed their specificity and biological relevance. Using a hypothesis-driven mass spectrometric approach, we also mapped in vivo phosphorylation at Cdk consensus motif-containing peptides within several cyclin-associated candidate Cdk substrates. Our results demonstrate that this approach can be used to detect a host of transient and dynamic protein associations within a biological module.