Exploiting Drug Resistance for Science♦ : Discovery and Exploitation of Inhibitor-resistant Aurora and Polo Kinase Mutants for the Analysis of Mitotic Networks
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PROVIDER: S-EPMC2708906 | biostudies-literature | 2009 Jun
REPOSITORIES: biostudies-literature
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Project description:The Aurora and Polo-like kinases are central components of mitotic signaling pathways, and recent evidence suggests that substantial cross-talk exists between Aurora A and Plk1. In addition to their validation as novel anticancer agents, small molecule kinase inhibitors are increasingly important tools to help dissect clinically relevant protein phosphorylation networks. However, one major problem associated with kinase inhibitors is their promiscuity toward "off-target" members of the kinome, which makes interpretation of data obtained from complex cellular systems challenging. Additionally, the emergence of inhibitor resistance in patients makes it clear that an understanding of resistance mechanisms is essential to inform drug design. In this study, we exploited structural knowledge of the binding modes of VX-680, an Aurora kinase inhibitor, and BI 2536, a Polo-like kinase inhibitor, to design and evaluate drug-resistant kinase mutants. Using inducible stable human cell lines, we authenticated mitotic targets for both compounds and demonstrated that Aurora A mutants exhibit differential cellular sensitivity toward the inhibitors VX-680 and MLN8054. In addition, we validated Aurora B as an important anti-proliferative target for VX-680 in model human cancer cells. Finally, this chemical genetic approach allowed us to prove that Aurora A activation loop phosphorylation is controlled by a Plk1-mediated pathway in human cells.
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Project description:Mitosis is choreographed by a number of protein kinases including polo-like kinases and Aurora kinases. As these kinases are frequently dysregulated in cancers, small-molecule inhibitors have been developed for targeted anticancer therapies. Given that PLK1 and Aurora kinases possess both unique functions as well as co-regulate multiple mitotic events, whether pharmacological inhibition of these kinases together can enhance mitotic catastrophe remains an outstanding issue to be determined. Using concentrations of inhibitors that did not induce severe mitotic defects on their own, we found that both the metaphase arrest and mitotic slippage induced by inhibitors targeting Aurora A and Aurora B (MK-5108 and Barasertib respectively) were enhanced by a PLK1 inhibitor (BI 2536). We found that PLK1 is overexpressed in cells from nasopharyngeal carcinoma, a highly invasive cancer with poor prognosis, in comparison to normal nasopharyngeal epithelial cells. Nasopharyngeal carcinoma cells were more sensitive to BI 2536 as a single agent and co-inhibition with Aurora kinases than normal cells. These observations underscore the mechanism and potential benefits of targeting PLK1 and Aurora kinases to induce mitotic catastrophe in cancer cells.
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Project description:Mitosis is a process involving a complex series of events that require careful coordination. Protein phosphorylation by a small number of kinases, in particular Aurora A, Aurora B, the cyclin-dependent kinase-cyclin complex Cdk1/cyclinB, and Polo-like kinase 1 (Plk1), orchestrates almost every step of cell division, from entry into mitosis to cytokinesis. To discover more about the functions of Aurora A, Aurora B, and kinases of the Plk family, we mapped mitotic phosphorylation sites to these kinases through the combined use of quantitative phosphoproteomics and selective targeting of kinase activities by small-molecule inhibitors. Using this integrated approach, we connected 778 phosphorylation sites on 562 proteins with these enzymes in cells arrested in mitosis. By connecting the kinases to protein complexes, we associated these kinases with functional modules. In addition to predicting previously unknown functions, this work establishes additional substrate-recognition motifs for these kinases and provides an analytical template for further use in dissecting kinase signaling events in other areas of cellular signaling and systems biology.
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Project description:The kinase Aurora-A (Aur-A), which is enriched at centrosomes, is required for centrosome maturation and accurate chromosome segregation, and recent work implicates centrosomes as sites where the earliest activation of cyclin B1-cdc2 occurs. Here, we have used Xenopus egg extracts to investigate Aur-A's contribution to cell cycle progression and spindle morphology in the presence or absence of centrosomes. We find that addition of active Aur-A accelerates cdc2 activation and mitotic entry. Depletion of endogenous Aur-A or addition of inactive Aur-A, which lead to monopolar spindles, delays but does not block mitotic entry. These effects on timing and spindle structure do not require the presence of centrosomes or chromosomes. The catalytic domain alone of Aur-A is sufficient to restore spindle bipolarity; additional N-terminal sequences function in mitotic timing.
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Project description:Cdc7p-Dbf4p is a conserved protein kinase required for the initiation of DNA replication. The Dbf4p regulatory subunit binds Cdc7p and is essential for Cdc7p kinase activation, however, the N-terminal third of Dbf4p is dispensable for its essential replication activities. Here, we define a short N-terminal Dbf4p region that targets Cdc7p-Dbf4p kinase to Cdc5p, the single Polo kinase in budding yeast that regulates mitotic progression and cytokinesis. Dbf4p mediates an interaction with the Polo substrate-binding domain to inhibit its essential role during mitosis. Although Dbf4p does not inhibit Polo kinase activity, it nonetheless inhibits Polo-mediated activation of the mitotic exit network (MEN), presumably by altering Polo substrate targeting. In addition, although dbf4 mutants defective for interaction with Polo transit S-phase normally, they aberrantly segregate chromosomes following nuclear misorientation. Therefore, Cdc7p-Dbf4p prevents inappropriate exit from mitosis by inhibiting Polo kinase and functions in the spindle position checkpoint.
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Project description:SignificanceMitosis is an essential process in all eukaryotes, but paradoxically, genes required for mitosis vary among species. The essentiality of many mitotic genes was bypassed by activating alternative mechanisms during evolution. However, bypass events have rarely been recapitulated experimentally. Here, using the fission yeast Schizosaccharomyces pombe, the essentiality of a kinase (Plo1) required for bipolar spindle formation was bypassed by other mutations, many of which are associated with glucose metabolism. The Plo1 bypass by the reduction in glucose uptake was dependent on another kinase (casein kinase I), which potentiated spindle microtubule formation. This study illustrates a rare experimental bypass of essentiality for mitotic genes and provides insights into the molecular diversity of mitosis.
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Project description:Bipolar mitotic spindle organization is fundamental to faithful chromosome segregation. Furry (Fry) is an evolutionarily conserved protein implicated in cell division and morphology. In human cells, Fry localizes to centrosomes and spindle microtubules in early mitosis, and depletion of Fry causes multipolar spindle formation. However, it remains unknown how Fry controls bipolar spindle organization. This study demonstrates that Fry binds to polo-like kinase 1 (Plk1) through the polo-box domain of Plk1 in a manner dependent on the cyclin-dependent kinase 1-mediated Fry phosphorylation at Thr-2516. Fry also binds to Aurora A and promotes Plk1 activity by binding to the polo-box domain of Plk1 and by facilitating Aurora A-mediated Plk1 phosphorylation at Thr-210. Depletion of Fry causes centrosome and centriole splitting in mitotic spindles and reduces the kinase activity of Plk1 in mitotic cells and the accumulation of Thr-210-phosphorylated Plk1 at the spindle poles. Our results suggest that Fry plays a crucial role in the structural integrity of mitotic centrosomes and in the maintenance of spindle bipolarity by promoting Plk1 activity at the spindle poles in early mitosis.
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Project description:During cell division, duplicated genetic material is separated into two distinct daughter cells. This process is essential for initial tissue formation during development and to maintain tissue integrity throughout an organism's lifetime. To ensure the efficacy and efficiency of this process, the cell employs a variety of regulatory and signaling proteins that function as mitotic regulators and checkpoint proteins. One vital mitotic regulator is polo-like kinase 1 (PLK1), a highly conserved member of the polo-like kinase family. Unique from its paralogues, it functions specifically during mitosis as a regulator of cell division. PLK1 is spatially and temporally enriched at three distinct subcellular locales; the mitotic centrosomes, kinetochores, and the cytokinetic midbody. These localization patterns allow PLK1 to phosphorylate specific downstream targets to regulate mitosis. In this review, we will explore how polo-like kinases were originally discovered and diverged into the five paralogues (PLK1-5) in mammals. We will then focus specifically on the most conserved, PLK1, where we will discuss what is known about how its activity is modulated, its role during the cell cycle, and new, innovative tools that have been developed to examine its function and interactions in cells.
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Project description:During large geomagnetic storms, red auroras are typically observed from low-latitude countries such as Japan. The color arises from a specific emission line of oxygen atoms at high altitudes. However, during the May 10-11th 2024 superstorm, magenta auroras were observed above Japan instead of the typical red. In this study, we demonstrate that the magenta hue is created by a mixture of red (O) and a blue (N2+) aurora at extremely high altitudes. The blue color originates from the N2+ first negative emission band caused by both resonant scattering of the upwelling molecular ions and heavy particle precipitation during the storm. This study is primarily driven by observations from citizen scientists, and confirmed and quantified with observations from spacecraft and modeling techniques. Additionally, we show that high solar activity, terrestrial season, and the preheating of the atmosphere all contribute to the occurrence of magenta aurora. This study showcases the value and richness of citizen science, and we anticipate that such approaches will become increasingly important in the future.
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Project description:At the onset of mitosis, the Golgi complex undergoes a multistep fragmentation process that is required for its correct partitioning into the daughter cells. Inhibition of this Golgi fragmentation results in cell cycle arrest at the G2 stage, suggesting that correct inheritance of the Golgi complex is monitored by a "Golgi mitotic checkpoint." However, the molecular basis of this G2 block is not known. Here, we show that the G2-specific Golgi fragmentation stage is concomitant with centrosome recruitment and activation of the mitotic kinase Aurora-A, an essential regulator for entry into mitosis. We show that a block of Golgi partitioning impairs centrosome recruitment and activation of Aurora-A, which results in the G2 block of cell cycle progression. Overexpression of Aurora-A overrides this cell cycle block, indicating that Aurora-A is a major effector of the Golgi checkpoint. Our findings provide the basis for further understanding of the signaling pathways that coordinate organelle inheritance and cell duplication.
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