240 research outputs found

    Aurora-A expressing tumour cells are deficient for homology-directed DNA double strand-break repair and sensitive to PARP inhibition.

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    The protein kinase Aurora-A is a major regulator of the cell cycle that orchestrates mitotic entry and is required for the assembly of a functional mitotic spindle. Overexpression of Aurora-A has been strongly linked with oncogenesis and this has led to considerable efforts at therapeutic targeting of the kinase activity of this protein. However, the exact mechanism by which Aurora-A promotes oncogenesis remains unclear. Here, we show that Aurora-A modulates the repair of DNA double-strand breaks (DSBs). Aurora-A expression inhibits RAD51 recruitment to DNA DSBs, decreases DSB repair by homologous recombination and sensitizes cancer cells to PARP inhibition. This impairment of RAD51 function requires inhibition of CHK1 by Polo-like kinase 1 (PLK1). These results identify a novel function of Aurora-A in modulating the response to DNA DSB that likely contributes to carcinogenesis and suggest a novel therapeutic approach to the treatment of cancers overexpressing this protein

    The enzymatic activity of the VEGFR2-receptor for the biosynthesis of dinucleoside polyphosphates

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    The group of dinucleoside polyphosphates encompasses a large number of molecules consisting of two nucleosides which are connected by a phosphate chain of variable length. While the receptors activated by dinucleoside polyphosphates as well as their degradation have been studied in detail, its biosynthesis has not been elucidated so far. Since endothelial cells released the dinucleoside polyphosphate uridine adenosine tetraphosphate (Up4A), we tested cytosolic proteins of human endothelial cells obtained from dermal vessels elicited for enzymatic activity. When incubated with ADP and UDP, these cells showed increasing concentrations of Up4A. The underlying enzyme was isolated by chromatography and the mass spectrometric analysis revealed that the enzymatic activity was caused by the vascular endothelial growth factor receptor 2 (VEGFR2). Since VEGFR2 but neither VEGFR1 nor VEGFR3 were capable to synthesise dinucleoside polyphosphates, Tyr-1175 of VEGFR2 is most likely essential for the enzymatic activity of interest. Further, VEGFR2-containing cells like HepG2, THP-1 and RAW264.7 were capable of synthesising dinucleoside polyphosphates. VEGFR2-transfected HEK 293T/17 but not native HEK 293T/17 cells synthesised dinucleoside polyphosphates in vivo too. The simultaneous biosynthesis of dinucleoside polyphosphates could amplify the response to VEGF, since dinucleoside polyphosphates induce cellular growth via P2Y purinergic receptors. Thus the biosynthesis of dinucleoside polyphosphates by VEGFR2 may enhance the proliferative response to VEGF. Given that VEGFR2 is primarily expressed in endothelial cells, the biosynthesis of dinucleoside polyphosphates is mainly located in the vascular system. Since the vasculature is also the main site of action of dinucleoside polyphosphates, activating vascular purinoceptors, blood vessels appear as an autocrine system with respect to dinucleoside polyphosphates. We conclude that VEGFR2 receptor is capable of synthesising dinucleoside polyphosphates. These mediators may modulate the effects of VEGFR2 due to their proliferative effects

    Dissecting mitosis by RNAi in Drosophila tissue culture cells

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    Here we describe a detailed methodology to study the function of genes whose products function during mitosis by dsRNA-mediated interference (RNAi) in cultured cells of Drosophila melanogaster. This procedure is particularly useful for the analysis of genes for which genetic mutations are not available or for the dissection of complicated phenotypes derived from the analysis of such mutants. With the advent of whole genome sequencing it is expected that RNAi-based screenings will be one method of choice for the identification and study of novel genes involved in particular cellular processes. In this paper we focused particularly on the procedures for the proper phenotypic analysis of cells after RNAi-mediated depletion of proteins required for mitosis, the process by which the genetic information is segregated equally between daughter cells. We use RNAi of the microtubule-associated protein MAST/Orbit as an example for the usefulness of the technique

    Vertical-external-cavity surface-emitting lasers and quantum dot lasers

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    The use of cavity to manipulate photon emission of quantum dots (QDs) has been opening unprecedented opportunities for realizing quantum functional nanophotonic devices and also quantum information devices. In particular, in the field of semiconductor lasers, QDs were introduced as a superior alternative to quantum wells to suppress the temperature dependence of the threshold current in vertical-external-cavity surface-emitting lasers (VECSELs). In this work, a review of properties and development of semiconductor VECSEL devices and QD laser devices is given. Based on the features of VECSEL devices, the main emphasis is put on the recent development of technological approach on semiconductor QD VECSELs. Then, from the viewpoint of both single QD nanolaser and cavity quantum electrodynamics (QED), a single-QD-cavity system resulting from the strong coupling of QD cavity is presented. A difference of this review from the other existing works on semiconductor VECSEL devices is that we will cover both the fundamental aspects and technological approaches of QD VECSEL devices. And lastly, the presented review here has provided a deep insight into useful guideline for the development of QD VECSEL technology and future quantum functional nanophotonic devices and monolithic photonic integrated circuits (MPhICs).Comment: 21 pages, 4 figures. arXiv admin note: text overlap with arXiv:0904.369

    A Method for Analyzing the Ubiquitination and Degradation of Aurora-A

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    The cell cycle machinery consists of regulatory proteins that control the progression through the cell cycle ensuring that DNA replication alternates with DNA segregation in mitosis to maintain cell integrity. Some of these key regulators have to be degraded at each cell cycle to prevent cellular dysfunction. Mitotic exit requires the inactivation of cyclin dependent kinase1 (cdk1) and it is the degradation of the cyclin subunit that inactivates the kinase. Cyclin degradation has been well characterized and it was shown that it is ubiquitin proteasome pathway that leads to the elimination of cyclins. By now, many other regulatory proteins were shown to be degraded by the same pathway, among them members of the aurora kinase family, degraded many other regulatory proteins. Aurora kinases are involved in mitotic spindle formation as well as in cytokinesis. The abundance and activity of the kinase is precisely regulated during the cell cycle. To understand how proteolysis regulates transitions through the cell cycle we describe two assays for ubiquitination and degradation of xenopus aurora kinase A using extracts from xenopus eggs or somatic cell lines

    A Translational Regulator, PUM2, Promotes Both Protein Stability and Kinase Activity of Aurora-A

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    Aurora-A, a centrosomal serine-threonine kinase, orchestrates several key aspects of cell division. However, the regulatory pathways for the protein stability and kinase activity of Aurora-A are still not completely understood. In this study, PUM2, an RNA-binding protein, is identified as a novel substrate and interacting protein of Aurora-A. Overexpression of the PUM2 mutant which fails to interact with Aurora-A, and depletion of PUM2 result in a decrease in the amount of Aurora-A. PUM2 physically binds to the D-box of Aurora-A, which is recognized by APC/CCdh1. Overexpression of PUM2 prevents ubiquitination and enhances the protein stability of Aurora-A, suggesting that PUM2 protects Aurora-A from APC/CCdh1-mediated degradation. Moreover, association of PUM2 with Aurora-A not only makes Aurora-A more stable but also enhances the kinase activity of Aurora-A. Our study suggests that PUM2 plays two different but important roles during cell cycle progression. In interphase, PUM2 localizes in cytoplasm and plays as translational repressor through its RNA binding domain. However, in mitosis, PUM2 physically associates with Aurora-A to ensure enough active Aurora-A at centrosomes for mitotic entry. This is the first time to reveal the moonlight role of PUM2 in mitosis

    Two TPX2-Dependent Switches Control the Activity of Aurora A

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    Aurora A is an important oncogenic kinase for mitotic spindle assembly and a potentially attractive target for human cancers. Its activation could be regulated by ATP cycle and its activator TPX2. To understand the activation mechanism of Aurora A, a series of 20 ns molecular dynamics (MD) simulations were performed on both the wild-type kinase and its mutants. Analyzing the three dynamic trajectories (Aurora A-ATP, Aurora A-ADP, and Aurora A-ADP-TPX2) at the residue level, for the first time we find two TPX2-dependent switches, i.e., switch-1 (Lys-143) and switch-2 (Arg-180), which are tightly associated with Aurora A activation. In the absence of TPX2, Lys-143 exhibits a “closed” state, and becomes hydrogen-bonded to ADP. Once TPX2 binding occurs, switch-1 is forced to “open” the binding site, thus pulling ADP away from Aurora A. Without facilitation of TPX2, switch-2 exits in an “open” conformation which accompanies the outward-flipping movement of P·Thr288 (in an inactive conformation), leading to the crucial phosphothreonine exposed and accessible for deactivation. However, with the binding of TPX2, switch-2 is forced to undergo a “closed” movement, thus capturing P·Thr288 into a buried position and locking its active conformation. Analysis of two Aurora A (K143A and R180A) mutants for the two switches further verifies their functionality and reliability in controlling Aurora activity. Our systems therefore suggest two switches determining Aurora A activation, which are important for the development of aurora kinase inhibitors
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