The Importance of Kinase-Phosphatase Integration:Lessons from Mitosis

status: publishe

Biogenesis and activity regulation of protein phosphatase 1

Protein phosphatase 1 (PP1) is expressed in all eukaryotic cells and catalyzes a substantial fraction of phosphoserine/threonine dephosphorylation reactions. It forms stable complexes with PP1-interacting proteins (PIPs) that guide the phosphatase throughout its life cycle and control its fate and function. The diversity of PIPs is huge (≈200 in vertebrates), and most of them combine short linear motifs to form large and unique interaction interfaces with PP1. Many PIPs have separate domains for PP1 anchoring, PP1 regulation, substrate recruitment and subcellular targeting, which enable them to direct associated PP1 to a specific subset of substrates and mediate acute activity control. Hence, PP1 functions as the catalytic subunit of a large number of multimeric holoenzymes, each with its own subset of substrates and mechanism(s) of regulation

A Sephin1-insensitive tripartite holophosphatase dephosphorylates translation initiation factor 2α.

The integrated stress response (ISR) is regulated by kinases that phosphorylate the α subunit of translation initiation factor 2 and phosphatases that dephosphorylate it. Genetic and biochemical observations indicate that the eIF2αP-directed holophosphatase, a therapeutic target in diseases of protein misfolding, is comprised of a regulatory subunit, PPP1R15, and a catalytic subunit, protein phosphatase 1 (PP1). In mammals, there are two isoforms of the regulatory subunit, PPP1R15A and PPP1R15B, with overlapping roles in the essential function of eIF2αP dephosphorylation. However, conflicting reports have appeared regarding the requirement for an additional co-factor, G-actin, in enabling substrate-specific dephosphorylation by PPP1R15-containing PP1 holoenzymes. An additional concern relates to the sensitivity of the holoenzyme to the [(o-chlorobenzylidene)amino]guanidines Sephin1 or guanabenz, putative small-molecule proteostasis modulators. It has been suggested that the source and method of purification of the PP1 catalytic subunit and the presence or absence of an N-terminal repeat-containing region in the PPP1R15A regulatory subunit might influence the requirement for G-actin and sensitivity of the holoenzyme to inhibitors. We found that eIF2αP dephosphorylation by PP1 was moderately stimulated by repeat-containing PPP1R15A in an unphysiological low ionic strength buffer, whereas stimulation imparted by the co-presence of PPP1R15A and G-actin was observed under a broad range of conditions, low and physiological ionic strength, regardless of whether the PPP1R15A regulatory subunit had or lacked the N-terminal repeat-containing region and whether it was paired with native PP1 purified from rabbit muscle or recombinant PP1 purified from bacteria. Furthermore, none of the PPP1R15A-containing holophosphatases tested were inhibited by Sephin1 or guanabenz.Supported by a Wellcome Trust Principal Research Fellowship to D.R. (Wellcome 200848/Z/16/Z) and a Wellcome Trust Strategic Award to the Cambridge Institute for Medical Research (Wellcome 100140). M.B. was supported by a Flemish Concerted Research Action (GOA15/016). W.P. was supported by National Institute of Health R01NS091336 and the American Diabetes Association Pathway to Stop Diabetes Grant 1-14-ACN-31. Z.C. is a PhD fellow of the Fund for Scientific Research - Flanders

Intracellular delivery of protein drugs with an autonomously lysing bacterial system reduces tumor growth and metastases

Critical cancer pathways often cannot be targeted because of limited efficiency crossing cell membranes. Here we report the development of a Salmonella-based intracellular delivery system to address this challenge. We engineer genetic circuits that (1) activate the regulator flhDC to drive invasion and (2) induce lysis to release proteins into tumor cells. Released protein drugs diffuse from Salmonella containing vacuoles into the cellular cytoplasm where they interact with their therapeutic targets. Control of invasion with flhDC increases delivery over 500 times. The autonomous triggering of lysis after invasion makes the platform self-limiting and prevents drug release in healthy organs. Bacterial delivery of constitutively active caspase-3 blocks the growth of hepatocellular carcinoma and lung metastases, and increases survival in mice. This success in targeted killing of cancer cells provides critical evidence that this approach will be applicable to a wide range of protein drugs for the treatment of solid tumors

Cancer Cell Expression of Autotaxin Controls Bone Metastasis Formation in Mouse through Lysophosphatidic Acid-Dependent Activation of Osteoclasts

Bone metastases are highly frequent complications of breast cancers. Current bone metastasis treatments using powerful anti-resorptive agents are only palliative indicating that factors independent of bone resorption control bone metastasis progression. Autotaxin (ATX/NPP2) is a secreted protein with both oncogenic and pro-metastatic properties. Through its lysosphospholipase D (lysoPLD) activity, ATX controls the level of lysophosphatidic acid (LPA) in the blood. Platelet-derived LPA promotes the progression of osteolytic bone metastases of breast cancer cells. We asked whether ATX was involved in the bone metastasis process. We characterized the role of ATX in osteolytic bone metastasis formation by using genetically modified breast cancer cells exploited on different osteolytic bone metastasis mouse models.Intravenous injection of human breast cancer MDA-B02 cells with forced expression of ATX (MDA-B02/ATX) to immunodeficiency BALB/C nude mice enhanced osteolytic bone metastasis formation, as judged by increased bone loss, tumor burden, and a higher number of active osteoclasts at the metastatic site. Mouse breast cancer 4T1 cells induced the formation of osteolytic bone metastases after intracardiac injection in immunocompetent BALB/C mice. These cells expressed active ATX and silencing ATX expression inhibited the extent of osteolytic bone lesions and decreased the number of active osteoclasts at the bone metastatic site. In vitro, osteoclast differentiation was enhanced in presence of MDA-B02/ATX cell conditioned media or recombinant autotaxin that was blocked by the autotaxin inhibitor vpc8a202. In vitro, addition of LPA to active charcoal-treated serum restored the capacity of the serum to support RANK-L/MCSF-induced osteoclastogenesis.Expression of autotaxin by cancer cells controls osteolytic bone metastasis formation. This work demonstrates a new role for LPA as a factor that stimulates directly cancer growth and metastasis, and osteoclast differentiation. Therefore, targeting the autotaxin/LPA track emerges as a potential new therapeutic approach to improve the outcome of patients with bone metastases

Identification of Linearized RMS-Voltage Dip Patterns Based on Clustering in Renewable Plants

[EN] Generation units connected to the grid are currently required to meet low-voltage ride-through (LVRT) requirements. In most developed countries, these requirements also apply to renewable sources, mainly wind power plants and photovoltaic installations connected to the grid. This study proposes an alternative characterisation solution to classify and visualise a large number of collected events in light of current limits and requirements. The authors' approach is based on linearised root-mean-square-(RMS)-voltage trajectories, taking into account LRVT requirements, and a clustering process to identify the most likely pattern trajectories. The proposed solution gives extensive information on an event's severity by providing a simple but complete visualisation of the linearised RMS-voltage patterns. In addition, these patterns are compared to current LVRT requirements to determine similarities or discrepancies. A large number of collected events can then be automatically classified and visualised for comparative purposes. Real disturbances collected from renewable sources in Spain are used to assess the proposed solution. Extensive results and discussions are also included in this study.The authors thank the financial support from the 'Ministerio de Economia y Competitividad' (Spain) and the European Union - ENE2016-78214-C2-2-R, Fulbright/Spanish Ministry of Education Visiting Scholar - PRX14/00694. This work was also supported by the US Department of Energy under contract no. DE-AC36-08-GO28308 with the National Renewable Energy LaboratoryGarcía-Sánchez, TM.; Gómez-Lázaro, E.; Muljadi, E.; Kessler, M.; Muñoz-Benavente, I.; Molina-García, A. (2018). Identification of Linearized RMS-Voltage Dip Patterns Based on Clustering in Renewable Plants. IET Generation Transmission & Distribution. 12(6):1256-1262. https://doi.org/10.1049/iet-gtd.2017.0474S12561262126Craciun B. Kerekes T. Sera D.et al.: ‘Overview of recent grid codes for PV power integration’.13th Int. Conf. on Optimization of Electrical and Electronic Equipment (OPTIM) 2012 May2012 pp.959–965‘World Energy Outlook 2012’. Technical Report International Egency Agency (IEA) 2012. Available atwww.iea.orgBehrens C.E.: ‘Energy policy: 113th congress issues’.Congressional Research Service 2013Lopes, J. A. P., Hatziargyriou, N., Mutale, J., Djapic, P., & Jenkins, N. (2007). Integrating distributed generation into electric power systems: A review of drivers, challenges and opportunities. Electric Power Systems Research, 77(9), 1189-1203. doi:10.1016/j.epsr.2006.08.016Glassmire, J., Komor, P., & Lilienthal, P. (2012). Electricity demand savings from distributed solar photovoltaics. Energy Policy, 51, 323-331. doi:10.1016/j.enpol.2012.08.022Carvalho, D., Wemans, J., Lima, J., & Malico, I. (2011). Photovoltaic energy mini-generation: Future perspectives for Portugal. Energy Policy, 39(9), 5465-5473. doi:10.1016/j.enpol.2011.05.016Battaglini, A., Komendantova, N., Brtnik, P., & Patt, A. (2012). Perception of barriers for expansion of electricity grids in the European Union. Energy Policy, 47, 254-259. doi:10.1016/j.enpol.2012.04.065‘European Commission 2010a. Energy 2020. A strategy for competitive sustainable and secure energy’. Technical Report Brussels November2011Beurskens P.V.L.W.M. Hekkenberg M.: ‘Renewable energy projections as published in the national renewable energy action plans of the european member states’. Technical Report European Environment Agency (EEA) November2011Coll-Mayor, D., Paget, M., & Lightner, E. (2007). Future intelligent power grids: Analysis of the vision in the European Union and the United States. Energy Policy, 35(4), 2453-2465. doi:10.1016/j.enpol.2006.09.001Passey, R., Spooner, T., MacGill, I., Watt, M., & Syngellakis, K. (2011). The potential impacts of grid-connected distributed generation and how to address them: A review of technical and non-technical factors. Energy Policy, 39(10), 6280-6290. doi:10.1016/j.enpol.2011.07.027Sangroniz N. Mora J.A. Teixeira M.D.: ‘Review of international grid codes for wind generation’ 2009‘Global Market Outlook for Photovoltaics Until 2016’. Technical Report European Photovoltaic Industry Association 2012. Available atwww.epia.orgKim S. Bollen M.: ‘Towards the development of a set of grid code requirements for wind farms: transient reactive power requirements’. Technical Report Available as Elforsk Report 13 : 04. Part 3 Report of Vindforsk Project V‐369 Vindforsk III January2013Tsili, M., & Papathanassiou, S. (2009). A review of grid code technical requirements for wind farms. IET Renewable Power Generation, 3(3), 308. doi:10.1049/iet-rpg.2008.0070Hossain, J., & Mahmud, A. (Eds.). (2014). Renewable Energy Integration. Green Energy and Technology. doi:10.1007/978-981-4585-27-9Sourkounis C. Tourou P.: ‘Grid code requirements for wind power integration in Europe’.Conf. Papers in Energy 2013 pp.1–9Voltage Ride-Through Capability Verification of Wind Turbines With Fully-Rated Converters Using Reachability Analysis. (2014). IEEE Transactions on Energy Conversion, 29(2), 392-405. doi:10.1109/tec.2013.2295168Mohseni, M., & Islam, S. M. (2012). Review of international grid codes for wind power integration: Diversity, technology and a case for global standard. Renewable and Sustainable Energy Reviews, 16(6), 3876-3890. doi:10.1016/j.rser.2012.03.039‘Royal Decree 1565/2010 by which regulates and modifies certain aspects of the activity of production of electric energy in special regime. (In spanish)’. Technical Report November2010Sourkounis C. Tourou P.: ‘Grid code requirements for wind power integration in Europe’.Conf. Papers in Science 2013Jiménez, F., Gómez-Lázaro, E., Fuentes, J. A., Molina-García, A., & Vigueras-Rodríguez, A. (2011). Validation of a double fed induction generator wind turbine model and wind farm verification following the Spanish grid code. Wind Energy, 15(4), 645-659. doi:10.1002/we.498Montoro D.: ‘Recommendations for unified technical regulations for grid‐connected PV systems’. Technical Report SUNRISE project – European Photovoltaic Industry Association the European Construction Industry Federation the European Association of Electrical Contractors International Union of Architects 2009. Available athttp://www.pvsunrise.eu/Merino, J., Mendoza-Araya, P., & Veganzones, C. (2014). State of the Art and Future Trends in Grid Codes Applicable to Isolated Electrical Systems. Energies, 7(12), 7936-7954. doi:10.3390/en7127936deAlmeida P. Barbosa P. Duque C.et al.: ‘Grid connection considerations for the integration of PV and wind sources’.IEEE 16th Int. Conf. on Harmonics and Quality of Power (ICHQP) May2014 pp.6–9‘Network code requirements for grid connection applicable to all generators’. Technical Report European Network of Transmission System Operators for Electricity ENTSO‐E October2013. Available athttps://www.entsoe.eu/Kagan N. Ferrari E. Matsuo N.et al.: ‘Influence of rms variation measurement protocols on electrical system performance indices for voltage sags and swells’.Proc. Ninth Int. Conf. on Harmonics and Quality of Power 2000 2000 vol.3 pp.790–795Bollen, M. H. J. (2003). Algorithms for characterizing measured three-phase unbalanced voltage dips. IEEE Transactions on Power Delivery, 18(3), 937-944. doi:10.1109/tpwrd.2003.813879Bollen M.H.: ‘Comparing voltage dip survey results’.Power Engineering Society Winter Meeting 2002 2002 vol.2 pp.1130–1134Moreno‐Muñoz A. de laRosa J.: ‘Voltage sag in highly automated factories’.Industry Applications Society Annual Meeting IAS'08 2008 pp.1–6Gomez-Lazaro, E., Fuentes, J. A., Molina-Garcia, A., & Canas-Carreton, M. (2009). Characterization and Visualization of Voltage Dips in Wind Power Installations. IEEE Transactions on Power Delivery, 24(4), 2071-2078. doi:10.1109/tpwrd.2009.2027513Gunther, E. W., & Mebta, H. (1995). A survey of distribution system power quality-preliminary results. IEEE Transactions on Power Delivery, 10(1), 322-329. doi:10.1109/61.368382Belloni F. Chiappa C. Chiumeo R.et al.: ‘Voltage dip measurements along MV lines vs primary substations measurements’.Int. Conf. on Renewable Energies and Power Quality (ICREPQ'12) March2012 pp.28–30Garcia-Sanchez, T., Gomez-Lazaro, E., Muljadi, E., Kessler, M., & Molina-Garcia, A. (2016). Statistical and Clustering Analysis for Disturbances: A Case Study of Voltage Dips in Wind Farms. IEEE Transactions on Power Delivery, 31(6), 2530-2537. doi:10.1109/tpwrd.2016.2522946Barrera Nunez V. Melendez Frigola J. Herraiz Jaramillo S.: ‘A survey on voltage sag events in power systems’.IEEE/PES Transmission and Distribution Conf. and Exposition: Latin America 2008 August2008 pp.1–3‘IEEE Standard 519‐1992: Recommended practices and requirements for harmonic control in electrical power systems’. Technical Report 1993. Available athttp://ieeexplore.ieee.org/servlet/opac?punumber=2227‘1159‐2009‐IEEE Recommended practice for monitoring electric power quality’. Technical Report June2009. Available athttp://ieeexplore.ieee.org/servlet/opac?punumber=515405

The phosphatase interactor NIPP1 regulates the occupancy of the histone methyltransferase EZH2 at Polycomb targets

Polycomb group (PcG) proteins are key regulators of stem-cell and cancer biology. They mainly act as repressors of differentiation and tumor-suppressor genes. One key silencing step involves the trimethylation of histone H3 on Lys27 (H3K27) by EZH2, a core component of the Polycomb Repressive Complex 2 (PRC2). The mechanism underlying the initial recruitment of mammalian PRC2 complexes is not well understood. Here, we show that NIPP1, a regulator of protein Ser/Thr phosphatase-1 (PP1), forms a complex with PP1 and PRC2 components on chromatin. The knockdown of NIPP1 or PP1 reduced the association of EZH2 with a subset of its target genes, whereas the overexpression of NIPP1 resulted in a retargeting of EZH2 from fully repressed to partially active PcG targets. However, the expression of a PP1-binding mutant of NIPP1 (NIPP1m) did not cause a redistribution of EZH2. Moreover, mapping of the chromatin binding sites with the DamID technique revealed that NIPP1 was associated with multiple PcG target genes, including the Homeobox A cluster, whereas NIPP1m showed a deficient binding at these loci. We propose that NIPP1 associates with a subset of PcG targets in a PP1-dependent manner and thereby contributes to the recruitment of the PRC2 complex

Protein phosphatase 1 regulates atypical mitotic and meiotic division in Plasmodium sexual stages

PP1 is a conserved eukaryotic serine/threonine phosphatase that regulates many aspects of mitosis and meiosis, often working in concert with other phosphatases, such as CDC14 and CDC25. The proliferative stages of the malaria parasite life cycle include sexual development within the mosquito vector, with male gamete formation characterized by an atypical rapid mitosis, consisting of three rounds of DNA synthesis, successive spindle formation with clustered kinetochores, and a meiotic stage during zygote to ookinete development following fertilization. It is unclear how PP1 is involved in these unusual processes. Using real-time livecell and ultrastructural imaging, conditional gene knockdown, RNA-seq and proteomic approaches, we show that Plasmodium PP1 is implicated in both mitotic exit and, potentially, establishing cell polarity during zygote development in the mosquito midgut, suggesting that small molecule inhibitors of PP1 should be explored for blocking parasite transmission