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Universidad Autónoma De Madrid Programa De UNIVERSIDAD AUTÓNOMA DE MADRID PROGRAMA DE DOCTORADO EN BIOCIENCIAS MOLECULARES FUNCTIONAL RELEVANCE OF PP2A-B55 PHOSPHATASES IN THE MAMMALIAN CELL CYCLE TESIS DOCTORAL María Sanz Flores Madrid, 2017 DEPARTAMENTO DE BIOLOGÍA MOLECULAR FACULTAD DE CIENCIAS UNIVERSIDAD AUTÓNOMA DE MADRID FUNCTIONAL RELEVANCE OF PP2A-B55 PHOSPHATASES IN THE MAMMALIAN CELL CYCLE María Sanz Flores Licenciada en Biología Director: Marcos Malumbres Martínez Codirectora: Mónica Álvarez Fernández Centro Nacional de Investigaciones Oncológicas (CNIO) Madrid, 2017 Mónica Álvarez Fernández, Investigadora del Grupo de División Celular y Cáncer del Centro Nacional de Investigaciones Oncológicas (CNIO) y, Marcos Malumbres Martínez, Jefe del Grupo de División Celular y Cáncer del Centro Nacional de Investigaciones Oncológicas (CNIO) Certifican: que María Sanz Flores ha realizado bajo su dirección el trabajo de Tesis Doctoral titulado: FUNCTIONAL RELEVANCE OF PP2A-B55 PHOSPHATASES IN THE MAMMALIAN CELL CYCLE Revisado el presente trabajo, consideran que reúne todos los méritos necesarios para su presentación y defensa con el fin de optar al grado de Doctor por la Universidad Autónoma de Madrid. Mónica Álvarez Fernández Marcos Malumbres RESUMEN/SUMMARY Resumen RESUMEN La fosforilación reversible de proteínas es un mecanismo esencial en la regulación del ciclo celular. Mientras que el papel llevado a cabo en mitosis por las proteínas quinasa ha sido profundamente caracterizado, la identidad y la función específica de las fosfatasas en la mitosis de mamíferos está aún por determinar. La proteína fosfatasa 2A (PP2A) es una fosfatasa de residuos serina y treonina de gran importancia en células eucarióticas. Existen evidencias de que los complejos formados por PP2A con subunidades reguladoras de la familia B55 juegan un importante papel en la desfosforilación de sustratos de quinasas dependiente de ciclina (CDKs) durante mitosis en diferentes organismos. Este hecho sugiere su posible relevancia también en la mitosis de mamíferos. Con el objetivo de dilucidar la función de estos complejos PP2A-B55 en mitosis en mamíferos, hemos generado modelos de ratón deficientes para los genes Ppp2r2a (B55α) y Ppp2r2d (B55δ) que codifican dos de las cuatro isoformas de esta familia de subunidades reguladoras; en particular, aquellas consideradas ubicuas y previamente relacionadas con el ciclo celular. El estudio de estos modelos ha revelado que B55α, pero no B55δ, es necesaria durante el desarrollo embrionario y resulta esencial para la supervivencia del animal. Además, a nivel celular, ambas isoformas tienen funciones específicas y redundantes. Las células deficientes para los complejos PP2A-B55 presentan alteraciones en la duración de mitosis y en la segregación cromosómica que ocurre en esta etapa del ciclo celular, dando lugar a defectos de proliferación. El análisis detallado de la progresión del ciclo celular en células deficientes para B55 ha revelado un nuevo papel de estos complejos en la agrupación cromosómica durante mitosis, mediada al menos parcialmente por la proteína pericromosómica Ki-67. El tratamiento con compuestos que afectan la polimerización de microtúbulos, en las células deficientes para B55, provoca dispersión cromosómica durante mitosis, una acumulación de la proteína Ki-67 y, en algunos casos, muerte celular. Estos datos ponen de manifiesto la importancia de estos complejos en la regulación de mitosis en mamíferos y en la respuesta a los fármacos que afectan la polimerización de microtúbulos, los cuales se utilizan frecuentemente en tratamientos contra el cáncer. 9 Summary SUMMARY Reversible protein phosphorylation is an essential mechanism of cell cycle regulation. Whereas the role of mitotic kinases has been deeply characterized, the identity and specific functions of mitotic phosphatases in mammalian cells has not been fully resolved yet. Protein phosphatase 2A (PP2A) is a major serine/threonine phosphatase in eukaryotic cells and there is evidence that PP2A complexes containing the B55 family of regulatory subunits play a key role in dephosphorylating CDK substrates during mitosis in different organisms. This fact makes these phosphatase complexes might be important for mitosis in mammals. To address the functional relevance of those PP2A-B55 complexes in mammalian cell cycle, we have generated loss-of-function mouse models for Ppp2r2a (B55α) and Ppp2r2d (B55δ), which encode the ubiquitous and cell cycle-related isoforms out of the four existing ones in mammals (B55α, β, γ, δ). Using these models we have found that B55α, but not B55δ, is required during late embryonic development and therefore essential for mouse survival. Moreover, at the cellular level, both isoforms have specific and overlapping roles in cell cycle regulation. PP2A-B55-null cells display defects in timing and chromosome segregation during mitosis resulting in impaired proliferation. Interestingly, analysis of cell cycle progression in B55-null cells has also revealed a new role for PP2A/B55 complexes in chromosome clustering during mitosis, which is mediated through the perichromosomal protein Ki-67. Treatment of B55 deficient cells with microtubule depolymerizing drugs leads to massive chromosome scattering in mitosis, excessive Ki67 accumulation and eventually mitotic cell death. These data highlight the importance of these phosphatase complexes in regulating mammalian mitosis and the response to microtubule poisons, a common chemotherapeutic reagent used for cancer treatment. 11 Index INDEX RESUMEN .................................................................................................................................... 9 SUMMARY ................................................................................................................................ 11 ABBREVIATIONS ..................................................................................................................... 17 1. INTRODUCTION ............................................................................................................... 21 1.1. Mitosis in the mammalian cell division cycle ................................................................. 21 1.1.1. Phases of mitosis ..................................................................................................... 21 1.1.2. Molecular mechanisms of mitosis ....................................................................... 23 1.1.2.1. Mitotic entry and progression to metaphase ................................................ 23 1.1.2.2. Metaphase to anaphase transition and mitotic exit ...................................... 24 1.1.2.3. Dephosphorylation as an essential step of mitotic exit ............................... 25 1.2. Protein phosphatase 2A (PP2A) ...................................................................................... 26 1.2.1. PP2A structure and function ............................................................................... 26 1.2.2. Mouse models of PP2A ....................................................................................... 28 1.2.3. PP2A and cancer ................................................................................................. 29 1.3. PP2A-B55 complexes ..................................................................................................... 30 1.3.1. Function of PP2A-B55 in cell cycle progression ................................................ 31 1.2.1. Regulation of PP2A-B55 complexes by the kinase MASTL (Greatwall) ....... 32 1.2.2. Role of PP2A-B55 in other cell cycle phases .................................................. 33 1.2.3. Cell cycle independent functions of PP2A-B55 .............................................. 33 1.3. The mitotic chromosome ................................................................................................. 34 1.3.1. The perichromosomal layer ................................................................................. 36 1.3.2. Ki67: more than a proliferation marker ........................................................... 36 2. OBJECTIVES ..................................................................................................................... 41 3. MATERIALS AND METHODS ........................................................................................ 45 3.1. Genetically modified mouse models ............................................................................... 45 3.1.1. Animal housing ................................................................................................... 45 3.1.2. Generation of genetically modified mouse models ............................................. 45 3.1.2.1. Construction of targeting vectors ................................................................ 45 3.1.2.2. Generation of quimeras ............................................................................... 45 3.1.2.3. Generation of Ppp2r2a and Ppp2r2d alleles ................................................ 46 13 Index 3.1.3. Mouse genotyping ............................................................................................... 46 3.1.4. Histological and immunohistochemical analysis ................................................ 47 3.2. Generation of PP2A-B55 antibodies ............................................................................... 47 3.3. Cell culture ...................................................................................................................... 50 3.3.1. MEFs extraction and culture ..............................................................................
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