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Role of MASTL in Mammals: Molecular Functions and Physiological Relevance

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Role of MASTL in Mammals: Molecular Functions and Physiological Relevance Departamento de Biología Molecular Facultad de Ciencias Universidad Autónoma de Madrid Role of MASTL in mammals: Molecular functions and physiological relevance Belén Sanz Castillo Degree in Biotechnology Thesis Directors: Dr. Marcos Malumbres Dra. Mónica Álvarez Fernández Centro Nacional de Investigaciones Oncológicas (CNIO) Madrid, 2017 Marcos Malumbres Martínez, Jefe del Grupo de División Celular y Cáncer del Centro Nacional de Investigaciones Oncológicas (CNIO) y, Mónica Álvarez Fernández, Investigadora del Grupo de División Celular y Cáncer del CNIO. Certifican: que Belén Sanz Castillo ha realizado bajo su dirección el trabajo de Tesis Doctoral titulado: ”Role of MASTL in mammals: Molecular functions and physiological relevance” en el Centro Nacional de Investigaciones Oncológicas y tutelada en el departamento de Biología Molecular de la Universidad Autónoma de Madrid. Revisado el presente trabajo, considera que reúne todas las condiciones requeridas por la legislación vigente y la originalidad y calidad científica para su presentación y defensa con el fin de optar al grado de Doctor. Y para que conste donde proceda, firmamos el presente certificado. Dr. Marcos Malumbres Martínez Dra. Mónica Álvarez Fernández 3 A mi familia “Tu llegada allí es tu destino. Mas no apresures nunca el viaje. Mejor que dure muchos años y atracar, viejo ya, en la isla, enriquecido de cuanto ganaste en el camino” Constantino Cavafis 5 Acknowledgements Esta tesis hubiera sido completamente imposible sin la ayuda y apoyo de muchas personas. En primer lugar Marcos, muchas gracias por haberme acogido en tu laboratorio, aún recuerdo cuando llegué cargada de ilusión y ahora pienso que no podría haber escogido un sitio mejor. Gracias por tu entusiasmo, pasión y fuerza que nos transmites a cada uno de los que trabajamos contigo. Por supuesto quiero agradecerte haberme dado la oportunidad de trabajar en este apasionante proyecto junto a Mónica. No podía haber tenido una mejor co-directora de tesis, ella ha sido mi gran apoyo y todo esto hubiera sido completamente imposible sin tu trabajo, ayuda, compresión, y apoyo incondicional. Gracias por escucharme, por nuestras innumerables discusiones, ideas, y por estar siempre ahí. No tengo palabras para agradecerte todo lo que me has apoyado, así que simplemente mil gracias y porque has conseguido sacar lo mejor de mí. No ha sido fácil encontrar el camino pero al final, como dice Guille, “todo esfuerzo tiene su recompensa”. Y es verdad, Guille es una fuente de sabiduría, en todos los sentidos. El otro gran pilar de este laboratorio, incansable e insaciable por la ciencia, siempre dispuesto a ayudar, a colaborar y a tenderte una mano en todo lo que necesitas. María Salazar, gran científica y mejor persona. Aportas serenidad, siempre son brillantes ideas, muchas gracias por tus consejos y tu ayuda en este proyecto. Bego, siempre con una sonrisa dispuesta a ayudar, por la alegría que transmites, y por todo lo que me has enseñado y ayudado también con los ratoncillos. Carol, muchas gracias por escucharme, ayudarme y preocuparte por mí. De vosotros he aprendido que no hay que rendirse nunca. A María Guirola gracias por cuidar del labo y hacernos el trabajo mucho más sencillo. Y a la juventud del laboratorio, por el buen ambiente que creáis y vuestra alegría. Filipa, María Maroto, María Sanz y Diego, compañeros de tesis, juntos hemos aprendido mucho, gracias por vuestro apoyo y por ser como sois! Eli siempre con una sonrisa y por el buen rollo que transmites. No podía olvidarme de Aisha, la persona con más mano para los ratoncillos que conozco!, sabes que sin tu ayuda los experimentos de los ratoncillos hubiera sido imposible, hasta a mi me salvaste alguna vez ☺. Paloma, tu eres una más del labo, ha sido muy divertido pero por supuesto gracias siempre por tu ayuda. Alejandro por tu granito de arena con los mutantes, buen trabajo! Y a los que estáis recién llegados, Pepe y Bea, muchas gracias también. Que voy a deciros, creáis el mejor ambiente para trabajar y ha sido un placer trabajar con vosotros, esta tesis hubiera sido imposible sin vuestro apoyo! No podía olvidarme de los que ya se fueron, pero que pasamos mucho tiempo juntos. Manu, Elena, Ale, Marianna, creía que no iba a llegar este momento pero ya estoy aquí! 9 Vosotros me acogisteis cuando llegué al labo y aprendí mucho de todos vosotros. Elena, gracias por tus consejos y Marianna, por tu fuerza, y consejos. A David, Iñaki y Eva, fue un placer trabajar con vosotros, al final estuvimos juntos casi 3 años, como pasa el tiempo… David, siempre dispuesto a ayudar, quiero agradecerte todo lo que nos ayudaste y a mi especialmente también con los ratoncillos. Iñaki por tu serenidad y Eva por esas risas. Y también me acuerdo de esas cervezas, botellines, cenas y esas risas que, de vez en cuando, nos hemos echado. Gracias a todos! 10 Summary/Resumen Summary/Resumen MASTL is a Ser/Thr kinase first identified in Drosophila and Xenopus as a protein required for mitosis. MASTL inhibits, through phosphorylation of ENSA and ARPP19, the phosphatase PP2A in complex with the B55 family of regulatory subunits. In this work, we describe that Mastl is essential for mitotic progression in mouse embryonic fibroblasts (MEFs) derived from a conditional loss-of-function mouse model for Mastl, through inhibition of PP2A/B55 complexes. Mastl mediated inhibition of PP2A/B55 prevents premature dephosphorylation of CDK1 phosphosubstrates that are essential for chromosome condensation and segregation, and proper progression through mitosis. This proliferative role of Mastl is essential in vitro in cell culture and in vivo. Work done in a mouse model for Mastl shows that ablation of Mastl in young mice results in mitotic aberrations, severe proliferative defects and rapid death after Mastl depletion. Deletion of Mastl in elder mice, in contrast, causes less severe proliferative problems and improved survival. Nevertheless, Mastl depletion in adult mice also leads, in the long term, to impaired tissue regeneration, altered tissue homeostasis and eventual death of the mice. Although PP2A/B55 is a phosphatase that regulates multiple cellular processes, the only function attributed so far to the PP2A/B55-inhibitory kinase MASTL in mammals is its role in mitosis. Aiming to identify new functions of MASTL, we have explored other potential cell cycle- dependent and independent functions of MASTL. Whereas Mastl was not required for S-phase entry or maintenance of the quiescence state, we have found a new and unexplored role for the MASTL-PP2A/B55 pathway in the control of insulin signaling downstream of mTORC1. The mTORC1/S6K1 axis triggers a negative feedback loop that inhibits the upstream PI3K pathway, which in turn, is also important for controlling mTORC1 activity. We have found that MASTL-mediated PP2A/B55 inhibition prevents the activating dephosphorylation of the feedback target proteins, IRS1 and GRB10, and is required to fine-tune the feedback-mediated inhibition of the PI3K/AKT pathway and its metabolic consequences. Interestingly, MASTL activity is also modulated downstream of the mTORC1/S6K1 axis in conditions of feedback activity. This feedback loop is physiologically relevant in vivo for metabolic diseases involving insulin resistance, such as obesity or type 2 diabetes. Importantly, we have found that depletion of Mastl improves glucose tolerance in a mouse model of high fat diet-induced obesity, and lowers basal glycaemia in old mice. This data together indicate that MASTL, through the inhibition of PP2A/B55, has a new mitotic-independent function in mammals in the control of PI3K/AKT- mTORC1 signaling and cooperates to control glucose homeostasis in vivo. 14 Summary/Resumen MASTL fue identificada por primera vez en Drosophila y Xenopus como una quinasa requerida para mitosis. MASTL inhibe, a través de la fosforilación de ENSA y ARPP19, los complejos de la fosfatasa PP2A con subunidades reguladoras de la familia B55. En este trabajo hemos demostrado que MASTL también es esencial para la progresión a través de mitosis en fibroblastos embrionarios de ratón del modelo condicional de pérdida de función de Mastl, mediante la inhibición de PP2A/B55. La inhibición de PP2A/B55 por MASTL previene la desfosforilación prematura de los epítopos fosforilados por CDK1, los cuales son esenciales para la condensación y segregación de los cromosomas, y la progresión a través de mitosis. Los estudios realizados en el modelo condicional demostraron que la eliminación de Mastl en ratones jóvenes resulta en aberraciones mitóticas y defectos proliferativos severos que comprometen la supervivencia rápidamente después de la eliminación de Mastl. Por el contrario, la depleción de Mastl en ratones adultos, resulta en defectos proliferativos menos severos y mayor supervivencia. La eliminación de Mastl a largo plazo resulta, sin embargo, en pérdida de capacidad regenerativa y homeostasis de los tejidos que finalmente provoca la muerte. Aunque PP2A/B55 es una fosfatasa que regula múltiples procesos, la única función atribuida hasta la fecha a su quinasa inhibitoria, MASTL, es por ahora su papel en mitosis. Mientras que MASTL no es esencial para la entrada en fase S o para el mantenimiento de quiescencia, hemos encontrado una nueva función de la vía MASTL-PP2A/B55 en el control de la ruta de señalización de insulina regulada por mTORC1. El módulo PI3K/AKT-mTORC1 acopla la presencia de nutrientes, energía y factores de crecimiento con el crecimiento, proliferación y metabolismo celular. mTORC1/S6K1 lanza un circuito de retroalimentación negativo (negative feedback loop) que inhibe la vía de PI3K/AKT después de su activación. Hemos descubierto que la inhibición de PP2A/B55 mediada por MASTL previene la desfosforilación de las proteínas fosforiladas por mTORC1/S6K1, IRS y GRB10, y es necesaria para regular la inhibición de AKT mediada por mTORC1 y sus consecuencias metabólicas. Además, la actividad catalítica de MASTL está regulada de manera dependiente de mTORC1/S6K1 en condiciones de inhibición de AKT.
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