Spatiotemporal regulation of the Greatwall : PP2A axis is required for mitotic progression

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(1)Université de Montréal. Spatiotemporal regulation of the Greatwall ─ PP2A axis is required for mitotic progression par Peng Wang Département de Biochimie et Médecine Moléculaire Institut de Recherche en Immunologie et en Cancérologie (IRIC) Faculté de Médecine Thèse présentée à la Faculté de Médecine en vue de l’obtention du grade de PhD en Biochimie et Médecine Moléculaire. Septembre, 2014. © Peng Wang, 2014.

(2) Résumé Le cycle cellulaire est hautement régulé par la phosphorylation réversible de plusieurs effecteurs. La kinase dépendante des cyclines Cdk1 déclenche la mitose en induisant le bris de l’enveloppe nucléaire, la condensation des chromosomes et la formation du fuseau mitotique. Chez les animaux métazoaires, ces évènements sont contrés par la protéine phosphatase PP2AB55, qui déphosphoryle plusieurs substrats de Cdk1. La kinase Greatwall (Gwl) est activée par le complexe cycline B-Cdk1 en début de mitose et induit ensuite l’inhibition de PP2A-B55 via Endos/Arpp19. Toutefois, les mécanismes moléculaires qui régulent Gwl sont encore peu connus. Nous avons montré que Gwl a une activité s’opposant à PP2A-B55, qui collabore avec la kinase Polo pour assurer l’attachement du centrosome au noyau et la progression du cycle cellulaire dans le syncytium de l’embryon de la drosophile. Ensuite, nous avons trouvé dans des cellules de drosophile que Gwl est localisée au noyau pendant l’interphase, mais qu’elle se relocalise au cytoplasme dès la prophase, avant le bris de l’enveloppe nucléaire. Nous avons montré que cette translocation de Gwl est cruciale pour sa fonction et qu’elle dépend de la phosphorylation de plusieurs résidus de la région centrale de Gwl par les kinases Polo et Cdk1. Cette région centrale contient également deux séquences de localisation nucléaire (respectivement NLS1 et NLS2). De plus, nos résultats suggèrent que la phosphorylation de Gwl par la kinase Polo promeut sa liaison avec la protéine 14-3-3İ, ce qui favorise la rétention cytoplasmique de Gwl. Le rôle de Cdk1 dans cette translocation reste quant à lui inconnu. De plus, nous avons montré que le complexe cycline B-Cdk1 entre dans le noyau avant que Gwl ne soit transportée dans le cytoplasme. Cdk1 pourrait donc activer Gwl et phosphoryler ses substrats nucléaires, à l’abri de PP2A-B55 qui est largement cytoplasmique. Gwl est ensuite exclue du noyau et relocalisée dans le cytoplasme afin d’induire l’inhibition de PP2A-B55. Cela permet de synchroniser les événements de phosphorylation se produisant dans le noyau et dans le cytoplasme. Fait intéressant, un mécanisme de régulation de la localisation de Gwl similaire à cela a été découvert chez l’humain et chez la levure, suggérant que ce mécanisme est conservé entre différentes espèces.. Mots clés: Greatwall (Gwl), Cycline B-Cdk1, PP2A-B55, Cycle cellulaire, Mitose. i .

(3) Abstract: Reversible phosphorylation of proteins, triggered by cyclically activated kinases and phosphatases, is a key mechanism to control cell cycle progression. CyclinB-Cdk1 is a crucial kinase phosphorylating a large number of substrates to trigger mitotic entry. However, in metazoans, it is counteracted mainly by a Protein Phosphatase 2A carrying the B55 regulatory subunit (PP2A-B55). On the other hand, the Greatwall (Gwl) kinase is activated by CyclinBCdk1 upon mitotic entry and subsequently induces the inhibition of PP2A-B55 by Endos/Arpp19, thus promoting mitotic entry and maintenance. Nonetheless, the regulatory mechanisms of Gwl are less clear. We demonstrated that in Drosophila syncytial embryos, PP2A-B55 is negatively regulated by Gwl, but collaborates with Polo kinase to ensure both nucleus attachment of centrosome and faithful cell cycle progression. Later, we discovered that in Drosophila, the subcellular localization of Gwl changes dramatically throughout the cell cycle. Gwl is nuclear in interphase but suddenly becomes mostly cytoplasmic in prophase before nuclear envelope breakdown. Such translocation is important for Gwl’s function and requires the phosphorylation of Gwl by both Polo kinase and Cdk1 in the region containing two Nuclear Localization Signals (NLSs). Phosphorylation of Gwl by Polo likely promotes its association with14-3-3İ thereby promoting Gwl cytoplasmic retention, whereas Cdk1’s role in this translocation remains elusive. Moreover, I found that most cyclin B is imported into the nucleus before Gwl translocates to the cytoplasm. Therefore, Cdk1 can activate Gwl and phosphorylate its nuclear substrates without the perturbation of PP2A-B55 which is largely cytoplasmic. Subsequently, Gwl translocates into cytoplasm to mediate the inhibition of PP2A-B55 so that the phosphorylation events can be synchronized between the nucleus and the cytoplasm. Interestingly, similar spatial regulation of Gwl was also uncovered in mammal cells and in yeast, implying a conserved regulatory mechanism across species. Key words: Greatwall (Gwl), CyclinB-Cdk1, PP2A-B55, Cell cycle, Mitosis.. ii .

(4) Table of Contents Résumé ............................................................................................................................................ i Abstract.......................................................................................................................................... ii Figures list .................................................................................................................................. viii Tables list ...................................................................................................................................... xi Abbreviations list ........................................................................................................................ xii Acknowledgements .................................................................................................................... xiv Chapter 1: Introduction ...............................................................................................................1 Backgrounds and general organization of chapters ...........................................................2 1.1 The cell cycle .....................................................................................................................4 1.1.1 Interphase ....................................................................................................................4 1.1.2 M phase .......................................................................................................................6 1.1.3 Cytokinesis..................................................................................................................8 1.2 Cell cycle regulation .........................................................................................................8 1.2.1 The cyclins and cyclin-dependent kinases (Cdks) are the central components of the cell cycle control system ......................................................................................................9 1.2.1.1 Cdks and cyclins ..........................................................................................9 1.2.1.2 Cyclins regulate the substrate specificity of cyclin-Cdk1 complexes .......11 1.2.1.3 Compensatory mechanisms among cyclins and Cdks ..............................12 1.2.1.4 Reversible phosphorylation is an important mechanism which regulates Cdk1 activity ..........................................................................................................13 1.2.1.5 Other mechanisms regulating Cdks activity .............................................15 1.2.2 The cell cycle control system is cyclical proteolysis dependent...............................16 1.2.3 Cell cycle checkpoints ..............................................................................................19 1.2.3.1 G1/S checkpoint .........................................................................................20 1.2.3.2 Intra-S phase checkpoint ............................................................................20 1.2.3.3 G2/M checkpoint .......................................................................................21 1.2.3.4 M phase checkpoint ...................................................................................22 1.3 Drosophila melanogaster as a model..............................................................................23 1.4 Mitotic progression is tightly regulated by reversible phosphorylations ..................27 iii .

(5) 1.5 Greatwall kinase ņ PP2A-B55 phosphatase axis in mitotic progression control .....31 1.5.1 PP2A-B55, a multifunctional phosphatase, is implicated in a broad variety of cellular processes ...............................................................................................................31 1.5.1.1 PP2A structure ...........................................................................................31 1.5.1.2 PP2A-B55 assembly regulation and substrate recruitment ........................32 1.5.1.3 PP2A-B55 and cancers ..............................................................................34 1.5.1.4 PP2A-B55 and cytoskeleton stability ........................................................34 1.5.1.5 PP2A-B55 and centrosome maturation......................................................35 1.5.1.6 PP2A-B55, as a major opponent of cyclin B-Cdk1 in higher eukaryotes, must be inhibited at mitotic onset and reactivated upon mitotic exit ....................36 1.5.2 The Greatwall kinase, a defense system, is set up by Cdk1 upon mitotic entry and disabled during M phase exit .............................................................................................38 1.5.2.1 The role of Greatwall in mitotic progression .............................................38 1.5.2.2 Greatwall substrates and non-mitotic functions.........................................39 1.5.2.3 The regulation of Greatwall .......................................................................40 1.6 References........................................................................................................................45 Chapter 2: PP2A-Tws Is Antagonized by Greatwall and Collaborates with Polo for Cell Cycle Progression and Centrosome Attachment to Nuclei in Drosophila Embryos .............57 2.1 Abstract ...........................................................................................................................58 2.2 Author summary.............................................................................................................59 2.3 Introduction ....................................................................................................................59 2.4 Results ..............................................................................................................................61 2.4.1 Polo function is required for proper cohesion between centrosomes and nuclei ......61 2.4.2 Transiently detached centrosomes are recaptured by mitotic spindles .....................64 2.4.3 A genetic screen identifies PP2A-Tws as a strong functional interactor of Polo and Gwl in the syncytial embryo ..............................................................................................66 2.4.4 Greatwall antagonizes PP2A-Tws in M-phase .........................................................69 2.4.5 PP2A-Tws collaborates with Polo to ensure centrosome cohesion to nuclei and nuclear divisions in the syncytial embryo ..........................................................................73 iv .

(6) 2.5 Discussions.......................................................................................................................74 2.5.1 Regulation of mitosis and centrosome attachment in the rapid embryonic cell cycles ............................................................................................................................................74 2.5.2 A conserved pathway controls M-phase entry and exit ............................................76 2.6 Materials and methods ...................................................................................................77 2.6.1 Fly husbandry, genetic screen and fertility tests .......................................................77 2.6.2 Immunofluorescence and confocal microscopy........................................................78 2.6.3 Time-lapse microscopy .............................................................................................78 2.6.4 Chemical inhibition of Polo ......................................................................................78 2.7 Acknowledgements .........................................................................................................79 2.8 References........................................................................................................................79 Chapter 3: Cell Cycle Regulation of Greatwall Kinase Nuclear Localization Facilitates Mitotic Progression .....................................................................................................................83 3.1 Abstract ...........................................................................................................................85 3.2 Introduction ....................................................................................................................85 3.3 Results ..............................................................................................................................87 3.3.1 The localization of Gwl is cell-cycle regulated ........................................................87 3.3.2 Gwl contains two functional NLS motifs in its central region .................................89 3.3.3 Nuclear localization of Greatwall is required for its function ..................................90 3.3.4 Polo kinase interacts with and phosphorylates Gwl .................................................93 3.3.5 Polo activity promotes the cytoplasmic localization of Gwl in prophase.................96 3.3.6 14-3-3İ collaborates with Polo promote the cytoplasmic localization of Gwl .......100 3.3.7 Cdk1 may contribute to regulate Gwl localization .................................................102 3.3.8 Failure to exclude Gwl from the nucleus leads to delays in mitosis .......................102 3.3.9 Excessive Polo activity in Gwl-compromised embryos leads to defective mitotic entry .................................................................................................................................103 3.4 Discussion ......................................................................................................................105 3.4.1 A new level of regulation of Gwl in the cell cycle .................................................105 v .

(7) 3.4.2 A mechanism directly linking Gwl and Polo ..........................................................107 3.4.3 A mode of regulation that could be generally conserved .......................................108 3.5 Materials and methods .................................................................................................109 3.5.1 Fly culture and transgenesis ....................................................................................109 3.5.2 DNA constructs.......................................................................................................109 3.5.3 Cell culture ..............................................................................................................110 3.5.4 Immunofluorescence and Western blotting ............................................................110 3.5.5 Microscopy .............................................................................................................110 3.5.6 Affinity purifications ..............................................................................................111 3.5.7 Kinase assays ..........................................................................................................111 3.5.8 Phosphorylation site mapping .................................................................................112 3.5.9 RNA interference ....................................................................................................112 3.5.10 Preparation of GST-fusion proteins ......................................................................112 3.5.11 GST-pulldown assay .............................................................................................113 3.5.12 Online supplemental Materials .............................................................................113 3.6 Acknowledgements .......................................................................................................114 3.7 Supplementary figures .................................................................................................115 3.8 References......................................................................................................................123 Chapter 4: Discussion and perspectives ..................................................................................126 4.1 Do PP2A-Tws and Polo function in the same or parallel pathway ..........................127 4.2 Do Gwl and Polo collaborate with or antagonize each other ...................................128 4.3 How important is Gwl for mitotic progression ..........................................................131 4.4 Spatiotemporal regulation of kinases and phosphatases is required for mitotic progression ..........................................................................................................................133 4.5 The regulation of Gwl, Endos/Arpp19, and PP2A-B55 at mitotic exit ....................137 4.6 Perspectives ...................................................................................................................140. vi .

(8) 4.7 References .....................................................................................................................140. vii .

(9) FIGURES LIST Figure 1.1. The cell cycle phases. Figure 1.2. Different stages of mitosis followed by cytokinesis. Figure 1.3. Cyclin and cyclin-dependent kinase. Figure 1.4. Each of the main phases of the cell cycle ņ G1, S (when DNA synthesis occurs), G2 and mitosis ņ is controlled by a cyclin ņ cyclin-dependent kinase (CDK) complex. Figure 1.5. The main reversible phosphorylation mechanisms required for Cdk1 activation upon mitotic entry. Figure 1.6. The ubiquitination pathway. Figure 1.7. Global roles for APC/C and SCF1 in the core cell cycle. Figure 1.8. A screen for the interacting genes with polo identified PP2A regulatory subunit twins. Figure 1.9. The principle of PrA affinity purification coupled to mass spectrometry (MS) of protein complex from Drosophila cultured cells and syncytial embryos. Figure 1.10. Theoretical prediction of the modes of Cdk1 activation controlled by different mechanisms. Figure 1.11. Overall structure of the PP2A holoenzyme involving the BĮ subunit. Figure 1.12. Mechanisms of activation of the Gwl kinase. Figure 1.13. A model for Gwl activation. Figure 1.14. Spatial model for the Gwl-PP2A axis in the control of mitotic entry. Figure 2.1. The Polo kinase is required for proper centrosome attachment to nuclei in syncytial embryos. Figure 2.2. Detached centrosomes from nuclei can be recaptured by the mitotic spindles. viii .

(10) Figure 2.3. A screen for genes functioning with polo identifies the PP2A subunit genes twins and microtubule star. Figure 2.4. Greatwall antagonizes PP2A-Tws in meiosis and mitosis. Figure 2.5. PP2A-Tws collaborates with Polo to promote cell cycle progression and centrosome cohesion to nuclei. Figure 3.1. The localization of Gwl is cell-cycle regulated. Figure 3.2. Gwl contains two essential NLS motifs in its central region. Figure 3.3. Nuclear localization of Greatwall is required for its function. Figure 3.4. Polo interacts with and phosphorylates Gwl. Figure 3.5. Polo activity promotes the cytoplasmic localization of Gwl in prophase. Figure 3.6. Exclusion of Gwl from the nucleus in prophase requires Polo and Cdk consensus sites and is required for timely mitotic progression. Figure 3.7. 14-3-3İ collaborates with Polo to promote the cytoplasmic localization of Gwl. Figure 3.8. Misregulation of Gwl by Polo interferes with cell cycle progression. Figure 3.9. An integrative model for the spatial regulation of Gwl. Figure S3.1. The localization of Gwl is cell cycle regulated (complement to Fig.1). Figure S3.2. Sequence alignment between Gwl orthologues from Drosophila and mosquito species. Figure S3.3. Phosphorylation site mapping. Figure S3.4. Polo phosphorylation of Gwl promotes its cytoplasmic localization (complement to Fig.3.5). Figure S3.5. Cyclin B is imported in the nucleus while Gwl is still in the nucleus (red arrowhead). ix .

(11) Figure 4.1. A model for spatiotemporal regulation of the Gwl ņ PP2A axis in the control of mitotic entry and exit.. x .

(12) TABLE LIST Table 1.1. The major cyclins and Cdks in vertebrates.. xi .

(13) ABBREVIATIONS LIST Arpp: cAMP-regulated phosphoprotein APC/C: Anaphase Promoting Complex/Cyclosome ATM: Ataxia Telangiectasia Mutated ATP: Adenosine Triphosphate ATR: Ataxia Telangiectasia Related CAK: Cdk Activating Kinase cAMP: cyclic Adenosine MonoPhosphate Cdc: Cell division cycle Cdh1: Cdc20 homolog 1 Cdk: Cyclin dependent kinase Chk: Checkpoint Protein Kinase CKI: Cdk Inhibitor DNA: DeoxyriboNucleic Acid Endos/Ensa: Į-Endosulfine/Endosulfine alpha FEAR: Cdc Fourteen Early Anaphase Release GFP: Green fluorescent protein Gwl: Greatwall kD: kiloDalton MEN: Mitotic Exit Network MPF: Maturation Promoting Factor MTOC: Micro Tubule Organizing Center NEBD: Nuclear Envelop BreakDown NES: Nuclear Export Signal NLS: Nuclear Localization Signal OA: Okadaic Acid PLK: Polo-Like Kinase xii .

(14) PP2A: Protein Phosphatase 2A PP1: Protein Phosphatase 1 pRb: Retinoblastoma Protein RNA: RiboNucleic Acid SAC: Spindle Assembly Checkpoint SCF: Skp/Cullin/F-Box TORC1: Tor complex 1. xiii .

(15) ACKNOWLEDGEMENTS First, I would like to thank Dr Vincent Archambault, not just because he gave me the opportunity to perform my PhD training in his laboratory, he also helped me so much throughout the course of my PhD projects. He not only taught me almost all the techniques frequently used during my PhD training but also guided me in my fellowship applications, my postdoctoral application, my English, my French, writing techniques…… Without his generous help, I need certainly several years more to complete this thesis. I am also thankful to all the past and present members of the Archambault’s laboratory. Especially, I want to thank to very kind Xavier Pinson and adorable Karine Normandin, they helped me so much and it is always very enjoyable to talk with them. I want to thank to very talented David Kachaner, nice Maxime Cormier, hard-working Haytham Mehsen, and young elites Myreille Larouche, Élizabeth Michaud and Guillaume Lépine. It is very agreeable to work with all these wonderful people and I am grateful for their support and friendship. Thanks Haytham for his very helpful advices for my English writing. I would like to thank the members of my thesis committee, Dr Gerardo Ferbeyre, Dr Eric Lécuyer, Dr Philip Branton, for taking time to evaluate my work. I would like to thank to Dr Jacques Montagne, Dr Jean-Philippe Parvy, Dr Annie Sainsard-Chanet, Dr Christian Vélot, without their help and support I couldn’t get the chance to continue my PhD study. I would like to thank Dr Marc Therrien, Dr Sébastien Carréno, Dr Gregory Emery, Dr Benjamin Kwok, Dr Damien D’Amours and all fly-lab members, for their constructive advices and guidance for my research projects. Finally, I would like to thank to my loving wife Haijin, she encouraged me to continue my study in biology. I am so sorry to have left her alone for numerous weekends and evenings because of lab work, her support and consideration have been essential in the last five years. I also want to thank my lovely daughter Emma Lujia for her sweet smile what’s her special encouragement to me. I thank my father Chuanfa and my mother Guizhi for their selfless love and support.. xiv .

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