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The Functional Characterisation of MOB1-Regulated Hippo Core Cassette Kinase Signalling

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The Functional Characterisation of MOB1-Regulated Hippo Core Cassette Kinase Signalling The functional characterisation of MOB1-regulated Hippo core cassette kinase signalling Yavuz Kulaberoglu University College London Cancer Institute PhD Supervisor: Dr. Alexander Hergovich A thesis submitted for the degree of Doctor of Philosophy 2017 Declaration I, Yavuz Kulaberoglu, have read and understood the University Regulations on Cheating and Plagiarism and by submitting this work confirm that it is my own and does not contain any unacknowledged work from any other handwritten, printed or Internet source. I further confirm that I have acknowledged all sources used and quoted in this work. I understand that penalties can be applied in cases of proven cheating and plagiarism. If this work is to be submitted in both electronic and paper copy I certify that both copies are the same. I understand that this work may now or in future be submitted for checking by the TurnitIn plagiarism detection service. i | P a g e Abstract The Hippo tumour suppressor pathway regulates tissue growth by co-ordinating cell death, proliferation and differentiation. The central Hippo core cassette consists of MST1/2, LATS1/2, NDR1/2 and MOB1, with recent studies of MOB1 knockout mice indicating MOB1 functions as the central hub of Hippo signalling. However, it has remained unknown which interactions of MOB1 with the Hippo core cassette kinases MST1/2, LATS1/2 and NDR1/2 are required for normal life and tumour suppression. Therefore, my PhD project focused on deciphering the complex protein-protein interactions of MOB1 with MST1/2, LATS1/2 and NDR1/2. To do so, we generated a series of MOB1 variants that are selectively impaired in their binding to NDR1/2 (Trc), MST1/2 (Hippo), or LATS1/2 (Warts) in human (fly) cells. Using these selective loss- of-interaction (SLOI) variants we studied the effects of MOB1 overexpression on the proliferation and anchorage-independent growth of human cancer cell lines, thereby establishing which MOB1 interactions are required for the tumour suppressive role of MOB1. Equally importantly, we found that human MOB1 can restore the survival of mats loss-of-function (LOF) in Drosophila. By generating and studying transgenic flies expressing our SLOI MOB1 variants combined with Mats LOF, we discovered that the Hippo/MOB1A interaction is dispensable for fly development and reproduction, while the Trc/MOB1A and Wts/MOB1A interactions are essential. Taken together, my PhD thesis defined a completely novel panel of MOB1 SLOI variants to study the importance of MOB1 interactions. By studying transgenic human cells lines and flies expressing these SLOI variants we delineated which MOB1 interactions are essential for life of a complex multicellular organisms and for tumour suppression in human cancer cells. Collectively, our findings suggest that the Hippo/MOB1 interaction is neither required for life nor for tumour suppression, while the other interactions of MOB1 with Hippo core cassette kinases are essential in a context-dependent manner. ii | P a g e Acknowledgements First of all, I would like to thank so much to Alex, for being such a supportive, motivational supervisor, for giving me an opportunity I would have never obtained in my life to work in such a motivating scientific environment and for being such a patient and tolerant to my “weak” English at the very beginning. I remember the first time I came in to meet Alex about my PhD project. At that time, Alex was outlining the PhD project and I remember well that I did not understand even one single word. Then, I remembered the famous saying from Richard Branson “If someone offers you an amazing opportunity and you are not sure you can do it, say YES, and then learn how to do it later”. All of a sudden, someone came in and asked Alex “Who is he”, after seeing me. He replied, “He is a new PhD student”. She answered, “Oooo, he is a new victim, then”, while smiling Alex replied again, “No, he is a new victor”. And, I started thinking, like “Am I going to achieve it?”. My gut feeling was telling me, “Yes, you can”. And I listened to my gut feeling. Also, I would like to thank all the members of the Hergovich Lab, especially to Ramazan, for being such a friendly and supportive. Also, my big thanks go to Nic Tapon, Ivana Bjedov, and Maxine Holder for being such a helpful in my Drosophila studies. Lastly, my big thanks go to the folks of Arnos grove house for being supportive. It would be shame for me if I forget to thank Ufuk Ucar for supporting me physiologically. iii | P a g e Turkish Acknowledgements Değerli Annecim, Babacım ve Amcacım (Metin Kulaberoglu), Size nasıl teşekkür edeceğimi gerçekten bilmiyorum. Daha doğrusu size ne kadar teşekkür etsem, bu yeterli olmaz. Anadolu lisesine kazandığımda, daha iyi eğitim alabilmem için, yediklerinden kısarak beni özel okula gönderdiğinizi, anneciğimin altınlarını satıp, bizlerin eğitimine verdiğini unutamam. Alinlerin terleriyle kazandıkları kazançları, eğitimime yatirdiğiniz hiç unutulur mu? Yaptığınız bu fedakârlıklar her zaman aklımda ve hiç unutmayacağım. Bende sizlerin bu fedakârlıklarına karşılık, Allah şahittir, canimi dişime takip calistim ve elimden geleni yaptım. Hiç şüpheniz olmasın, size utandıracak hiçbir şey yapmadım. Dünyanın en iyi 25. Üniversitesinde yüksek lisans eğitimimi tamamladım ve dünyanın en iyi 10. Üniversitesinde, an itibariyle doktora eğitimimi bitirmek üzereyim. Ve dünyanın en iyi 4. Üniversitesi olan Cambridge Üniversitesinden 3 sene, post-doc teklifi aldım. Bu basarilari elde ederken, Allah`tan başka kimseden yârdim talep etmedim. Baba yarim olan, Metin amcamın, ailemin bu zorlu süreçlerinde hem maddi hem de manevi olarak yanında olmasını da unutmak çok büyük vefasızlık olur. Amca, bu elde edilmiş başarılarda, senin de çok büyük payın var ve sana nasıl teşekkür edeceğimi bilmiyorum!!! Ve bu basarilari elde etmemde, maddi ve manevi olarak yanımda olan, Yasin abimi, kardeşim Beyza’yı, babaannemi, Gülay yengemi unutmak ise büyük vefasızlık olur. Hepinizi çok seviyorum ve teşekkür ediyorum… Son olarak, Aziz Türkiye Cumhuriyeti Devleti`ne, bana sponsorluk yaptığı için çok teşekkür ediyorum. Aziz milletimin, alin terlerinden akıtarak vermiş olduğu vergilerle almış olduğum eğitimle, basta insanlığa sonra Aziz Türk milletine hizmet edeceğimden kimsenin kuskusu olmasın. iv | P a g e Abbreviations AGC…………………………………… Protein kinase A (PKA)/PKG/PKC-like. AS…………………………………….... Activation segment motif CRB……………………………………. Crumbs DKO……………………………………. Double knock-out EMT…………………………………….. Epithelial-mesenchymal transition ErbB4……………………………………. Receptor tyrosine-protein kinase erbB-4 Ex………………………………………… Expanded GPCR…………………………………….. G-protein coupled receptor HID……………………………………….. Head involution defective HIPK……………………………………… Homeodomain interacting protein kinase HM………………………………………... Hydrophobic motif HP…………………………………………. Hematoporphrin Hpo………………………………………... Hippo Hppy………………………………………. Happyhour IAP……………………………………….... Inhibitors of apoptosis protein KO…………………………………………. Knock-out LATS1/2…………………………………… Large tumour suppressor 1/2 LIFR……………………………………….. Leukemia inhibitory factor receptor LPA………………………………………... serum-borne lysophosphatidic acid MAP……………………………………… Microtubule affinity-regulating kinase 1 MAP4K…………………………………... Mitogen-activated protein kinase mats………………………………………. Mob as a tumour suppressor MEN……………………………………… Mitotic exit network v | P a g e MOB1…………………………………….. Mps-one binder 1 MST1/2………………………… Mammalian Ste20-like serine/threonine kinases 1/2 NDR……………………………………… Nuclear dbf2 related NF2……………………………………….. Neurofibromatosis-2 NTR………………………………………. N-terminal regulatory domain OA………………………………………… Okadaic acid PAR……………………………………….. Protease-activated receptors PP2A………………………………………. Protein phosphatase type 2A PPI…………………………………………. Protein-protein interaction RASSF……………………………………... RAS association domain family RUNX1/2…………………………………... Runt-related transcription factor 1 S1P…………………………………………. Sphingosine 1-phosphate SAV………………………………………... Salvador SIK…………………………………………. Salt-inducible kinase SIN…………………………………………. Septation initiation network TAO1………………………………………. Serine/threonine-protein kinase TAZ………………………… Transcriptional co-activator with PDZ-binding motif TBX5……………………………………… T-box transcription factor 5 TEAD……………………………………… TEA domain family member Trc………………………………………….. Tricornered TSG………………………………………… Tumour suppressor gene VGLL4……………………………………... Vestigial-like family member 4 VP………………………………………….. Verteporfin Wts………………………………………….. Warts vi | P a g e YAP………………………………………… Yes associated protein Yki…………………………………………... Yorkie β-TrCP………………. beta-transducin repeat containing E3 ubiquitin protein ligase vii | P a g e Table of Contents 1. Introduction ......................................................................................................................... 1 1.1. General introduction ..................................................................................................... 1 1.2. The Core kinase network of the Hippo Pathway .......................................................... 4 1.3. Upstream regulators of the Hippo pathway ............................................................... 12 1.4. Hippo signalling in diseases ........................................................................................ 14 1.4.1. The Hippo pathway and cancer ........................................................................... 14 1.4.2. Hippo signalling in non-cancer pathologies ......................................................... 18 1.4.3. Hippo Signalling as a therapeutic target .............................................................
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