The role of the C-terminus Merlin in its tumor suppressor function Vinay Mandati To cite this version: Vinay Mandati. The role of the C-terminus Merlin in its tumor suppressor function. Agricultural sciences. Université Paris Sud - Paris XI, 2013. English. NNT : 2013PA112140. tel-01124131 HAL Id: tel-01124131 https://tel.archives-ouvertes.fr/tel-01124131 Submitted on 19 Mar 2015 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. 1 TABLE OF CONTENTS Abbreviations ……………………………………………………………………………...... 8 Resume …………………………………………………………………………………… 10 Abstract …………………………………………………………………………………….. 11 1. Introduction ………………………………………………………………………………12 1.1 Neurofibromatoses ……………………………………………………………………….14 1.2 NF2 disease ………………………………………………………………………………15 1.3 The NF2 gene …………………………………………………………………………….17 1.4 Mutational spectrum of NF2 gene ………………………………………………………..18 1.5 NF2 in other cancers ……………………………………………………………………...20 2. ERM proteins and Merlin ……………………………………………………………….21 2.1 ERMs ……………………………………………………………………………………..21 2.1.1 Band 4.1 Proteins and ERMs …………………………………………………………...21 2.1.2 ERMs structure ………………………………………………………………………....23 2.1.3 Sub-cellular localization and tissue distribution of ERMs ……………………………..25 2.1.4 ERM proteins and their binding partners ……………………………………………….25 2.1.5 Assimilation of ERMs into signaling pathways ………………………………………...26 2.1.5. A. ERMs and Ras signaling …………………………………………………...26 2.1.5. B. ERMs in membrane transport ………………………………………………29 2.1.6 ERM functions in metastasis …………………………………………………………...30 2.1.7 Regulation of ERM proteins activity …………………………………………………...31 2.1.7. A. Conformational regulation of ERMs ……………………………………….31 2.1.7. B. ERMs regulation by phosphorylation ………………………………………33 2 2.2 Merlin ……………………………………………………………………………………..34 2.2.1 Merlin: structure ………………………………………………………………..34 2.2.2 Isoforms of Merlin ……………………………………………………………..35 2.2.3 Tissue distribution and sub cellular localization of Merlin …………………….37 2.2.4 Functional analysis of Merlin in different model organisms …………………..37 3. Merlin functions and its regulation ……………………………………………………..39 3.1 Contact dependent inhibition ……………………………………………………………..39 3.2 Apoptosis/survival ………………………………………………………………………..42 3.3 Regulation of Merlin activity ……………………………………………………………..43 3.3.1 Conformational regulation of Merlin activity ………………………………….43 3.3.2 Merlin regulation by Phosphorylation …………………………………………44 3.3.3 Merlin phosphorylation by Akt ………………………………………………...47 3.3.4 Phosphatases and Merlin ……………………………………………………….50 3.4 Proteasomal mediated degradation of Merlin …………………………………………….50 4. Merlin controls cell growth by regulating various signaling pathways at the plasma membrane and in the nucleus ……………………………………………………………...52 4.1 Regulation of mitogenic signaling pathways by Merlin ………………………………….52 4.2 PKA pathway ……………………………………………………………………………..55 4.3 Merlin role in Receptor Tyrosine Kinase (RTK) regulation ……………………………...58 4.4 The Hippo pathway ……………………………………………………………………….59 4.4.1 Core components of the Hippo pathway ……………………………………….59 4.4.2 Merlin, an upstream regulator of Hippo pathway ……………………………...62 4.4.3 Regulation of the Hippo pathway in physiological and pathological conditions 63 3 4.5 Merlin inhibits the E3 ubiquitin ligase, CRL4-DCAF1, in the nucleus …………………..64 4.6 Merlin in mTOR signaling pathway ………………………………………………….......65 Objectives of the thesis ……………………………………………………………………..68 Part 1. Identification of novel interacting partners of the C-terminus Merlin important for its tumor suppressor function ………………………………………………………….71 1. Introduction……………………………………………………………………………….72 1.1 Merlin and its interacting partners ………………………………………………………..72 1.2 Hepatocyte growth factor-regulated tyrosine kinase substrate …………………………...73 2. Results …………………………………………………………………………………….74 2.1 Interaction of Merlin with HRS …………………………………………………………..74 2.2 Experimental strategy to fish for novel interacting partners of C-terminus Merlin ……...76 2.3 AMOT family proteins …………………………………………………………………...77 2.3.1 Structure of Amot family proteins ……………………………………………..78 2.3.2 AMOT family protein functions ……………………………………………….79 2.4 Merlin interacts with Amot family proteins through its coil-coiled domain ……………..82 2.5 Merlin phosphorylation (S518) does not affect its interaction with Amot and AmotL1 85 2.6 Merlin binds to the coiled-coiled domain of AmotL1 ……………………………………87 2.7 Merlin and AmotL1 co-localize at plasma membrane and as well as in the cytosol ……..89 2.8 Merlin affects AmotL1 functions at different levels ……………………………………...93 2.8.1 Merlin regulates AmotL1 at transcriptional level ……………………………...94 2.8.2 Merlin regulates AmotL1 at protein level ……………………………………...98 2.9 Merlin regulates proliferation and migration of breast cancer cells through AmotL1 ….101 4 3. Interactions between Hippo core components ………………………………………...102 3.1 Interaction between YAP and AMOT family proteins ………………………………….102 3.2 Interaction between Merlin, AmotL1 and KIBRA ……………………………………...105 3.3 Interaction of Merlin with LATS and YAP ……………………………………………..108 3.4 Merlin, a magnet of Hippo pathway …………………………………………………….111 Part 2. Identification of novel phosphorylation sites on C-terminus Merlin …………..116 1. Introduction ……………………………………………………………………………..117 1.1 Generalized view of cell cycle and its role in cancer development ……………………..117 1.2 Control of the cell cycle …………………………………………………………………119 1.3 Mitotic kinases in cell cycle regulation and cancer ……………………………………..120 1.3.1Aurora kinases …………………………………………………………………120 1.3.2 Aurora A functions and its role in cancer development ………………………121 2. ERM proteins and Merlin in cell cycle progression …………………………………..124 2.1 Phosphorylation mediated activation of ERMs is important for the mitotic progression 124 2.2 Role of Merlin in cell cycle regulation ………………………………………………...126 3. Results …………………………………………………………………………………...127 3.1 Role of novel phosphorylation sites of C-terminus Merlin in its tumor suppressor… 127 3.2 Identification of a novel phosphorylation at C-terminus of Merlin ……………………..127 3.3 Generation and characterization of polyclonal ab against Merlin T581…………………130 3.4 Phosphorylation of Merlin at T581 does not affect its subcellular localization ………...131 3.5 Merlin is hyper-phosphorylated during mitosis at T581 and S518 ……………………...132 3.6 Aurora Kinase A phosphorylates Merlin at S518 but not T581 ………………………...134 3.7 Aurora kinase A binds to FERM domain of Merlin …………………………………….137 5 3.8 Mitotic kinases (LATS, SLIK) and PKA, PAK do not seem to phosphorylate…. 139 3.9 Phosphorylation of S518 facilitates the phosphorylation of T581 during the mitosis …..140 3.10 Coordinated phosphorylation of Merlin at S518 and T581 ……………………………142 3.11 Phosphorylation of Merlin at Threonine 581 inhibits Merlin and Ezrin binding ……...144 3.12 Aurora A phosphorylates Merlin in interphasic breast cancer cells…………………....147 3.13 Aurora kinase A co-localize with Merlin in the cytosol as well as in the nucleus …….151 3.14 Aurora kinase A phosphorylates Merlin at S518 residue inside the nucleus …………..153 3.15 Consequences of Merlin phosphorylation by Aurora A kinase on Yap activity ………155 Discussion and Perspectives ………………………………………………………………157 Materials and Methods ……………………………………………………………………169 Acknowledgements ………………………………………………………………………...173 References ………………………………………………………………………………… 179 6 7 ABBREVIATIONS ABS actin binding site AJ adherens junction AKAP A-kinase anchoring protein AKT AKT8 virus oncogene cellular homolog BB blue box= dominant negative Merlin C-ter carboxyl terminal CTM c-terminus of Merlin CD44 cluster of differentiation 44 CRL4 cullin-RING-ligase 4 DCAF1 DDB1- and Cul4-associated factor D-merlin Drosophila melanogaster Merlin Dox Doxycycline ECM extracellular matrix ERM exrin-radixin-moesin EGFR epidermal growth factor ERK extracellular regulated kinase F-actin filamentous actin FAK focal adhesion kinase FERM four.1 protein, ezrin, radixin, moesin FRET fluorescence resonance energy transfer GEF guanine nucleotide exchange factor HEI10 human enhancer of invasion clone 10 HRS hepatocyte growth factor-regulated kinase substrate LATS large tumor suppressor 8 Merlin moesin-ezrin-radixin-like protein Mer merlin NF1 neurofibromatosis 1 NF2 neurofibromatosis 2 PAK p21-activated kinase Rac 1 Ras related C3 botulinum toxin substrate 1 Ras rat sarcoma RTK receptor tyrosine kinase UPP ubiquitin proteasome pathway VprBP Vpr-binding protein VS vestibular schwannoma WT wild type 9 RESUME La neurofibromatose de type 2 (NF2) est une maladie autosomique causée soit par l'inactivation du gène NF2, soit par la perte de la proteine issue due ce gène, Merline. Cela entraîne à son tour la formation de plusieurs tumeurs nerveuse bénignes (non invasives) comme les schwannomes, méningiomes et les épendymomes. De plus, une diminution de l'expression de Merline est observée dans les cancers du sein invasifs, toutefois le rôle de Merline dans ces tumeurs invasives est peu étudié. Merline est la seule protéine ayant un rôle de suppresseur de tumeur dans la famille des ERM (Ezrin / Radixin / Moesin). Nous,