Phosphorylation of Filamin A by Cdk1/cyclin B1 Regulates Filamin A Subcellular Localization and is Important for Daughter Cell Separation

ii Acknowledgements . iii Table of Contents . v List of Abbreviations . viii List of Figures . xi List of Tables . xiv Chapter 1 General Introduction . 1 Rationale and hypothesis . 1 1.1 Actin cytoskeleton . 2 1.1.1 Actin dynamics . 2 1.1.2 Vertebrate actin filament organization . 7 1.1.3 Actin-binding proteins . 17 1.2 Filamin A . 23 1.2.1 Discovery of filamin . 23 1.2.2 Filamin structure . 23 1.2.3 Mammalian filamin isoforms and their cellular and tissue distribution . 27 1.2.4 Filamins in amoeba, fruit-fly and nematode . 28 1.2.5 Physiological significance of FLNa . 32 1.2.6 Biological filamin A functions . 36 1.2.7 Regulation of filamin A . 46 1.2.8 Mouse models of FLNa-deficiency . 49 1.3 Cell cycle . 51 1.3.1 Stages of cell cycle . 51 1.3.2 Mitosis . 54 1.3.3 Cyclin-dependent kinases . 57 1.3.4 Regulation of Cdk1 . 57 1.3. 5 Cdk1/cyclin B1 substrates . 59 1.3. 6 Cytokinesis . 62 1.3. 7 Actin cytoskeleton and mitotic cell rounding . 67 1.3. 8 Actin-regulated cell cycle checkpoints . 68 Objectives . 70 Chapter 2 Materials and Methods . 71 2.1 Cell culture, drug treatments and transfection . 71 2.2 Flow cytometry . 74 2.3 Plasmids, site-directed mutagenesis and cloning . 74 2.4 Preparation of lysate, SDS-PAGE and Western blot . 75 2.5 Antibodies . 76 2.6 His-FLNa expression and purification . 77 2.7 Immunoprecipitation . 78 2.7. 1 Immunoprecipitation of Cdk1/cyclin B1 . 78 2.7. 2 Immunoprecipitation of FLNa . 78 vi 2.7. 3 Co-immunoprecipitation to validate FLNa-interactors identified from mass spectrometry . 79 2.8 Quantification of total p-FLNa from mitotic HeLa cells . 80 2.9 In vitro phosphorylation of FLNa with Cdk1 . 80 2.9. 1 Detection of phosphorylated HeLa FLNa, recombinant His-FLNa and FLAGFLNa with p-FLNa antibody . 80 2.9. 2 Detection of phosphorylated recombinant His-FLNa with radioactive ATP . 81 2.10 In vitro dephosphorylation assay . 81 2.11 F-actin cosedimentation assays . 82 2.12 Preparation of fixed cells for immunofluorescence . 82 2.13 Immunofluorescence confocal microscopy . 83 2.14 Phase contrast microscopy . 84 2.15 Time-lapse imaging . 84 2.16 Covalently coupling antibodies to sepharose beads . 85 2.17 Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) . 85 2.18 In-gel digestion, extraction of peptides and phosphopeptide enrichment . 89 2.19 Liquid chromatography tandem mass spectrometry . 89 2.20 Cell proliferation assay . 90 Chapter 3 Filamin A is phosphorylated by Cdk1 in vivo . 91 3.1 Abstract . 91 3.2 Introduction . 92 3.3 Results . 94 3.3.1 FLNa is phosphorylated in mitosis by Cdk1/cyclin B1 . 94 3.3.2 FLNa is phosphorylated in intact mitotic HeLa cells . 105 3.3.3 The majority of FLNa is phosphorylated in mitosis . 108 3.3.4 Quantitative mass spectrometry shows FLNa is phosphorylated on serines 1084, 1459 and 1533 in mitotic cells . 111 3.3. 5 FLNa is phosphorylated by Cdk1 on serines 1084, 1459 and 1533 . 116 3.3. 6 Summary . 120 3.4 Discussion. 121 3.4.1 Cdk1/cyclin B1 phosphorylates FLNa on serines 1084, 1459 and 1533 . 121 3.4.2 Literature on FLNa phospho-sites in mitotic HeLa cells . 121 3.4.3 Discrepancy between in vitro and in vivo FLNa phosphorylation sites in mitosis 122 3.4. 4 The p-FLNa antibody recognizes multiple mitotic epitopes on FLNa . 123 3.4. 5 The p-FLNa antibody likely has nonspecific binding to p-FLNb . 123 3.4. 6 We estimate that FLNa is stoichiometrically phosphorylated in mitosis . 124 3.4. 7 p-FLNa is dephosphorylated at the end of mitosis . 125 Chapter 4 Filamin A phosphorylation regulates its subcellular localization and is important for cell separation and interphase cell behaviour . 127 4.1 Abstract . 127 4.2 Introduction . 128 4.3 Results . 131 4.3.1 Actin structures are different in interphase and mitotic HeLa cells . 131 vii 4.3.2 Effect of FLNa phosphorylation by Cdk1/cyclin B1 on F-actin binding . 134 4.3.3 FLNa-WT GFP and FLNa-AAA GFP localize to prominent actin structures in interphase M2 and HeLa cells. 146 4.3.4 FLNa-WT G