FUNCTIONAL CONSEQUENCES OF COMPLETE GSK-3 ABLATION IN MOUSE EMBRYONIC FIBROBLASTS By Ioana M Miron A thesis submitted in conformity with the requirements for the degree of Master of Science, Graduate Department of Medical Biophysics in the University of Toronto ©Copyright by Ioana M Miron 2008 Abstract Functional consequences of complete GSK-3 ablation in mouse embryonic fibroblasts Master of Science, 2008. Ioana M. Miron, Department of Medical Biophysics, University of Toronto Glycogen Synthase Kinase-3 (GSK-3) is a highly conserved serine/threonine kinase comprised of two mammalian homologues, GSK-3α and β, encoded by independent genes. This thesis reports the characterization of GSK-3-null primary mouse embryonic fibroblasts (MEFs) generated by gene targeting to gain insight into the physiological functions of this protein kinase. Combined inactivation of both alleles of GSK-3α and GSK-β led to elevated sensitivity to TNFα-induced apoptosis, altered organization of focal adhesion complexes, defects in cell spreading on fibronectin, decreased cell growth associated with altered cell cycle progression through the G2/M phase and increased spontaneous apoptosis. Future work will focus on unraveling the molecular mechanisms responsible for these effects and identifying the common and distinct cellular roles for GSK-3α and β, and specific variants of these isoforms. ii Table of Contents Abstract ____________________________________________________________________ ii Table of Contents ___________________________________________________________ iii List of Tables_______________________________________________________________ vi List of Figures ______________________________________________________________ vii Abbreviations _____________________________________________________________ viii Chapter 1: Introduction_______________________________________________________ 1 1.1 Therapeutic Potential of Protein Kinases ______________________________________ 2 1.2 Glycogen Synthase Kinase-3 Isoforms________________________________________ 2 1.3 GSK-3 Substrates ________________________________________________________ 4 1.4 Regulation of GSK-3 _____________________________________________________ 6 1.5 Role of GSK-3 in Cellular Functions _________________________________________ 9 1.5.1 Cell Cycle Progression ________________________________________________ 9 1.5.2 Intrinsic and Extrinsic Apoptosis________________________________________ 11 1.5.3 Cellular Architecture and Motility_______________________________________ 14 1.5.4 Mitogen-Activated Protein Kinase (MAPK) Signaling_______________________ 16 1.6 GSK-3 and Disease______________________________________________________ 17 1.8 Rationale and Thesis Objective ____________________________________________ 19 Chapter 2: Materials and Methods_____________________________________________ 21 2.1 Cell Culture____________________________________________________________ 22 2.2 Adenovirus Production ___________________________________________________ 22 2.3 Generation of Embryos Harbouring Conditional Alleles of GSK-3α and β __________ 22 2.4 Isolation of Embryonic Fibroblasts from GSK-3 αFL/FL/βFL/FL Mice ________________ 23 2.5 Preparation of Lysates and Cytosolic β-Catenin Isolation ________________________ 24 2.6 TCF-Reporter Assay_____________________________________________________ 24 2.7 Immunoblotting ________________________________________________________ 25 2.8 Immunofluorescence_____________________________________________________ 25 2.9 Microarray Analysis of GSK-3 Knockout MEFs _______________________________ 26 2.10 Alamar Blue Proliferation Assay __________________________________________ 27 2.11 Senescence Associated β-Galactosidase Staining _____________________________ 27 2.12 Cell Cycle Analysis and BrdU Incorporation_________________________________ 27 2.13 Annexin V Assay ______________________________________________________ 28 iii 2.14 Time-Lapse Microscopy_________________________________________________ 28 2.15 Adhesion Assay _______________________________________________________ 29 2.16 Cell Spreading Assay ___________________________________________________ 29 2.17 Cell Migration Assay ___________________________________________________ 30 2.18 Rho Activity Assay_____________________________________________________ 30 2.19 MAPK Activation______________________________________________________ 31 2.20 Statistical Analysis _____________________________________________________ 31 Chapter 3: Results __________________________________________________________ 32 3.1 Generation of GSK-3 Conditional Mouse Embryonic Fibroblasts__________________ 32 3.2 Microarray Analysis _____________________________________________________ 36 3.3 Effect of GSK-3 Deficiency on Cell Proliferation ______________________________ 36 3.4 Cell Cycle Regulation in GSK-3 Depleted Cells _______________________________ 36 3.5 Effect of GSK-3 Depletion on Apoptosis_____________________________________ 38 3.6 TNF sensitivity in GSK-3 Deficient Cells ____________________________________ 40 3.7 Cellular Morphology and Focal Adhesion Formation in the Absence of GSK-3 ______ 43 3.8 The Role of GSK-3 in Cell Adhesion, Spreading and Migration___________________ 47 3.9 Effect of Loss of GSK-3 on MAPK signaling _________________________________ 51 Chapter 4: Discussion and Future Directions ____________________________________ 54 4.1 Summary______________________________________________________________ 55 4.2 Proliferation ___________________________________________________________ 56 4.3 Cell Cycle Progression ___________________________________________________ 57 4.4 Stimuli-Induced Apoptosis________________________________________________ 58 4.5 Actin Organization and Focal Adhesion assembly______________________________ 58 4.6 MAPK Signaling _______________________________________________________ 61 4.7 Adipocyte Differentiation_________________________________________________ 62 4.8 GSK-3 Isoforms and Mutants______________________________________________ 62 References _________________________________________________________________ 65 Appendix: Identification of Novel GSK-3 Substrates ______________________________ 82 A.1 Introduction ___________________________________________________________ 83 A.2 Materials and Methods __________________________________________________ 85 iv A.2.1 Immunoprecipitation of BCLAF1 ______________________________________ 85 A.2.2 In-gel Enzymatic Cleavage and Extraction of Peptides ______________________ 86 A.2.3 Quantification of BCLAF1 Phosphorylation ________________________________ 86 A.3 Results _______________________________________________________________ 87 A.4 Discussion ____________________________________________________________ 90 A.4 References ____________________________________________________________ 91 v List of Tables Table 1.1: Putative GSK-3 substrates_______________________________________________5 Table 4.1 List of GSK-3α and β variants ___________________________________________64 Table A.1: Predicted GSK-3 phosphorylation sites on BCLAF1 identified using NetworKin.__84 vi List of Figures Figure 1.1: A schematic representation of the mammalian GSK-3 isoforms, α and β. ________ 3 Figure 1.2: Schematic representation of the Wnt signaling pathway. _____________________ 8 Figure 3.1: Treatment of GSK-3 α(FL/FL) / β(FL/FL) MEFs with AdCre results in compound knockouts of GSK-3α and β, stabilization of β-catenin and β-catenin/TCF-transactivation activity. ____________________________________________________________________ 35 Figure 3.2: Functional analysis of microarray data. __________________________________ 37 Figure 3.3: GSK-3 is critical for primary MEF proliferation. __________________________ 37 Figure 3.4: Role of GSK-3 in cell cycle regulation in MEFs. __________________________ 39 Figure 3.5: GSK-3 deletion induces spontaneous apoptosis. ___________________________ 41 Figure 3.6: Enhanced TNFα-induced apoptosis in GSK-3 deficient MEFs. _______________ 42 Figure 3.7: Morphology of GSK-3 DKO MEFs in regular culture conditions. _____________ 44 Figure 3.8: F-actin distribution and focal complex assembly in GSK-3-null cells in normal culture conditions. ___________________________________________________________ 45 Figure 3.9: Normal Rho activity in GSK-3 DKO MEFs after LPA stimulation. ____________ 46 Figure 3.10: Deletion of GSK-3 does not cause defective cell adhesion. _________________ 48 Figure 3.11: GSK-3-null MEFs exhibit abnormal spreading on fibronectin._______________ 50 Figure 3.12: Migration of MEFs is not impeded by lack of GSK-3. _____________________ 52 Figure 3.13: Effects of loss of GSK-3 expression on ERK, JNK/SAPK and p38 MAPK activities.___________________________________________________________________ 53 Figure A.1: BCLAF1 phosphorylation sites identified using mass spectrometry.___________ 88 Figure A.2: Quantitative measurement of GSK-3-dependent phosphorylation of BCLAF1. __ 89 vii Abbreviations 7-AAD 7-amino-actinomycin D AdCre adenovirus encoding cre recombinase AdLacZ adenovirus expressing LacZ ADD1 adipocyte determination- and differentiation-dependent factor 1 AIP Aurora-A-interacting protein Ala alanine ANOVA analysis of variance APC adenomatous polyposis coli Apo apoptosis-inducing ligand ATP adenosine triphosphate BCLAF1 Bcl-2-associated transcription factor 1 BCL-2 B-cell lymphoma-2 BCL-3 B-cell lymphoma 3-encoded protein BrdU 5-bromo-2-deoxyuridine BSA bovine serum albumin BUB1 budding uninhibited by benzimidazole