Characterization of Cell Biological and Physiological Functions of the Phosphoglycolate Phosphatase AUM
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Characterization of cell biological and physiological functions of the phosphoglycolate phosphatase AUM Charakterisierung zellbiologischer und physiologischer Funktionen der Phosphoglykolat-Phosphatase AUM Dissertation for a doctoral degree at the Graduate School of Life Sciences, Julius-Maximilians-Universität Würzburg (Section Biomedicine) submitted by Gabriela Segerer from Kitzingen am Main Würzburg, November 2015 Submitted on: ……………………………………………………………… Members of the Promotionskomitee : Chairperson: Prof. Dr. Manfred Gessler Primary Supervisor: Prof. Dr. Antje Gohla Supervisor (Second): Prof. Dr. Dr. Manfred Schartl Supervisor (Third): PD Dr. Heike Hermanns Date of Public Defence: …………………………………………… Date of Receipt of Certificates: …………………………………………. Table of contents Table of contents 1 INTRODUCTION 1 1.1 Phospho-regulation by kinases and phosphatases 1 1.2 Classification of phosphatases 2 1.3 Haloacid dehalogenase (HAD) phosphatases 3 1.3.1 Structural features of HAD phosphatases 3 1.3.2 HAD phosphatases in health and diseases 5 1.4 Characterization of phosphoglycolate phosphatase PGP 6 1.4.1 PGP dephosphorylates phosphoglycolate in vitro 7 1.4.1.1 Source of mammalian phosphoglycolate (PG) 7 1.4.1.2 Function of mammalian phosphoglycolate (PG) 8 1.4.1.3 TPI controls a branch point between glucose- and lipid metabolism 10 1.4.2 PGP acts as a tyrosine-directed phosphatase in vitro 11 1.4.3 PGP is a regulator of integrin-dependent cell adhesion 13 1.5 Integrins 14 1.5.1 Integrin signaling 15 1.5.2 Integrin-dependent cell adhesion, spreading and migration 16 1.6 Cytoskeletal rearrangements during cell adhesion and migration 17 1.6.1 Regulation of actin remodeling 19 1.6.2 Actin-based membrane structures 19 1.6.3 Circular dorsal ruffles (CDRs) 21 1.6.3.1 Functions of CDRs 21 1.6.3.2 CDR formation signaling 22 1.7 Role of PGP in vivo 23 2 AIM OF THE STUDY 25 3 MATERIALS AND METHODS 27 3.1 Materials 27 3.1.1 Chemicals and reagents 27 3.1.2 Technical equipment 30 3.1.3 Consumable supplies 30 I Table of contents 3.1.4 DNA- and protein ladders 31 3.1.5 Commercial kits 31 3.1.6 Commercial buffers 32 3.1.7 Cell lines and mouse models 32 3.1.8 Cell culture medium 32 3.1.9 Antibodies 33 3.1.10 Immunocytochemistry reagents 34 3.1.11 Pharmacological inhibitors and negative controls 34 3.1.12 Inhibitors for metabolic studies 35 3.1.13 Enzymes and purified proteins 35 3.1.14 Radioactive nucleotides 35 3.1.15 Plasmids 35 3.1.16 Solutions and buffers 36 3.1.17 Software 41 3.2 Methods 43 3.2.1 Generation and breeding of Pgp knockout PGP D34N knockin mice 43 3.2.2 Genotyping of mice 43 3.2.3 Agarose gel electrophoresis 44 3.2.4 Embryo explants and isolation of primary cells 44 3.2.5 Cell culture techniques 46 3.2.5.1 Cell lines, primary cells and cell culture 46 3.2.5.2 Freezing cells 47 3.2.5.3 Thawing cells 47 3.2.5.4 Transfection 47 3.2.5.5 Coating of cell culture dishes, endothelial cell sorting and lymphocyte activation 48 3.2.5.6 Immunocytochemistry 49 3.2.6 Cellular assays 49 3.2.6.1 Pharmacological inhibitors 49 3.2.6.2 Proliferation assays 49 3.2.6.3 Quantification of circular dorsal ruffles (CDR) 49 3.2.6.4 Cell spreading assays 50 3.2.6.5 PGP-rescue experiments 50 3.2.6.6 Endothelial cell spreading assays 51 3.2.6.7 Time lapse cell migration assays 51 3.2.6.8 Transwell cell migration assays 51 3.2.6.9 PKC activity assay 52 II Table of contents 3.2.6.10 Flow cytometry 53 3.2.7 Metabolic studies 53 3.2.7.1 Determination of glycerol 3-phosphate (G3P) levels 53 3.2.7.2 Lipidomics 53 3.2.7.3 Determination of ATP levels 54 3.2.7.4 Analysis of triose phosphate isomerase (TPI) activity 54 3.2.7.5 Analysis of cellular respiration 55 3.2.8 Protein analytics 56 3.2.8.1 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) 56 3.2.8.2 Western Blot analysis 56 3.2.8.3 Immunoblot stripping 57 3.2.8.4 Preparation of cell lysates 57 3.2.8.5 Immunoprecipitation 57 3.2.9 Experiments with purified proteins 58 3.2.9.1 Cellular PLC γ1 dephosphorylation assays 58 3.2.9.2 Recombinant PLC γ1 dephosphorylation assays 59 3.2.10 Image quantification 59 3.2.11 Statistical analysis 59 4 RESULTS 61 4.1 Role of phosphoglycolate phosphatase (PGP) for cell spreading and cell migration 61 4.1.1 Cellular effects of PGP 62 4.1.1.1 PGP activity regulates integrin- and RTK- induced circular dorsal ruffle formation 62 4.1.1.2 PGP localizes to circular dorsal ruffles 66 4.1.1.3 PGP regulates integrin- and RTK-induced cell spreading in a CDR- dependent manner 67 4.1.1.4 Loss of PGP activity increases cell spreading of mouse lung endothelial cells 70 4.1.1.5 PGP regulates cell migration 72 4.1.1.6 PGP inactivation in lymphocytes lead to an increase in cell migration 74 4.1.2 Molecular mechanisms of PGP-dependent effects on CDR formation, cell spreading and cell migration 76 4.1.2.1 Role of PGP in EGF-receptor signaling 77 4.1.2.2 PGP regulates CDR–formation and cell migration by modulating PLC γ1 activity downstream of Src kinase family signaling 80 4.1.2.3 Decrease in PLC γ1 activity leads to a reduction of PKC-mediated effects 84 III Table of contents 4.1.2.4 Loss of PGP catalyzes PKC-mediated effects on cytoskeleton 86 4.1.2.5 PLC γ1 is not a direct protein substrate of PGP 88 4.1.2.6 PGP inactivation leads to altered membrane composition 90 4.2 The role of phosphoglycolate phosphatase in vivo 93 4.2.1 Whole-body PGP inactivation (EIIa-mouse) is embryonic lethal 93 4.2.2 Deletion of PGP activity in endothelial and hematopoietic cells 96 4.2.3 Effects of whole-body PGP inactivation on the cardiovascular system 96 4.2.4 Loss of PGP activity causes an oxygen-dependent proliferation defect 97 4.2.5 Loss of PGP activity leads to a metabolic shift towards increased lipogenesis 100 4.2.6 PGP loss elevates energy-producing metabolism in an oxygen-dependent manner 106 5 DISCUSSION 113 5.1 Role of PGP for cell spreading and cell migration 114 5.1.1 Does PGP primarily act as a PG phosphatase and/or as a tyrosine-directed phosphatase during cell spreading and migration? 114 5.1.2 What is the link between upregulated PS levels and PGP-dependent effects on cell spreading and cell migration? 116 5.1.2.1 Functions of PS 116 5.1.2.2 PS accumulation leads to catalyzed PKC-mediated signal transduction 117 5.1.3 Untargeted lipidomic analysis of GC-1 cells 119 5.1.4 Inhibitor studies 120 5.1.5 Do elevated cellular ATP levels affect actin reorganization? 120 5.1.6 CDR formation 122 5.1.6.1 Signaling pathways involved in CDR formation 122 5.1.6.2 CDR functions 123 5.2 Physiological functions of PGP 125 5.2.1 PGP inactivation leads to cell proliferation arrest 125 5.2.2 Is PGP a PG phosphatase in vitro and in vivo? 126 5.2.3 Analysis of the link between DNA damage-derived PG and inhibition of TPI activity 127 5.2.4 Ablation of PGP activity only moderately inhibits TPI activity 127 5.2.5 Glycolysis, gluconeogenesis and glyceroneogenesis as potential PG-regulated processes 128 5.2.6 TPI inhibition results in the accumulation of long-chain polyunsaturated fatty acids 130 5.2.7 PG inactivation leads to elevated lipogenesis and lipolysis 131 IV Table of contents 6 SUMMARY 135 7 REFERENCES 141 8 APPENDIX 159 8.1 Curriculum vitae 159 8.2 Publication list and conference contributions 161 8.3 Affidavit 163 8.4 Acknowledgments 165 V VI Figure and table index Figure and table index Figure 1: Human phosphatome. ......................................................................................... 3 Figure 2: Structural diversity of HAD phosphatase cap domains. ................................... 4 Figure 3: PGP is a PG phosphatase in vitro . ..................................................................... 7 Figure 4: Origin of mammalian phosphoglycolate (PG). .................................................. 8 Figure 5: TPI inhibition by phosphoglycolate. .................................................................. 9 Figure 6: Carbohydrate- and lipid metabolism. ................................................................10 Figure 7: PGP is implicated in growth factor signaling. ..................................................11 Figure 8: Simplified model of EGF receptor signaling. ....................................................12 Figure 9: Role of PGP for cell adhesion. ...........................................................................14 Figure 10: Integrin activation signaling. ...........................................................................15 Figure 11: Actin dynamics and focal adhesion turnover during cell spreading and migration. ..........................................................................................................18 Figure 12: Protrusive actin structures. .............................................................................20 Figure 13: CDR formation signaling. .................................................................................23 Figure 14: Pgp targeting strategy. .....................................................................................24 Figure 15: Lymph node atlas. ............................................................................................45 Figure 16: Transwell assay based on the Boyden chamber method. .............................52 Figure 17: Mitochondrial stress assay. .............................................................................55 Figure 18: Western blot sandwich. ....................................................................................56 Figure 19: Spermatogonial GC1 cell line. .........................................................................61