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Molecular Mechanisms Regulating Phospholipase C-Γ2 Activity

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Molecular Mechanisms Regulating Phospholipase C-Γ2 Activity Molecular mechanisms regulating phospholipase C‐2 activity Dissertation zur Erlangung des Doktorgrades Dr. rer. nat. der Fakultät für Naturwissenschaften der Universität Ulm vorgelegt von Anja Jasmin Bühler aus Ulm Universität Ulm Institut für Pharmakologie and Toxikologie Ulm 2013 Current Dean of the Faculty of Natural Sciences: Prof. Dr. Joachim Ankerhold First Supervisor: Prof. Dr. Peter Gierschik Second Supervisor: Prof. Dr. Ralf Marienfeld Day Doctorate Awarded: 24th March 2014 Table of contents Table of Contents List of Figures............................................................................................................................... v Abbreviations and Units............................................................................................................ vii 1 Introduction .................................................................................................................. 1 1.1 Signal transduction......................................................................................................... 1 1.2 Phospholipases C............................................................................................................. 2 1.3 Regulation of Phospholipases C..................................................................................... 5 1.4 Role of PLC2 in inflammatory and autoimmune diseases ....................................... 14 1.5 Anaplastic lymphoma kinase ....................................................................................... 17 1.6 Aim of the work............................................................................................................. 23 2 Materials and Methods............................................................................................... 25 2.1 Materials........................................................................................................................ 25 2.2 Microbiology and Molecular Biology.......................................................................... 40 2.3 Cell culture and DNA transfection.............................................................................. 48 2.4 Protein Biochemistry .................................................................................................... 49 2.5 Measurement of phospholipase C activity.................................................................. 53 2.6 Thermodynamic calculations....................................................................................... 55 2.7 Cluster robustness analysis.......................................................................................... 56 2.8 Statistical analysis......................................................................................................... 56 3 Results .........................................................................................................................58 3.1 Autoinhibitory regulation of PLC2 ............................................................................ 58 3.2 Functional reassembly of PLC2 by separately expressed fragments ...................... 65 3.3 Phosphorylation induced activation of PLC2............................................................ 69 3.4 Anaplastic lymphoma kinase ....................................................................................... 73 3.5 Characterization of PLC2 deletion mutants associated with PLAID ..................... 84 4 Discussion ................................................................................................................. 110 4.1 Autoinhibitory and phosphorylation-induced regulation of PLC2 activity ......... 111 iii Table of contents 4.2 Molecular mechanisms underlying the human hereditary disease PLAID........... 123 5 Summary ................................................................................................................... 131 6 References................................................................................................................. 133 Acknowledgements ........................................................................................................... 165 Curriculum Vitae.............................................................................................................. 166 Publications ...................................................................................................................... 167 Appendix ........................................................................................................................... 168 Statutory Declaration ....................................................................................................... 169 Sections 2.1.1, 2.6, 2.7, 3.5.1, 3.5.2, 3.5.3,.3.5.4, 3.5.5, 3.5.6, 3.5.7,.3.5.10, 3.5.11, and 4.2 of this dissertation have been used for a publication and are taken from the corresponding manuscript [26]. iv List of Figures List of Figures Figure 1-1: Domain organization of mammalian PLC isozymes ...................................................................... 4 Figure 1-2: Oncogenic signalling of NPM-ALK ............................................................................................. 21 Figure 3-1: Role of 2 specific array on the autoinhibitory regulation of PLC2............................................ 59 G12V Figure 3-2: Effect of Rac2 and constitutively active Rac2 on the activity of PLC2SH and PLC2SA . 60 Figure 3-3: Inhibitory effect of 2 SH domains on the activity of PLC2SH.................................................. 62 Figure 3-4: Role of the SH2C and SH3 domain in autoinhibitory regulation of PLC2 .................................. 64 G12V Figure 3-5: Effect of Rac2 and constitutively active Rac2 on the activity of PLC2SH mutants............. 65 Figure 3-6: Functional reassembly of truncated PLC2 fragments................................................................. 67 H327A/H372A F897Q Figure 3-7: Functional reassembly of truncated PLC2 fragments with PLC2 and PLC2 ..... 68 Figure 3-8: Effect of mimicking phosphorylation of different tyrosine residues on the activity of PLC2 ...... 71 G12V Figure 3-9: Effect of Rac2 and constitutively active Rac2 on the activity of PLC2 phosphomimetic mutants ............................................................................................................................................................ 73 Figure 3-10: Effect of ALK and its fusion proteins NPM-ALK and EML4-ALK on the activity of PLC isozymes, PLC2 phosphomimetic mutant, and PLC2.................................................................................... 75 Figure 3-11: Effect of the constitutively active ALK mutants ALKF1174L and ALKR1275Q on the activity of PLC isozymes........................................................................................................................................................... 76 Figure 3-12: Association of ALK and NPM-ALK with PLC isozymes in intact cells .................................... 78 Figure 3-13: Effect of ALK and NPM-ALK kinase-dead mutants on the activity of PLC isozymes ................ 79 I310T Figure 3-14: Association of NPM-ALK and NPM-ALK with PLC in intact cells .................................. 81 Y1604F Y664F Figure 3-15: Effect of ALK and NPM-ALK on the activity of PLC isozymes.................................. 82 Y664F Figure 3-16: Association of NPM-ALK with PLC1 ................................................................................. 83 Figure 3-17: Specific activation of PLC219 and PLC220-22 at subphysiological temperatures............. 85 Figure 3-18: Q10 analysis of the activities of wild-type PLC2, PLC219, and PLC220-22. ..................... 86 Figure 3-19: Effect of subphysiological temperatures on cellular inositol phospholipid levels ..................... 87 Figure 3-20: Effect of cycloheximide on the activity of PLC219 and PLC220-22 at 31 °C ..................... 88 Figure 3-21: The activation of PLC219 and PLC220-22 by subphysiological temperatures is reversible ......................................................................................................................................................................... 89 Figure 3-22: Time course of the specific activation and inactivation of PLC219 and PLC220-22 by changes to and from a subphysiological temperatur....................................................................................... 90 Figure 3-23: Effect of deleting exon 19 to 22 of PLCG2 on the enzymatic activity at 37 °C and 31 °C......... 91 Figure 3-24: Effect of the deletion of PLC1 residues corresponding to exon 19 or exon 20-22 of PLCG2 on the activity of PLC1 at subphysiological temperatures .................................................................................. 93 Figure 3-25: Effect of subphysiological temperatures on the activities of PLC2PCI, PLC2SA, PLC2SH and PLC2SH2C............................................................................................................................................. 94 Figure 3-26: Effect of subphysiological temperatures on the activities of PLC2 deletion mutants................ 96 v List of Figures Ali5 Ali14 Figure 3-27: Effect of subphysiological temperatures on the activities of PLC2 , PLC2 , and Ali5/Ali14 PLC2 .................................................................................................................................................... 97 Figure 3-28: Summary and clustering of the effects of the PLC2 mutations.................................................
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