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Regulation of Atypical Pkcs Regulation of Atypical PKCs Jessie Ann Le Good This thesis is submitted in partial fulfilment of the requirements for the degree of Doctor of Philosophy from the University of London February 1999 Imperial Cancer Research Fund 44 Lincoln's Inn Fields London WC2A 3PX ProQuest Number: 10609017 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 10609017 Published by ProQuest LLC(2017). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 Abstract Protein phosphorylation plays an essential role in a diverse array of signalling cascades and regulates many cellular processes. Protein kinase Cs (PKCs) constitute one of the families of kinases involved in phosphorylating substrates on serine or threonine residues. These kinases were initially identified as being receptors for tumour promoters (phorbol esters) and the conditions required to activate different isoforms determine the subgroup classification of the 10 isoforms. Classical PKCs (a,pi, pH and y) depend upon Ca2+ and lipids (DAG, PS- phospholipids.) Novel PKCs ( 8,e,ri, j x and 0 ) are insensitive to Ca2+ but are activated by lipids, DAG and phospholipids. The atypical PKCs (£,i and X) differ greatly. These proteins are insensitive to Ca2+ and phorbol ester binding. The lack of knowledge on the control of the atypical PKCs has made the role of the atypical PKCs more elusive. Nevertheless, PKC £ has been implicated in cell growth and differentiation. Moreover, PKC £ is thought to be involved in a plethora of signal transduction pathways, including the Ras and MEK/MAPK pathways. The related atypical PKC i may be involved in UV induced apoptosis and insulin signalling. The aims of this thesis are to define the control and biological role of the atypical PKCs - primarily focusing on PKC As one approach, the project attempted to create a knockout mouse. This would help define a biological end point and therefore permit elucidation of the inputs. This study led to the identification of a pseudogene and its origin is described. As a second approach to investigate PKC £ control, various direct paths were followed - ranging from searching for potential binding proteins and cellular localisation, to analysis of activation by lipids and phosphorylation. These studies have provided evidence for the dynamic control of PKC £ (and PKC i) through a kinase cascade involving the lipid kinase PI3-kinase, the lipid responsive PDK1 and finally phosphorylation of PKC £ at a site defined as threonine 410. The operation of this pathway and its influence on PKC £ autophosphorylation (in vivo) and activity (in vitro) are presented. Acknowledgements So many people have helped me during the course of this thesis - far too many to name in person. Of course without the intellectual input and laboratory of Peter Parker, none of this work would have been possible. Many thanks to everyone in the lab, past and present members, especially to my close bench neighbours for putting up with and helping me. I really want to thank all my fellow students and friends at the ICRF, in particular Kate and Chris, for their moral support and encouragement. Many thanks to Claire and all my friends for entertaining me and listening to the moans. This thesis is dedicated to my family, in appreciation of their continual support, encouragement and positive thinking. iii Table of Contents Title P age ............................................................................................................................... i A bstract................................................................................................................................ ii Acknowledgements .........................................................................................................iii Table of Contents .............................................................................................................iv List of Tables and Figures .............................................................................................ix Abbreviations ..................................................................................................................xiii Chapter 1 Introduction 1.1 General concepts in signalling pathways ...........................................................1 1.2 Signalling Cell Surface Receptors ............................................................................1 1.3 Eukaryotic Protein Kinases ........................................................................................4 1.4 PKC introduction ...........................................................................................................5 1.5 The PKC Family......................................................................................................... 6 1.6 C1 Domain .....................................................................................................................7 1.7 C2 Domain ...................................................................................................................11 1.8 Pseudosubstrate Region ..........................................................................................13 1.9 Kinase domain ......................................................................................................... 14 1.10 V5 Domain .................................................................................................................17 1.11 Mechanisms of Activation ...................................................................................18 1.12 Phosphorylation ....................................................................................................22 1.13 What is the Role of Phosphorylation? ............................................................ 26 1.14 PKC dephosphorylation - a role in inactivation ........................................... 26 1.15 Signalling Pathways involved in the Activation of PKCs ..........................27 1.16 The Role of the Atypical PKCs..........................................................................31 Chapter 2 Materials and Methods 2.1 Materials...................................................................................................................... 33 2.1.1 Chemicals and Radiochemicals ................................................................ 33 2.2 Methods .......................................................................................................................34 2.2.1 Library Screening .............................................................................................. 34 2.2.2 Membrane Hybridisation ..................................................................................34 2.2.3 Random Prime Labelling of a cDNA probe ............................................34 2.2.4 Southern blotting ............................................................................................35 2.2.5 Double-Stranded DNA Sequencing ............................................................. 35 2.2.6 Phage DNA purification ................................................................................... 35 2.2.7 Molecular Biology ..............................................................................................35 2.2.8 Cloning .............................................................................................................. 36 2.2.9 Polymerase Chain Reaction (PCR) .............................................................. 36 2.2.10 Mutagenesis .....................................................................................................37 2.2.11 In vitro transcription-translation ............................................................... 38 2.2.12 In vivo transfection of COS and 293 cells ................................................ 38 2.2.13 Immunoprecipitation ................................................................................... 38 2.2.14 35S-methionine labelling ............................................................................39 2.2.15 Total lysates .................................................................................................. 39 2.2.16 Polyacrylamide Gel Electrophoresis (PAGE) ..........................................39 2.2.17 Western Blotting ...........................................................................................40 2.2.18 Coomassie Staining and Destaining of SDS-PAGE G els ..............40 2.2.19 Antisera production .........................................................................................40 2.2.20 Immunofluorescence .................................................................................. 40 2.2.21 Kinase Assays ..............................................................................................41 v 2.2.22 PDK kinase assays ..................................................................................... 41 2.2.23 Bacterial protein ex p ressio n .....................................................................41 2.2.24 Isolation of HIS/GST tagged proteins ........................................................42
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