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Kinetics and Mechanism of the Interaction of Protein UC San Diego UC San Diego Electronic Theses and Dissertations Title Kinetics and mechanism of the interaction of the C1 domain of protein kinase C with lipid membranes Permalink https://escholarship.org/uc/item/1vw5x6bm Author Dries, Daniel Robert Publication Date 2007 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO KINETICS AND MECHANISM OF THE INTERACTION OF THE C1 DOMAIN OF PROTEIN KINASE C WITH LIPID MEMBRANES A Dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biomedical Sciences by Daniel Robert Dries Committee in charge: Professor Alexandra C. Newton, Chair Professor Joseph A. Adams Professor Edward A. Dennis Professor Elizabeth A. Komives Professor Palmer W. Taylor 2007 The Dissertation of Daniel Robert Dries is approved, and it is acceptable in quality and form for publication on microfilm. _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ Chair University of California, San Diego 2007 iii Dedicated to Bob and Lisa iv TABLE OF CONTENTS Page SIGNATURE PAGE......................................................................................................iii DEDICATION ...............................................................................................................iv TABLE OF CONTENTS ................................................................................................v LIST OF ABBREVIATIONS .........................................................................................x LIST OF FIGURES........................................................................................................xi LIST OF TABLES .......................................................................................................xiv ACKNOWLEDGMENTS.............................................................................................xv CURRICULUM VITA...............................................................................................xviii ABSTRACT ................................................................................................................xxii CHAPTER 1: INTRODUCTION...................................................................................1 Protein Kinase C: The Early Years .....................................................................1 The Structure of Protein Kinase C.......................................................................2 Translocation to Membranes and PKC’s Membrane-Targeting Modules ..........6 The Structure of the C1 Domain .........................................................................7 Binding of Phospholipids to the C1 Domain.......................................................8 Binding of DG and Phorbol Esters to the C1 Domain ......................................10 Inequality Among C1 Domains.........................................................................11 Atypical, Non-DG/Phorbol Ester-Binding C1 Domains...................................12 Questions Addressed in the Following Chapters...............................................14 v CHAPTER 2: KINETICS AND MECHANISM OF THE INTERACTION OF THE C1B DOMAIN OF PROTEIN KINASE C WITH LIPID MEMBRANES..................................................................................................16 Questions Addressed in Chapter 2 ....................................................................19 Design of a Fluorescence-Based Probe for the Interaction of the C1b Domain of PKCβI/II with Lipid Membranes ........................................19 Binding of C1bβ-Y123W to Vesicles Containing Increasing Mole Percent Phosphatidylserine.................................................................................23 Sensitivity to Ionic Strength of the Interaction of C1bβ-Y123W with Lipid Vesicles..................................................................................................27 Interaction of C1bβ-Y123W with Phosphatidylserine (PS) and Phosphatidylglycerol (PG) ....................................................................29 Interaction of C1bβ-Y123W with Phorbol Ester versus Diacylglycerol...........38 Conclusions .......................................................................................................41 Acknowledgement.............................................................................................55 CHAPTER 3: A SINGLE RESIDUE IN THE C1 DOMAIN SENSITIZES NOVEL PROTEIN KINASE C ISOFORMS TO CELLULAR DIACYLGLYCEROL PRODUCTION............................................................56 Questions addressed in Chapter 3......................................................................57 Sequence alignment of various C1 domains .....................................................57 Binding of C1bβ and C1bδ Domains to Lipid Vesicles in vitro .......................58 vi Binding of C1bβ and C1bδ Domains to Lipid Vesicles Containing Phosphatidylserine versus Phosphatidylglycerol ..................................61 Translocation of C1bβ and C1bδ Domains to Membranes in Response to Natural Agonist in vivo..........................................................................62 Ca2+-Independent Protein Kinase C Activity for Wild-Type and Mutant PKCβ and Wild-Type PKCδ .................................................................64 Basal Localization of Wild-Type and Mutant C1bβ and C1bδ Domains and Full-Length Proteins in vivo ...........................................................66 Structure and Surface Properties of Representative C1 Domains.....................67 Conclusions .......................................................................................................71 Acknowledgement.............................................................................................74 CHAPTER 4: THE INCREASED ELECTROSTATIC POTENTIAL OF THE ATYPICAL C1 DOMAIN OF PROTEIN KINASE C ζ DOES NOT INCREASE ITS AFFINITY FOR ANIONIC LIPIDS RELATIVE TO TYPICAL C1 DOMAINS.................................................................................75 Regulation of Atypical PKCs by Phospholipids ...............................................75 Structures of Typical and Atypical C1 domains................................................77 Molecular Modeling and Electrostatic Potential Maps of the C1b Domain of PKCγ and the C1 Domain of PKCζ ..................................................79 Binding of C1bβ and C1ζ to Phosphatidylserine-Containing Membranes .......81 Binding of C1ζ to a Panel of Phosphoinositides and Phospholipids.................83 vii Conclusions .......................................................................................................85 Acknowledgement.............................................................................................90 CHAPTER 5: SUMMARY AND CONCLUSIONS ...................................................91 Significance .......................................................................................................93 APPENDIX: EXPERIMENTAL..................................................................................98 I. Materials......................................................................................................98 II. Methods .......................................................................................................99 Cloning of Protein Kinase C and C1 Domains......................................99 Mutagenesis...........................................................................................99 Bacterial Production of Recombinant Protein and Bacterial Cell Lysis .........................................................................................99 Purification of C1 Domains.................................................................100 Lipids...................................................................................................101 Preparation of Lipid Vesicles for Stopped-Flow Spectroscopy ..........102 Preparation of Sucrose-Loaded Lipid Vesicles (SLVs) ......................103 Incorporation of PMA into Vesicles....................................................104 Steady-State Fluorimetry.....................................................................104 Sucrose-Loaded Vesicle (SLV) Assay................................................105 Silver Staining of SDS-PAGE Gels ....................................................106 Data Analysis for Sucrose-Loaded Vesicle Assay ..............................107 Stopped-Flow Fluorescence Spectroscopy .........................................107 Cell Culture.........................................................................................109 viii Protein Kinase C Activity Assay.........................................................110 Cell Imaging ........................................................................................110 Phospholipid Dot Blot.........................................................................111 Sequence Alignment and Phylogenetic Analysis................................112 Modeling..............................................................................................112 REFERENCES............................................................................................................114
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