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UC San Diego UC San Diego Electronic Theses and Dissertations UC San Diego UC San Diego Electronic Theses and Dissertations Title The mechanics of SR protein phosphorylation by the splicing kinases SRPK1 and Clk/Sty Permalink https://escholarship.org/uc/item/03n8h3g9 Author Hagopian, Jonathan Charles Publication Date 2008 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO The Mechanics of SR protein Phosphorylation by the Splicing Kinases SRPK1 and Clk/Sty A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biomedical Sciences by Jonathan Charles Hagopian Committee in charge: Professor Joseph Adams, Chair Professor Xiang-Dong Fu, Co-Chair Professor Pieter Dorrestein Professor Gourisankar Ghosh Professor Susan Taylor 2008 i Copyright Jonathan Charles Hagopian, 2008 All Rights Reserved ii The dissertation of Jonathan Charles Hagopian is approved, and acceptable in quality and form for publication electronically and on microfilm: ______________________________________________ ______________________________________________ ______________________________________________ ______________________________________________ Co-Chair ______________________________________________ Chair University of California, San Diego 2008 iii TABLE OF CONTENTS Signature Page.............................................................................................. iii Table of Contents.......................................................................................... iv List of Figures............................................................................................... vii Acknowledgments....................................................................................... xi Vita................................................................................................................ xiii Publications................................................................................................... xiv Abstract of the Dissertation........................................................................... xv Chapter I General Introduction................................................................................ 1 A. Pre-mRNA splicing.................................................................................. 2 B. SR Protein Localization........................................................................... 7 C. Splicing Kinases are Structurally and Catalytically Unique Enzymes................................................................................................... 9 D. Processive Phosphorylation by Protein Kinases...................................... 11 E. SR Proteins and SR Protein Kinases in Disease and Tumorigenesis.......................................................................................... 15 F. Focus of Study......................................................................................... 18 Chapter II Assay Development and Sequence Analysis of Investigated Proteins.............................................................................. 20 A. Introduction............................................................................................ 21 B. Assay Development................................................................................. 22 1. The Kinase-Dead Start-Trap Experiment for Mechanistic Analysis of Polyphosphorylation by SR Protein Kinases............ 22 2. The Competition Assay Yields True Dissociation Constants for SRPK1 Substrates.................................................. 27 C. Sequence Analysis of Investigated Proteins............................................ 34 Chapter III Kinetic Analysis of ASF/SF2 Phosphorylation by SRPK1 and Clk/Sty........................................................................... 44 iv A. Abstract................................................................................................... 45 B. Introduction............................................................................................. 45 C. Results..................................................................................................... 49 1. Phosphorylation Kinetics of ASF/SF2 by SRPK1 and Clk/Sty.................................................................................. 49 2. Dissociation Constants of SRPK1 and Clk/Sty........................... 51 3. SRPK1 Phosphorylates the N-terminal Region of the ASF/SF2 RS Domain.............................................................. 52 4. Stepwise Phosphorylation of ASF/SF2 by SRPK1 and Clk/Sty.................................................................................. 52 5. Processive Phosphorylation of ASF/SF2 by SRPK1.................... 56 6. Clk/Sty Catalyzes Processive Phosphorylation of the Entire RS Domain.............................................................. 57 7. Sequential Phosphorylation of RS Domain Segments By Clk/Sty.................................................................................... 59 8. Contribution of SRPK1 Spacer Domain to Catalysis and Affinity for ASF/SF2............................................................. 62 D. Discussion................................................................................................ 64 1. Partial Phosphorylation of the RS Domain By SRPK1................ 64 2. Modifying the Entire RS Domain without Enzyme Dissociation.................................................................................. 66 3. Cellular Control of Phosphorylation Specificity......................... 66 4. Signals in the Spacer for Cytoplasmic Localization of SRPK1..................................................................................... 67 E. Materials and Methods............................................................................. 68 1. Expression and Purification of Recombinant Proteins................ 68 2. Phosphorylation Reactions.......................................................... 70 3. Data Analysis............................................................................... 71 Chapter IV Adaptable Molecular Interactions Guide Phosphorylation of the SR Protein ASF/SF2 by SRPK1.......................................................... 73 A. Abstract................................................................................................... 74 B. Introduction............................................................................................. 74 C. Results..................................................................................................... 80 1. RS domain is Sufficient for High-Affinity Binding of ASF/SF2.................................................................................. 80 2. RS Domain can Initiate, but not Sustain, Processive Phosphorylation........................................................................... 82 3. High-Affinity ASF/SF2 Binding is Resistant to RS Domain Charge Alterations....................................................................... 86 4. Redundant Binding Determinants Revealed Through C-terminal Deletions................................................................... 90 v 5. Docking Groove in SRPK1 Controls Initiation of Processive Phosphorylation.................................................... 94 D. Discussion.............................................................................................. 96 1. SRPK1 Uses an Atypical Docking Mechanism for ASF/SF2 Recognition............................................................ 97 2. Redundant Binding Modules Guide Processivity Through a Strain Mechanism...................................................... 98 3. Model for Processive Phosphorylation....................................... 100 E. Materials and Methods............................................................................ 102 1. Peptide Synthesis 2. Expression and Purification of Recombinant Proteins................ 102 3. Phosphorylation Reactions........................................................... 103 4. Mass Spectrometric Analyses...................................................... 104 5. Data Analysis............................................................................... 104 Chapter V FT-ICR MS Analysis of ASF/SF2 Protein Phosphorylation by SRPK1.............................................................................................. 106 A. Abstract..................................................................................................... 107 B. Introduction............................................................................................... 108 C. Results....................................................................................................... 110 1. The Mysterious Phospho-Serine of the SRPK1S:ASF/SF2(RRM1RS2) Crystal Structure................ 110 2. Analysis of ASF/SF2RRM1 Phosphorylation by SRPK1.......... 114 3. Analysis of ASF/SF2 Phosphorylation by SRPK1....................... 118 D. Discussion................................................................................................ 127 1. Understanding the SRPK1S: ASF/SF2(RRM1RS2) Phospho-serine.............................................................................. 127 2. Insights Gained by FTMS Analysis of ASF/SF2 Phosphorylation by SRPK1.......................................................... 128 Chapter VI Future Directions................................................................................. 133 A. Arginine-Serine
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