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