UC San Diego Electronic Theses and Dissertations
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UC San Diego UC San Diego Electronic Theses and Dissertations Title Isolation and characterization of neuronal substrates of the ubiquitin proteasome system Permalink https://escholarship.org/uc/item/7jg5g3qg Author Keil, Jeffrey McCartney Publication Date 2011 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Isolation and Characterization of Neuronal Substrates of the Ubiquitin Proteasome System A dissertation submitted in partial satisfaction of the requirements for the degree Doctor of Philosophy in Biology by Jeffrey McCartney Keil Committee in charge: Professor Gentry Patrick, Chair Professor Michael Burkart Professor Randolph Hampton Professor Terunaga Nakagawa Professor Yimin Zou 2011 Copyright Jeffrey McCartney Keil, 2011 All rights reserved The dissertation of Jeffrey McCartney Keil is approved, and it is acceptable in quality and form for publication on microfilm and electronically: ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ ________________________________________________________________________ Chair University of California, San Diego 2011 iii DEDICATION To my loving parents, Richard and Mary, who made this all possible. iv EPIGRAPH You could tell by the way he talked, though, that he had gone to school a long time. That was probably what was wrong with him. John Kennedy Toole (A Confederacy of Dunces) v TABLE OF CONTENTS Signature Page ............................................................................................................. iii Dedication .................................................................................................................... iv Epigraph ....................................................................................................................... v Table of Contents ......................................................................................................... vi List of Figures .............................................................................................................. viii List of Tables ............................................................................................................... x Acknowledgements ...................................................................................................... xi Vita and Publications ................................................................................................... xiii Abstract of the Dissertation ......................................................................................... xiv Chapter I Isolation and Identification of Novel, Synaptic Substrates of the Ubiquitin Proteasome System ..................................................................................................... 1 Introduction ...................................................................................................... 2 Results .............................................................................................................. 8 Discussion ........................................................................................................ 13 Materials and Methods ..................................................................................... 17 Figures .............................................................................................................. 23 Chapter II Characterization of Stromal Interaction Molecule I (STIM1) .................. 44 Introduction ...................................................................................................... 45 Results .............................................................................................................. 51 vi Discussion ........................................................................................................ 63 Materials and Methods ..................................................................................... 70 Figures .............................................................................................................. 76 Chapter III Ubiquitin-Mediated Regulation of Actin Dynamics ............................... 92 Introduction ...................................................................................................... 93 Results .............................................................................................................. 97 Discussion ........................................................................................................ 103 Materials and Methods ..................................................................................... 106 Figures .............................................................................................................. 109 References .................................................................................................................... 113 vii LIST OF FIGURES Chapter I Figure 1. Isolation and identification of synaptic ubiquitinated proteins ...................... 23 Figure 2. Identification of synaptic ubiquitinated proteins ............................................ 24 Figure 3. Pie chart showing function classification of synaptic candidate ubiquitinated proteins (synCUP’s) purified by NiNTA affinity chromatography and then identified by mass spec. .................................................................................................................... 26 Figure 4. S5A enrichment of rat P2 lysates for mass spectrometry ............................... 40 Figure 5. Scatterplot of top 150 overlapping targets between DMSO and MG-132 treated samples ........................................................................................................................... 43 Chapter II Figure 6. STIM1 is ubiquitinated in the rat brain and HEK-293T cells ........................ 76 Figure 7. Biochemical examination of spatial and temporal expression patterns of STIM1 in the rat brain ................................................................................................................ 77 Figure 8. Immunohistochemical examination of spatial and temporal expression patterns of endogenous STIM1 in the rat brain ........................................................................... 78 Figure 9. Immunohistochemical examination of spatial and temporal expression patterns of exogenous STIM1 in the rat brain ............................................................................. 80 Figure 10. STIM1 is present at the surface of mature and developing hippocampal neurons ........................................................................................................................... 81 Figure 11. Inhibition of SOCE causes decreased neurite outgrowth in developing hippocampal neurons ..................................................................................................... 82 viii Figure 12. Store-depletion induced rapid redistribution of STIM1-GFP in hippocampal neurons ........................................................................................................................... 84 Figure 13. Effects of proteasome inhibition on store-operated calcium (SOC) influx, STIM1 subcellular localization and STIM1 stability ..................................................... 86 Figure 14. Store-depletion increases population of proteasome-dependent STIM1 ubiquitination ................................................................................................................. 87 Figure 15. Overexpression of the E3 ligase POSH decreases surface STIM1-GFP levels… .......................................................................................................................... 88 Figure 16. Predicted ubiquitination sites on mouse STIM1 .......................................... 89 Figure 17. Lysine mutant of STIM1 shows defects in SOCE, with concomitant loss in stability and surface down-regulation ............................................................................ 90 Figure 18. Mitosis-dependent posttranslational modifications of STIM1 ..................... 91 Chapter III Figure 19.Actin based spine motility is proteasomal-dependent… ............................... 109 Figure 20. Kalirin-5 and kalirin-7 are ubiquitinated ...................................................... 110 Figure 21. PP1/2 and PKA activity affect the ubiquitination and stability of kalirin-7 and kalirin-5 .......................................................................................................................... 111 Figure 22. Kalirin-5 is ubiquitinated by Cul4B ............................................................. 112 ix LIST OF TABLES Chapter I Table 1. Unfiltered, non-redundant list of proteins identified via proteomic screen for synaptic ubiquitinated proteins ...................................................................................... 19 Table 2. Top 40 synaptic ubiquitinated proteins by ratio .............................................. 25 Table 3. Unfiltered, non-redundant list of proteins identified via proteomic screen for synaptic ubiquitinated proteins ...................................................................................... 27 Table 4. Top 50 hits from S5A directed screen for synaptic poly-ubiquitinated