IDENTIFICATION OF NOVEL SUBSTRATES OF LRRK2, A PARKINSON’S DISEASE ASSOCIATED KINASE By CAROLINE H. LEE Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Dissertation Adviser: Dr. Shu G. Chen Department of Biochemistry CASE WESTERN RESERVE UNIVERSITY May 2014 Case Western Reserve University School of Graduate Studies We hereby approve the thesis/dissertation of CAROLINE H. LEE Candidate for the degree of Doctor of Philosophy Committee Chair Dr. Michael E. Harris Committee Members Dr. Shu G. Chen Dr. Robert Petersen Dr. Hung-ying Kao Dr. Neena Singh Date of Defense December 18, 2013 *We also certify that written approval has been obtained for any proprietary material contained therein. 1 Table of Contents Table of Contents ...................................................................................................................... 2 List of Tables .............................................................................................................................. 4 List of Figures ............................................................................................................................. 5 List of Abbreviations ............................................................................................................... 6 Acknowledgements .................................................................................................................. 7 Abstract ..................................................................................................................................... 10 Chapter 1: Review .................................................................................................................. 12 Parkinson’s Disease ........................................................................................................................ 13 Clinical features of Parkinson’s disease and its prevalence ............................................................ 13 Pathological features of Parkinson’s disease ........................................................................................ 13 Genetics of Parkinson’s disease ............................................................................................................... 14 Leucine­rich repeat kinase 2 (LRRK2) ..................................................................................... 16 Prevalence of LRRK2 mutations in PD ................................................................................................. 16 LRRK2-associated neuropathology ........................................................................................................ 17 Structure of LRRK2 ..................................................................................................................................... 18 Biological function of LRRK2 ................................................................................................................. 19 Animal models of LRRK2­linked PD ......................................................................................... 21 Identifying substrates of LRRK2 ................................................................................................ 22 Chemical genetic approach ........................................................................................................................ 22 Current findings on proposed substrates ................................................................................................ 24 Chapter 2: Chemical Genetic Identification of LRRK2 Substrates ........................ 28 Abstract ............................................................................................................................................... 29 Introduction ...................................................................................................................................... 29 Experimental Procedures ............................................................................................................. 32 Generation of analog-sensitive LRRK2 variants ................................................................................ 32 Immunoprecipitation of flag-tagged LRRK2 ....................................................................................... 33 In vitro kinase assay ..................................................................................................................................... 33 Indirect competition assay .......................................................................................................................... 34 Synthesis of N6-cPe-ATPγS using Ynk1p ............................................................................................ 34 In vivo Substrate labeling using digitonin ............................................................................................. 35 Immunoprecipitation of in vivo labeled substrates ............................................................................. 36 Mass spectrometry analysis ....................................................................................................................... 36 Generation of flag-tagged eIF4G1 variants .......................................................................................... 37 Results ................................................................................................................................................. 38 Characterization of analog-sensitive LRRK2 ...................................................................................... 38 In vivo substrate phosphorylation by AS-G2019S.............................................................................. 41 Global analysis of potential LRRK2 substrates ................................................................................... 43 Validation of eIF4G1 as a LRRK2 substrate ........................................................................................ 44 Discussion .......................................................................................................................................... 45 Chapter 3: Future Directions ............................................................................................. 51 2 Tables ......................................................................................................................................... 58 Table 1. LRRK2 mutations and clinical phenotypes, as reported in various populations. ....................................................................................................................................... 59 Table 2. List of small molecule LRRK2 inhibitors. There are several LRRK2 inhibitors with different molecular structures. ................................................................... 59 Table 3. List of proposed LRRK2 substrates. ......................................................................... 60 Figures ....................................................................................................................................... 61 Figure 1................................................................................................................................................ 62 Figure 2................................................................................................................................................ 62 Figure 3................................................................................................................................................ 63 Figure 4................................................................................................................................................ 64 Figure 5................................................................................................................................................ 65 Figure 6................................................................................................................................................ 66 Figure 7................................................................................................................................................ 67 Figure 8................................................................................................................................................ 68 Figure 9................................................................................................................................................ 70 Figure 10 ............................................................................................................................................. 71 Figure 11 ............................................................................................................................................. 72 Appendix ................................................................................................................................... 73 Appendix I .......................................................................................................................................... 74 References ................................................................................................................................ 81 3 List of Tables Table 1. LRRK2 mutations and clinical phenotypes…………………………………60 Table 2. List of small molecule LRRK2 inhibitors…………………………………...60 Table 3. List of proposed LRRK2 substrates…………………………………………61 4 List of Figures Figure 1. Lewy bodies in PD brains………......................................................................63