Wayne State University Wayne State University Dissertations 1-1-2017 The evelopmeD nt Of Chemical Methods To Discover Kinase Substrates And Map Cell Signaling With Gamma-Modified Atp Analog- Dependent Kinase-Catalyzed Phosphorylation Dissanayaka Mudiyanselage Maheeka Madhubashini Embogama Wayne State University, Follow this and additional works at: https://digitalcommons.wayne.edu/oa_dissertations Part of the Analytical Chemistry Commons, and the Biochemistry Commons Recommended Citation Embogama, Dissanayaka Mudiyanselage Maheeka Madhubashini, "The eD velopment Of Chemical Methods To Discover Kinase Substrates And Map Cell Signaling With Gamma-Modified Atp Analog-Dependent Kinase-Catalyzed Phosphorylation" (2017). Wayne State University Dissertations. 1698. https://digitalcommons.wayne.edu/oa_dissertations/1698 This Open Access Dissertation is brought to you for free and open access by DigitalCommons@WayneState. It has been accepted for inclusion in Wayne State University Dissertations by an authorized administrator of DigitalCommons@WayneState. THE DEVELOPMENT OF CHEMICAL METHODS TO DISCOVER KINASE SUBSTRATES AND MAP CELL SIGNALING WITH GAMMA-MODIFIED ATP ANALOG-DEPENDENT KINASE-CATALYZED PHOSPHORYLATION by DISSANAYAKA M. MAHEEKA M. EMBOGAMA DISSERTATION Submitted to the Graduate School of Wayne State University, Detroit, Michigan in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY 2017 MAJOR: CHEMISTRY (Biochemistry) Approved By: Advisor Date DEDICATION To my beloved mother, father, husband, daughter and sister. ii ACKNOWLEGEMENTS Many people have helped me during the past five years of earning my PhD. I would like to take this opportunity to convey my gratitude to them. First and foremost, I would like to thank my research supervisor Dr. Mary Kay Pflum for being the greatest mentor that I have met so far. Her positive attitude, unlimited support and guidance with extreme patience and encouragement have helped me to complete my PhD successfully. My sincere gratitude goes to my thesis committee members, Dr. Louis J. Romano, Dr. Sarah Trimpin and Dr. Russell L. Finley, for their time, helpful suggestions and support. I would also like to thank all the Pflum lab members that I have worked with over the last five years. My special appreciation goes to, Charmara, Todd, Satish, Magdalene, Thilani, Ahmed Fouda, Pavithra, Dhanusha, Ahmed Negmeldin, Inosha, Vindya, Nuwan and Aparni. Thank you all for giving wonderful experiences about research and life in general. Further, I would like to convey my gratitude to Dr. Joseph Caruso and Namhee Shrin from the Proteomic core facility at Wayne State University, for their valuable support for mass spectrometry analysis. Finally, I would like to thank my mother, husband, daughter, family and friends for their love and encouragement to thrive in my graduate studies. iii TABLE OF CONTENTS Dedication ........................................................................................................................... ii Acknowledgements ............................................................................................................ iii List of Tables ................................................................................................................... viii List of Figures .................................................................................................................... ix List of Schemes ................................................................................................................. xii List of Abbreviations ....................................................................................................... xiii CHAPTER 1: Introduction ..............................................................................................1 1.1 Kinases ...........................................................................................................................1 1.2 Phosphorylation .............................................................................................................2 1.3 Protein kinase structure and mechanism ........................................................................4 1.4 Kinase consensus sites ...................................................................................................7 1.5 Kinase-mediated cell signaling ......................................................................................8 1.5.1 PKA signaling .....................................................................................................9 1.6 Kinase-mediated pathological conditions ....................................................................11 1.7 Kinase inhibitors for therapeutics ................................................................................12 1.7.1 Monoclonal antibodies and vaccines as kinase inhibitors ..................................12 1.7.2 Small molecule kinase inhibitors ........................................................................13 1.7.3 Structure based drug design ................................................................................13 1.8 Kinase substrate profiling and mapping signaling pathway ........................................15 1.9 Kinase cosubstrate promiscuity with gamma-phosphate modified ATP analogs ........15 1.9.1 ATP- γS ...............................................................................................................16 iv 1.9.2 ATP-dansyl .........................................................................................................17 1.9.3 ATP-aryl azide ....................................................................................................18 1.9.4 ATP-BODIPY and ATP-ferrocene .....................................................................18 1.9.5 ATP-biotin ..........................................................................................................21 1.10 Thesis Projects ...........................................................................................................23 CHAPTER 2: Characterization of Kinase-catalyzed biotinylation ............................25 2.1 Introduction ..................................................................................................................25 2.1.1 Crystal structures of protein kinase active site ...................................................25 2.1.2 Human kinome ....................................................................................................27 2.1.3 Characterization of generality of kinase-catalyzed biotinylation by qualitative, quantitative, and kinetic methods ......................................................................................29 2.2 Results and Discussion ................................................................................................31 2.2.1 Synthesis of ATP-biotin ......................................................................................31 2.2.2 Quantitative analysis of kinase-catalyzed biotinylation using gel methods .......31 2.2.3 Kinetic analysis of kinase-catalyzed biotinylation .............................................33 2.3 Discussion and conclusions .........................................................................................38 2.4 Experimental ...............................................................................................................39 2.4.1 Materials .............................................................................................................39 2.4.2 Instrumentation ....................................................................................................39 2.4.3 Synthesis of ATP-biotin .......................................................................................40 2.4.3.1 Synthesis of biotin-amine ..............................................................................40 2.4.3.2 Synthesis of ATP-biotin .................................................................................40 2.4.4 Quantitative Kinase-catalyzed biotinylation of protiens.......................................41 v 2.4.5 SDS-PAGE ...........................................................................................................41 2.4.6 Pro-Q staining .......................................................................................................42 2.4.7 Sypro Ruby Staining .............................................................................................43 2.4.8 Gel Image Quantification ......................................................................................43 2.4.9 NADH-coupled enzyme assay ..............................................................................44 CHAPTER 3: Kinase-catalyzed biotinylation with inactivated lysates for discovery of substrates (K-BILDS) ..................................................................................................46 3.1 Introduction to current methods available to identify kinase-substrates ......................46 3.1.1 Kinase substrate detection by in-vitro assays using (γ-32P) ATP ......................47 3.1.2 MS based substrate detection after an enrichment ..............................................48 3.1.3 ASKA for kinase substrate discovery .................................................................48 3.1.4 Kinase-substrate detection by protein-protein interaction assays .......................49 3.2 Results and Discussion ................................................................................................50