©2018 NA CAI ALL RIGHTS RESERVED THE FUNCTIONAL INTERPLAY BETWEEN TRPM7 CHANNEL-KINASE AUTOPHOSPHORYLATION AND ITS CELLULAR REGULATION by NA CAI A dissertation submitted to the School of Graduate Studies Rutgers, The State University of New Jersey In partial fulfillment of the requirements For the degree of Doctor of Philosophy Graduate Program in Cellular and Molecular Pharmacology Written under the direction of Loren W. Runnels, Ph.D. And approved by ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ ________________________________________________ New Brunswick, New Jersey May, 2018 ABSTRACT OF THE DISSERTATION THE FUNCTIONAL INTERPLAY BETWEEN TRPM7 CHANNEL-KINASE AUTOPHOSPHORYLATION AND ITS CELLULAR REGULATION By NA CAI Dissertation Director: Loren W. Runnels, Ph.D. As a member of the transient receptor potential ion channel subfamily, TRPM7 is a remarkable ion channel in possession of its own functional kinase domain. TRPM7 is 2+ 2+ ubiquitously expressed and permeable to divalent cations, allowing Mg , Ca , and trace metals ions such as Zn2+ to constitute the channel’s characteristic small inward current. The channel’s functional kinase domain is located at the protein’s cytosolic COOH terminus, placing TRPM7 also into a family of serine/threonine-phosphorylating alpha- kinases. It is not intuitively clear why a channel is covalently linked to a kinase, especially as it has been found that the kinase activity of TRPM7 is not required for channel gating. Previous studies have shown that TRPM7 is autophosphorylated, and yet the functional outcome of this autophosphorylation remain unknown. Motivated to understand the impact of phosphorylation on the function and regulation of this channel-kinase, I performed a comprehensive phosphoproteomic analysis of TRPM7 by mass spectrometry to identify ii the major in vivo phosphorylation sites on TRPM7. The results of the mass spectrometry study uncovered potential mechanisms by which the catalytic activity of TRPM7 kinase is regulated through autophosphorylation. My experiments also revealed a significant role of TRPM7’s kinase activity in regulating the posttranslational processing of TRPM7. Utilizing the TRPM7-K1646R kinase-inactive mutant, I discovered that TRPM7 kinase inactivation leads to faster protein degradation and intracellular retention of the channel in polarized epithelial cells compared to the wildtype protein. Mutational analysis of TRPM7 autophosphorylation sites further revealed a role for S1360 as a key residue mediating both protein stability and intracellular trafficking of TRPM7. In addition, I discovered that the intrinsic kinase activity of TRPM7 mediates the interaction of the channel with the signaling protein 14-3-3θ, whose binding sites on TRPM7 also contribute to the regulation of TRPM7 trafficking. Overall, these findings expand our knowledge of the in vivo phosphorylation profile of TRPM7 and, more importantly, increase our understanding of the significance of TRPM7’s kinase for functional regulation of the channel. iii ACKNOWLEDGMENTS It is a great pleasure to express my gratitude to many people who helped in making this thesis possible. First and foremost, I would like to thank my supervisor Dr. Loren W. Runnels for all of his guidance and support. I am deeply honored to become a student of Loren’s and carry on the scientific inquiry he started twenty years ago. I admire Loren for his passion for science, his dedication to students, and his kindness and generosity to everyone surrounding. Loren has taught me more than just research but more importantly about how to recognize and trust my potential. His continuous encouragement and support guided me through every step of the journey, especially at some of the more difficult moments. I am deeply grateful for his support and mentorship. I would also like to express my sincere gratitude to my committee members, Dr. Nancy C. Walworth, Dr. Ann M. Stock, Dr. Victor Jin and Dr. Kiran Madura, whose advice and support are invaluable to my progress. Their passion for research inspires me to continue pursuing my own. I wish to thank Dr. Vikas Nanda at the Department of Biochemistry for his helpful discussion on the kinase project and expertise in molecular modeling. I am also grateful to Dr. Peter Lobel, director of the Biological Mass Spectrometry Facility, for giving me introduction lectures on mass spectrometry that allowed me to take on my project. I am thankful to my lab members who accompanied me in the past years. I am lucky to have Liping Lou as my twin sister in the lab. I thank her for always being so cheerful and putting up with my stress temperament from time to time. I would also like to thank Dr. Yuko Komiya for sharing with me many of her research techniques. I enjoyed her company and learned a great deal from her strong work ethics. I appreciate Namariq Al- iv Saadi for helping me with experiments in the past a couple of months as I was racing across the finish line. Other current and former members of the lab, Sandra Tetteh, Dr. Zhiyong Bai, Dr. Jeffrey Overton, and Shufei Tao, have also offered me help and support. I would like to express my gratitude to all the faculty, students, and staff members of the Pharmacology Department. The department has become my second home because of all you lovely people, and I will miss our monthly gatherings and Christmas parties. I want to say thanks to my close friends, Chen Wang and Liping Lou, for standing by my side all the way through and giving me unconditional support. I see them as my sisters and will always cherish our friendships. I am also grateful for the company of many good friends: Yijun Zhou, Jing Lin, Huirong Zhang, Wurihan, Yuanwang Pan, Chengshen Zhu, Zhichao Song, and many more. Their presence made my graduate life joyful and unforgettable. I sincerely wish every one of them a very bright future. I want to dedicate this thesis especially to my grandfather, who had great faith in me since I was little. He installed me with the idea of growing up and becoming a female scientist. I am sure he would be very proud of me now. Lastly, and most importantly, I want to thank my parents for their endless love, encouragement, and support. I have been away from home for too long, but there was not a single day that I don't feel their unconditional love for me. I want to say “mom and dad, thank you for your sacrifices. I am proud to be your daughter, and I love you two so deeply.” Na Cai New Jersey, United States March 2018 v TABLE OF CONTENTS ABSTRACT OF THE DISSERTATION ............................................................................. ii ACKNOWLEDGMENTS ..................................................................................................... iv LIST OF FIGURES ............................................................................................................. viii LIST OF TABLES ................................................................................................................. ix INTRODUCTION ................................................................................................................... 1 1. TRPM7 .............................................................................................................................. 1 2. The functional interplay between TRPM7 channel and kinase domain .................... 3 3. Cellular and physiological functions of TRPM7 kinase ............................................... 4 4. Functional significance of TRPM7 phosphorylation .................................................... 6 5. Regulation of TRPM7 kinase activity ............................................................................ 7 6. Rationale and hypothesis ................................................................................................ 9 MATERIALS AND METHODS .......................................................................................... 10 SECTION I: MASS SPECTROMETRIC ANALYSIS OF TRPM7 AND TRPM6 IN VIVO PHOSPHORYLATION ......................................................................................... 10 1.1 Constructs .................................................................................................................. 10 1.2 Cell lines .................................................................................................................... 10 1.3 Expression and purification of TRPM7 kinases for MS analysis .............................. 11 1.4 Phosphorylation sites identification by LC-MS/MS ................................................. 13 1.5 In vitro kinase assay for TRPM7 proteins expressed in HEK-293 cells ................... 14 1.6 Immunoblotting ......................................................................................................... 14 SECTION II: TRPM7 KINASE ACTIVITY IS REGULATED BY AUTOPHOSPHORYLATION ......................................................................................... 16 2.1 Constructs .................................................................................................................. 16 2.2 Sumo-TRPM7-kinase purification ............................................................................ 16 2.3 MS analysis of Sumo-TRPM7-kinase ...................................................................... 17 2.4 In vitro kinase assay of Sumo-TRPM7-kinase .........................................................