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AKAP18: Bidirectional Regulator of Protein Phosphorylation" (2013)

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AKAP18: Bidirectional Regulator of Protein Phosphorylation University of Connecticut OpenCommons@UConn Doctoral Dissertations University of Connecticut Graduate School 7-1-2013 AKAP18: idirB ectional Regulator Of Protein Phosphorylation Arpita Singh University of Connecticut Health Center, [email protected] Follow this and additional works at: https://opencommons.uconn.edu/dissertations Recommended Citation Singh, Arpita, "AKAP18: Bidirectional Regulator Of Protein Phosphorylation" (2013). Doctoral Dissertations. 193. https://opencommons.uconn.edu/dissertations/193 AKAP18: Bidirectional Regulator of Protein Phosphorylation Arpita Singh, Ph.D. University of Connecticut, 2013 (Dissertation Completed: May 2013) The thesis work presented here focuses on AKAP18γ mediated multivalent complex formation and its oligomerization. AKAP18γ belongs to a family of structurally diverse but functionally analogous scaffolding proteins called A-Kinase Anchoring Proteins (AKAPs). These scaffolds specifically target Protein Kinase A (PKA) holoenzyme to a distinct cellular compartment to regulate PKA activity and substrate specificity. Previously, the long isoforms of an AKAP called AKAP18 (AKAP18γ/δ) were established as important cardiac calcium regulating proteins due to their ability to mediate phosphorylation of a protein called Phospholamban (PLN). My dissertation work shows that AKAP18γ directly interacts with Inhibitior-1 (I-1) and enhances its phosphorylation under stimulatory conditions. The AKAP18γ-PKA-I-1 complex together regulates Protein Phosphatase 1 (PP1), also found to be associated with this complex. PP1 is a negative regulator of cardiac function due to its ability to dephosphorylate PLN. Based on these results, we propose a model in which AKAP18γ acts as multivalent complex organizing scaffold that may be targeted to the sarcoplasmic reticulum in cardiac myocytes for PLN phosphorylation regulation. This complex may be found in other cell types to regulate PP1 function alone as I-1, PP1 and AKAP18γ have been found in other tissue. Arpita Singh- University of Connecticut, 2013 Moreover, I also discovered that AKAP18γ is an oligomerizing scaffold and the interaction is isoform specific. A shorter isoform of this gene, AKAP18α does not oligomerize and there is no hetero oligomerization between these isoforms (AKA18α and AKAP18γ). Live cell microscopy shows that AKAP18γ can form heterogenous oligomers ranging from monomers to tetramers in cells. Furthermore, in order to understand the physiological implication of this oligomerization, we developed a computational model. The model was based on activation and release of PKA catalytic subunit, which requires PKA regulatory subunit phosphorylation. Presence of multiple PKA holoezymes will potentiate release of multiple PKA catalytic subunits due to increased PKA regulatory subunit phosphorylation. When this possibility was tested against a generic membrane compartmentalized substrate, the computational model revealed increased PKA mediated phosphorylation of the substrate. These results are suggestive of a mechanism in which AKAP18γ oligomerizes to mediate enhanced substrate phosphorylation by PKA. Based on the evidence presented in this thesis and work done by others, it appears that AKAP18γ not only regulates PLN phosphorylation but also its de-phosphorylation by regulating PP1 activity. Importantly, AKAP18γ can oligomerize which may be the potential mechanism by which AKAP18γ concentrates enzymes required for PLN regulation. The title of the thesis “AKAP18: bidirectional regulator of protein phosphorylation” reflects these properties of AKAP18γ. AKAP18: Bidirectional Regulator of Protein Phosphorylation Arpita Singh B.Sc, Banaras Hindu University, 2005 M.Sc., Pondicherry University, 2007 A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the University of Connecticut 2013 Copyright by, Arpita Singh 2013 i APPROVAL PAGE Doctor of Philosophy Dissertation AKAP18: Bidirectional Regulator of Protein Phosphorylation Presented by Arpita Singh, B.Sc., M.Sc. Major Advisor Dr. Kimberly Dodge-Kafka Associate Advisor Dr. Asis Das Associate Advisor Dr. Bing Hao Associate Advisor Dr. Betty A. Eipper Associate Advisor Dr. Lixia Yue University of Connecticut 2013 ii ACKNOWLEDGEMENTS “I dedicate this thesis work to my grandparents, who were a source of motivation to me- their memories continue to inspire me” iii This thesis work is a culmination of five years of work, which I could not have finished without the support of these people whom I wish to acknowledge. I owe my deepest gratitude to my supervisor, Dr. Kimberly Dodge-Kafka, for providing me the opportunity to work in her laboratory. She has been a great teacher, a guide for navigating through graduate school and extremely supportive through highs and lows. I thank her for her dedication towards ensuring my success. I am extremely grateful to my PhD thesis committee members, Dr. Asis Das, Dr. Betty Eipper, Dr. Bing Hao, and Dr. Lixia Yue, for their advice and guidance during various stages of my PhD. I am indebted to them for helping me develop qualities that are required to be a good scientist. My current and previous lab mates, Dr. Max Vargas, Dr. Andrew Le, John Redden, and Marc Riggati, also share the credit for my success. They have been extremely supportive both in and out of the lab. I am very thankful for the light moments we have shared and the tough ones we have endured together. I would like to thank the departments of Molecular, Microbial and Structural Biology and Calhoun Cardiology Center for extending their support and help. For the various seminars, journal clubs, retreats and holiday parties that I have attended and had a great experience at, I thank you. I also express my gratitude to Dr. Roger Thrall for his guidance and continued support. My special thanks to Dr. John Carson for helping me hone my presentation skills, to Dr. Ann Cowan for always being here to help with microscopy questions and to Dr. Chris Heinen for all his advice during the various department presentations and journal clubs. iv I am indebted to UConn Health Center for inviting me to pursue my PhD studies at the institute. I also thank Dr. Barbara Kream and Dr. Lawrence A. Klobutcher who have been great help in understanding the nitty-gritty’s of graduate school. The graduate school office, the registrar’s office, the human resources office and the bursar’s office for helping me navigate through the formailities and requirements that needed to be fulfilled from time to time over the past five years. My success at every stage of my life would be attributed to my family. My mother, who has been my pillar of strength, has made my success the mission of her life. I cannot imagine coming this far without her. My father, who inculcated in me the love of biology and medicine, has been a role model for me. I thank my loving brother, who always had words of encouragement during the toughest times. I cannot find words to express my gratitude to my in-laws who have been very understanding and supportive through this journey. They truly have been my second set of parents. This thesis would have remained a dream had it not been for my husband who had motivated me to embark on this endeavor and has been constantly by my side from the start to the finish line. It is hard to imagine how things would have turned out without his constant encouragement and cheer. A special thanks to all my friends, for sharing my joys and sorrows, for being there for me always. Finally, I would like to thank all the people who have touched my life, in ways big and small, and have encouraged and appreciated my efforts. v TABLE OF CONTENTS APPROVAL PAGE ......................................................................................................... II ACKNOWLEDGEMENTS ...........................................................................................III TABLE OF CONTENTS ...............................................................................................VI 1. INTRODUCTION ..................................................................................................... 1 1.1. SCAFFOLDING PROTEINS IN CELL FUNCTION.......................................... 1 1.2. AKAPS AND THEIR ROLE IN PKA SIGNAL INTEGRATION AND REGULATION............................................................................................................... 4 1.3. REGULATION OF PP1 FOR PKA SIGNAL PROPAGATION....................... 13 1.4. REGULATION OF PP1 BY I-1 ......................................................................... 20 1.5. REGULATION OF PP1 BY AKAPS................................................................. 23 1.6. IMPORTANCE OF PP1 AND I-1 IN DISEASE ............................................... 26 1.7. AKAP18 AS A MODEL AKAP......................................................................... 32 2. THE LARGE ISOFORMS OF AKAP18 MEDIATE THE PHOSPHORYLATION OF INHIBITOR-1 BY PKA AND THE INHIBITION OF PP1 ACTIVITY............................................................................................................... 38 2.1. ABSTRACT ........................................................................................................ 39 2.2. INTRODUCTION............................................................................................... 39 2.3. MATERIALS AND METHODS ........................................................................ 41 2.3.1. Antibodies ....................................................................................................
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