Structural basis for phosphatase regulation by the anchoring protein AKAP79 Patrick J. Nygren A dissertation submitted in partial fulfillment of the requirement for the degree of Doctor of Philosophy University of Washington 2015 Reading Committee: John Scott, Chair Stan McKnight Rich Gardner Program authorized to offer degree: Pharmacology © Copyright 2015 Patrick J. Nygren University of Washington Abstract Structural basis for phosphatase regulation by the anchoring protein AKAP79 Patrick J. Nygren Chair of the Supervisory Committee: Professor John D. Scott Department of Pharmacology The calcium/calmodulin-activated protein phosphatase 2B (PP2B, or calcineurin) regulates diverse biological processes including glucose homeostasis and synaptic plasticity. PP2B is targeted to specific substrates, such as the GluRI subunit of the AMPA receptor, by the scaffolding molecule A-kinase anchoring protein 79 (AKAP79), which also interacts with protein kinase A (PKA). AKAP79 contains a short linear motif that is the primary site of interaction with PP2B. We have identified an auxiliary interaction site for PP2B at the N-terminal region of AKAP79. Using hybrid approaches including single particle EM, chemical crosslinking/mass spectrometry, biophysical techniques, and in vitro protein- protein interaction assays, I show that this additional interaction exclusively binds activated PP2B through a conserved LxVP mechanism. I mapped a 16 amino acid region in AKAP79 that is necessary for the LxVP interaction and show that the peptide can effectively compete for binding. I use FRET reporters for PP2B activity to show that this auxiliary interaction is important for tuning the activity of PP2B towards specific substrates. Preliminary negative stain EM studies reveal that AKAP79 forms multiple simultaneous contacts with PP2B in the presence of calcium and calmodulin. This structural model shows that AKAP79 has a greater role in regulating PP2B activity and targeting than previously appreciated. This work describes a dynamic model of PP2B anchoring in which the active i phosphatase is anchored differently than the inactive form. A bipartite interaction allows control of localization through the PxIxIT motif, while simultaneously allowing fine control of PP2B sensitivity to calcium through the dynamic LxVP motif. ii Table of Contents Abstract ............................................................................................................ i Table of Contents .......................................................................................... iii List of Figures and Tables ............................................................................. iv Table of Abbreviations .................................................................................... v Acknowledgements ......................................................................................... 1 Chapter 1 – Introduction to AKAPs and phosphatase anchoring ............. 4 Chapter 2 – Purification and electron microscopy of AKAP79/PP2B ..... 20 Chapter 3 – Identification and characterization of an LxVP motif in AKAP79 ......................................................................................................... 36 Chapter 4 – Experimental methods ............................................................. 49 References ..................................................................................................... 58 iii List of Figures 1.1 – Structural basis for protein kinase A (PKA) holoenzyme formation and anchoring ................................................................................................. 7 1.2 – Structural basis for phosphatase regulation and anchoring .......... 15 2.1 – Characterizing disorder and short linear motifs in AKAP79 complexes ..................................................................................................... 26 2.2 – Analysis of compositional and conformational changes in the presence and absence of calcium .............................................................. 28 2.3 – Random conical tilt analysis of inactive complexes ........................ 31 2.4 – Random conical tilt analysis of active complexes ........................... 32 3.1 – Crosslinking/mass-spectrometry of AKAP79/PP2B interactions ... 38 3.2 – Mapping and characterization of the LxVP binding interfaces for AKAP79 and PP2B ....................................................................................... 40 3.3 – Use of AKAP79-CaNAR biosensors for anchored PP2B activity reveal that AKAP79 fine-tunes PP2B sensitivity towards physiological ranges of calcium ......................................................................................... 43 iv Table of Abbreviations Abbreviation Full name AKAP A-kinase anchoring protein AMPA α-amino-3-hydroxy-5- methylisooxazole-4-propionic acid CaM calmodulin CaMKII Ca2+ /calmodulin-dependent protein kinase II cAMP Cyclic adenosine mono phosphate CaNAR Calcineurin activity reporter Cav1.2 Voltage-gated calcium channel 1.2 Cryo-EM Cryo-electron microscopy DARPP32 Dopamine- and cAMP-regulated phosphoprotein, Mr 32 kDa D/D domain Docking/dimerization domain diFMUP 6,8-difluoro-4-methylumbelliferyl phosphate EDTA Ethylenediaminetetraacetic acid FK506 Tacrolimus FKBP FK506-binding protein FMP-API-1 3,3'-diamino-4,4'- dihydroxydiphenylmethane FRET Förster resonance energy transfer GluA1 Glutamate AMPA receptor subunit 1 GPCR G-protein coupled receptor GST Glutathione s-transferase Ht31 Human thyroid clone 31 IPTG Isopropyl β-D-1-thiogalactopyranoside MAGUK Membrane-associated guanylate kinase mAKAP Muscle-specific AKAP MAP2 Microtubule-associated protein 1 MBBR Membrane binding basic region MBP Maltose binding protein NFAT Nuclear factor activator of T cells NMDA N-methyl D-aspartate NMR Nuclear magnetic resonance PDE Phosphodiesterase PIP2 Phosphoinositol 2-phosphate PKA Protein kinase A PKC Protein kinase C pNPP para-Nitrophenylphosphate PNUTS PP1 nuclear targeting subunit v PP1 Protein phosphatase 1 PP2B Protein phosphatase 2B PSD-95 Postsynaptic density protein 95 RI PKA regulatory subunit isoform I RII PKA regulatory subunit isoform II RCaMP Red genetically encoded calcium indicator RCT Random conical tilt SAP97 Synapse associated protein 97 SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SEC-MALS Size-exclusion chromatography- multiangle light scattering SLiM Short linear motif superAKAPis Super AKAP in silico XL-MS Crosslinking-mass spectrometry vi Acknowledgments First I want to thank John Scott, for guiding and mentoring me through this project. John, without your investment in my development as a scientist, my thesis would have been a shadow of itself. I am grateful for the opportunity to work in your group and learn from you. In addition, I am indebted to Lorene for her help with formatting and thinking through how to best layout figures and organize my manuscripts. Mel has been invaluable in keeping our lab group coordinated and on top of administrative details. I would also like to thank my graduate committee for their guidance and support through this multi-faceted project, and in particular my reading committee, John, Stan, and Rich, for taking time to review this thesis. My fellow lab members, past and present, have been the best day-to-day support I could ask for. Senior members such as Donelson, Matt, Dave, Simon, Heidi, Emily, Catherine, and Chris offered valuable input and advice almost daily, whilst also being available to commiserate over a beer when their advice went awry. Junior members such as Laura, Rigney, Paula, and Leah, shared their infectious youthful optimism, and also commiserated over beers. However, the most special friendships were shared with Bret and Jen, who were with me for nearly all of my graduate career, and supported me in personal and professional times through the hardest and the best situations alike, always making sure I had my head on straight. And also commiserated over beers. In addition, I have had the tremendous privilege of being surrounded by numerous friends in Seattle, who’ve helped me relax and enjoy this time in my life. I have decompressed and expressed my creativity by being in a band with Max, Cody, and Stephen. I’ve dreamed about the future over beers and video games with Mario. I’ve stayed active by climbing with Jessie, Cory, Allison, 1 Hanna, David, Jacob, and many others. I’ve done a lot of idiot things with Kristine, Avery, Josh, and Max. I’ve had some of the most fun roommates anyone could ask for, especially Claire, Bob, and Nina, and the rest have been awesome and understanding as well. I also thank the entire Holladay family, Lindsay, Meredith, Mallory and Ginny, and their parents Rick and Mary Beth for their support and love. Finally, and most importantly, I want to thank my family, especially my parents Chris and Julie, and my brother and sister-in-law Mark and Robin, who have supported me through difficult times, while sharing joy in successes and fun times as well. 2