Cyclic AMP dependent Protein Kinase A (PKA) mutant associated with Fibrolamellar Hepatocellular carcinoma (FLHCC): Structure, dynamics and in cell studies. A Dissertation SUBMITTED TO THE FACULTY OF THE UNIVERSITY OF MINNESOTA Adak Karamafrooz IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY ■ David D Thomas Laurie Parker March 2020 © Adak N Karamafrooz ALL RIGHTS RESERVED This work is dedicated to: All genuine souls who have remained loyal to their integrity, despite all odds. …and to my dear ones who bear with me all the way through. “To strive, to seek, to find, and not to yield…” ALFRED, LORD TENNYSON i List of Tables 3.1 Michaelis-Menten Kinetics constants for PKA-CDNAJB1and PKAWT .................... 110 3.2 Midpoint of thermal denaturation (Tm) for PKA-CDNAJB1and PKAWT................... 110 µ 3.3 Dissociation constants ( M) of nucleotides and inhibitor peptide PKI5-24 .......... 110 5.1 Subset of PRKACA-Dnajb1 direct Peptide Substrates identified by KALIP ...... 127 5.2 subset of PRKACA-Dnajb1 direct Protein Substrates identified by KALIP ....... 128 5.3 Canonical pathways that are enriched in both protein and peptide KALIP ....... 128 5.4 Subset of PRKACA-Dnajb1 substrates affected by the two PKA inhibitors ....... 129 6.2 Catalytic parameters derived from coupled enzyme assay for Human PKA and Cushing’s disease related PKA mutants W196R and L205R ........................... 142 6.3 Comparison of the Thermodynamic parameters for the two Cushing's disease mutant compared to the wild type PK ............................................................... 143 ii List of Figures CHAPTER 1 1.1 Position of the catalytic and glycine rich loop in the conformation of the PRKACA. ........................................................................................................................ 102 1.2 Hydrophobic C-spine and R-spine in the conformation of PRKACA and their relative position compared to important loops in the PKA catalytic subunit ...... 102 1.3 Glycine-rich loop of the C subunit in its closed and open conformation ........... 103 1.4 Post translational modifications in the N-terminal tail of PKA-Cα...................... 103 1.5 hydrogen bonding of the tip of the loop in the PKA-C:PKI ............................... 103 1.6 Reaction pathway for catalysis ........................................................................ 103 1.7 Regulatory and catalytic subunit complex in PKA ............................................ 104 1.8 Sequence alignment of the hinge of different R subunit isoforms .................... 104 1.9 A kinase anchoring protein (AKAP) bound to the regulatory subunit ............... 104 1.10 A schematic representation of cAMP signaling pathway inside the cell ........... 105 1.11 A simplified scheme of the possible role of cAMP/PKA pathway in cancer ...... 105 CHAPTER 3 3.1 PKA DANJB1 (Cyan, PDB:4WB7) crystal structure superimposed on PKAWT ...... 106 3.2 Statistical analysis of the chemical shift changes in PKA DANJB1 ....................... 106 DANJB1 3.3 A. Chemical shift analysis of PKA in complex with PKI5-24 vs PKIFL ......... 107 WT 3.3 B. Chemical shift analysis of PKA in complex with PKI5-24 vs PKIFL .............. 107 3.4 Mutual information and allosteric networks in PKAWT and PKA DANJB1 .............. 108 3.5 Emergence of allosteric hubs upon ATP binding in PKAWT and PKA DANJB1 ...... 109 CHAPTER 4 4.1 Structures of J-PKAcα chimera and wild-type PKAcα. ..................................... 111 4.2 RMSF per residue. RMSF for both J-PKACα and wild-type PKACα ................ 112 4.3 Movement of the J-domain in J-PKAcα chimera. .............................................. 113 4.4 Top four clusters by population from cluster analysis ...................................... 114 4.5 Top clusters from cluster analysis modeled into the RIIβ holoenzyme ............ 115 iii 4.6 Residue-specific T1/T2 ratio of NMR relaxation of J-PKACα ........................... 116 S.1 Backbone assignment of PKA-DNAJB1 ........................................................... 117 S.2 1H-15N TROSY-HSQC spectra for PKADNAJB1 .................................................... 118 S.3 Mutual information and allosteric networks in the binary and ternary forms of PKA WT .............................................................................................................................. 119 CHAPTER 5 5.1 A. Schematic presentation of Kinase assay linked with phosphoproteomic (KALIP). .......................................................................................................... 120 5.1 B. Overlap of the in-cell phospho-proteins ....................................................... 120 5.2 Comparison of some of enriched canonical pathways derived from phosphorylation analysis of in-cell PRKACA and PRKACA-Dnajb1 ................. 121 5.3 Comparison of Phosphorylation sites in each of PRKACA and its mutant PRKACA-Dnajb1.............................................................................................. 122 5.4 Comparison of canonical pathways derived from IPA phosphorylation analysis of PRKACA and PRKACA-Dnajb1 Peptide direct substrates .............................. 123 5.5 Comparison of canonical pathways derived from IPA phosphorylation analysis of PRKACA and PRKACA-Dnajb1 Protein direct substrates ............................... 124 5.6 A. Pattern of downregulated phosphorylation sites in each species ................ 125 5.6 B. Comparison of phosphorylated substrates percentage in PRKACA-Dnajb1 in the presence of inhibitors PKI and rp-cAMP ..................................................... 125 5.7 Inhibition of canonical pathways derived from IPA phosphorylation analysis of PRKACA and PRKACA-Dnajb1 ....................................................................... 126 S.1 Western blot of kinases expressed in HEK293 cells ......................................... 130 CHAPTER 6 6.1 Location of the Cushing’s disease mutations at the interface of R-Cα binding site ........................................................................................................................ 142 6.2 Affinity pull-down assay using His-tagged RIIB and untagged W196R ............ 143 6.3 Inhibitory mechanism using Non-Radioactive Protein Kinase Assays .............. 143 6.4 TROSY-HSQC for PKA-Human Isoform II (Apo form) ...................................... 144 6.5 TROSY-HSQC of Isoform 2 in APO and AMPPNP bound form overlaid ......... 144 6.6 PKA-Human isoform II in complex with nucleotide and PKI ............................ 145 iv 6.7 TROSY-HSQC spectra of 15N, 13C perdeuterated Human PKA bound to ATPγN and PKI Ternary complex) ......................................................................... 145 5-24 ( 6.8 PKA mutant “L205R” in the apo and in ATP-γN bound (binary) and PKI5-25 (ternary) form ................................................................................................... 146 6.9 CONCISE analysis applied to PKA mutant “L205R” ........................................ 147 6.10 TROSY-HSQC spectra of 15N-labeled W196R ................................................. 147 6.11 Comparison of correlation matrix of the sidechain movements in W196R with that of PKA-WT ....................................................................................................... 148 v List of Abbreviations: ADP: Adenosine Diphosphate AKAP: A kinase anchoring protein ATP: Adenosine Triphosphate ATPγN: Adenylyl-imidodiphophate cAMP: cyclic adenosine monophosphate CHESCA: chemical shift covariance analysis CNC: Carney complex CONCISE: COordinated ChemIcal Shifts bEhavior CREB: cAMP Response Element-binding protein SCP: chemical shift perturbation EPK: Eukaryotic Protein kinase EPR: Electron Paramagnetic Relaxation ERK: Extracellular signal-Regulated Kinase FL-HCC: Fibrolamellar Hepatocellular Carcinoma FLC: Fibrolamellar Hepatocellular Carcinoma FPLC: Fast protein liquid chromatography FRET: Förster resonance energy transfer HSQC: Heteronuclear Single Quantum Correlation Spectroscopy ITC: isothermal titration calorimetry KALIP: Kinase Assay Linked with Phosphoproteomic LC-MS: Liquid Chromatography-Mass spectrometry MAPK: Mitogen-Activated Protein Kinase mTOR: mammalian target of rapamycin MD: Molecular Dynamics NMR: Nuclear Magnetic Resonance NMT: N-myristoyltransferase NOE: Nuclear Overhauser Effect vi NOESY: Nuclear Overhauser Enhancement Spectroscopy PCR: Polymerase Chain Reaction PKA: cAMP-dependent protein kinase A PKA-C: catalytic subunit of cAMP-dependent protein kinase A PKA-Cα: catalytic subunit of cAMP-dependent protein kinase A PKI: Protein Kinase Inhibitor PKI5-24: 20 amino acid fragment of Protein Kinase Inhibitor PTMs: post-translational modifications RMSD: Root Mean Square Deviation RMSF: Root Mean Square Fluctuation SAXS: Small angle x-ray scattering SMOAC: Sequential enrichment by Metal Oxide Affinity Chromatography TMS: Tetramethylsilane TOCSY: TOtal Correlated SpectroscopY TROSY: Transverse Optimized Spectroscopy vii Contents Dedication .......................................................................................................................i List of Tables .................................................................................................................ii List of Figures ...............................................................................................................iii List of Abbreviations ....................................................................................................v