AMP-activated protein kinase : Screening for novel membrane substrates and creatine kinase phosphorylation linked to specific subcellular compartment Sacnicte Ramirez Rios To cite this version: Sacnicte Ramirez Rios. AMP-activated protein kinase : Screening for novel membrane substrates and creatine kinase phosphorylation linked to specific subcellular compartment. Cellular Biology. Université de Grenoble, 2010. English. tel-00641109 HAL Id: tel-00641109 https://tel.archives-ouvertes.fr/tel-00641109 Submitted on 14 Nov 2011 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE Pour obtenir le grade de DOCTEUR DE L’UNIVERSITÉ DE GRENOBLE Spécialité : Biologie Cellulaire Arrêté ministériel : 7 août 2006 Présentée par Sacnicte RAMIREZ RIOS Thèse dirigée par Uwe SCHLATTNER préparée au sein du Laboratoire de Bioénergétique Fondamentale et Appliquée dans l'École Doctorale Chimie et Sciences du Vivant La protéine kinase activée par AMP : Criblage de nouveaux substrats membranaires et phosphorylation de la créatine kinase liée à une compartimentation subcellulaire Thèse soutenue publiquement le « 20 décembre 2010 », devant le jury composé de : Pr. Theo WALLIMANN Professeur à l’ETH Hönggerberg HPM, (Rapporteur) Pr. Bé WIERINGA Professeur, Department of Cell Biology, Nijmegen University (Rapporteur) Dr. Marie-Lise LACOMBE Professeur INSERM UMRS (Examinateur) Pr. Uwe SCHLATTNER Professeur au Laboratoire de Bioénergétique Fondamentale et Appliquée (Membre) Table of Contents AIM OF THE THESIS ........................................................................................................... III 1. Regulation of cellular and whole body energy homeostasis ........................................ 1 1.1. Cellular energy metabolism ............................................................................................. 2 1.2. Cellular energy homeostasis ............................................................................................. 2 1.3. Phosphotransfer systems .................................................................................................. 3 1.4. Creatine kinase ................................................................................................................. 4 1.4.1. Temporal and spatial buffering functions of creatine kinases .......................................... 5 1.4.2. Creatine metabolism and brain energetics ....................................................................... 6 1.4.3. Subcellular localization of CK isoforms .......................................................................... 8 1.4.4. BCK structure ................................................................................................................. 11 1.4.5. MtCK structure ............................................................................................................... 13 1.4.6. Creatine kinase regulation .............................................................................................. 14 1.5. AMP-activated protein kinase: A key-regulator of energy metabolism ........................ 15 1.5.1. AMPK as a multifunctional metabolic sensor ................................................................ 15 1.5.2. Heterotrimeric structure and expression ........................................................................ 16 1.6. AMPK regulation ........................................................................................................... 17 1.6.1. Molecular regulation ...................................................................................................... 17 Regulation by phosphorylation ................................................................................. 17 Allosteric regulation .................................................................................................. 18 1.6.2. Cellular regulation .......................................................................................................... 19 1.6.3. Pharmacological activation ............................................................................................ 19 1.7. AMPK downstream signaling ........................................................................................ 20 1.7.1. AMPK regulation of carbohydrate metabolism ............................................................. 20 1.7.2. AMPK regulation of lipid metabolism ........................................................................... 23 1.7.3. AMPK regulation of protein metabolism, cell proliferation and other pathways .......... 23 1.8. AMPK role of AMPK in human physiopathology ......................................................... 25 1.8.1. AMPK and the metabolic syndrome .............................................................................. 25 1.8.2. AMPK and cancer .......................................................................................................... 26 1.8.3. AMPK mutation in human pathology ............................................................................ 26 1.9. References ...................................................................................................................... 27 2. A versatile multidimensional protein purification system with full Internet remote control based on a standard HPLC system ................................................... 39 2.1. Introduction .................................................................................................................... 41 2.2. Experimental procedures ................................................................................................ 41 2.2.1. System hardware ............................................................................................................ 41 2.2.2. System software ............................................................................................................. 43 2.2.3. 4-D chromatography implementation ............................................................................ 43 2.3. Results and discussion .................................................................................................... 45 2.4. Acknowledgments .......................................................................................................... 48 2.5. Supplementary material and methods ............................................................................ 49 2.6. System software ............................................................................................................. 49 2.7. References ...................................................................................................................... 51 3. AMP-activated protein kinase phosphorylates brain-type creatine kinase in vivo to regulate subcellular localization, not enzyme activity .................................. 52 3.1. Introduction .................................................................................................................... 54 3.2. Results ............................................................................................................................ 55 3.2.1. BCK is phosphorylated by AMPK in vitro .................................................................... 55 3.3. BCK displays two putative AMPK phosphosites .......................................................... 56 3.3.1. Mutation of BCK at Ser6 prevents phosphorylation by activated AMPK Į1 and Į2. ... 57 3.3.2. Ser6 is phosphorylated rapidly and with high stoichiometry ......................................... 59 3.3.3. Active AMPK interacts transiently with BCK wild-type via its Į subunit N-terminal domain ........................................................................................................................ 60 3.3.4. BCK wild-type and phospho-mimetic mutant S6D do not differ in enzymatic activity ........................................................................................................................ 60 3.3.5. Specificity of phospho-BCK detection using a purified polyclonal anti P-Ser6-BCK antibody ...................................................................................................................... 61 3.3.6. BCK is phosphorylated at Ser6 in astrocytes treated with A769662 or AICA-riboside ............................................................................................................ 62 3.3.7. Ser6-phosphorylated BCK localizes to perinuclear regions in astrocytes and fibroblasts ................................................................................................................... 64 3.3.8. Phospho-BCK is targeted to the endoplasmic reticulum. .............................................. 65 3.4. Discussion ...................................................................................................................... 66 3.5. Experimental procedures ................................................................................................ 71 3.5.1. Materials ........................................................................................................................