Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Genetic analysis of the catalytic activity of Integrin-linked kinase (ILK) in vivo Anika Lange aus Osterburg/Altmark 2010 Erklärung Diese Dissertation wurde im Sinne von § 13 Abs. 3 der Promotionsordnung vom 29. Januar 1998 von Herrn Prof. Dr. Reinhard Fässler betreut. Ehrenwörtliche Versicherung Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet. München, am ---------------------------- Anika Lange Dissertation eingereicht am: 22.01.2010 1. Gutachter Prof. Dr. Reinhard Fässler 2. Gutachter Prof. Dr. Christian Wahl-Schott Mündliche Prüfung am: 04.03.2010 Für meine lieben Eltern Table of Contents Table of Contents Table of Contents ............................................................................................................... I List of Publications ......................................................................................................... III Abbreviations .................................................................................................................. IV Summary.......................................................................................................................... VI Introduction....................................................................................................................... 1 1. The integrin receptor family.................................................................................... 1 1.1 Structure of integrins.......................................................................................... 1 1.2 Integrins and their ligands ................................................................................. 4 1.3 Bidirectional regulation of integrin signaling................................................... 8 1.3.1 Inside-out signaling...................................................................................... 8 1.3.2 Outside-in signalling .................................................................................. 12 1.4 Assembly of integrin-dependent adhesion structures.................................... 16 1.4.1 The integrin-actin connection ................................................................... 18 1.5 The ILK/PINCH/parvin (IPP) complex.......................................................... 20 1.5.1 The molecular composition of the IPP complex...................................... 21 1.5.2 The biological functions of the IPP complex ........................................... 22 1.5.3 The putative kinase activity of ILK.......................................................... 24 2. Kidney physiology................................................................................................... 26 2.1 Kidney development in mice............................................................................ 30 2.1.1 Kidney morphogenesis – pronephros, mesonephros and metanephros 30 2.1.2 Mesenchymal signals initiate kidney development ................................. 33 2.1.3 Ureteric bud outgrowth and branching................................................... 36 2.1.4 Tubulogenesis – MET................................................................................ 42 2.1.5 Role of stroma in kidney development..................................................... 44 2.2 Renal abnormalities in humans ....................................................................... 46 2.2.1 The glomerulus and proteinuria............................................................... 49 2.2.2 Cystic kidney diseases................................................................................ 54 2.3 Integrins and kidney......................................................................................... 56 2.3.1 ECM and its receptors in mammalian nephrogenesis............................ 56 I Table of Contents 2.3.2 Role of integrins and their binding partners in the development of the collecting system.................................................................................................. 59 2.3.3 Role of integrins and their binding partners in the development and function of the glomerulus.................................................................................. 60 Aim of the Thesis............................................................................................................. 63 Short Summaries of Publications .................................................................................. 64 Publication I: Local call: from integrins to actin assembly .................................... 64 Publication II: How ILK and kindlins cooperate to orchestrate integrin signaling ....................................................................................................................................... 65 Publication III: Integrin-linked kinase is an adaptor with essential functions during mouse development ........................................................................................ 66 Publication IV: The ILK/PINCH/parvin complex: the kinase is dead, long live the pseudokinase! .............................................................................................................. 67 Publication V: Integrin-mediated signals control microtubule dynamics required for plasma membrane targeting of caveolae ............................................................ 68 Publication VI: Bacteria hijack integrin-linked kinase to stabilize focal adhesions and block cell detachment.......................................................................................... 68 References........................................................................................................................ 69 Acknowledgements ......................................................................................................... 83 Curriculum Vitae............................................................................................................ 84 Supplements..................................................................................................................... 85 II List of Publications List of Publications This thesis is based on the following publications, which are referred to in the text by their Roman numerals (I-VI): I. Wiesner S, Lange A and Fässler R. Local call: from integrins to actin assembly. Trends in Cell Biology 2006, 16 II. Böttcher RT, Lange A and Fässler R. How ILK and kindlins cooperate to orchestrate integrin signaling. Current Opinion in Cell Biology 2009, 21(5): 670-5 III. Lange A, Wickström SA, Jakobson M, Zent R, Sainio K and Fässler R. Integrin- linked kinase is an adaptor with essential functions during mouse development. Nature 2009, 461: 1002-1006 . IV. Wickström SA, Lange A, Montanez E and Reinhard Fässler. The ILK/PINCH/parvin complex: the kinase is dead, long live the pseudokinase! EMBO J 2010, 29(2): 281-91. The following publications were not the focus of my project but I contributed to them: V. Wickström SA, Lange A, Hess MW, Krüger M, Pfaller K, Mann M, Bloch W, Huber LA and Fässler R. Integrin-mediated signals control microtubule dynamics required for plasma membrane targeting of caveolae. Submitted manuscript VI. Kim M, Ogawa M, Fujita Y, Yoshkawa Y, Nagai T, Koyama T, Nagai S, Lange A, Fässler R and Sasakawa C. Bacteria hijack integrin-linked kinase to stabilize focal adhesions and block cell detachment. Nature 2009, 459: 578- 583. III Abbreviations Abbreviations ADAM a disintegrin and metalloproteinase ADP adenosine diphosphate Akt RAC-alpha serine/threonine protein kinase APC adenomatous polyposis coli Arp2/3 actin-related protein 2/3 complex ATP adenosine triphosphate BMP bone morphogenetic protein Cdc42 cell division cycle 42 CH calponin homology Cre cyclization recombinase DAG diacylglycerol E embryonic day ECM extracellular matrix EGF epidermal growth factor ERK extracellular signal-regulated kinase FA focal adhesion F-actin filamentous actin FAK focal adhesion kinase FERM 4.1, ezrin, radixin, moesin FGF fibroblast growth factor FGFR fibroblast growth factor receptor FP foot process GAP GTPase activating protein GBM glomerular basement membrane GDI GDP dissociation inhibitor GDNF glial-cell-derived neurotrophic factor GDP guanosine diphosphate GEF guanine nucleotide exchange factor GFR growth factor receptor GPCR G protein coupled receptor Gsk-3β glycogen synthase kinase-3β HGF hepatocyte growth factor ICAM intercellular adhesion molecule ILK integrin-linked kinase IP3 inositol triphosphate IPP ILK/PINCH/parvin JNK c-jun N-terminal kinase MAPK mitogen-activated protein kinase MET mesenchymal-to-epithelial transition MIDAS metal-ion-dependent adhesion site MLC myosin light chain MM metanephric mesenchyme MMP matrix metalloproteinase Nck2 noncatalytic region of tyrosine kinase, adaptor protein 2 NS nephrotic syndrome IV Abbreviations PCP planar cell polarity PH pleckstrin homology PI3K phosphatidylinositol 3-kinase PINCH particularly interesting new Cys-His rich protein PKC protein kinase C PKD polycystic kidney disease PLC phospholipase C PTB phosphotyrosine binding Ret rearranged during transformation RGD arginine, glycine, aspartate RhoA Ras homology gene family, member A ROCK Rho-associated protein kinase SD slit diaphragm SH2 src homology 2 Src Rous sarcoma oncogene TGFβ transforming growth factor β UB ureteric bud VCAM vascular cell-adhesion molecule WD wolffian duct Wnt wingless-type MMTV integration site family member WT1 wilms tumour