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Casein Kinase 1 Isoforms in Degenerative Disorders

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Casein Kinase 1 Isoforms in Degenerative Disorders CASEIN KINASE 1 ISOFORMS IN DEGENERATIVE DISORDERS DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Theresa Joseph Kannanayakal, M.Sc., M.S. * * * * * The Ohio State University 2004 Dissertation Committee: Approved by Professor Jeff A. Kuret, Adviser Professor John D. Oberdick Professor Dale D. Vandre Adviser Professor Mike X. Zhu Biophysics Graduate Program ABSTRACT Casein Kinase 1 (CK1) enzyme is one of the largest family of Serine/Threonine protein kinases. CK1 has a wide distribution spanning many eukaryotic families. In cells, its kinase activity has been found in various sub-cellular compartments enabling it to phosphorylate many proteins involved in cellular maintenance and disease pathogenesis. Tau is one such substrate whose hyperphosphorylation results in degeneration of neurons in Alzheimer’s disease (AD). AD is a slow neuroprogessive disorder histopathologically characterized by Granulovacuolar degeneration bodies (GVBs) and intraneuronal accumulation of tau in Neurofibrillary Tangles (NFTs). The level of CK1 isoforms, CK1α, CK1δ and CK1ε has been shown to be elevated in AD. Previous studies of the correlation of CK1δ with lesions had demonstrated its importance in tau hyperphosphorylation. Hence we investigated distribution of CK1α and CK1ε with the lesions to understand if they would play role in tau hyperphosphorylation similar to CK1δ. The kinase results were also compared with lesion correlation studies of peptidyl cis/trans prolyl isomerase (Pin1) and caspase-3. Our results showed that among the enzymes investigated, CK1 isoforms have the greatest extent of colocalization with the lesions. We have also investigated the distribution of CK1α with different stages of NFTs that follow AD progression. It was observed that CK1α follows AD progression, establishing the importance of CK1 isoforms in AD. Correlation of CK1 isoforms with tau pathology led us to investigate the presence of the isoforms in a muscle degenerative disorder, Inclusion ii Body Myositis (IBM) containing tau inclusions. CK1α was found in the tau inclusions of IBM, demonstrating the importance of CK1 isoforms in degenerative disorders in general. Since CK1 is established in both maintenance of the cell and pathogenesis of degenerative diseases, we investigated the regulation and protein substrate recognition of the kinase domain of the enzymes. Using structure and sequence comparison with other conserved protein kinases, we identified and mutated the essential residues involved in regulation and protein substrate recognition. Examination of the mutants with kinase assay revealed that T166 is important for regulation while R183, K222, K229 are important for protein substrate recognition. Thus, these various studies establish the importance of the CK1 family in degenerative disorders. iii To my husband Lal Jose and to my parents, Joseph K. David and P.V. Poulina iv ACKNOWLEDGMENTS I wish to thank my adviser, Dr. Jeff Kuret, for intellectual support, constant guidance, encouragement, enthusiasm and his patience all through my graduate career, which made this thesis possible. I wish to thank my committee members, Dr. John Oberdick, Dr. Brad Stokes, Dr. Dale Vandre and Dr. Mike Zhu for stimulating discussions and for providing encouragement. I am grateful to Dr. Leyla de Toledo-Morrell (Rush Alzheimer's Disease Center, Rush University, Chicago, Illinois), Dr. Paul D. Coleman (University of Rochester, Rochester, NY), Dr. Maria Santi (Dept. Pathology, The Ohio State University School of Medicine, Columbus, OH) and Harvard Brain Bank (Boston, MA) for providing human autopsy Alzheimer’s disease as well as control brains tissues and Dr. Jerry Mendell (Dept. Neurology, The Ohio State University School of Medicine, Columbus, OH) for providing human biopsy Inclusion Body Myositis muscle tissue as well as control muscle tissues. I am greatly thankful to Dr. Dale Vandre (Ohio State University, Columbus, OH) for providing Pin1 antisera, to Dr. Peter Davies, (Albert Einstein College of Medicine, NY) v for providing Alz-50 antibody and to ICOS corporation (Icos Corporation, Bothell, WA) for providing IC128A antibody. I am indebted to the staff of campus microscope facility, Dr. Dick Burry, Kathy Wolken and Brian Kemmenoe for their incredible skills and persistence. I am fortunate to have the opportunity to work with a group of energetic people Carmen Chirita, Erin Congdon, Guibin Li, Mihaela Necula, Kelly Threm, Haishan Yin and Qi Zhong. Finally, it is impossible to have my research career without love and support of my husband and my parents, as well as the encouragement of my family and friends. I am forever indebted to my in-laws: Jose Kokkat and Mary Jose, to my brothers: Davis, Antony and Thomas, my sister’s family: Lawrence Jacob, Vimala and Amala Lawrence, to my foster family: Stanley and Tanya Mathew, to my Jones group: Swarnali Acharyya, Rinku Jain, Senthil Meenrajan, and Soja Sekharan. To all of you, Thanks. vi VITA August 27, 1976............................................................................….....Born, Chennai, India 1996 B.Sc ..................................................................................…..….Stella Maris College, (Physics) Madras University, India 1998 M.Sc............................................................... ...........Department of Crystallography, (Biophysics & Crystallography) Madras University, India 2001 M.S ...............................................................................................Biophysics Program, (Biophysics) The Ohio State University (OSU), USA 2001-Present..................................................................….....................Doctoral candidate, (Biophysics) Biophysics Program, OSU, USA FIELD OF STUDY Major Field: Biophysics vii TABLE OF CONTENTS Page ABSTRACT.........................................................................................................……...... ii DEDICATION................................................................................................…....…..… iv ACKNOWLEDGMENTS.............................................................................…...….........v VITA.................................................................................................................……….....vii LIST OF TABLES.................................................................................................…..... xii LIST OF FIGURES................................................................................................….... xiii LIST OF ABBREVIATIONS……....................................................................…...….. xv CHAPTERS: 1. Introduction............................................................…..................................….......…....1 1.1 Alzheimer’s Disease…………..................................................................……..1 1.1.1 Hypotheses for occurrence of AD lesions.................................…..….2 1.1.2 Phosphorylation and tau…………..............................................…….4 1.2 Casein Kinase 1. ……………….........................................................................7 1.2.1 CK1 in lower eukaryotes…....................................................………..7 1.2.2 CK1 in higher eukaryotes….......................................................……..8 1.2.3 Conserved residues in CK1....................………………………..…..10 1.3 Pin1 overview…….........................……...........................................................11 viii 1.4 Caspase-3……………………………………………………………………..13 1.5 Inclusion Body Myositis……………………………………………………...14 1.6 Immunoshistochemical technique…………………………………………….17 1.7 Epi-Fluorescence/Confocal Microscopy……………………………………...19 2. Vulnerability of lesion affected neurons to the presence of enzymes involved in Alzheimer’s Disease…………………………………………………………………..…21 2.1 Summary …………………………………...………………………………...21 2.2 Introduction………………………………………………………....………..22 2.3 Materials and methods…………………………………………………….….29 2.3.1 Antibodies……………………………………………………….….29 2.3.2 Fluorophores…………………………………………………….....31 2.3.3 Human Tissue……………………………………………….…..….31 2.3.4 Immunohistochemistry……………………………….………….....35 2.3.5 Analytical Methods…………………………............………......….36 2.3.6 Statistical analysis…………………………………………… . .….36 2.4 Results. …………………………………………………………………….... 36 2.4.1 Western Blots…………………………………………………..…....36 2.4.2 Histochemistry of control cases………………………………...…...38 2.4.3 CK1 isoforms correlate well with NFTs……………………...…..…38 ix 2.4.4 Pin1 granules might represent new lesion. ………………………...39 2.4.5 Caspase-3 is present in GVB containing neurons more than in NFTs…………………………………………………….…..39 2.4.6 CK1α and different stages of NFTs………………………………....40 2.4.7 GVBs and NFTs do not coexist in the same neuron.…….……….....40 2.4.8 Pre-tangle neurons and GVBs.…….………………………………..41 2.5 Discussion…………………………………………………………..….……..53 3. Casein Kinase 1 α found in the tau inclusions of Inclusion Body Myositis…..…..59 3.1 Summary ………………………………………………………………….….59 3.2 Introduction…………………………………………………….………….….60 3.3 Materials and methods. …………………………………………………..…..63 3.3.1 Patients and controls………….…………………………………….63 3.3.2 Antibodies…………………………………………………………..63 3.3.3 Fluorescence immunohistochemistry…………………………..…...64 3.4 Results …………………………………………………………….………….64 3.4.1 H&E stainings of control and s-IBM muscle fibers………….……..64 3.4.2 Control muscle sections………………………………………….....65 3.4.3 s-IBM muscle………………………………………………..……...65
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