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Functional Characterization of the Kinase and Pseudokinase Domains in the Janus Tyrosine Kinase (JAK) 2

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Functional Characterization of the Kinase and Pseudokinase Domains in the Janus Tyrosine Kinase (JAK) 2 YASHAVANTHI NIRANJAN Functional Characterization of the Kinase and Pseudokinase Domains in the Janus Tyrosine Kinase (JAK) 2 ACADEMIC DISSERTATION To be presented, with the permission of the Board of the BioMediTech of the University of Tampere, for public discussion in the Auditorium of Finn-Medi 5, Biokatu 12, Tampere, on April 25th, 2014, at 12 o’clock. UNIVERSITY OF TAMPERE YASHAVANTHI NIRANJAN Functional Characterization of the Kinase and Pseudokinase Domains in the Janus Tyrosine Kinase (JAK) 2 Acta Universitatis Tamperensis 1922 Tampere University Press Tampere 2014 ACADEMIC DISSERTATION University of Tampere, BioMediTech Finland Supervised by Reviewed by Professor Olli Silvennoinen Professor Gerhard Müller-Newen University of Tampere Universitätsklinirum der RWTH Aachen Finland Germany Professor Jari Yli-Kauhaluoma University of Helsinki Finland Copyright ©2014 Tampere University Press and the author Cover design by Mikko Reinikka Distributor: [email protected] http://granum.uta.fi Acta Universitatis Tamperensis 1922 Acta Electronica Universitatis Tamperensis 1406 ISBN 978-951-44-9416-1 (print) ISBN 978-951-44-9417-8 (pdf) ISSN-L 1455-1616 ISSN 1456-954X ISSN 1455-1616 http://tampub.uta.fi Suomen Yliopistopaino Oy – Juvenes Print Tampere 2014 441 729 Painotuote This thesis is dedicated to my grandparents, beloved parents and my little sister उ配साहो बलवानर्य नास्त配र्配ु साहा配परं बलम ्| सो配साहस्तर् च लोके ष ु न ककञ्चचदपप दलु भय म ्|| Nothing is unachievable for a person with enthusiasm and commitment 3 TABLE OF CONTENTS 1. LIST OF ORIGINAL COMMUNICATIONS ............................................................... 7 2. LIST OF ABBREVIATIONS ............................................................................................. 8 3. ABSTRACT .......................................................................................................................... 11 4. INTRODUCTION ............................................................................................................. 13 5. REVIEW OF LITERATURE ........................................................................................... 15 5.1 Cytokines and the cytokine receptor family ....................................................... 15 5.2 Protein kinases ........................................................................................................ 18 5.2.1 Classification ............................................................................................ 18 5.2.2 Structural features of protein kinases ................................................... 20 5.2.3 Common regulatory mechanisms in tyrosine kinases ....................... 24 5.3 Family of pseudokinases ....................................................................................... 26 5.3.1 History, evolution and properties of pseudokinases ......................... 26 5.3.2 Structural insights into pseudokinases ................................................. 28 5.3.3 Structural variation in non-pseudokinases .......................................... 33 5.3.4 Determining the ATP binding characteristics and roles of pseudokinases .................................................................................. 34 5.3.5 Regulation of catalytically active pseudokinases – intra/inter domain regulation ........................................................................... 37 5.4 JAK tyrosine kinases .............................................................................................. 38 5.4.1 General overview of JAK kinases ........................................................ 38 5.4.2 Domain organization of JAKs .............................................................. 39 5.4.3 Wild type JAK2 - emphasis on the kinase and pseudokinase domains ............................................................................................ 42 5.4.4 JAK/STAT pathway – canonical and non-canonical functions ........................................................................................... 45 5.5 Pathophysiology of JAK2 ..................................................................................... 49 5.5.1 Mutational studies in JAK2, the discovery of the V617F pathogenic allele .............................................................................. 49 5.5.2 Regulation of JAK2 ................................................................................ 54 5.5.2.1 Mechanisms of JAK2 V617F activation ........................... 54 5.5.2.2 Role of pseudokinase domain in JAK2 regulation ........................................................................ 55 4 5.5.2.3 Modeling studies on JH1JH2 regulation......................... 56 5.5.3 JAK2 deficient mouse models .............................................................. 57 5.5.4 JAK2 and cancer ..................................................................................... 58 5.6 Biochemical properties of JAK2 .......................................................................... 59 5.6.1 Kinetic parameters .................................................................................. 59 5.6.2 JAK2 inhibitors and their IC50 specificity .......................................... 61 6. OBJECTIVES OF THE (PRESENT) STUDY............................................................. 63 7. MATERIALS AND METHODS ..................................................................................... 65 7.1 DNA constructs and cloning ............................................................................... 65 7.2 Sequencing and sequence analysis ....................................................................... 67 7.3 Cell culture .............................................................................................................. 67 7.4 Antibodies and cytokines ...................................................................................... 68 7.5 Cell line transfections ............................................................................................ 69 7.6 Cell lysis, Immunoprecipitation and Immunoblotting ..................................... 70 7.6.1 Cell lysis .................................................................................................... 70 7.6.2 Immunoprecipitation .............................................................................. 70 7.6.3 Immunoblotting and analysis of protein phosphorylation ............... 70 7.7 Autophosphorylation studies and in vitro kinetic assay..................................... 71 7.8 In vitro translation.................................................................................................... 71 7.9 MANT-ATP direct binding studies .................................................................... 72 7.10 Protein expression and recombinant purification ........................................... 72 7.11 PamChip® peptide microarrays .......................................................................... 74 7.12 Luciferase reporter gene assay ........................................................................... 76 8. RESULTS .............................................................................................................................. 77 8.1 Catalytic activity of the JAK2 pseudokinase domain (Article I) ..................... 77 8.1.1 Serine 523 and Tyrosine 570 are putative phosphorylation sites in JAK2 JH2. ........................................................................... 77 8.1.2 Phosphorylation of JH2 in JAK2 deficient γ2A fibrosarcoma cells ........................................................................... 78 8.1.3 Impact of JH2 activity on cytokine receptor-mediated signaling ............................................................................................ 78 8.1.4 Role of MPN-causing mutations on the catalytic activity of JH2 .................................................................................................... 79 8.2 Determination of the binding affinity of ATP to the JAK2 kinase domain (Article II) ............................................................................................ 79 8.2.1 Common factors that affect the intensity of fluorescence resonance energy transfer (FRET) ............................................... 79 8.2.2 Determination of the instrument parameters lp and ly 5 facilitates the correction of the primary and secondary inner filter effect .............................................................................. 80 8.2.3 Use of high concentrations of MANT-ATP is possible only when the primary inner filter effect is corrected ........................ 81 8.2.4 Effect of protein fluorescence emission on MANT-ATP and vice versa ...................................................................................... 81 8.2.5 Fluorescence measurement of nucleotide binding to the JAK2 kinase domain ....................................................................... 82 8.3 Effect of JH2 and the SH2-JH2 linker on JAK2 activity (Article III) ........... 84 8.3.1 JAK2 JH1 and the tandem JH1JH2 domains follow a random Bi-Bi mechanism .............................................................. 84 8.3.2 Kinetic properties of the JAK2 JH2 domain ...................................... 85 8.3.3 Effect of JH2 on JH1JH2 kinetics ....................................................... 87 8.3.4 Effect of inhibitors on JAK2 activity ................................................... 88 9. DISCUSSION .....................................................................................................................
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