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FINAL THESIS 2014 for PRINT And Biochemical Analysis of Human Cancer-Associated Pseudokinases Fiona P. Bailey A thesis submitted to the University of Sheffield for the degree of Doctor of Philosophy June 2014 Department of Oncology 1 Declaration I declare that no portion of this work has been submitted to this, or any other University for the award of a degree. The work described below is my own, except where referenced. 2 Acknowledgments I would like to thank Dr. Patrick Eyers for the fantastic opportunity of undertaking a PhD in his laboratory. Moreover, I cannot thank him enough for the constant support and guidance provided at all times throughout this PhD. I also want to thank everybody who has collaborated with this project and has kindly contributed reagents, materials or time towards supporting this work. I would like to acknowledge everybody at The University of Sheffield and The University of Liverpool for their support throughout the project, including Dr. Michael Trikic and Dr. Dominic Byrne for their assistance, invaluable input and thought-provoking discussions over the years. Finally, a special note of thanks goes to my parents Pat and Richard, and brother James, who have never doubted my abilities and always provided encouragement and support. 3 Abstract Mutation or aberrant expression of protein kinase domains can lead to the development of a variety of cancers, which is why these proteins have become the focus of many clinical therapies. ~10% of the human kinome have been classified as pseudokinases, because they lack at least one of the highly conserved motifs that were originally used to help define the protein kinase superfamily. Due to these deviations from the normal situation, pseudokinases were originally predicted to be catalytically inactive, often based solely upon bioinformatic sequence alignment evidence. However, the discovery of pseudokinases that can hydrolyze or bind to ATP either at, or below, the nucleotide concentrations typically found in cells, suggests that each pseudokinase requires a thorough and individual biochemical analysis using a combination of techniques in order to reveal its biological function. Trib2 and NOK are two pseudokinases that contain a highly atypical ‘DFG’ metal ion binding motif, when compared to canonical kinase domains, and both are implicated in a variety of human cancers. Trib2 is predicted to be a Ser/Thr pseudokinase and is very closely related to the Trib1 and Trib3 pseudokinases in humans, and to the Tribbles pseudokinase that was originally characterized in the model fruit fly Drosophila Melanogaster. Along with an E3-ligase, Trib2 is required to target the tumor-suppressing transcription factor C/EBP for degradation in cells. The results presented in this thesis include the first demonstration of a Mg2+ ion- independent ATP binding and autophosphorylation function for Trib2 in vitro. NOK is a pseudokinase that exhibits similarity to the RPTK sub-family, however it does not have a detectable ligand-binding domain, but does contain a putative transmembrane domain that lies adjacent to the pseudokinase domain. Overexpression of NOK mRNA has been observed in a variety of cancers, but its mode of action is still unclear. I describe in this thesis that NOK localizes to the nuclear membrane of cells and contains a localization sequence that resembles the EGFR Nuclear Localization Sequence, which translocates to the nucleus via the Inner Nuclear Membrane. Work described in this thesis also describes the development of tools to pave the way for future cellular analysis of both Trib2 and NOK, which will reveal whether these pseudokinases could be exciting, and novel drug targets in human diseases. 4 Contents Contents ....................................................................................................................... 5 List of Figures and Tables .......................................................................................... 10 List Of Abbreviations ................................................................................................. 14 CHAPTER 1. Introduction ................................................................................... 20 1.1 Protein Kinases ............................................................................................... 20 1.2 Common phosphate acceptor residues of protein substrates .......................... 26 1.3 Kinase domain activation ............................................................................... 27 1.4 Protein kinases and cancer ............................................................................. 29 1.5 Protein kinases as drug targets ....................................................................... 30 1.6 Protein pseudokinases .................................................................................... 31 1.7 Pseudokinases act in a variety of ways to alter cell signaling ........................ 38 1.8 CASK ............................................................................................................. 40 1.9 ErbB3 ............................................................................................................. 41 1.10 STRADα ......................................................................................................... 42 1.11 The ‘With No Lysine’ (WNK) family of kinases .......................................... 43 1.12 JAK2 JH2 pseudokinase domain .................................................................... 43 1.13 VRK3 ............................................................................................................. 44 1.14 KSR1/2 ........................................................................................................... 45 1.15 MLKL ............................................................................................................. 46 1.16 Tribbles pseudokinases .................................................................................. 46 1.17 Trib2 ............................................................................................................... 49 1.18 Predicted structure of Trib2 in three dimensions ........................................... 55 1.19 Trib2 is predicted to assemble Regulatory and Catalytic ‘spines’ ................. 58 1.20 Trib2 is not essential for mouse development ................................................ 60 1.21 Trib2 is associated with COP1-mediated protein degradation ....................... 60 1.22 Trib2 binding to COP1 is required for C/EBP degradation and AML onset ........................................................................................................................ 60 1.23 Trib2 and ALL ................................................................................................ 65 1.24 Trib2 regulates the stability of C/EBP in NSCLC ....................................... 65 1.25 Trib2 levels are regulated by -TRCP and Smurf1 ........................................ 66 1.26 Trib2 regulates FOXO activity in malignant melanoma ................................ 67 1.27 Mutational analysis of Trib2 confirms a cancer association .......................... 67 1.28 Trib2 and Inflammatory Bowel Disease ........................................................ 71 5 1.29 Trib2 and Trib3 target multiple proteins for degradation ............................... 71 1.30 Trib2 autoantigens are linked to narcolepsy ................................................... 72 1.31 Trib2 autoantibodies associated with uveitis .................................................. 72 1.32 Trib2 can inhibit MKK7 and MEK1 activity ................................................. 72 1.33 Trib1 can hyperactivate the ERK pathway..................................................... 73 1.34 Trib1 and fatty liver disease ........................................................................... 73 1.35 Trib3 does not induce AML, but is a cancer-associated protein .................... 74 1.36 C/EBP regulation is evolutionary conserved in Trib proteins ..................... 75 1.37 SgK495 is required for lung development in murine embryos ...................... 75 1.38 NOK (Novel Oncogene with Kinase domain) ............................................... 77 1.39 NOK catalytic activity has not been demonstrated convincingly .................. 80 1.40 NOK activates the Akt and MAPK pathways ................................................ 80 1.41 NOK is implicated in cancer progression ...................................................... 81 1.42 Aims of this Thesis ......................................................................................... 82 CHAPTER 2. Materials and Methods.................................................................. 83 2.1 Chemicals and Reagents ................................................................................. 83 2.2 Antibodies described in this thesis ................................................................. 84 2.3 Plasmid vectors .............................................................................................. 85 2.4 Primer design .................................................................................................. 86 2.5 PCR cycling parameters used to generate LIC-compatible ends ................... 89 2.6 PCR products for blunt-end ligation into pCR2.1-TOPO vector using an overhanging adenine and Topoisomerase I (‘TOPO’ cloning) ...................... 89 2.7 Site-Directed Mutagenesis PCR procedure ...................................................
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