Regulation of ULK1 in autophagy A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Life Sciences 2012 Stefan Ludwik Loska Table of Contents List of Figures...................................................................................................................6 Abstract.............................................................................................................................8 Declaration........................................................................................................................9 Copyright statement........................................................................................................10 Acknowledgements.........................................................................................................11 Autobiographical statement............................................................................................12 Abbreviations..................................................................................................................13 1. Introduction................................................................................................................19 1.1. Autophagy...........................................................................................................19 1.1.1. Role.............................................................................................................19 1.1.2. Mechanism..................................................................................................23 1.1.3. Molecular machinery of autophagy............................................................25 1.1.4. Initiation......................................................................................................27 1.1.5. Nucleation...................................................................................................28 1.1.6. Expansion and closure.................................................................................30 1.1.7. Maturation...................................................................................................33 1.2. ULK1..................................................................................................................35 1.2.1. Domain architecture of ULK1....................................................................35 1.2.2. Function of ULK1 in axon formation.........................................................38 1.2.3. Function of ULK1 in autophagy.................................................................41 1.2.4. ULK1 complex............................................................................................47 1.2.5. Upstream regulation of ULK1....................................................................50 1.2.5.1. mTOR signalling.................................................................................50 1.2.5.2. Role of mTOR and AMPK in regulation of ULK1.............................54 1.3. Conclusions and aims of the project...................................................................61 2. Materials and Methods...............................................................................................65 2.1. DNA Constructs..................................................................................................65 2.1.1. Introducing TEV site into pAc5.1/V5-HisB................................................65 2.1.2. pAc5.1/V5-TEV-HisB-ULK1 and mutants................................................65 2.1.3. pDEST-HisMBP-ULK1 1-278 K46N.........................................................67 2 2.1.4. pcDNA3-myc-ULK1...................................................................................68 2.1.5. pEF-myc-B-RAF D594A............................................................................68 2.1.6. pET28a-LIC-MERTK-571-864wt and K619N...........................................68 2.1.7. pc5FLAG-Pim1 wt and K67M....................................................................69 2.2. Buffers.................................................................................................................69 2.2.1. SDS-PAGE and western blotting................................................................69 2.2.2. Protein purification......................................................................................69 2.2.3. Cell Lysis.....................................................................................................70 2.2.4. Kinase assay:...............................................................................................70 2.2.5. In-gel kinase assay:.....................................................................................71 2.2.6. Isoelectric Focusing:...................................................................................71 2.3. Recombinant proteins.........................................................................................71 2.4. His-MBP-ULK1 1-278 K46N purification.........................................................72 2.4.1. Pilot experiment..........................................................................................72 2.4.2. Column purification....................................................................................73 2.5. His-MERTK 571-864 purification......................................................................74 2.6. Bradford assay....................................................................................................75 2.7. SDS-PAGE and western blotting........................................................................75 2.8. Cell culture, transfection, treatment and lysis.....................................................76 2.8.1. S2 cells........................................................................................................76 2.8.2. Mammalian cells lines (MEF, HEK293, Cos7, HeLa)................................77 2.9. ULK1 expression in Drosophila S2 cells............................................................77 2.10. Pim1/ULK1 pull down assay............................................................................78 2.11. Kinase assay......................................................................................................78 2.11.1. Kinase........................................................................................................79 2.11.1.1. Recombinant kinases.........................................................................79 2.11.1.2. V5-His-ULK1....................................................................................79 2.11.1.3. myc-ULK1.........................................................................................79 2.11.2. Substrate....................................................................................................80 2.11.2.1. Recombinant substrates.....................................................................80 2.11.2.2. B-RAF...............................................................................................80 2.12. Phospho-site mapping by mass spectrometry...................................................80 3 2.12.1. Kinase assay and sample preparation........................................................80 2.12.2. Mass Spectrometry....................................................................................81 2.12.3. Data Analysis............................................................................................81 2.13. In-gel kinase assay............................................................................................82 2.14. 2D in-gel kinase assay......................................................................................82 2.15. Ion exchange chromatography..........................................................................83 2.16. Pim1/ULK1/MBP kinase assay........................................................................83 2.17. Microarray screening........................................................................................84 3. ULK1 substrate screening..........................................................................................87 3.1. Purification of ULK1..........................................................................................88 3.2. Probing the microarray.......................................................................................93 3.3. Testing the candidates.........................................................................................97 3.3.1. MERTK.......................................................................................................97 3.3.2. B-RAF.........................................................................................................99 3.4. Discussion.........................................................................................................100 3.4.1. Obtaining active ULK1.............................................................................100 3.4.2. Screening results.......................................................................................102 4. Upstream regulation of ULK1..................................................................................108 4.1. ULK1 1-278 K46N purification........................................................................110 4.2. In-gel kinase assay............................................................................................112 4.3. Identification of the 34 kDa kinase...................................................................113