........................................................................................................................................vi Table of contents.........................................................................................................................vii List of figures..............................................................................................................................xiii List of tables...............................................................................................................................xvi Chapter 1: Introduction ..................................................................................................................................... 1 1.1 Signal Transduction ................................................................................................................................... 1 1.2 Shc General...........................................................................................................................5 1.3 ShcA .................................................................................................................................................................. 6 1.3.1 Isoforms ....................................................................................................................................................... 6 1.3.2 Domain architecture ............................................................................................................................... 6 1.3.2.1 PTB domain ............................................................................................................................................ 6 1.3.2.2 CH1 domain ............................................................................................................................................ 8 1.3.2.3 SH2 domain ............................................................................................................................................ 9 1.3.2.4 CH2 domain ......................................................................................................................................... 10 1.3.2.5 Cytochrome C binding region ....................................................................................................... 11 viii 1.4 ShcA in signaling ....................................................................................................................................... 11 1.4.1 Mitogen-activated protein kinase (MAPK) signaling .............................................................. 11 1.4.1.1 Extracellular signal-regulated kinase (Erk) pathway ......................................................... 11 1.4.1.2 c-Jun N-terminal kinase pathway.................................................................................13 1.4.2 Phosphatidylinositol-4, 5-bisphosphate 3-kinase (PI3K) pathway..............................13 1.4.3 Mammalian target of rapamycin (mTOR) signaling ................................................................ 14 1.4.4 Redox signaling ...................................................................................................................................... 14 1.5 ShcA in Development............................................................................................................15 1.5.1 Cardiovascular system ........................................................................................................................ 15 1.5.2 Muscular tissue ...................................................................................................................................... 16 1.5.3 Immune system ..................................................................................................................................... 17 1.5.4 Neuronal tissue ...................................................................................................................................... 18 1.6 ShcA in human disease ........................................................................................................................... 20 1.6.1 Neurodegenerative disease .............................................................................................................. 20 1.6.2 Cancer ........................................................................................................................................................ 20 1.6.2.1 Breast Cancer ...................................................................................................................................... 20 1.7 Summary ....................................................................................................................................................... 24 1.8 Research objectives .................................................................................................................................. 25 Chapter 2: Materials and Methods ............................................................................................................. 26 2.1 Materials ....................................................................................................................................................... 26 ix 2.1.1 Chemicals .................................................................................................................................................. 26 2.1.2 Enzymes and growth factor ............................................................................................................... 26 2.1.3 Kits and other materials ...................................................................................................................... 26 2.1.4 Bacterial cell culture reagents .......................................................................................................... 27 2.1.5 Mammalian cell culture reagents..................................................................................................... 28 2.1.5 Composition of complete growth media ....................................................................................... 28 2.1.6 Antibodies..........................................................................................................................29 2.1.7 Plasmids ..................................................................................................................................................... 30 2.1.8 Stock solutions and buffers ................................................................................................................ 31 2.2 Molecular biology ...................................................................................................................................... 37 2.2.1 Restriction cloning................................................................................................................................. 37 2.2.2 Mutagenesis ............................................................................................................................................. 39 2.2.3 Transformation ....................................................................................................................................... 40 2.2.4 DNA extraction ........................................................................................................................................ 40 2.2.5 Generation of competent cells .......................................................................................................... 40 2.3 Cell biological methods ........................................................................................................................... 41 2.3.1 Maintenance of mammalian cell lines ............................................................................................ 41 2.3.2 Transfection of mammalian cell lines ............................................................................................ 41 2.3.3 Lentiviral infection of mammalian cell lines ............................................................................... 41 2.3.4 Generation of stable cell lines ........................................................................................................... 42 x 2.3.5 Preparation and recovery of frozen stocks of mammalian cells....................................42 2.3.6 Fluorescence lifetime imaging microscopy (FLIM) .................................................................. 42 2.3.7 Immunoprecipitation ........................................................................................................................... 43 2.4 Protein expression and purification ................................................................................................... 43 2.4.3 GST-tagged ShcPTB and ShcSH2 proteins .................................................................................... 44 2.4.4 His-tagged Erk2 protein ...................................................................................................................... 45 2.4.5 GST-tagged Erk2 ..................................................................................................................................... 45 2.4.6 GST-tagged Pin1 proteins ................................................................................................................... 45 2.4.7 His-tagged Pin1 ....................................................................................................................................... 45 2.4.8 GFP-14-3-3 zeta ...................................................................................................................................... 46 2.4.9 GST-tagged 14-3-3 Tau and Epsilon proteins ............................................................................. 46 2.4.10 HA-tagged Nedd4 ................................................................................................................................