Dissertation submitted to the Combined Faculties for the Natural Sciences and for Mathematics of the Ruperto-Carola University of Heidelberg, Germany for the degree of Doctor of Natural Sciences presented by Meher Vinay Krishna Mohan Majety, M.Sc. in Biotechnology Born in Vijayawada, India Oral-examination: Development and application of a high throughput cell based assay to identify novel modulators of ERK1/2 activation and, Functional characterisation of the candidate Radial spokehead like (Rshl1) Referees: PD. Dr. Stefan Wiemann Prof. Dr. Ingrid Grummt To my Grandfather Contents SUMMARY............................................................................................................................... 1 ZUSAMENFASSUNG ............................................................................................................. 2 1 INTRODUCTION............................................................................................................ 3 1.1 The Mitogen Activated Protein Kinase pathway ................................................................................. 3 1.1.1 The Extra-cellular signal regulated Kinase (ERK) pathway and its mediators.................................... 4 1.1.2 Cytosolic substrates ............................................................................................................................. 6 1.1.3 Nuclear Targets of ERK1/2.................................................................................................................. 7 1.2 Regulation of ERK1/2 pathway ............................................................................................................. 8 1.2.1 Regulation via stimulus intensity and duration .................................................................................... 8 1.2.2 Regulation by Raf specificity............................................................................................................... 8 1.2.3 Regulation by cellular localisation....................................................................................................... 9 1.2.4 Regulation by scaffolding proteins ...................................................................................................... 9 1.2.5 Regulation by phosphatases ................................................................................................................. 9 1.2.6 Regulation by feed back inhibition .................................................................................................... 11 1.2.7 Cross talk between signalling pathways............................................................................................. 11 1.3 Physiological roles of ERK1/2 cascade................................................................................................ 11 1.3.1 Proliferation and Cell cycle................................................................................................................ 11 1.3.2 Differentiation.................................................................................................................................... 13 1.3.3 Apoptosis ........................................................................................................................................... 13 1.3.4 Cell Adhesion and Migration............................................................................................................. 13 1.4 ERK pathway and disease.................................................................................................................... 13 1.4.1 Cancer ................................................................................................................................................ 13 1.4.2 ERK pathway and cardiovascular diseases ........................................................................................ 14 1.4.3 Neuronal disorders............................................................................................................................. 14 1.5 Overview of the project ........................................................................................................................ 14 1.5.1 Detection of perturbations in ERK1/2 activity................................................................................... 16 1.7 High throughput cell based assay for identification of novel modulators in MAPK signalling..... 18 2 MATERIALS AND METHODS................................................................................... 19 2.1 Materials................................................................................................................................................ 19 2.1.1 Instruments and Equipment ............................................................................................................... 19 2.1.2 Plastics and Glassware....................................................................................................................... 20 2.1.3 Chemicals, Reagents and Media ........................................................................................................ 20 2.1.4 Kits..................................................................................................................................................... 22 2.1.5 Antibodies.......................................................................................................................................... 23 2.1.6 Peptides used for antibody generation ............................................................................................... 24 2.1.7 Buffers and Media.............................................................................................................................. 24 2.1.8 Antibiotics.......................................................................................................................................... 27 2.1.9 Restriction enzymes ........................................................................................................................... 27 2.1.10 Bacterial Strains ............................................................................................................................ 27 2.1.11 Vectors .......................................................................................................................................... 28 2.1.11.1 Gateway entry vector................................................................................................................ 28 2.1.12 Cell lines ....................................................................................................................................... 29 2.2 Methods ................................................................................................................................................. 30 2.2.1 Polymerase Chain Reaction (PCR) .................................................................................................... 30 2.2.1.1 Generation of Entry clones....................................................................................................... 30 2.2.1.1.1 BP reaction ............................................................................................................................... 32 Contents 2.2.1.1.2 LR reaction............................................................................................................................... 33 2.2.2 Preparation of electro competent cells ............................................................................................... 34 2.2.3 Transformation of bacteria by electroporation................................................................................... 34 2.2.4 Isolation of plasmid DNA (Mini-prep) .............................................................................................. 35 2.2.5 Large scale preparation of plasmid DNA (Maxi prep)....................................................................... 35 2.2.6 Measuring the concentration of DNA................................................................................................ 36 2.2.7 Restriction digest ............................................................................................................................... 36 2.2.8 Agarose gel electrophoresis ............................................................................................................... 37 2.2.9 Cell culture......................................................................................................................................... 37 2.2.9.1 Sub-culturing and maintenance of mammalian cells................................................................ 37 2.2.1.1 Cell counting using a Neuberger chamber................................................................................ 37 2.2.1.2 Transfection of mammalian cells ............................................................................................. 38 2.2.10 ß-galactosidase assay..................................................................................................................... 39 2.2.11 Protein extraction from mammalian cells...................................................................................... 39 2.2.12 Protein quantification .................................................................................................................... 40 2.2.12.1 Measurement of protein concentration at UV 280.................................................................... 40 2.2.12.2 Estimation of protein concentration