Developing quantitative GTPase affinity purification (qGAP) to identify interaction partners of Rho GTPases Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) eingereicht im Fachbereich Biologie, Chemie, Pharmazie der Freien Universität Berlin vorgelegt von FLORIAN ERNST RUDOLF BENJAMIN PAUL (DIPL.-BIOCHEM.) Januar 2014 ii Die vorliegende Arbeit wurde von Oktober 2008 bis Januar 2014 am Max-Delbrück-Centrum für Molekulare Medizin unter der Anleitung von Prof. Dr. MATTHIAS SELBACH angefertigt. 1. Gutachter: Prof. Dr. MATTHIAS SELBACH Cell Signalling and Mass Spectrometry Max-Delbrück-Centrum, Berlin 2. Gutachter: Prof. Dr. UDO HEINEMANN Institut für Chemie / Biochemie Freie Universität Berlin / Max-Delbrück-Centrum, Berlin Disputation am 8. Mai 2014 iii iv “Study hard what interests you the most in the most undisciplined, irreverent and original manner possible.” ― Richard P. Feynman v vi Contents 1 Introduction ............................................................................................................................ 1 1.1 Rho GTPases ..................................................................................................................... 2 1.1.1 The GDP/GTP cycle .......................................................................................................... 2 1.1.2 Rho GTPases as part of the Ras superfamily ................................................................... 3 1.1.3 The family of Rho GTPases .............................................................................................. 4 1.2 Regulation of Rho GTPases ............................................................................................... 7 1.2.1 Guanine nucleotide exchange factors ............................................................................. 7 1.2.2 GTPase activating proteins .............................................................................................. 8 1.2.3 Guanine dissociation inhibitors ....................................................................................... 9 1.2.4 Subcellular localization .................................................................................................... 9 1.3 General concepts of Rho GTPase signaling ...................................................................... 10 1.3.1 Cell polarity .................................................................................................................... 10 1.3.2 Actin cytoskeleton ......................................................................................................... 10 1.3.3 Gene expression ............................................................................................................ 12 1.3.4 Enzymatic activity and cell cycle.................................................................................... 12 1.4 Disease relevance of Rho GTPases .................................................................................. 13 1.5 Mass spectrometry for Protein identification ................................................................. 14 1.5.1 Quantitative mass spectrometry ................................................................................... 15 1.5.2 Protein Identification and Quantification ..................................................................... 17 1.6 Protein Interactions and q-AP/MS .................................................................................. 17 1.6.1 Identification of Rho GTPase interaction partners ........................................................ 20 1.7 Objectives ...................................................................................................................... 21 2 Materials & Methods ............................................................................................................. 23 2.1 Materials ........................................................................................................................ 23 2.1.1 Chemicals ....................................................................................................................... 23 2.1.2 Media and buffers ......................................................................................................... 23 2.1.3 Enzymes/Proteins .......................................................................................................... 27 2.1.4 Antibodies ...................................................................................................................... 27 2.1.5 Kits ................................................................................................................................. 28 2.1.6 Bacteria strains .............................................................................................................. 28 2.1.7 Plasmids ......................................................................................................................... 28 2.1.8 Cell lines ......................................................................................................................... 28 2.1.9 cDNA clones ................................................................................................................... 29 2.2 Molecular biology methods ............................................................................................ 29 2.2.1 Primer design and Oligonucleotides ............................................................................. 29 2.2.2 Polymerase Chain Reaction ........................................................................................... 29 vii 2.2.3 DNA purification ............................................................................................................ 29 2.2.4 Restriction digest ........................................................................................................... 29 2.2.5 Agarose gel electrophoresis .......................................................................................... 29 2.2.6 DNA extraction .............................................................................................................. 29 2.2.7 Ligation .......................................................................................................................... 30 2.2.8 Transformation of chemically competent E. coli........................................................... 30 2.2.9 Long term storage of E. coli cultures ............................................................................ 30 2.2.10 Plasmid preparation ...................................................................................................... 30 2.3 Biochemical Methods ..................................................................................................... 30 2.3.1 Antibiotics ...................................................................................................................... 30 2.3.2 Protein expression in E. coli .......................................................................................... 31 2.3.3 E. coli cell lysis and preparation of the cytosolic fraction ............................................. 31 2.3.4 Glutathione affinity chromatography ........................................................................... 31 2.3.5 Size exclusion chromatography ..................................................................................... 31 2.3.6 Nucleotide loading ........................................................................................................ 32 2.3.7 Determination of protein-bound nucleotide ................................................................ 32 2.3.8 Protein concentration determination ........................................................................... 32 2.3.9 Protein concentration and storage ............................................................................... 32 2.3.10 Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) .................... 32 2.3.11 Western blotting ........................................................................................................... 33 2.4 Cell cultivation, fractionation and lysis ........................................................................... 33 2.4.1 Cell media/SILAC............................................................................................................ 33 2.4.2 Cell cultivation ............................................................................................................... 33 2.4.3 Preparation of cytosolic extracts ................................................................................... 34 2.4.4 Transient transfection of mammalian cells ................................................................... 34 2.4.5 Lysis of adherent mammalian cells ............................................................................... 34 2.4.6 Preparation of mice brain .............................................................................................. 34 2.4.7 Immunoprecipitation (GFP-fusion proteins) ................................................................. 35 2.4.8 Pull down assay ............................................................................................................. 35 2.4.9 Protein ethanol precipitation ........................................................................................ 36 2.4.10 Proximity ligation assay ................................................................................................