Identification and functional studies of two novel serine phosphorylation sites of insulin receptor substrate (IRS)-2: Ser 675 and Ser 907 [Elektronische Ressource] / vorgelegt von Louise Fritsche

Imfritl1

Sujets

• Biologie

Informations

• Publié par: universitat_hohenheim
• Publié le: 01 janvier 2010
• Nombre de lectures: 29
• Langue: Deutsch
• Poids de l'ouvrage: 3 Mo

Extrait

Identification and functional studies of two novel
serine phosphorylation sites of insulin receptor
substrate (IRS)-2: Ser 675 and Ser 907


Dissertation zur Erlangung des Doktorgrades
der Naturwissenschaften (Dr. rer. nat.)

Fakultät Naturwissenschaften
Universität Hohenheim

Institut für Biologische Chemie und Ernährungswissenschaft
Universität Hohenheim

und

Universitätsklinikum Tübingen
Abteilung für Innere Medizin IV-
Pathobiochemie und klinische Chemie


vorgelegt von
Louise Fritsche


aus Potsdam
2010 3








Dekan: Prof. Dr. rer. nat. Heinz Breer
1. berichtende Person: Prof. Dr. rer. nat. Erwin D. Schleicher
2. berichtende Person: Prof. Dr. rer. nat. Lutz Graeve
Eingereicht am: 15.06.2010
Mündliche Prüfung am: 12.10.2010




Die vorliegende Arbeit wurde am 29.07.2010 von der Fakultät Naturwissenschaften
der Universität Hohenheim als „Dissertation zur Erlangung des Doktorgrades der
Naturwissenschaften“ angenommen. Table of contents 5
Table of contents
Table of contents ........................................................................................................ 5
1 Introduction...................................................................................................... 9
1.1 Insulin signaling ................................................................................................. 9
1.1.1 Insulin ................................................................................................................ 9
1.1.2 The insulin receptor (IR)..................................................................................... 9
1.1.3 Insulin signal transduction................................................................................ 10
1.1.4 PI-3 kinase pathway and PKB/Akt dependent signaling ................................... 12
1.1.5 MAP kinase pathway ....................................................................................... 14
1.2 IRS proteins..................................................................................................... 15
1.2.1 Protein structure of IRS-1 and -2...................................................................... 16
1.2.2 Regulation of IRS proteins ............................................................................... 19
1.3 Insulin resistance and type II diabetes mellitus ................................................ 25
1.4 Aims of the thesis............................................................................................. 26
2 Materials ......................................................................................................... 29
2.1 Chemicals........................................................................................................ 29
2.2 Buffers and solutions ....................................................................................... 31
2.3 Gels ................................................................................................................. 34
2.4 Culture media and supplements....................................................................... 35
2.5 Kits .................................................................................................................. 36
2.6 Enzymes and molecular markers..................................................................... 36
2.7 Consumables................................................................................................... 36
2.8 Laboratory equipment ...................................................................................... 37
2.9 Software .......................................................................................................... 38
2.10 Primers and siRNA oligonucleotides ................................................................ 39
2.10.1 Primers for real time PCR ................................................................................ 39
2.10.2 Primers for PCR-mutagenesis.......................................................................... 39
2.10.3 Sequencing primers......................................................................................... 40
2.10.4 siRNA oligonucleotides .................................................................................... 40
2.11 Antibodies........................................................................................................ 41
2.11.1 Primary antibodies ........................................................................................... 41
2.11.2 Secondary antibodies ...................................................................................... 42
2.12 Plasmids .......................................................................................................... 42
2.13 Cells, bacterial strains and animals.................................................................. 43
2.13.1 Cells................................................................................................................. 43
2.13.2 Bacteria Strains ............................................................................................... 43
2.13.3 Animals............................................................................................................ 43 Table of contents 6
3 Methods.......................................................................................................... 45
3.1 Cell culture....................................................................................................... 45
3.1.1 Cultivation, passaging and seeding for experiments ........................................ 45
3.1.2 Cryopreservation of cell lines ........................................................................... 46
3.1.3 Electroporation of Fao cells with siRNA ........................................................... 46
3.1.4 Transient transfection ...................................................................................... 47
3.1.5 Generation of HEK293 and Huh-7 cells stably expressing IRS-2 mutants........ 48
3.2 Animal studies ................................................................................................. 49
3.3 Protein biochemical methods ........................................................................... 50
3.3.1 Cell lysis .......................................................................................................... 50
3.3.2 Liver tissue lysis............................................................................................... 50
3.3.3 Protein assay for determination of protein concentration (Bradford assay)....... 50
3.3.4 Immunoprecipitation (IP).................................................................................. 51
3.3.5 SDS-PAGE ...................................................................................................... 52
3.3.6 Western blotting............................................................................................... 52
3.3.7 Immunodetection ............................................................................................. 52
3.3.8 Stripping of membranes................................................................................... 53
3.4 Generation of monoclonal phospho-specific antibodies.................................... 53
3.5 Molecular methods........................................................................................... 54
3.5.1 PCR-mutagenesis and DpnI digestion ............................................................. 54
3.5.2 Transformation................................................................................................. 55
3.5.3 Miniprep/Maxiprep for the isolation of plasmid-DNA from transformed E. coli .. 56
3.5.4 Sequencing of plasmid-DNA ............................................................................ 56
3.5.5 Digestion with restriction enzymes and separation of DNA fragments with agarose
gel 57
3.5.6 Generation of competent E. coli....................................................................... 58
3.5.7 RNA extraction from cultured cells ................................................................... 58
3.5.8 Reverse transcriptase (RT)-reaction ................................................................ 59
3.5.9 Real time quantitative PCR on Lightcycler 480 ................................................ 60
3.6 Data analysis ................................................................................................... 63
3.6.1 Quantification of immunoblots by scanning densitometry................................. 63
3.6.2 Statistics .......................................................................................................... 63
4 Results............................................................................................................ 65
4.1 Testing of antibodies........................................................................................ 65
4.1.1 Prescreening in Fao rat hepatoma cells ........................................................... 65
4.1.2 Antibody specificity .......................................................................................... 66
4.1.3 IRS-2 phosphorylation in primary huma