Identification of the novel Akt2 interacting proteins RPS25 and eIF3c [Elektronische Ressource] : characterization of interaction and physiological consequences / von Xuehui He

Xuehui He

Sujets

• Biologie

Informations

• Publié par: eberhard_karls_universitat_tubingen
• Publié le: 01 janvier 2007
• Nombre de lectures: 45
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Identification of the novel Akt2
interacting proteins RPS25 and eIF3c:
characterization of interaction and
physiological consequences




der Fakultät für Biologie
der EBERHARD KARLS UNIVERSITÄT TÜBINGEN

zur Erlangung des Grades eines Doktors
der Naturwissenschaften



von


Xuehui He
aus Henan/China
vorgelegte


D i s s e r t a t i o n



2007





























Tag der mündlichen Prüfung: 25 June 2007
Dekan: Prof. Dr. Friedrich Schöffl
1. Berichterstatter: PD Dr. Reiner Lammers
2. Berichterstatter: Prof. Dr. Hans-Georg Rammensee Contents - 1 -
Contents
Contents.............................................................................................................................. - 1 -
1 Introduction .................................................................................................................... - 6 -
1.1 The structure of Akt...................................................................................................... - 6 -
1.2 The regulation of Akt.................................................................................................... - 8 -
1.2.1 PI3K dependent activation of Akt ........................................................................ - 8 -
1.2.2 Akt activation in lymphocytes............................................................................ - 10 -
1.2.3 Negative feedback control of Akt....................................................................... - 10 -
1.3 Akt substrates and physiological functions ............................................................ - 11 -
1.3.1 Regulation of cell metabolism - 11 -
1.3.2 Regulation of cell survival and apoptosis........................................................... - 12 -
1.3.3 Regulation of cell proliferation........................................................................... - 15 -
1.3.4 Regulation of protein translation ........................................................................ - 15 -
1.4 Akt isoform specific functions................................................................................... - 17 -
1.5 Akt functions in lymphocytes.................................................................................... - 18 -
1.6 Deregulation of Akt in human diseases .................................................................. - 19 -
1.7 Akt interacting proteins.............................................................................................. - 19 -
1.7.1 Binding proteins for the Akt PH domain............................................................ - 20 -
1.7.2 Binding proteins for the Akt kinase domain....................................................... - 20 -
1.7.3 Binding proteins for the carboxyl-terminus of Akt ............................................ - 21 -
2 Aim of the doctoral thesis......................................................................................... - 23 -
3 Materials and methods - 24 -
3.1 Materials ...................................................................................................................... - 24 -
3.1.1 Reagents ............................................................................................................. - 24 -
3.1.2 Kits ..................................................................................................................... - 25 -
3.1.3 Buffers ................................................................................................................ - 26 -
3.1.4 Yeast, bacteria and mammalian cell lines .......................................................... - 30 -
3.1.5 Media.................................................................................................................. - 30 -
3.1.6 Antibodies........................................................................................................... - 31 -
3.1.7 Oligonucleotides................................................................................................. - 32 -
3.1.8 Vectors and expression plasmids........................................................................ - 33 -
3.2 Methods ....................................................................................................................... - 34 -
3.2.1 Yeast two-hybrid system .................................................................................... - 34 -
3.2.2 Polymerase chain reaction (PCR)....................................................................... - 39 -
3.2.3 Isolation of plasmid DNA from E. coli .............................................................. - 39 - Contents - 2 -
3.2.4 Cloning procedures and transformation of E. coli.............................................. - 40 -
3.2.5 Site-directed mutagenesis................................................................................... - 40 -
3.2.6 DNA gel electrophoresis .................................................................................... - 41 -
3.2.7 Glutathione S-transferase fusion protein purification ........................................ - 41 -
3.2.8 Affinity purification of antibodies...................................................................... - 42 -
3.2.9 Cell culture ......................................................................................................... - 43 -
3.2.10 Cell treatment ................................................................................................... - 44 -
3.2.11 Transfection of mammalian cells ..................................................................... - 44 -
3.2.12 Cell lysis and co-immunoprecipitation............................................................. - 45 -
3.2.13 SDS polyacrylamide gel electrophoresis.......................................................... - 46 -
3.2.14 Immunokinase activity assay............................................................................ - 47 -
3.2.15 Western blotting ............................................................................................... - 47 -
3.2.16 GST pull-down assay ....................................................................................... - 48 -
3.2.17 Determination of protein and DNA concentrations.......................................... - 48 -
3.2.18 Immunofluorescence ........................................................................................ - 48 -
3.2.19 Determining cell growth curve using trypan blue staining............................... - 49 -
3.2.20 Luciferase activity assay................................................................................... - 49 -
4 Results ........................................................................................................................... - 51 -
4.1 Identification of proteins interacting with Akt2 ....................................................... - 51 -
4.1.1 Yeast two-hybrid screen ..................................................................................... - 51 -
4.1.2 Interaction of Akt2 and new candidate binding proteins.................................... - 53 -
4.1.3 Expression of RPS25 in mammalian cells.......................................................... - 56 -
4.1.4 Interaction of RPS25 with endogenous Akt2 ..................................................... - 57 -
4.1.5 RPS25 associates with unphosphorylated Akt2 ................................................. - 58 -
4.2 Detection of tyrosine phosphorylation of RPS25 .................................................. - 59 -
4.3 Mapping the RPS25 binding site of Akt2................................................................ - 60 -
4.4 Mapping the Akt2 binding site of RPS25 - 64 -
4.5 RPS25 improves the interaction of Akt2 and eIF3c.............................................. - 65 -
4.6 Intracellular localization of RPS25........................................................................... - 69 -
4.6.1 RPS25 localizes in the nucleus of C2C12 cells.................................................. - 69 -
4.6.2 RPS25 increases the nuclear translocation of Akt2............................................ - 70 -
4.6.3 eIF3c does not affect cellular distribution of Akt2 or RPS25 ............................ - 71 -
4.7 RPS25 regulates the growth of Jurkat cells........................................................... - 74 -
4.8 Interaction of Akt2 and RPS25 has no effect on the IL-2 promoter ................... - 76 -
4.9 Regulation of protein translation by the complex of Akt2, RPS25 and eIF3c... - 77 -
5 Discussion .................................................................................................................... - 82 -
5.1 Yeast two-hybrid screen for Akt2 interacting proteins.......................................... - 82 - Contents - 3 -
5.2 Confirmation of interactions outside the yeast system......................................... - 83 -
5.3 Characterization of the interaction of Akt2 and RPS25........................................ - 84 -
5.4 A protein complex of eIF3c, Akt2 and RPS25 ...........................................