Overexpression and analysis on posttranslational modification of the retinoic acid related orphan receptor {α4 [alpha-4] [Elektronische Ressource] / von Adriane Lechtken

Overexpression and analysis on posttranslational modification
of the Retinoic Acid Related Orphan Receptor α4



Dissertation zur
Erlangung des Doktorgrades
der Naturwissenschaften



vorgelegt beim Fachbereich
Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe-Universität
in Frankfurt am Main






von
Adriane Lechtken
aus Erlangen




Frankfurt am Main (2007)
(D30)







Vom Fachbereich Biochemie, Chemie und Pharmazie
der Johann Wolfgang Goethe-Universität als Dissertation angenommen.




















Dekan: Prof. Dr. Harald Schwalbe
Gutachter: Prof. Dr. Dieter Steinhilber
Prof. Dr. Oliver Werz
Datum der Disputation: 30.08.2007








“There is a theory which states that if ever anybody discovers exactly what the Universe is for
and why it is here, it will instantly disappear and be replaced by something even more bizarre
and inexplicable. There is another theory which states that this has already happened.”

(Douglas Adams)



CONTENTS
1 INTRODUCTION ........................................................................1
1.1 Nuclear Receptors......................................................................................................1
1.1.1 Structure of Nuclear Receptors .......................................................................... 3
1.1.2 Hormone Response Elements (HREs) ............................................................... 6
1.1.3 Regulation of Nuclear Receptor Activity........................................................... 7
1.2 The Retinoic Acid Related Orphan Receptor (ROR α)............................................. 13
1.2.1 ROR α Isoforms ................................................................................................ 13
1.2.2 ROR Response Element (RORE)..................................................................... 14
1.2.3 ROR α Activity ................................................................................................. 15
1.2.4 Physiological Functions...................................................................................16
1.3 Circadian Rhythmicity.............................................................................................19
1.3.1 Core-Clock.......................................................................................................19
1.3.2 Phosphorylation of Core-Clock Members........................................................ 24
1.4 Mitogen-Activated Protein Kinases (MAPKs) 27
2 AIM OF THE PRESENT INVESTIGATION.........................30
3 MATERIALS AND METHODS...............................................31
3.1 Western Blot.............................................................................................................31
3.2 Coomassie Staining..................................................................................................31
3.3 Bradford Protein Determination............................................................................... 32
3.4 Electrophoretic Mobility Shift Assay (EMSA)........................................................ 32
3.5 In-Gel Kinase Assay (IGKA)................................................................................... 33
3.6 In Vitro Kinase Assay (IVKA)................................................................................. 34
3.7 Cell Culture..............................................................................................................34
3.8 Transient Transfection..............................................................................................34
3.9 Reportergene Assay35
3.9.1 Luciferase Assay35
3.9.2 SEAP Assay.....................................................................................................36
3.10 Analysis of EGFP-ROR α4 Subcellular Distribution................................................ 36
3.11 Genetic Engineering and Plasmid Construction....................................................... 37
3.11.1 ROR α4 Protein Overexpression in E. coli: pET28a-ROR α4-6xHisN ............. 37

3.11.2 hBmal1 Promoter: pGL3-Bmal1...................................................................... 38
3.11.3 hBmal1 Isoform A and B Protein Expression: pSG5-Bmal1-A, B.................. 39
3.11.4 Determination of Anti-ROR α Antibody Epitope: pEGFP-C2-HI-LBD-1-6.... 40
3.11.5 Ca/Dn-MEK1 Expression Plasmid: pcDNA3.1(+)-ca/dn-MEK1.................... 41
3.11.6 SUMO1 Expression Plasmid: pSG5-SUMO1.................................................. 42
4 RESULTS....................................................................................43
4.1 Overexpression and Purification of Human ROR α4 in E. coli ................................ 43
4.1.1 ROR α4 Overexpression ................................................................................... 43
4.1.2 Separation of Inclusion Bodies and Refolding................................................. 45
4.1.3 Nickel Affinity Chromatography ..................................................................... 46
4.1.4 DNA Binding Activity of Refolded ROR α4.................................................... 48
4.1.5 Protein Recovery..............................................................................................49
4.1.6 Overexpression of Human ROR α4 in HeLa 50
4.2 Generation of an Anti-ROR α Antibody ................................................................... 51
4.2.1 Preparation of Protein and Immunization ........................................................ 51
4.2.2 Control of Specificity....................................................................................... 52
4.2.3 Purification of Anti-ROR α Antibody Clone 6E8............................................. 53
4.2.4 Determination of Epitope ................................................................................. 53
4.3 Phosphorylation of ROR α4...................................................................................... 56
4.3.1 In-Gel Kinase Assay......................................................................................... 56
4.3.2 In Vitro Phosphorylation by ERK-2................................................................. 58
4.3.3 p38 and PKA .................................................................................................... 59
4.3.4 Receptor-RORE Complex Formation .............................................................. 60
4.3.5 Transcriptional Activation................................................................................61
4.3.6 Replacement by RevErb α................................................................................. 63
4.3.7 PMA and U0126............................................................................................... 64
4.3.8 Ca/Dn-MEK1...................................................................................................65
4.3.9 Interaction with Bmal1.....................................................................................66
4.3.10 Cellular Distribution.........................................................................................67
4.3.11 Degradation by the Proteasome........................................................................ 68
4.4 Further Posttranslational Modifications ................................................................... 69
4.4.1 Sumoylation.....................................................................................................69
4.4.2 Protein Cleavage..............................................................................................70
4.5 RevErb α.................................................................................................................... 72
4.5.1 Overexpression and Purification ...................................................................... 72
4.5.2 Phosphorylation of RevErb α ............................................................................ 72
5 DISCUSSION..............................................................................76
6 SUMMARY.................................................................................86
7 ZUSAMMENFASSUNG............................................................89
8 ABBREVIATIONS ....................................................................94
9 REFERENCES ...........................................................................97
10 APPENDIX ...............................................................................118
11 PUBLICATIONS......................................................................122
12 CURRICULUM VITAE ...........................................................123
13 DANKSAGUNG ........................................................................125 Introduction
1 INTRODUCTION
1.1 Nuclear Receptors

Nuclear receptors are ligand-inducible transcription factors that modulate gene expression in
response to a wide range of developmental, physiological, and environmental cues. They are
involved in numerous p