Analysis of the binding properties of the kinase C-RAF to mitochondria and characterization of its effects on the cellular and molecular level [Elektronische Ressource] = Untersuchung der Bindungseigenschaften der Kinase C-RAF an Mitochondrien und Charakterisierung der Effekte auf zellulärer und molekulare Ebene / submitted by Jochen Füller

Sujets

• Biologie

Informations

• Publié par: julius-maximilians-universitat_wurzburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 6
• Langue: English
• Poids de l'ouvrage: 4 Mo

Extrait

Analysis of the binding properties of the kinase C-RAF to
mitochondria and characterisation of its effects on the cellular
and molecular level


Untersuchung der Bindungseigenschaften der Kinase C-RAF an
Mitochondrien und Charakterisierung der Effekte auf zellulärer
und molekulare Ebene





Doctoral thesis for submission to a doctoral degree
at the Graduate School of Life Sciences,
Julius Maximilian University Würzburg,
Section Infection and Immunity


submitted by



Jochen Füller



from

Bamberg





Würzburg, 2008



































Submitted on: …………………………………………………………….
Office stamp


Members of the Promotionskomitee:

Chairperson: …………………………………………………………………
to be completed by the Office

Primary Supervisor: Prof. Dr. UR Rapp

Supervisor (second): Prof. Dr. R. Benz

Supervisor (third):



Day of Rigorosum: ……………………………………………………….



Certificates were handed-out on: …………………………………….. Contents

Contents

Summary.............................................................................................................................. 6
Zusammenfassung............................................................................................................ 7
1. Introduction
1.1. RAF kinases as proto-oncogenes and signaling through the RAS-RAF-MEK-
ERK cascade.............................................................................................................. 10
1.2. Structure of RAF kinases......................................................................................... 13
1.3. Regulation of RAF kinases....................................................................................... 15
1.3.1. Membrane recruitment..................................................................................... 15
1.3.2. Phosphorylation............................................................................................... 16
1.3.2.1.Direct regulatory role............................................................................... 16
1.3.2.2.Indirect regulatory role............................................................................. 17
1.3.3. Oligemerization and complex formation with other proteins......................... 19
1.4. Anti-apoptotic functions of RAF............................................................................ 19
1.4.1. General considerations on apoptosis................................................................ 19
1.4.2. Role of the Bcl-2 family in apoptosis............................................................. 22
1.4.3. BH3-only proteins regulate the apoptotic pathway......................................... 24
1.4.4. Control of the apoptotic machinery by RAF kinases...................................... 27
1.4.4.1.Direct regulation...................................................................................... 29
1.4.4.2.Transcriptional regulation of apoptosis.................................................. 29
1.4.4.3.Transformation, regulation of cell differentiation
and survival regulation.............................................................................. 30
1.5. RAF kinases at the level of mitochondria............................................................. 31
1.6. General regulation of the BH3-only protein BAD............................................... 32
1.6.1. BAD as a substrate for RAF-MEK-ERK cascade.......................................... 35
1.7. Aim of the work....................................................................................................... 36
2. Materials and Methods
2.1. Materials................................................................................................................... 38
2.1.1. Instruments...................................................................................................... 38
2.1.2. Chemical reagents........................................................................................... 38
2.1.3. Cell culture compounds.................................................................................. 39
2.1.4. Enzymes.......................................................................................................... 40 Contents

2.1.5. Kits.................................................................................................................. 40
2.1.6. Antibodies....................................................................................................... 40
2.1.7. DNA-constructs.............................................................................................. 41
2.1.8. Cell lines......................................................................................................... 44
2.1.9. Solutions and Buffers..................................................................................... 45
2.2. Methods
2.2.1. Competent bacteria...................................................................................... 46
2.2.1.1.Preparation of competent bacteria........................................................... 46
2.2.1.2.Transformation of competent bacteria..................................................... 46
2.2.2. DNA-methods............................................................................................... 46
2.2.2.1.DNA-isolation from competent bacteria.................................................. 46
2.2.2.2.Electrophoresis on agarose gel................................................................ 47
2.2.2.3.Cloning of expression vectors.................................................................. 47
2.2.3. RNA-method: real-time PCR...................................................................... 49
2.2.4. Protein methods............................................................................................ 49
2.2.4.1.Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE)............................................................................................... 49
2.2.4.2.Immunoblotting......................................................................................... 50
2.2.4.3.Immunoblot stripping................................................................................ 50
2.2.4.4.Immunoprecipitation................................................................................. 50
2.2.4.5.Rabbit Reticulocyte Lysates...................................................................... 51
2.2.5. Cell culture..................................................................................................... 51
2.2.5.1.Transfections............................................................................................. 51
2.2.5.2.Immunofluorescent labelling..................................................................... 52
2.2.5.3.Videomicroscopy....................................................................................... 53
2.2.5.4.Electron microscopy................................................................................. 53
2.2.6. Experimental assays and treatments.......................................................... 53
2.2.6.1.Mitochondrial purification........................................................................ 53
2.2.6.2.In vitro assay for mitochondrial binding and import................................ 54
2.2.6.3.Mitochondrial treatments.......................................................................... 55
2.2.6.4.Anthrax Lethal Toxin (LT) purification and use on cells.......................... 55
2.2.6.5.Analysis of BAD phosphorylation in NIH3T3 C-RAF BxB-ER cells........ 55
2.2.6.6.Analysis of BAD ubiquitylation.................................................................. 56
2.2.6.7.Statistics..................................................................................................... 56 Contents

3. Results
3.1. C-RAF specifically interacts with mitochondria................................................... 57
3.1.1. RAF kinases exhibit distinct localizations on cellular membrane organelles.. 57
3.1.1.1.Immunofluorescence and cell fractionation............................................... 57
3.1.1.2.C-RAF efficiently binds to purified mitochondria in vitro........................ 59
3.2. Cellular effects of C-RAF at the level of mitochondria: Active C-RAF
specifically changes mitochondria’s subcellular distribution in a MEK-
dependent fashion.................................................................................................... 65
3.3. Molecular effects of C-RAF activation: Acute C-RAF activation phosphorylates
BAD in a fast and potent fashion at Ser-112......................................................... 71
3.3.1. C-RAF acts indirectly in BAD phosphorylation: involvement of the
downstream cascade..........................