BAD phosphorylation [Elektronische Ressource] : a novel link between apoptosis and cancer = BAD-Phosphorylierung / submitted by Lisa Polzien

BAD Phosphorylation:
A Novel Link between Apoptosis and Cancer

BAD Phosphorylierung:
Eine Neue Verbindung zwischen Apoptose und Krebs


Doctoral Thesis for Submission to a Doctoral Degree
at the Graduate School of Life Sciences,
Julius Maximilian University of Würzburg


Sections
Infection & Immunity
and
Biomedicine











submitted by
Lisa Polzien
from
Würzburg

Würzburg, 2011


thSubmitted on: February 18 , 2011
Office stamp

Members of the Promotionskomitee:
Chairperson: Prof. Dr.Thomas Dandekar


Primary Supervisor: Prof. Dr. Dr. h.c. Roland Benz
Supervisor (second): Prof. Dr. Thomas Rudel
Supervisor (third): PD Dr. Mirko Hekman


Day of Rigorosum: 25.05.2011


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












The thesis is based on the following manuscripts:
1. Polzien L, Baljuls A, Rennefahrt UE, Fischer A, Schmitz W, Zahedi RP, Sickmann A, Metz R,
Albert S, Benz R, Hekman M, Rapp UR (2009) Identification of novel in vivo phosphorylation
sites of the human pro-apoptotic protein BAD: pore-forming activity of BAD is regulated by
phosphorylation. J. Biol. Chem. 284, 28004-28020

2. Polzien L, Benz R, Rapp UR (2010) Can BAD pores be good? New insights from examining
BAD as a target of RAF kinases. Adv. Enzyme Regul. 50, 147-159

3. Polzien L, Baljuls A, Roth HM, Kuper J, Benz R, Schweimer K, Hekman M, Rapp UR (2010)
Pore-forming activity of BAD is regulated by specific phosphorylation and structural transitions
of the C-terminal part. Biochim. Biophys Acta. 1810, 162-169

4. Polzien L, Baljuls A, Albrecht M, Hekman M, Rapp UR (2011) BAD contributes to RAF-
mediated proliferation and cooperates with B-RAF-V600E in cancer signaling. J. Biol. Chem.
[Epub ahead of print]



„Dissertation unter Einschluss mehrerer Manuskripte“ in der GSLS –
Erklärung zu Eigenanteilen an Publikationen und Zweitpublikationsrechten
(ggf. weitere Blätter dieses Formblatts verwenden)

Publikation (Vollständiges Zitat): Polzien L, Baljuls A, Rennefahrt UE, Fischer A, Schmitz W, Zahedi
RP, Sickmann A, Metz R, Albert S, Benz R, Hekman M, Rapp UR (2009) Identification of novel in
vivo phosphorylation sites of the human proapoptotic protein BAD: pore-forming activity of BAD is
regulated by phosphorylation. J. Biol. Chem. 284, 28004-28020
Autoren-Initialen, Verantwortlichkeit abnehmend von links nach rechts Beteiligt an
Planung der Untersuchungen MH LP URR, RB UER AB
Datenerhebung LP UER RM AF, SA WS, RPZ, AS
Daten-Analyse und Interpretation LP MH URR, RB UER AB
Schreiben des Manuskripts MH LP URR AB

Publikation (Vollständiges Zitat): Polzien L, Benz R, Rapp UR (2010) Can BAD pores be good? New
insights from examining BAD as a target of RAF kinases. Adv. Enzyme Regul. 50, 147-159
Beteiligt an Autoren-Initialen, Verantwortlichkeit abnehmend von links nach rechts
Planung der Untersuchungen LP RB URR
Datenerhebung LP
Daten-Analyse und Interpretation LP RB URR
Schreiben des Manuskripts LP URR RB

Publikation (Vollständiges Zitat): Polzien L, Baljuls A, Roth HM, Kuper J, Benz R, Schweimer K,
Hekman M, Rapp UR (2010) Pore-forming activity of BAD is regulated by specific phosphorylation
and structural transitions of the C-terminal part. Biochim. Biophys. Acta. 1810,162-169
Beteiligt an Autoren-Initialen, Verantwortlichkeit abnehmend von links nach rechts
Planung der Untersuchungen LP MH AB, RB URR
Datenerhebung LP HMR JK KS
Daten-Analyse und Interpretation LP MH, RB AB HMR JK, KS
Schreiben des Manuskripts MH LP AB

Publikation (Vollständiges Zitat): Polzien L, Baljuls A, Albrecht M, Hekman M, Rapp UR (2011)
BAD contributes to RAF-mediated proliferation and cooperates with B-RAF-V600E in cancer
signaling. J. Biol. Chem. [Epub ahead of print]
Autoren-Initialen, Verantwortlichkeit abnehmend von links nach rechts Beteiligt an
Planung der Untersuchungen LP MH MA AB URR
Datenerhebung LP MA
Daten-Analyse und Interpretation LP MH MA
Schreiben des Manuskripts LP MH MA AB

Für alle in dieser „Dissertation unter Einschluss mehrerer Manuskripte“ verwendeten Manuskripte liegen die
notwendigen Genehmigungen der Verlage und Co-Autoren für die Zweitpublikation vor.
Mit meiner Unterschrift bestätige ich die Kenntnisnahme und das Einverständnis meines direkten Betreuers.

Lisa Polzien, 18.02.2011, ___________________
Name Kandidat(in), Datum, Unterschrift




Table of Content
Content
Summary .................................................................................................................................... 4
Zusammenfassung ...................... 6
1. General Introduction .............................................................................................................. 8
1.1. Apoptosis .................................... 8
1.2. The Bcl-2 Family of Proteins .................................... 10
1.2.1. Pore-Forming Activity of the Bcl-2 Family Proteins ...................... 12
1.2.2. BH3-only Proteins as Sensors for Distinct Apoptotic Pathways ..................................... 13
1.2.3. The BH3-only Protein BAD ............................................................................................ 15
1.3. RAF Kinases ............................................................. 16
1.3.1. Structure of the RAF Kinases .......................... 17
1.3.2. Regulation of RAF Kinase Activity ................................................................................. 19
1.3.2.1. Regulation of C-RAF Activation .............. 19
1.3.2.2. Regulation of A-RAF Activation ............. 20
1.3.2.3. Regulation of B-RAF Activation .............................................................................. 20
1.4. The Family of 14-3-3 Proteins .................................. 21
1.4.1. 14-3-3 Proteins as Key Regulators of RAF Kinases ........................ 22
1.4.2. 14-3-3 Proteins Control Apoptosis .................................................................................. 23
1.5. Bcl-2 Proteins are Substrates of RAF and Play a Role in Human Diseases ............................. 25
1.6. Aim of the Project ..................................................... 26
2. General Experimental Procedures ....................................................................................... 28
2.1. Materials ................................................................... 28
2.1.1. Instruments ...................... 28
2.1.2. Chemical Reagents and General Materials ...................................... 29
2.1.3. Cell Culture Materials ...................................................................... 31
2.1.4. Antibodies used for Western Blotting and Immunoprecipitation .... 32
2.1.5. Enzymes ........................................................................................... 32
2.1.6. Kits ................................................................... 33
2.1.7. Plasmids 33
2.1.8. Oligonucleotides .............................................. 35
2.1.9. siRNAs for RNA Interference ......................................................... 36
2.1.10. Cell Lines and Bacterial Strains ....................... 36
2.2. Solutions and Buffers ................................................................................ 37
2.3. Methods..................................................................... 42
2.3.1. Microbiological Methods ................................................................. 42
2.3.1.1. Preparation of Chemocompetent Bacteria (CaCl Method) ..... 42 2
2.3.1.2. Transformation of Chemocompetent Bacteria .......................... 42
2.3.2. Molecular Biological Methods ......................................................................................... 42
2.3.2.1. Amplification of DNA by PCR ................ 42
2.3.2.2. Agarose Gel Electrophoresis of DNA ...................................................................... 44
2.3.2.3. Isolation of DNA Fragments from an Agarose Gel .................. 44
2.3.2.4. Purification of DNA Fragments ............... 45
2.3.2.5. Digestion of DNA with Restriction Endonucleases ................................................. 45
1 Table of Content
2.3.2.6. DNA Ligation ........................................................................................................... 45
2.3.2.7. Purification of Plasmid DNA ................... 45
2.3.2.8. Determination of DNA Concentration and Quality .................. 45
2.3.2.9. Site-Directed Mutagenesis ........................................................................................ 46
2.3.3. Biochemical Methods ....................................................................................................... 46
2.3.3.1. Preparation of Cell Lysates ....................... 46
2.3.3.2. Determination of Protein Concentration (Bradford Assay) ...... 46
2.3.3.3. Sodium Dodecy