Struktur-Funktionsanalyse der {Gßγ-vermittelten [Gbetagamma-vermittelten] Aktivierung der {Phosphoinositid-3-Kinase -γ [Phosphoinositid-3-Kinase-gamma] [Elektronische Ressource] = Structural and functional analysis of {Gßγ-induced [Gbetagamma-induced] stimulation of {phosphoinositide 3-kinase γ [phosphoinositide 3-kinase gamma] / vorgelegt von Aliaksei Shymanets

Shymanets

Sujets

• Biologie

Informations

• Publié par: heinrich-heine-universitat_dusseldorf
• Publié le: 01 janvier 2007
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Struktur-Funktionsanalyse der G βγ-vermittelten
Aktivierung der Phosphoinositid 3-Kinase γ

(Structural and functional analysis of G βγ-induced
stimulation of phosphoinositide 3-kinase γ)








Inaugural-Dissertation

zur

Erlangung des Doktorgrades der
Mathematisch-Naturwissenschaftlichen Fakultät
der Heinrich-Heine-Universität Düsseldorf

vorgelegt von

Aliaksei Shymanets

aus Baranovichi (Belarus)



2007 Aus dem Institut für Biochemie und Molekularbiologie II

der Heinrich-Heine Universität Düsseldorf
























Gedruckt mit der Genehmigung der

Mathematisch-Naturwissenschaftlichen Fakultät der

Heinrich-Heine-Universität Düsseldorf




Referent: Prof. Dr. Dr. Bernd Nürnberg

Koreferent: Prof. Dr. Claudia Staudt

Tag der mündlichen Prüfung: 21.06.2007 Contents I


CONTENTS
1 INTRODUCTION 1
1.1 General principles of transmembrane signal transduction 1
1.2 G-protein-dependent signal transduction 3
1.2.1 Structure and function of G-proteins 3
1.2.1.1 G α subunits 4
1.2.1.2 Structure of G βγ dimer 7
1.2.1.3 G βγ effectors 9
1.2.2 Molecular interaction of G βγ dimer with G α subunit and effectors 9
1.3 Phosphoinositide 3-kinases (PI3Ks) 12
1.3.1 The PI3K family 12
1.3.1.1 Class I PI3Ks 12
1.3.1.1.1 Class I PI3Ks 14 A
1.3.1.1.2 Class I15 B
1.3.1.2 Class II PI3Ks 16
1.3.1.3 Class III PI3Ks 17
1.3.2 Regulation of PI3K γ activity 17
1.3.2.1 G βγ-dependent stimulation of lipid and protein kinase activity of PI3K γ 17
1.3.2.2 PI3K γ inhibitors 18
1.3.2.3 The role of PTEN and SHIP in regulation of PI3K γ activity 21
1.3.3 The targets and biological significance of 3-phosphoinositides 21
2 AIMS OF THE STUDY 24
3 MATERIALS 25
3.1 List of manufacturers and distributors 25
3.2 Chemicals 25
3.3 Enzymes, proteins, peptides and other biological active substances 27
3.4 Non-radioactively labeled nucleotides 27
3.5 Radioactively labeled nucleotides 28
3.6 Cell cultures, cell culture mediums, and supplements 28
3.7 Protein standards 28
3.8 Chromatography and separation materials
3.9 Membranes and filters 29
3.10 Autoradiography films, screens, and accessories 29 Contents II


4 EXPERIMENTAL PROCEDURES 30
4.1 Standard biochemical methods 30
4.1.1 Measurement of protein concentration 30
4.1.2 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 30
4.1.3 Coomassie staining of SDS-polyacrylamide gels 31
4.1.4 Immunoblotting 31
4.1.5 Stripping and reprobing of membranes 33
4.1.6 Immunoprecipitation of recombinant monomeric and dimeric PI3K γ 33
4.1.7 Determination of nucleotide concentrations 33
4.2 Expression of recombinant proteins in insect cells 34
4.2.1 Culture of Sf9 cells 35
4.2.2 Recombinant virus amplification 35
4.2.3 Estimation of the virus titer (Plaque Assay) 36
4.2.4 Recombinant protein expression 36
4.3 Protein purification 37
4.3.1 Purification of recombinant membrane-attached
His-tagged G β γ dimers and their mutants from Sf9-cells 37 1 2
4.3.1.1 Preparation of plasma membrane extracts 37
4.3.1.2 Affinity-chromatography 37
4.3.1.3 Anion exchange chromatography 38
4.3.1.4 Gel filtration (Size exclusion chromatography) 38
4.3.2 Purification of cytosolic His- and GST-tagged PI3K γ 39
4.3.3 Copurification of G β γ with PI3K γ subunits 40 1 2
4.4 Limited trypsin proteolysis of G β γ 40 1 2
4.5 Determination of PI3K γ activity 41
4.5.1 Measurement of lipid kinase activity 41
4.5.2 ent of protein kinase activity 42
4.6 Lipid vesicle pull-down assay 42
4.7 Generation and presentation of the dose-response curves of
PI3K γ stimulation induced by G β γ mutants 42 1 2-HisContents III


5 RESULTS 45
5.1 Purification of recombinant G β γ from Sf9 cells 45 1 2-His
5.2 Characterization of purified recombinant G β γ and PI3K γ 48 1 2
5.2.1 Purity and trypsin sensitivity of G β γ variants 48 1 2-His
5.2.2 Influence of N-terminal His- and GST-tags and N-terminal fragment
(amino acids 1 - 34) of p110 γ on the lipid kinase activity of PI3K γ 51
5.2.3 The non-ionic detergent C E affects the lipid kinase activity of PI3K γ 54 12 10
5.3 G β γ –induced autophosphorylation of PI3K γ 56 1 2-His
5.3.1 Autophosphorylation of PI3K γ catalytic subunit occurs at Ser-1101 56
5.3.2 Phospholipid vesicles are indispensable for G βγ-induced
stimulation of PI3K γ autophosphorylation 57
5.3.3 Protein kinase activity of the monomeric PI3K γ 58
5.4 G β residues relevant for interaction and stimulation 1
of PI3K γ enzymatic activities 61
5.4.1 G β amino acids essential for stimulation of monomeric and 1
dimeric PI3K γ enzymatic activities 61
5.4.2 G β alanine mutants relevant for discrimination 1
between different enzymatic qualities of PI3K γ 67
5.4.3 Monoclonal anti-p110 γ antibody (mAb 641) discriminates between
G β γ -induced stimulation of monomeric and dimeric PI3K γ activities 70 1 2
5.5 Role of G βγ dimer and non-catalytic subunit (p101)
in activation of PI3K γ 74
5.5.1 Effect of phosphatidylserine on association of G β γ to the 1 2-His
lipid compartment 74
5.5.2 Effect of G β γ on stimulation of lipid vesicle-bound PI3K γ 75 1 2-His
5.5.3 p101 serves as a potential activator of p110 γ 77
6 DISCUSSION 81
6.1 Purification and analysis of recombinant G β γ variants 1 2-His
from Sf9 cells 81
6.2 Autophosphorylation of class I PI3K 83 B
6.3 Role of p101 in the activation of PI3K γ 87
6.4 G β residues implicated in interaction with class I PI3K 90 1 B
Contents IV


6.5 Monoclonal anti-p110 γ antibody (mAb 641) differentially
affects PI3K γ enzymatic activities 95
7 SUMMARY 98
8 ZUSAMMENFASSUNG 100
9 REFERENCES 103
10 CURRICULUM VITAE 124
11 ACKNOWLEDGEMENTS 127
12 SUPPLEMENTARY MATERIAL 128
Abbreviations V


ABBREVIATIONS
β-AR β-adrenergic receptor
AC adenylyl cyclase
AMF chemical combination added to buffer: 40 µM aluminium chloride,
6 mM magnesium chloride and 10 mM sodium fluoride
APS ammonium persulfate
ATP adenosine-5’-triphosphate
BSA bovine serum albumin
cAMP cyclic adenosine-3’,5’-monophosphate
cGMP cyclic guanosine-3’,5’-m
CHAPS 3 [(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate
Ci Curie
cpm counts per minute
CTX cholera toxin secreted by Vibrio cholerae
C E polyoxyethylene-(10)-lauryl ether 12 10
DAG diacylglycerol
DMSO dimethylsulfoxide
DNA deoxyribonucleic acid
DTT dithiothreitol
EC the molar concentration of a substance, which produces 50 % of the 50
maximum possible response for that substance.
EDTA ethylenediamine-N,N,N’N’-tetraacetic acid
EGTA ethylene glycol bis(2-aminoethyl ether)-N,N,N'N'-tetraacetic acid
Fig. figure
2(x) g centrifugal force (9,81 m/s )
GABA γ-aminobutyric acid
GDP guanosine-5’-diphosphate
G-protein heterotrimeric guanine nucleotide binding protein
GST glutathione-S-transferase
GTP guanosine-5’-triphosphate
GTP γS guanosine-5’-[ γ-thio]- triphosphate
h hours
HEPES 4-2-hydroxyethyl-1-piperazineethanesulfonic acid
His-tag hexahistidine-tag
HPLC high pressure (performance) liquid chromatography
IP inositol-1,4,5-trisphosphate 3
kDa kilodalton (a unit of mass, being 1000 daltons)
Lubrol PX polyoxyethylene-(9)-lauryl ether
M mol per liter
MALDI matrix assisted laser desorption ionisation
min minutes
MOI multiplicity of infection
NAD reduced form of nicotinamide adenine dinucleotide
2+Ni -NTA nickel nitrilotriacetic acid Abbreviations VI


PIKAPp84 a novel regulatory subunit of class I PI3K γ (also known as p87 ) B
p85 non-catalytic subunit of class I PI3Ks α, β and δ A
PIKAPp87 PI3K γ (also known as p84) B
p101 PI3K γ B
p110 catalytic subunit of class I PI3Ks
PAGE polyacrylamide gel electrophoresis
PBS phosphate-buffered saline
pfu plaque-forming unit: a virus or group of viruses which cause a plaque
pH logarithmic measure of hydrogen ion concentration (a measure of the
acidity or alkalinity of a solution)
PI phosphatidylinositol
PI-4,5-P phosphatidylinositol-4,5-bisphosphate (PIP ) 2 2
PI-3,4,5-P phosphatidylinositol-3,4,5-triphosphate (PIP ) 3 3
PI3K phosphatidylinositol-3-kinase
PLC phospholipase C
PTX pertussis toxin secreted by Bordetella pertussis
PVDF polyvinylidene fluoride
rpm revolutions per minute
s seconds
S.D. standard deviation
SDS sodium dodecyl sulfate
S.E.M. standard error of the mean
Sf9 cells cells derived from the pupal ovary of Spodoptera frugiperda
TEMED N,N,N’,N’-tetramethylethylenediamine
Tris 2-amino-2-hydroxymethyl-1,3-propanediol
Tween 20 polyoxyethylene-(20)-monolaurate
UV ultraviolet
% (v/v) volume/volume percent