Functional analysis of RACK1 as a novel interaction partner of BMPRII in pulmonary arterial hypertension [Elektronische Ressource] / vorgelegt von Anna Zakrzewicz
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English
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Functional analysis of RACK1 as a novel interaction partner of BMPRII in pulmonary arterial hypertension [Elektronische Ressource] / vorgelegt von Anna Zakrzewicz

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128 Pages
English

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FUNCTIONAL ANALYSIS OF RACK1 AS A NOVELINTERACTION PARTNER OF BMPRII INPULMONARY ARTERIAL HYPERTENSIONANNA ZAKRZEWICZINAUGURAL-DISSERTATIONzur Erlangung des Grades einesédition scientifique Doktors der HumanbiologieVVB LAUFERSWEILER VERLAG des Fachbereichs Medizin derVVB LAUFERSWEILER VERLAG Justus-Liebig-Universität GießenISBN 3-8359-5186-6STAUFENBERGRING 15D-35396 GIESSENTel: 0641-5599888 Fax: -5599890redaktion@doktorverlag.dewww.doktorverlag.de 9 7 8 3 8 3 5 9 5 1 8 6 0édition scientifiqueVVB VVB LAUFERSWEILER VERLAGANNA ZAKRZEWICZ RACK1 IN PAH. Das Werk ist in allen seinen Teilen urheberrechtlich geschützt. Jede Verwertung ist ohne schriftliche Zustimmung des Autors oder des Verlages unzulässig. Das gilt insbesondere für Vervielfältigungen, Übersetzungen, Mikroverfilmungen und die Einspeicherung in und Verarbeitung durch elektronische Systeme.1. Auflage 2007All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the Author or the Publishers.st1 Edition 2007© 2007 by VVB LAUFERSWEILER VERLAG, GiessenPrinted in Germany VVB LAUFERSWEILER VERLAGédition scientifiqueSTAUFENBERGRING 15, D-35396 GIESSENTel: 0641-5599888 Fax: 0641-5599890 email: redaktion@doktorverlag.dewww.doktorverlag.

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FUNCTIONAL ANALYSIS OF RACK1 AS A NOVEL
INTERACTION PARTNER OF BMPRII IN
PULMONARY ARTERIAL HYPERTENSION
ANNA ZAKRZEWICZ
INAUGURAL-DISSERTATION
zur Erlangung des Grades eines
édition scientifique Doktors der Humanbiologie
VVB LAUFERSWEILER VERLAG des Fachbereichs Medizin der
VVB LAUFERSWEILER VERLAG Justus-Liebig-Universität GießenISBN 3-8359-5186-6
STAUFENBERGRING 15
D-35396 GIESSEN
Tel: 0641-5599888 Fax: -5599890
redaktion@doktorverlag.de
www.doktorverlag.de 9 7 8 3 8 3 5 9 5 1 8 6 0
édition scientifique
VVB VVB LAUFERSWEILER VERLAG
ANNA ZAKRZEWICZ RACK1 IN PAH. Das Werk ist in allen seinen Teilen urheberrechtlich geschützt.
Jede Verwertung ist ohne schriftliche Zustimmung des Autors
oder des Verlages unzulässig. Das gilt insbesondere für
Vervielfältigungen, Übersetzungen, Mikroverfilmungen
und die Einspeicherung in und Verarbeitung durch
elektronische Systeme.
1. Auflage 2007
All rights reserved. No part of this publication may be
reproduced, stored in a retrieval system, or transmitted,
in any form or by any means, electronic, mechanical,
photocopying, recording, or otherwise, without the prior
written permission of the Author or the Publishers.
st
1 Edition 2007
© 2007 by VVB LAUFERSWEILER VERLAG, Giessen
Printed in Germany
VVB LAUFERSWEILER VERLAG
édition scientifique
STAUFENBERGRING 15, D-35396 GIESSEN
Tel: 0641-5599888 Fax: 0641-5599890
email: redaktion@doktorverlag.de
www.doktorverlag.de
Functional analysis of RACK1 as a
novel interaction partner of BMPRII
in pulmonary arterial hypertension



Inaugural-Dissertation
zur Erlangung des Grades eines
Doktors der Humanbiologie
des Fachbereichs Medizin der
Justus-Liebig-Universität Gießen




vorgelegt von


Anna Zakrzewicz
aus Skarzysko-Kamienna, Polen



Gießen, 2006
Aus dem Zentrum für Innere Medizin
des Klinikums der Justus-Liebig-Universität Gießen
Director: Prof. Dr. W. Seeger



















Gutachter: Prof. Dr. W. Seeger / Dr. O. Eickelberg

Gutachter: PD Dr. S. Kanse




Tag der Disputation: 4. Juni 2007

Table of contents I


I Table of contents
I TABLE OF CONTENTS .................................................................................................I
II LIST OF FIGURES.................................................................................................... VII
III LIST OF TABLES ..................................................................................................... IX
IV ABBREVIATIONS ..................................................................................................... X
III SUMMARY.............................................................................................................. XIII
IV ZUSAMMENFASSUNG ..........................................................................................XIV
1 INTRODUCTION......................................................................................................1
1.1 Pulmonary arterial hypertension .......................................................................1
1.1.1 Characteristics of pulmonary arterial hypertension.........................................1
1.1.2 Histopathology of pulmonary arterial hypertension.........................................2
1.1.3 Genetic basis of pulmonary arterial hypertension...........................................3
1.1.4 Animal models of pulmonary arterial hypertension.........................................4
1.1.4.1 The monocrotaline rat model of pulmonary arterial hypertension................4
1.1.4.2 The hypoxia-induced model of pulmonary arterial hypertension .................5
1.1.4.3 Transgenic animals ....................................................................................5
1.2 BMP signalling ....................................................................................................6
1.2.1 Bone morphogenetic proteins ........................................................................6
1.2.2 BMP receptors...............................................................................................7
1.2.3 The BMP signalling pathways ........................................................................9
1.2.3.1 Smad-dependent pathways........................................................................9
1.2.3.2 BMP-MAPK dependent pathway ..............................................................11
1.3 BMP signalling in lung development and homeostasis .................................12
1.4 BMPRII and pulmonary arterial hypertension.................................................14
1.4.1 Genomic structure and function of BMPRII ..................................................14 Table of contents II


1.4.2 BMPR2 mutations in pulmonary arterial hypertension patients.....................15
1.4.3 Functional consequences of BMPR2 mutations...........................................16
1.4.3.1 Loss of transcriptional activitiy..................................................................16
1.4.3.2 Decreased ligand binding ability...............................................................17
1.4.3.3 Failure of BMPRII trafficking to the plasma membrane.............................17
1.4.3.4 Activation of Smad-independent BMP signalling pathways.......................17
1.4.3.5 Increased of BMP signalling .....................................................................18
1.4.3.6 Down-regulation of BMPRII expression....................................................18
1.4.3.7 Failure of antiproliferative effects on vascular cells...................................18
1.5 Experimental design and aim of the project ...................................................19
2 MATERIALS AND METHODS ...............................................................................20
2.1 Materials ............................................................................................................20
2.1.1 Equipment....................................................................................................20
2.1.2 Reagents .....................................................................................................21
2.1.3 Cell Lines.....................................................................................................23
2.1.3.1 Mammalian cell lines ................................................................................23
2.1.3.2 Yeast cells................................................................................................23
2.1.3.3 Prokaryotic cells .......................................................................................23
2.1.4 Animals........................................................................................................23
2.1.4.1 A monocrotaline rat model of pulmonary arterial hypertension .................23
2.1.4.2 Hypoxia mouse model of pulmonary arterial hypertension........................24
2.2 Methods.............................................................................................................24
2.2.1 RNA isolation ...............................................................................................24
2.2.2 Reverse Transcription..................................................................................24
2.2.2.1 RT - Mix....................................................................................................25
2.2.3 The Polymerase Chain Reaction (PCR).......................................................25
2.2.3.1 PCR - Mix.................................................................................................25
2.2.3.2 PCR program ...........................................................................................26
2.2.4 Site-directed mutagenesis............................................................................26
TM2.2.4.1 The QuikChange PCR-Mix....................................................................27
2.2.5 Gel electrophoresis......................................................................................27
2.2.5.1 Agarose gel electrophoresis .....................................................................28
2.2.5.2 SDS polyacrylamide gel electrophoresis (SDS-PAGE).............................28 Table of contents III


2.2.6 Recombinant DNA technology .....................................................................29
2.2.6.1 PCR product purification...........................................................................30
2.2.6.2 Ligation of PCR products into the pGEM-T Easy Vector...........................30
2.2.6.3 Ligation-Mix..............................................................................................30
2.2.7 Subcloning into expression vectors..............................................................30
2.2.7.1 DNA digestion using restriction endonucleases........................................31
2.2.8 Immunological methods ...............................................................................31
2.2.8.1 Immunoblot (Western blot) .......................................................................31
2.2.8.1.1 Protein extraction from mammalian cells ............................................31
2.2.8.1.2 Protein extraction from yeast cells ......................................................32
2.2.8.1.3 Protein bloting.....................................................................................32
2.2.8.1.4 Protein detection.................................................................................32
2.2.8.2 Co-immunoprecipitation............................................................................33
2.2.8.3 Glutathione S-transferase (GST) pull-down..............................................34
2.2.8.4 Immunohistochemistry..............................................................................35
2.2.8.5 Immunocytochemistry...............................................................................35
2.3 Methodology of the yeast two-hybrid system.................................................35
2.3.1.1 Bait plasmids constructions ......................................................................38
2.3.1.2 Verification of bait protein expression.......................................................39
2.3.1.2.1 Transformation of bait constructs into S. cerevisiae AH109 yeast strain
39
2.3.1.2.2 Extraction of yeast total protein...........................................................39
2.3.1.2.3 Detection of bait protein expression....................................................40
2.3.1.3 Test of the GAL4-DNA-BD/ bait protein for transcriptional autoactivation .40
2.3.1.4 Gal4-DNA-AD fusion cDNA library............................................................40
2.3.2 Screening the pretransformed cDNA library.................................................41
2.3.2.1 Yeast mating ............................................................................................41
2.3.2.2 Identification of positives colonies ............................................................42
2.4 Luciferase assay...............................................................................................42
2.4.1 Microbiological methods...............................................................................42
2.4.1.1 Cultivation of E. coli..................................................................................42
2.4.1.2 Preparation of competent E. coli cells for transformation..........................43
2.4.1.3 Transformation of plasmid DNA into competent E. coli cells.....................44
2.4.1.4 Plasmid minipreparation...........................................................................44 Table of contents IV


2.4.1.5 Plasmid midipreparation...........................................................................45
2.4.2 Cultivation of yeast.......................................................................................45
2.4.2.1 Preparation of competent S. cerevisiae cells for transformation ...............46
2.4.2.2 Transformation of bait constructs into AH109 yeast strain ........................46
2.4.3 Culture of mammalian cells and transfection technique................................46
2.4.3.1 Cell culture conditions ..............................................................................46
TM
2.4.3.2 Transient transfection using Lipofectamine 2000 reagent......................47
2.4.3.3 Transient transfection of SMC using Nucleofector technology..................47
2.4.3.4 Transfection with small interfering RNA (siRNA).......................................48
2.4.3.5 Proliferation assay....................................................................................48
3 RESULTS ..............................................................................................................49
3.1 Identification of new proteins interacting with the intracellular region of
BMPRII..........................................................................................................................49
3.1.1 Construction and expression of the BMPRII baits ........................................49
3.1.2 Test for autonomous reporter gene activation..............................................51
3.1.3 A yeast two-hybrid screen using kinase and total baits ................................52
3.1.3.1 Identification of RACK1 as novel interacting partner of BMPRII................54
3.1.4 Confirmation of the BMPRII interaction with full-length RACK1 using the
yeast two-hybrid system.............................................................................................56
3.1.5 Confirmation of the interaction between the BMPRII kinase domain and the
full-length RACK1 by GST pull-down .........................................................................57
3.1.5.1 Overexpression of GST-tagged kinase domain of BMPRII. ......................57
3.1.5.2 GST pull-down assay ...............................................................................59
3.1.6 Co-immunoprecipitation of BMPRII with RACK1 in a BMP2 ligand-
independent manner ..................................................................................................59
3.2 Mapping the region of BMPRII required for RACK1 binding..........................61
3.2.1 Construction and overexpression of truncated variants of the GST-tagged
kinase domain of BMPRII...........................................................................................61
3.2.2 Effect of BMPR2 mutations on the interaction with RACK1..........................62
3.3 RACK1 expression in different mouse tissues...............................................63
3.4 Expression of RACK1 in a rat model of monocrotaline-induced pulmonary
hypertension................................................................................................................63 Table of contents V


3.5 Expression of RACK1 in human lungs............................................................66

3.6 Immunolocalisation of BMPRII and RACK1 in the human lung sections......67
3.7 Co-localisation of endogenous BMPRII and RACK1 in human paSMC.........68
3.8 Effects of RACK1 on paSMC proliferation ......................................................69
3.9 Functional effect of RACK1 on BMP signalling ..............................................73
4 DISCUSSION.........................................................................................................75
4.1 The yeast two-hybrid system as a powerful but limited tool in our study ....75
4.2 Possible candidates from yeast two-hybrid screen .......................................76
4.2.1 PIASy...........................................................................................................77
4.2.2 RACK1 as BMPRII-interacting protein..........................................................78
4.3 Implications of RACK1-BMPRII interactions in human pathophysiology .....80
4.3.1 RACK1 function: possible implications for development of pulmonary arterial
hypertension ..............................................................................................................80
4.3.1.1 Regulation of G /S cell progression and cellular growth ...........................82 1
4.3.1.2 Regulation of integrin-mediated adhesion and chemotactic cell migration 82
4.3.1.3 Regulation of protein kinase C and integrin-dependent cell migration.......82
4.3.1.4 Regulation of cell proliferation ..................................................................83
4.3.2 The paSMC and RACK1..............................................................................83
4.4 Possible models for the involvement of RACK1 in the development of
pulmonary arterial hypertension ................................................................................84
4.4.1 BMP signalling and pulmonary arterial hypertension....................................85
4.4.2 BMP signalling and vascular remodelling.....................................................86
4.5 Conclusions and future perspectives .............................................................87
5 APPENDIX.............................................................................................................89
6 REFERENCES.......................................................................................................94
7 DECLARATION ...................................................................................................102