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«Von der Fakultät für Energie-, Verfahrens- und Biotechnik der Universität Stuttgart zur Erlangung der Würde eines Doktors der Naturwissenschaften ...»

-- [ Page 1 ] --

Entwicklung von molekularen Werkzeugen zur

Untersuchung einer Funktion der Proteinkinase D in der

Organisation des Golgi Komplexes

Von der Fakultät für Energie-, Verfahrens- und Biotechnik der

Universität Stuttgart zur Erlangung der Würde eines

Doktors der Naturwissenschaften (Dr. rer. nat.)

genehmigte Abhandlung

Vorgelegt von

Stephan Alexander Eisler

aus Karlsruhe

Hauptberichter: Prof. Dr. Klaus Pfizenmaier

Mitberichterin: Prof. Dr. Monilola Olayioye

Tag der mündlichen Prüfung: 01.08.2012 Institut für Zellbiologie und Immunologie Universität Stuttgart Inhaltsverzeichnis Inhaltsverzeichnis Inhaltsverzeichnis

Abkürzungsverzeichnis

Zusammenfassung

Summary

1 Einleitung

1.1 Proteinkinase D

1.1.1 Identifizierung, Klassifizierung und Strukturdomänen

1.1.2 Aktivierung von PKD

1.1.3 PKD-Funktionen und subzelluläre Lokalisierung

1.1.4 Rolle von PKD am Golgi Komplex

1.2 Genetisch kodierte Fluoreszenz-basierte Kinaseaktivitätsreporter

1.3 Stabiles Isotopen Labeling mit Aminosäuren in der Zellkultur (SILAC)............... 23

1.4 Zielsetzung

2 Ergebnisse und Diskussion

2.1 Generierung von Golgi-PKD-Aktivitätsreportern

2.2 Verbindung zwischen Mikrotubuli, PKD-Aktivität und Golgi Integrität............... 29

2.3 PKD kontrolliert die Fragmentierung des Golgi Komplexes beim Eintritt der Zelle in die Mitose

2.4 Globale Analyse PKD-abhängiger Phosphorylierungsereignisse in Nocodazolbehandelten Zellen

2.5 Schlussfolgerung

3 Publikationsmanuskripte

3.1 A Golgi PKD activity reporter reveals a crucial role of PKD in nocodazole-induced Golgi dispersal

3.1.1

Abstract

3.1.2 Introduction

3.1.3 Results and discussion

3.1.4 Materials and methods

3.1.5 Acknowledgements

3.1.6 References

3.1.7 Supplement

3.2 G-PKDrep-live, a genetically encoded FRET reporter to measure PKD activity at the trans-Golgi network

3.2.1 Abstract

3.2.2 Introduction

3.2.3 Material and Methods

3.2.4 Results and Discussion

3.2.5 Concluding Remarks

3.2.6 Acknowledgements

Inhaltsverzeichnis 3.2.7 References

3.3 Global detection of Protein Kinase D-dependent phosphorylation events in nocodazole-treated human cells

3.3.1 Abstract

3.3.2 Introduction

3.3.3 Experimental Procedures

3.3.4 Results

3.3.5 Discussion

3.3.6 Acknowledgements

3.3.7 References

3.3.8 Supplement

3.4 PKD controls interstack Golgi connections through a Raf-MEK1 pathway.........101 3.4.1 Abstract

3.4.2 Introduction

3.4.3 Material and Methods

3.4.4 Results

3.4.5 Discussion

3.4.6 Acknowledgments

3.4.7 References

3.4.8 Supplement

4 Gesamtliteraturverzeichnis

Danksagung

Abkürzungsverzeichnis Abkürzungsverzeichnis

–  –  –

PH-Domäne Pleckstrin-Homologie-Domäne PI4KIIIβ Phosphatidylinositol-4 Kinase IIIβ PI4P Phosphatidylinositol (4)-phosphat PIP2 Phosphatidylinositol (4,5)-bisphosphat PKA Proteinkinase A PKC Proteinkinase C PKD Proteinkinase D PKD1ca Constitutive active PKD1 PKD1kd Kinase dead PKD1 PLC Phospholipase C PLD Phospholipase D Plk1 Polo-like kinase 1 PMT Photo multiplier tube Rho Ras homologue RIN1 Rab interactor 1 SCX Strong cation exchange SILAC Stable isotope labeling with amino acids in cell culture siRNA Small interfering RNA SNX Sorting nexin SSH1L Slingshot-1-Like TGN Trans-Golgi Netzwerk TNF Tumor Nekrose Faktor VAP-B Vesicle-associated membrane protein-associated protein B VEGF Vascular endothelial growth factor YFP Yellow fluorescent protein Zusammenfassung Zusammenfassung Die Proteinkinase D (PKD) Familie der Serin/Threonin Kinasen umfasst in Säugerzellen drei Mitglieder: PKD1, PKD2 und PKD3. PKDs werden downstream von G-Protein gekoppelten Rezeptoren (GPCR) als Effektoren von Diacylglycerol (DAG) aktiviert. Sie sind an der Regulation zahlreicher zellulärer Prozesse, wie z.B. Zellmotilität, Überleben der Zelle bei oxidativem Stress und Aktivität von Transkriptionsfaktoren beteiligt. Am besten beschrieben ist die Funktion am Golgi Komplex. Hier kontrolliert PKD am Trans-Golgi Netzwerk (TGN) die Abschnürung von Transportvesikeln. Der Aktivierungsstatus von PKD ist stets streng reguliert und die jeweilige Funktion eng mit der subzellulären Lokalisierung verknüpft.

Genetisch codierte, Fluoreszenz-basierte Kinaseaktivitätsreporter sind wichtige molekulare Werkzeuge, um die räumliche und zeitliche Veränderung von Kinaseaktivität auf subzellulärer Ebene zu beobachten und somit die Antwort der Kinase auf Veränderungen der physiologischen Umgebung zu erfassen. Im Rahmen dieser Arbeit wurden zwei dieser Reporter, die spezifisch für die Messung von PKD-Aktivität am Golgi Komplex eingesetzt werden können, entwickelt. Bei beiden Reportern wird die PKD-Aktivität über den Phosphorylierungsstatus der PKD-Substratsequenz von Phosphatidylinositol-4 Kinase IIIβ (PI4KIIIβ) visualisiert. Der erste Reporter, G-PKDrep wurde für Aktivitätsmessungen in fixierten Zellen entwickelt, wobei der Phosphorylierungsgrad der Substratsequenz mit einem phosphospezifischen Antikörper quantifiziert wird. Mit G-PKDrep-live wurde G-PKDrep zu einem FRET-basierten Reporter weiterentwickelt, mit dem PKD-Aktivität am Golgi Komplex in lebenden Zellen über die Zeit gemessen werden kann.





Mit Hilfe von G-PKDrep konnte unter Verwendung der Mikrotubuli-zerstörenden Substanz Nocodazol eine Funktion von PKD in der Organisation des Golgi Komplex aufgezeigt werden. Hierbei wurde gezeigt, dass die durch Mikrotubuli-Verlust hervorgerufene GolgiFragmentierung abhängig von PKD-Aktivität ist. Zur Aufklärung der zugrunde liegenden Signalwege dieses Prozesses wurde eine globale SILAC-basierte Phosphoproteom-Studie durchgeführt. Durch Expression einer kinasetoten PKD wurden dabei 124 PKD-abhängige Phosphorylierungsstellen identifiziert, die unter Nocodazolbedingungen signifikant herabreguliert waren. Dabei wurden viele PKD-Zielsequenzen in Golgi-residenten Proteinen, z.B. Mitglieder des IGF2-Rezeptor Netzwerks, identifiziert. Zudem zeigte sich, dass PKD nach Nocodazolstimulation den MAP-Kinase Signalweg aktiviert, dessen downstream Effektoren zu Beginn der Mitose die Golgi-Fragmentierung initiieren.

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4 Gesamtliteraturverzeichnis

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