DARWIN Digitale Dissertationen German Version Strich

FU Berlin
Digitale Dissertation

Boris Radau :
Proteinkinase C-dependent Regulation of Vesicleformation at the Trans-Golgi-Network
Die Regulation der Vesikelbildung am trans-Golgi-Netzwerk durch Proteinkinase C

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Abstract

PKC is known to stimulate the formation of constitutive transport vesicles at the trans Golgi network [165]. In an attempt to understand the mechanism behind that process, two possible experimental approaches were followed: In the first approach PKC-binding proteins at the Golgi apparatus (GA) were analyzed according to a PKC-overlay assay technique. b -Actin was identified as a major binding protein of activated PKCa by high resolution 2D-electrophoresis and microsequencing techniques. In the second experimental setup isolated Golgi cisternae or permeabilized HepG2-cells were used to phosphorylate and separate Golgi proteins by high resolution 2D-electrophoresis according to Hartinger et al. [259] and Görg et al. [303] in the presence of PKCa [260]. Proteins not phosphorylated in the presence of Calphostin C, Ro 31-8220 and/or Gö 6976, were sequenced by mass spectroscopy (MALDI-MS and Q-TOF). For the identified in vitro PKC-substrates MARCKS, MacMARCKS, Myosin RLC, Cytokeratin 8 and Cytokeratin 18, it was possible to demonstrate their phosphorylation in permeabilized HepG2 cells too. Therefore, a biological relevance of this phosphorylation can be assumed. Other known Golgi associated proteins like Rab-6, Rab-8 and VAMP-2 were also identified or were shown to be Golgi associated for the first time as in the case of Annexin-IV, Profilin-I, Rab-7, GRP-78 and Endobrevin. Additionally Annexin IV and Profilin I were tested for their effect on in-vitro-vesicle biogenesis: Even though an Annexin IV-specific antibody did not influence the budding efficacy of HSPG in the cell free system at all [371], the addition of a Profilin I-specific antibody did actually reduce the budding efficiency [368]. Supporting the above result, Profilin I was also identified in post-Golgi-vesicle fractions biochemically. Generally the identification of the mayor PKC substrates MARCKS, MacMARCKS and Myosin RLC is pointing towards a new signal transduction pathway, by which PKC might regulate the vesicle biogenesis at the TGN

Table of Contents

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Titelblatt und Inhalt

1 Einleitung 1

2 Aufgabenstellung 25

3 Material 26

4 Methoden 33

5 Ergebnisse und Diskussion 50

6 Zusammenfassung 98

7 Summary 99

8 Tabellen 100

9 Literatur 106

More Information:

Online available: http://www.diss.fu-berlin.de/2001/89/indexe.html
Language of PhDThesis: german
Keywords: PKC ; Phosphorylierung ; Golgi-Apparat ; TGN
DNB-Sachgruppe: 30 Chemie
Date of disputation: 29-May-2001
PhDThesis from: Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee: Priv. Doz. Dr. Peter Westermann
Second Referee: Priv. Doz. Dr. Klaus Buchner
Contact (Author): clauborabla@web.de
Contact (Advisor): pwesterm@mdc-berlin.de
Date created:28-Jun-2001
Date available:29-Jun-2001

 

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