DARWIN Digitale Dissertationen German Version Strich

FU Berlin
Digitale Dissertation

Stefan Wagner :
Investigations on protein kinase C´s subcellular distribution
Untersuchungen zur subzellulären Verteilung von Proteinkinase C

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Abstract

Protein kinase C (PKC) is a family of serin/threonine kinases with important roles in many signal transduction processes. One important factor for the determination of the specific functions of protein kinase C´s isoforms is their specific subcellullar localisation, which changes following activation in the course of signal transduction events. In order to investigate PKCa´s subcellular distribution and especially its accumulation in the cell nucleus I used fusion proteins constisting of PKC and the green fluorescent protein (GFP).
In a first approach I expressed PKCa and fusion proteins constisting of glutathione-S-transferase and PKCa (GST-His6-PKCa) and GFP-PKCa in Sf9-insect cells. Using an activity assay, I could show that the proteins were catalytically active. Obviously, the N-terminal fusions GST-His6 and GFP do not influence the PKC-activity, since there is no signifikant difference between the activities of the fusion proteins and PKCa.
The purified GFP-PKCa was used in an assay with digitonin-permeabilised NIH 3T3 fibroblasts. The fusion protein undergoes nuclear accumulation without any further stimuli in a manner which is not simply due to diffusion. The strong inhibition of GFP-PKCa´s nuclear import following preincubation with reticulozyte lysate suggests the existence of a binding factor interfering with nuclear accumulation. Interestingly, permeabilisation appears to be a trigger for PKCa´s nuclear translocation, since the fusion protein accumulates in the nucleus in transiently transfected cells following permeabilisation, too.
Unexpectedly, transient overexpression of GFP-PKCa, PKCa-GFP and PKCa in fibroblasts favours translocation to the plasma membrane, whereas in nontransfected cells endogenous PKCa is translocated to the nucleus following activation with phorbol ester. This increased translocation to the plasma membrane seems to be favoured rather by overexpression than by GFP-fusion.
Use of GFP-PKCa deletion mutants suggests that PKCa´s first 187 amino acids play an important role in the protein´s nuclear import. In contrast to this, the catalytic domain is apparantly not involved in nuclear translocation. Conceivably, sequences between 252 and 289 and the V5-region inhibit PKCa´s nuclear accumulation.

Table of Contents

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Inhaltsverzeichnis
1.Einleitung 1
1.1. Kernhülle und Kernporenkomplexe 1
1.2. Transport vom Cytoplasma in den Kern 2
1.3. Proteinkinase C 4
1.4. Das grün fluoreszierende Protein (green fluorescent protein, GFP) 11
1.5. Zielsetzung der Arbeit 15
2. Ergebnisse 16
2.1. Expression verschiedener PKCa´s in Insektenzellen 16
2.2. In vitro Transporttest mit GFP-PKCa 19
2.3. Digitonin-Permeabilisierung transfizierter NIH 3T3-Fibroblasten 23
2.4. Subzelluläre Veteilung in transfizierten NIH 3T3-Fibroblasten 24
2.5. Subzelluläre Veteilung von GFP-PKCa in transfizierten Cos-7-, CHO- und Neuro 2a-Zellen 38
3. Diskussion 40
3.1. Expression in Sf9-Insektenzellen 40
3.2. Digitonin-Permebilisierung transfizierter NIH 3T3-Fibroblasten 41
3.3. Subzelluläre Verteilung von GFP-PKCa-Fusionsproteinen 42
3.4. Subzelluläre Verteilung von GFP-PKCa-Punktmutanten 43
3.5. Untersuchungen zu Kernlokalisationssequenzen von PKCa 44
3.6. Modell für die Kerntranslokation von Proteinkinase Ca 46
3.7. Zusammenfassung und Ausblick 48
3.8. Summary 50
4. Material & Methoden 51
4.1. Zellkultur 51
4.2. Proteinexpression in E. coli 54
4.3. Proteinreinigung 55
4.4. PKC-Aktivitätstest 57
4.5. In vitro Transporttest 58
4.6. Protein-Bestimmung 60
4.7. Gelelektrophorese von Proteinen 60
4.8. Western Blot 61
4.9. Fluoreszenzmikroskopie 62
4.10. Molekularbiologische Methoden 63
5. Literatur 72
6. Anhang 82
6.1. Lebenslauf 82
6.2. Veröffentlichungen 83
6.3. Danksagungen 84
6.4. Abkürzungen 85

More Information:

Online available: http://www.diss.fu-berlin.de/1999/58/indexe.html
Language of PhDThesis: german
Keywords: protein kinase C, nucleus, plasma membrane, green fluorescent protein
DNB-Sachgruppe: 30 Chemie
Date of disputation: 07-Oct-1999
PhDThesis from: Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee: Prof. Dr. F. Hucho
Second Referee: Prof. Dr. G. Schultz
Contact (Author): srw@chemie.fu-berlin.de
Contact (Advisor): klabuch@chemie.fu-berlin.de
Date created:13-Oct-1999
Date available:24-Aug-2000

 

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