DARWIN Digitale Dissertationen German Version Strich

FU Berlin
Digitale Dissertation

Olaf Schaefer :
Proteinkinase C in the signaltransduktion of promyeloide 32D cells
Proteinkinase C in der Signaltransduktion promyeloider 32D Zellen

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Abstract

Protein kinase C forms a group of Serine/Threonine kinases. The members of this family execute important tasks in the signal transduction in growth, differentiation, immune response and cancer. To properly asses their functions it is important to look at both, the PKC substrates and the impact of PKC activation on the intra cellular environment.
The first part of this work elucidated the role of the potential PKC substrates DAP1, Diff6, PDI and TCTP. In vivo all of them are phosphorylated after stimulation with the PKC activator phorbol ester.
While GST-PDI was available as a purified fusion protein, neither protein nor cDNA was available of the other proteins. So the cDNA of DAP1, Diff6 and TCTP was cloned out of mouse 32D cells and subcloned into expression plasmids containing the gene for GST. After expression and purification, all of the proteins were used in in vitro kinase assays. Here, all of them proved not to be substrates of PKCd, PKCe or PKCa. On the contrary these proteins are probably subtrates for other kinases downstream of PKC.
The second part dealt with the downstream effects of the PKC activation in promyelocytic mouse 32D cells. Here changes in the transkriptom of the cells were analyzed on a global basis.
First PKCd was shown to downregulate PKCe in the 32D PKCd cells. The mRNA for the translational regulated tumor protein TCTP on the other side was upregulated in both, 32D PKCd and 32D PKCe cells. In cDNA-Array studies other PKC regulated mRNAs were identified and proved by quantitative RT-PCR, consequently. The mRNA for the anti inflammatory protein Lipocortin 1 was downregulated by PKCd and PKCe. Of several members of the MAP kinase pathway, one, the mRNA of MAPKAPK2 was shown to be upregulated by the tested PKCs.
The depletion of Lipocortin 1 could lead to a continually activated MAPK/ERK signaling and increased inflammation. Therefore the PKC dependent upregulation of MAPKAPK2 and it?s simultaneous activation might develop a synergistic effect. This could be a step towards the coordinated induction of differentiation and immune response in the 32D cells.
 

Table of Contents

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Inhaltsverzeichnis 1
1 Einführung 5
1.1 Zellen reagieren spezifisch auf Signale aus ihrer Umgebung 5
1.2 Die Proteinkinase C Familie 6
1.3 Entwicklung und Differenzierung von 32D Zellen 15
1.4 Die Untersuchung differentieller Genexpression mit DNA Arrays 18
2 Zielsetzung der Arbeit 25
3 Material 24
4 Methoden 29
4.1 Kultivierung eukaryotischer Zellen 29
4.2 Proteinchemische Methoden 31
4.3 Molekularbiologische Methoden 37
4.4 cDNA-Array 49
4.5 TaqMan-Analyse 54
5 Ergebnisse 60
5.1 Überprüfung von DAP1, Diff6, PDI und TCTP auf ihre Phosphorylierbarkeit durch PKC 60
5.2 Proteinkinase C regulierte Genexpression 67
6 Diskussion 89
6.1 DAP1, Diff6, TCTP und PDI sind keine PKC-Substrate 89
6.2 PKCe in 32D Zellen 90
6.3 Erhöhte TCTP-Expression in 32D PKCd- und 32D PKCe-Zellen 91
6.4 PKC-vermittelte Expression von Adhäsionsmolekülen 93
6.5 PKC-vermittelte Reduktion der Lipocortin 1-Expression 93
6.6 PKC-vermittelte Steigerung der MAPKAPK2-Expression 95
6.7 Veränderungen im Transkriptom von 32D Zellen durch die Überexpression von PKC 98
6.8 Möglichkeiten der Expressionsanalyse mit cDNA-Arrays 99
7 Zusammenfassung 102
8 Abstract 103
9 Literaturverzeichnis 104
10 Abkürzungsverzeichnis 119
11 Abbildungsverzeichnis 123
12 Lebenslauf und Veröffentlichungen 125
13 Danksagungen 126

More Information:

Online available: http://www.diss.fu-berlin.de/2001/5/indexe.html
Language of PhDThesis: german
Keywords: PKC 32D signal transduction
DNB-Sachgruppe: 32 Biologie
Date of disputation: 15-Jan-2001
PhDThesis from: Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee: Prof. Dr. Brigitte Wittmann-Liebold
Second Referee: Prof. Dr. Ferdinand Hucho
Contact (Author): olaf_schaefer@gmx.net
Contact (Advisor): nephrologie@mh-hannover.de
Date created:16-Jan-2001
Date available:17-Jan-2001

 

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