FU Berlin Digitale Dissertation
Ingo Tobias Lehmann : Protein kinase Cα in the nucleus nuclear import, interacting proteins and substrates Proteinkinase Cα im Zellkern
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Abstract The family of protein kinase C (PKC) isozymes are key-players in many signaltransduction pathways. The classical isoform PKCα is able to translocate into the nucleus and probably to induce changes in gene expression. Therefore identification of its nuclear localization signal, interacting proteins and its substrates in the nucleus are of special interest. Based on earlier work in our laboratory which led to prediction of a nuclear localization signal NLS in the C1-region of PKCα (Wagner, 1999), fusion proteins of deletion mutants of PKCα and either GFP-β-Galactosidase or two GFP-molecules, respectively, were constructed and expressed in mammalian cell lines by transient expression to exactly define the NLS of PKC&alpha. The localisation pattern of the fusion proteins were analysed by confocal laser scan-ning microscopy. Surprisingly, the fusion proteins turned out to be cytotoxic, assembling in multiple intracellular aggregates with different shape and sizes. Based on these observations it was concluded that the approach of expressing fusion proteins cannot be used to find the exact NLS of PKCα. The second part of this work was aimed to identify interacting partners and substrates of nuclear PKCα. Nuclear extracts of mammalian cells were incubated with GST-PKCα, immuno-precipitated and subsequently analysed with western blot analysis, which reveals that PTB associated splicing factor (PSF) is a PKCα-interacting protein. This interaction depends on the activation status of PKCα. It was also shown that PSF is a weak substrate of PKCα. In search for additional substrates of PKCα nuclear membranes and their detergent insoluble fractions where phosphorylated in vitro. The proteins were separated via 2D BAC-SDS-PAGE and gel spots corresponding to the autoradiography were used to identify the proteins by MALDI-mass spectrometry. With this method, we identified substrates, which are already known in literature as well as 13 new substrates of PKC&alpha. Three of these proteins (heterochromatin 2, the Ott-protein and a putative protein) are known only from sequence data. The other newly identified substrates of PKCα, which include PSF, are known to be involved in many different processes including splicing, the export of mRNA, chromatin organization, regulation of gene-expression and biogenesis of ribosomes.

Table of Contents Download the whole PhDthesis as a zip-tar file or as zip-File For download in PDF format click the chapter title Titel und Inhaltsverzeichnis   1.   Einleitung 3 1.1.   Signaltransduktion zum Zellkern 3 1.2.  Der Zellkern 6 1.2.1. Die Kernmembran 6 1.2.2. Der Kernporenkomplex 9 1.2.3. Kernimport und Export 11 1.3. PKC im Zellkern 15 1.4. Substrate der PKC im Zellkern 17 1.5. Spleißen 19 1.6. Der PTB-assoziierte splicing Faktor (PSF) 20 1.7. Zielsetzung der Arbeit 22   2. Ergebnisse 24 2.1. Suche nach dem NLS der PKCα 24 2.1.1. Die pHMΔPKCα-Konstrukte 24 2.1.2. Die pHMΔPKCαDiGFP-Konstrukte 29 2.2. PSF als PKCα-bindendes Protein im Zellkern 35 2.2.1. Charakterisierung der verwendeten Antikörper 35 2.2.2. Untersuchung zum PTB-assoziierten Spleißfaktor (PSF) 36 2.3. Substrate der PKCα an der inneren Kernmembran 39 2.3.1. Phosphorylierung der Kernmembran 39 2.3.2. Phosphorylierung der Triton X-100-resistenten Fraktion der Kernmembran 42   3. Diskussion 44 3.1. Das Kernlokalisationssignal der PKCα 44 3.2. Physiologische Bedeutung der PSF-Bindung 49 3.3. Nucleäre Substrate der PKCα 52 3.4. Zusammenfassung 60 3.5. Ausblick 61 3.6. Summary 61   4. Material und Methoden 63 4.1. Analytik von Proteinen 63 4.1.1. Proteinbestimmung nach Bradford 63 4.1.2. Eindimensionale diskontinuierliche SDS-Polyacrylamid-Gelelektrophorese nach Laemmli 63 4.1.3. BAC-SDS-PAGE 64 4.1.4. Blotting von Proteinen nach dem „Semidry“-Verfahren 66 4.1.5. Ponceaufärbung von Proteinen auf der Nitrozellulosemembran 66 4.1.6. SYPRO-Färbung von Proteinen auf der Nitrozellulosemembran 66 4.1.7. Nachweis von Proteinen durch Immunoblotting (Western-Blotting) 67 4.1.8. „Strippen“ der Blotmembran 68 4.1.9. Immunpräzipitation 68 4.1.10. Bindungstest („Pull down assay“) 69 4.1.11. Phosphorylierung von Proteinen (in vitro) 69 4.1.12. In Gel-Verdau mit Trypsin 70 4.2. Molekularbiologische Methoden 72 4.2.1. Anzucht von Bakterien 72 4.2.2. Herstellung kompetenter E. coli 72 4.2.3. Transformation kompetenter E. coli 73 4.2.4. Mini/Midi-Präparation von Plasmid-DNA 73 4.2.5. Ethanolfällung von DNA 74 4.2.6. Konzentrationsbestimmung von DNA 74 4.2.7. DNA-Spaltung mit Restriktionsenzymen 75 4.2.8. DNA-Ligation 75 4.2.9. Sequenzierung von DNA: 75 4.2.10. Polymerase-Kettenreaktion (PCR) nach Arnheim und Ehrlich (1992) 76 4.2.11. Agarose-Gelelektrophorese 76 4.2.12. DNA-Elution aus Agarosegelen 77 4.2.13. Herstellung der Vektoren 77 4.3. Zellkultur 82 4.3.1. Sf9-Zellen 82 4.3.2. Transfektion von Sf9-Zellen 82 4.3.3. Bestimmung des Virustiters 83 4.3.4. Amplifikationsschritte und Expression des Fusionsproteins 84 4.3.5. Isolierung und Aufreinigung des GST-Fusionsproteins 85 4.3.6. Säugerzellen 86 4.3.7. Transfektion von Säugerzellen 86 4.3.8. Fluoreszenzmikroskopie 87 4.3.9. Subzelluläre Fraktionierung: Kernpräparation 87 4.3.10. Präparation von Kernmembranen und Nucleoplasma 88 4.3.11.  Tritonextraktion von Kernmembranen 89   5. Literatur 90   6. Anhang 101 6.1. Abkürzungen 101 6.2. Publikationen 102 6.3. Danksagungen 103

More Information:
Online available:	http://www.diss.fu-berlin.de/2002/259/indexe.html
Language of PhDThesis:	german
Keywords:	Protein kinase C; nucleus; signaltransduction; nuclear import; substrates, NLS
DNB-Sachgruppe:	30 Chemie
Date of disputation:	29-Oct-2002
PhDThesis from:	Fachbereich Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee:	Prof. Dr. Ferdinand Hucho
Second Referee:	PD Dr. Mathias Ziegler
Contact (Author):	lehmanni@chemie.fu-berlin.de
Contact (Advisor):	hucho@chemie.fu-berlin.de
Date created:	28-Nov-2002
Date available:	29-Nov-2002

 

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