Untersuchung der Regulationsproteine Geminin und Cdt1 in menschlichen Zellen

Kulartz, Monika

Untersuchung der Regulationsproteine Geminin und Cdt1 in menschlichen Zellen

Dateien

Dissertation_Kulartz_2004.pdfGröße: 2.03 MBDownloads: 1928

Datum

2004

Autor:innen

Kulartz, Monika

URI (zitierfähiger Link)

http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-13798

Link zur Lizenz

Urheberrechtlich geschützt

Open Access-Veröffentlichung

Open Access Green

Sammlungen

Biologie

Titel in einer weiteren Sprache

Investigation on the regulator proteins Geminin and Cdt1 in human cells

Publikationstyp

Dissertation

Publikationsstatus

Published

Zusammenfassung

One major function of Geminin is to inhibit replication-licensing by sequestration of the initiator protein Cdt1. Geminin is a cell-cycle regulated protein that is expressed during S and G2 phase, rapidly degraded at the end of mitosis and absent in G1 phase.
This work has shown that Geminin-mRNA is synthesized during the whole cell cycle in HeLa-cells with a slight increase at the beginning of S phase. Metabolic labelling experiments with synchronized HeLa-cells have shown that the protein Geminin is permanently synthesized during S phase. Furthermore, the fraction of Geminin that is synthesized in the first hours of S phase becomes then slowly degraded with a half-life of 3 to 4 hours.
A knockdown of Geminin with specific siRNA-sequences does neither affect DNA replication, cell-cycle progression nor expression and chromatin binding of the initiator-protein Cdt1.
Cell-cycle studies have further shown that Geminin and Cdt1 are only co-expressed in a short period of the cell cycle, bind together to chromatin at the G1-to-S transition and interact with each other during this period. Chromatin-immunoprecipitations have shown that this interaction takes place on chromatin and analysis of the MCM4-promtor region refers to the existence of Geminin and Cdt1 near the binding site of the origin recognition complex ORC. Geminin stays bound to chromatin during S phase, when Cdt1 is released and degraded. A stabilization of Cdt1 with the proteasome-inhibitor MG132 in S phase has led to an increase in the amount of chromatin-bound Geminin and an interaction of both proteins also during S phase.
In vitro and in vivo studies gave evidence that Geminin is phosphorylated in a cell-cycle dependent manner during S phase. Specific inhibitors have shown that protein-kinase CK2 is the major responsible kinase for the phosphorylation of Geminin, but glycogen-synthase kinase does also contribute. The phosphorylation with CK2 takes place in the C-terminal part of Geminin whereas GSK3 most likely phosphorylates N-terminal, probably at serine-45. Treating HeLa-cells with kinase-specific inhibitors does not influence the behaviour of Geminin, namely its interaction with Cdt1 or its subcellular localisation.

Zusammenfassung in einer weiteren Sprache

Eine bekannte Funktion von Geminin ist die Hemmung des Initiationsproteins Cdt1, die zur Inhibition der DNA-Replikation führen kann. Geminin wird während der S- und G2-Phase exprimiert und unterliegt am Ende der Mitose einem schnellen Abbau.
In dieser Arbeit konnte gezeigt werden, dass die Geminin-mRNA während des gesamten Zellzyklus gebildet wird, die Menge zu Beginn der S-Phase jedoch leicht ansteigt. Geminin wird während der S-Phase permanent neu synthetisiert, die Gesamtmenge in der Zelle nimmt jedoch nicht zu, da das neu gebildete Geminin mit einer Halbwertszeit von 3-4 h abgebaut wird.
Ein Knockdown von Geminin mittels siRNA hatte in HeLa-Zellen weder Auswirkungen auf die DNA-Replikation und das Fortschreiten im Zellzyklus, noch auf die Expression und Chromatinbindung des Initiationsproteins Cdt1.
Zellzyklusstudien haben weiter ergeben, dass Geminin und Cdt1 nur in einer kleinen Periode am G1-S-Übergang in HeLa-Zellen koexprimiert werden und zu diesem Zeitpunkt miteinander interagieren. ChIP-Assays haben erwiesen, dass diese Interaktion am Chromatin stattfindet und dass Geminin und Cdt1 in der Nähe einer bekannten ORC-Bindestelle am Chromatin vorliegen.
Auch während der S-Phase, wenn kein Cdt1 in der Zelle nachweisbar ist, befindet sich Geminin am Chromatin. Durch Stabilisierung von Cdt1 in der S-Phase mit dem Proteasomen-Inhibitor MG132 konnte die Menge an chromatingebundenem Geminin gesteigert werden und außerdem ein Komplex aus beiden Proteinen in den Extrakten MG132-behandelter Zellen nachgewiesen werden.
In vivo- und in vitro-Studien haben gezeigt, dass Geminin einer S-Phase-abhängigen Phosphorylierung unterliegt. Der Einsatz von spezifischen Kinase-Inhibitoren konnte die Proteinkinase CK2 als verantwortliche Kinase identifizieren und einen, etwas geringeren, Beitrag der Glycogen-Synthase-Kinase 3 (GSK3) zur Geminin-Phosphorylierung nachweisen. Beide Kinasen phosphorylieren in unterschiedlichen Bereichen von Geminin: die CK2- abhängige Phosphorylierung erfolgt im C-terminalen Bereich der Aminosäuren 176-210, wohingegen die GSK3 weiter N-terminal phosphoryliert, vermutlich am Serin 45. Es konnten keine Auswirkungen der Phosphorylierung auf das Verhalten von Geminin in der Zelle beobachtet werden.

Fachgebiet (DDC)

570 Biowissenschaften, Biologie

Schlagwörter

Geminin, Cdt1, replication, protein kinase CK2

Zitieren

ISO 690

KULARTZ, Monika, 2004. Untersuchung der Regulationsproteine Geminin und Cdt1 in menschlichen Zellen [Dissertation]. Konstanz: University of Konstanz

BibTex

@phdthesis{Kulartz2004Unter-7172,
  year={2004},
  title={Untersuchung der Regulationsproteine Geminin und Cdt1 in menschlichen Zellen},
  author={Kulartz, Monika},
  address={Konstanz},
  school={Universität Konstanz}
}

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Prüfungsdatum der Dissertation

October 22, 2004