Untersuchung der Regulationsproteine Geminin und Cdt1 in menschlichen Zellen

Kulartz, Monika

Startseite
→
Biologie
→
Biologie
→
Dokumentanzeige

Untersuchung der Regulationsproteine Geminin und Cdt1 in menschlichen Zellen

Publikationstyp:	Dissertation
URI (zitierfähiger Link):	http://nbn-resolving.de/urn:nbn:de:bsz:352-opus-13798
Autor/innen:	Kulartz, Monika
Erscheinungsjahr:	2004
Titel in einer weiteren Sprache:	Investigation on the regulator proteins Geminin and Cdt1 in human cells
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.
Prüfungsdatum (bei Dissertationen):	22. Okt 2004
Hochschulschriftenvermerk:	Konstanz, Univ., Diss., 2004
Fachgebiet (DDC):	570 Biowissenschaften, Biologie
Normierte Schlagwörter (GND):	Zellzyklus, Replikation, Phosphorylierung, Proteinkinase CK2
Schlagwörter:	Geminin, Cdt1, replication, protein kinase CK2
Link zur Lizenz:	Nutzungsbedingungen

Zitieren

Dateien zu dieser Ressource

Name: Dissertation_Kulartz_2004.pdf

Größe: 2.029Mb

Format: PDF

Öffnen

Prüfsumme: MD5:d43b9c7566e6a6a68dc631a3f3836101

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

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

Kulartz, Monika 2011-03-24T17:32:24Z 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.<br />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.<br />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.<br />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.<br />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. application/pdf deu Untersuchung der Regulationsproteine Geminin und Cdt1 in menschlichen Zellen Investigation on the regulator proteins Geminin and Cdt1 in human cells terms-of-use Kulartz, Monika 2004 2011-03-24T17:32:24Z