Untersuchung der Regulationsproteine Geminin und Cdt1 in menschlichen Zellen

Abstract

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.