DNA replication, regulation of the cell cycle, and cancer
Initial Award Abstract
The final decision that makes a cell to commit into another cell cycle and continue to proliferate or to stop proliferation is the result of a balance between different signal inputs. Therefore, to understand the mechanisms involved in the regulation of cell proliferation and its loss in cancer, it is crucial to understand how the eukaryotic cell cycle is regulated. Cdc6p is a very important protein in the cell cycle because it regulates the initiation of DNA replication and also the passage through mitosis. The crucial role of Cdc6 in the cell cycle is mirrored by its potential in cancer diagnosis. In fact, it has been recently shown that Cdc6, as well as other proteins working in S phase like the Mcms, are absent in quiescent and differentiated cells.These results offer potential for improved early diagnosis and differential diagnosis of cancer by using these proteins as markers for pre-malignant (dysplastic) and malignant cells in clinical samples.
Our long term goal is to understand why eukaryotic cells, from human to yeasts, need to get rid of Cdc6p at the onset of S phase or whenever cells are undergoing differentiation, and how this is achieved at the molecular level. We believe that the data obtained with this proposal will increase the knowledge of crucial parameters in the cell cycle regulation and cell proliferation and therefore its use for diagnostic and therapeutical purposes.
In S. cerevisiae, the initiation of DNA replication and the prevention of aberrant reinitiation are mediated by the S phase cyclins and Cdc28 kinase. Cdc6p is a very conserved protein from yeast to humans that participates in the formation of the pre-replicative complexes (preRC) at the end of mitosis and G1. At the transition of G1 to S, and until late mitosis, it becomes highly unstable and it is degraded via the SCF pathway. Cdc6p is a good candidate to be a substrate of Cdc28 kinase because Cdc6p associates strongly and specifically with Clb5,6p/Cdc28p through its N-terminal 48 amino acids. Recent work in my laboratory has shown that at least one of the roles of Cdc6p phosphorylation is to target Cdc6p for degradation by the ubiquitin-dependent 26S proteasome. On the other hand, previous work in my laboratory suggest that Cdc6p, a well established replication protein, may also have a role in G2 and M phases because the ectopic expression of Cdc6p in other cell cycle phases other than G1 leads to a cell cycle delay in G2/M.This observation was also reported long ago by others, but has not been extensively analyzed.
The specific aims of this proposal are: i)analyze the mechanism by which Cdc6p is targeted for destruction and how this is influenced by the interaction and phosphorylation by Clbp/Cdc28p ; ii) analyze the molecular events involved in the formation of the pre-replicative and replicative complexes and how they are influenced by Cdc6 phosphorylation ; iii) determine if and how other degradation pathways in yeast, in particular the APC, are implicated in Cdc6 function. We will characterize the role of Cdc6p phosphorylation in regulated degradation, and extend the study to subcellular localization, binding to origins and interaction with Cdc28p. For this purpose we will take advantage of a novel approach by using proteasome inhibitors in vivo. This will ensure that the ubiquitinated Cdc6 protein is not degraded and can be monitored in vivo. |