Person:

Yim, Hyungshin

The accurate division of duplicated DNA is essential for maintenance of genomic stability in proliferating eukaryotic cells. Errors in DNA replication and chromosomal segregation may lead to cell death or genomic mutations that lead to oncogenic properties. Thus, tight regulation of DNA replication and mitosis is essential for maintaining genomic integrity. Cell division cycle 6 (Cdc6) is an essential factor for initiating DNA replication. Recent work shows that phosphorylation of Cdc6 by polo-like kinase 1 (Plk1), one of the essential mitotic kinases, regulates mitotic exit mediated by Cdk1 and separase. Here we discuss how pre-replicative complex factors are connected with Plk1 and affect mitotic exit.

Polo-like kinase 1 (Plk1) plays several roles in mitosis, and it has been suggested to have a role in tumorigenesis. We have previously reported that Plk1 depletion results in cell death in cancer cells, whereas normal cells survive similar depletion. However, Plk1 depletion together with p53 depletion induces cell death in normal cells as well. This communication presents evidence on the sequence of events that leads to cell death in cancer cells. DNA damage is detected at the first S phase following Plk1 depletion and is more severe in Plk1-depleted p53-null cancer cells. As a consequence of Plk1 depletion using lentivirus-based small interfering RNA techniques, prereplicative complex (pre-RC) formation is disrupted at the (G_1/S) transition, and DNA synthesis is reduced during S phase of the first cycle after depletion. The levels of geminin, an inhibitor of DNA pre-RC, and Emi1, an inhibitor of anaphase-promoting complex/cyclosome, are elevated in Plk1-depleted cells. The rate of cell cycling is slower in Plk1-depleted cells than in control cells when synchronized by serum starvation. Plk1 depletion results in disrupted DNA pre-RC formation, reduced DNA synthesis, and DNA damage before cells display severe mitotic catastrophe or apoptosis. Our data suggest that Plk1 is required for cell cycle progression not only in mitosis but also for DNA synthesis, maintenance of DNA integrity, and prevention of cell death.

Defining the links between cell division and DNA replication is essential for understanding normal cell cycle progression and tumorigenesis. In this report we explore the effect of phosphorylation of cell division cycle 6 (Cdc6), a DNA replication initiation factor, by polo-like kinase 1 (Plk1) on the regulation of chromosomal segregation. In mitosis, the phosphorylation of Cdc6 was highly increased, in correlation with the level of Plk1, and conversely, Cdc6 is hypophosphorylated in Plk1-depleted cells, although cyclin A- and cyclin B1-dependent kinases are active. Binding between Cdc6 and Plk1 occurs through the polo-box domain of Plk1, and Cdc6 is phosphorylated by Plk1 on T37. Immunohistochemistry studies reveal that Cdc6 and Plk1 colocalize to the central spindle in anaphase. Expression of T37V mutant of Cdc6 (Cdc6-TV) induces binucleated cells and incompletely separated nuclei. Wild-type Cdc6 but not Cdc6-TV binds cyclin-dependent kinase 1 (Cdk1). Expression of wild-type Plk1 but not kinase-defective mutant promotes the binding of Cdc6 to Cdk1. Cells expressing wild-type Cdc6 display lower Cdk1 activity and higher separase activity than cells expressing Cdc6-TV. These results suggest that Plk1-mediated phosphorylation of Cdc6 promotes the interaction of Cdc6 and Cdk1, leading to the attenuation of Cdk1 activity, release of separase, and subsequent anaphase progression.