Abstract
Previously, we demonstrated that expression of polo-like kinase (PLK) is required for cellular DNA synthesis and that overexpression of PLK is sufficient to induce DNA synthesis. We now report that the endogenous levels of PLK, its phosphorylation status, and protein kinase activity are tightly regulated during cell cycle progression. PLK protein is low in G1, accumulates during S and G2M, and is rapidly reduced after mitosis. During mitosis, PLK is phosphorylated on serine, and its serine threonine kinase function is activated at a time close to that of p34cdc2. The phosphorylated form of PLK migrates with reduced mobility on SDS-polyacrylamide gel electrophoresis, and dephosphorylation by purified protein phosphatase 2A converts it to the more rapidly migrating form and reduces the total amount of PLK kinase activity. Purified p34cdc2-cyclin B complex can phosphorylate PLK protein in vitro but causes little increase in PLK kinase activity.
Highlights
We demonstrated that expression of pololike kinase (PLK) is required for cellular DNA synthesis and that overexpression of PLK is sufficient to induce DNA synthesis
We report that the endogenous levels of PLK, its phosphorylation status, and protein kinase activity are tightly regulated during cell cycle progression
In our initial report on the cloning of human and murine PLK, we showed evidence that PLK has a function in S phase [34] similar to the finding for the yeast PLK homologue cdc5, which apparently has roles during S phase and during mitosis [29]
Summary
CdK, cyclin-dependent protein kinases; PLK, polo-like kinase; PP2A, protein phosphatase 2A; PAGE, polyacrylamide gel electrophoresis. A number of cell cycle-regulated kinases unrelated to the CdKs have been identified in lower eukaryotes, including the homologous yeast CDC5 [29] and Drosophila polo [30]. Saccharomyces cerevisiae CDC5 mutants likewise are impaired in mitotic spindle formation, but the CDC5 kinase appears to have a function during S phase [29]. We investigate the cell cycle regulation of PLK by measuring endogenous PLK protein levels, phosphorylation state, and kinase activity. During the G2 to M phase transition, PLK protein is phosphorylated on serine, and its kinase function is stimulated. We further demonstrate that Cdc2/cyclin B can phosphorylate PLK but has little effect on PLK kinase activity, suggesting that mitotic activation of PLK will not be explained solely by Cdc
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