Cell Cycle Control of Cdc7p Kinase Activity through Regulation of Dbf4p Stability

In Saccharomyces cerevisiae, the heteromeric kinase complex Cdc7p-Dbf4p plays a pivotal role at replication origins in triggering the initiation of DNA replication during the S phase. We have assayed the kinase activity of endogenous levels of Cdc7p kinase by using a likely physiological target, Mcm2p, as a substrate. Using this assay, we have confirmed that Cdc7p kinase activity fluctuates during the cell cycle; it is low in the G(1) phase, rises as cells enter the S phase, and remains high until cells complete mitosis. These changes in kinase activity cannot be accounted for by changes in the levels of the catalytic subunit Cdc7p, as these levels are constant during the cell cycle. However, the fluctuations in kinase activity do correlate with levels of the regulatory subunit Dbf4p. The regulation of Dbf4p levels can be attributed in part to increased degradation of the protein in G(1) cells. This G(1)-phase instability is cdc16 dependent, suggesting a role of the anaphase-promoting complex in the turnover of Dbf4p. Overexpression of Dbf4p in the G(1) phase can partially overcome this elevated turnover and lead to an increase in Cdc7p kinase activity. Thus, the regulation of Dbf4p levels through the control of Dbf4p degradation has an important role in the regulation of Cdc7p kinase activity during the cell cycle.

在酿酒酵母（Saccharomyces cerevisiae）中，异源寡聚激酶复合物（heteromeric kinase complex）Cdc7p-Dbf4p在触发S期（S phase）DNA复制起始的DNA复制起点（replication origins）处发挥关键作用。我们以Mcm2p作为潜在生理底物（likely physiological target），检测了内源性水平的Cdc7p激酶的激酶活性。利用该检测体系，我们证实Cdc7p的激酶活性在细胞周期（cell cycle）中存在波动：G1期（G1 phase）时活性较低，随细胞进入S期活性升高，并在细胞完成有丝分裂（mitosis）前维持在较高水平。该激酶活性的变化无法通过催化亚基（catalytic subunit）Cdc7p的水平变化来解释，因为该亚基的水平在细胞周期中始终保持恒定。然而，激酶活性的波动却与调节亚基（regulatory subunit）Dbf4p的水平密切相关。Dbf4p水平的调控一定程度上可归因于G1期细胞中该蛋白的降解（degradation）增强。这种G1期的蛋白不稳定性依赖于cdc16，这提示后期促进复合物（anaphase-promoting complex）参与了Dbf4p的周转过程。在G1期过表达（overexpression）Dbf4p可部分抵消这种增强的降解作用，并使Cdc7p的激酶活性升高。因此，通过调控Dbf4p的降解来调节其水平，在细胞周期中Cdc7p激酶活性的调控中发挥重要作用。