Abstract
BackgroundCdc5 (polo kinase/Plk1) is a highly conserved key regulator of the S. cerevisiae cell cycle from S-phase until cytokinesis. However, much of the regulatory mechanisms that govern Cdc5 remain to be determined. Cdc5 is phosphorylated on up to 10 sites during mitosis. In this study, we investigated the function of phosphorylation site T23, the only full consensus Cdk1 (Cdc28) phosphorylation site present.FindingsCdc5T23A introduces a degron that reduces its cellular amount to undetectable levels, which are nevertheless sufficient for normal cell proliferation. The degron acts in cis and is reversed by N-terminal GFP-tagging. Cdk1 kinase activity is required to maintain Cdc5 levels during G2. This, Cdk1 inhibited, Cdc5 degradation is APC/CCdh1 independent and requires new protein synthesis. Cdc5T23E is hyperactive, and reduces the levels of Cdc5 (in trans) and drastically reduces Clb2 levels.ConclusionsPhosphorylation of Cdc5 by Cdk1 is required to maintain Cdc5 levels during G2. However, phosphorylation of T23 (probably by Cdk1) caps Cdc5 and other CLB2 cluster protein accumulation, preventing potential protein toxicity, which may arise from their overexpression or from APC/CCdh1 inactivation.
Highlights
Cdc5 is a highly conserved key regulator of the S. cerevisiae cell cycle from S-phase until cytokinesis
Cdc5/polo kinase is a crucial player in cell-cycle regulation from yeast to man, and the processes and substrates it regulates have been extensively investigated
Phosphorylation of T242 by Cdk1 has been reported to be essential for viability and mitotic activity [16], though this is disputed [1]
Summary
Cdc5 (polo kinase/Plk1) is a highly conserved key regulator of the S. cerevisiae cell cycle from S-phase until cytokinesis. 0 60 80 100 120 140 0 60 80 100 120 140 minutes after release from S-phase arrest (2hours 0.2M HU) into nocadazole (5μg/ml) plasmid cdc5Δ cells eGFP-cdc5WT B: Cdc5Δ cells [3] expressing Cdc5 from the indicated plasmids [16] were arrested in S-phase (0.2 M hydroxyurea), metaphase (5 μg/ml nocodazole) or early G1 (1 μg/ml rapamycin) for 2.5h.
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