Phosphorylation of Cdc5 regulates its accumulation

Cdc5T23A 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.Phosphorylation 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.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. Cdc5 regulates numerous cell cycle events, including promoting the destruction of the Cdk1-Clb2 inhibitor Swe1 [1], CLB2 cluster transcription (including itself) [2], spindle-pole body separation [3], spindle positioning [4], microtubule organization [5], recovery from hydroxyurea [6], APC/C activation [7], mitotic exit [8], cytokinesis [9,10], Cdc14 localization [11,12] and APC/CCdh1 inhibition [13]. However, the regulation of Cdc5 itself remains relatively uncharacterized. In response to DNA damage, Cdc5 is inhibited by Rad53 mediated phosphorylation at an unidentified site [14,15]. Phosphorylation of T242 by Cdk1 has been reported to be essential for viability and mitotic activity [16], though this is disputed [1]. Nine other sites have been reported to be phosphorylated during mitosis [16], of which one, T29, when mutated to alanine stabilizes Cdc5 during G1 [17]. Four of these sites are clustered within the first 70 amino acids of Cdc5, suggesting modification of this region may be of importance to the regulation of Cdc5.Cdc5 degra