Cell Cycle Laboratory, The Francis Crick Institute, London NW1 1AT, UK.
Cell Cycle Laboratory, The Francis Crick Institute, London NW1 1AT, UK.
Curr Biol. 2020 Mar 9;30(5):883-892.e4. doi: 10.1016/j.cub.2019.12.053. Epub 2020 Feb 20.
The cyclin-dependent kinases (CDKs) are the major cell-cycle regulators that phosphorylate hundreds of substrates, controlling the onset of S phase and M phase [1-3]. However, the patterns of substrate phosphorylation increase are not uniform, as different substrates become phosphorylated at different times as cells proceed through the cell cycle [4, 5]. In fission yeast, the correct ordering of CDK substrate phosphorylation can be established by the activity of a single mitotic cyclin-CDK complex [6, 7]. Here, we investigate the substrate-docking region, the hydrophobic patch, on the fission yeast mitotic cyclin Cdc13 as a potential mechanism to correctly order CDK substrate phosphorylation. We show that the hydrophobic patch targets Cdc13 to the yeast centrosome equivalent, the spindle pole body (SPB), and disruption of this motif prevents both centrosomal localization of Cdc13 and the onset of mitosis but does not prevent S phase. CDK phosphorylation in mitosis is compromised for approximately half of all mitotic CDK substrates, with substrates affected generally being those that require the highest levels of CDK activity to become phosphorylated and those that are located at the SPB. Our experiments suggest that the hydrophobic patch of mitotic cyclins contributes to CDK substrate selection by directing the localization of Cdc13-CDK to centrosomes and that this localization of CDK contributes to the CDK substrate phosphorylation necessary to ensure proper entry into mitosis. Finally, we show that mutation of the hydrophobic patch prevents cyclin B1 localization to centrosomes in human cells, suggesting that this mechanism of cyclin-CDK spatial regulation may be conserved across eukaryotes.
细胞周期蛋白依赖性激酶（CDKs）是主要的细胞周期调控因子，它们可以磷酸化数百种底物，控制 S 期和 M 期的起始[1-3]。然而，底物磷酸化的增加模式并不均匀，因为不同的底物在细胞通过细胞周期时会在不同的时间被磷酸化[4,5]。在裂殖酵母中，单个有丝分裂周期蛋白-CDK 复合物的活性可以确定 CDK 底物磷酸化的正确顺序[6,7]。在这里，我们研究了裂殖酵母有丝分裂周期蛋白 Cdc13 的底物结合区域，即疏水区，作为正确排列 CDK 底物磷酸化的潜在机制。我们表明，疏水区将 Cdc13 靶向酵母中心体等价物，纺锤体极体（SPB），并且破坏这个模体既阻止了 Cdc13 的中心体定位，也阻止了有丝分裂的开始，但不阻止 S 期。大约一半的有丝分裂 CDK 底物的 CDK 磷酸化受到影响，受影响的底物通常是那些需要最高水平的 CDK 活性才能被磷酸化的底物，以及那些位于 SPB 的底物。我们的实验表明，有丝分裂周期蛋白的疏水区通过将 Cdc13-CDK 引导到中心体来促进 CDK 底物的选择，并且这种 CDK 的定位有助于确保正确进入有丝分裂所需的 CDK 底物磷酸化。最后，我们表明疏水区的突变阻止了人细胞中环蛋白 B1 向中心体的定位，这表明这种细胞周期蛋白-CDK 空间调节的机制可能在真核生物中是保守的。
