Huang Jun-Yong, Morley Gary, Li Deyu, Whitaker Michael
Institute for Cell and Molecular Biosciences, Faculty of Medical Sciences, University of Newcastle upon Tyne, Catherine Cookson Building, Framlington Place, Newcastle upon Tyne, NE2 4HH, UK.
J Cell Sci. 2007 Jun 15;120(Pt 12):1990-7. doi: 10.1242/jcs.006833. Epub 2007 May 22.
Anaphase-promoting complex or cyclosome (APC/C) controls the metaphase-to-anaphase transition and mitosis exit by triggering the degradation of key cell cycle regulators such as securin and B-type cyclins. However, little is known about the functions of individual APC/C subunits and how they might regulate APC/C activity in space and time. Here, we report that two potential Cdk1 kinase phosphorylation sites are required for the chromosomal localisation of GFP::Cdc27 during mitosis. Either or both of the highly conserved proline residues in the Cdk1 phosphorylation consensus sequence motifs were mutated to alanine (Cdc27 P304A or P456A). The singly mutated fusion proteins, GFP::Cdc27P304A and GFP::Cdc27P456A, can still localise to mitotic chromosomes in a manner identical to wild-type GFP::Cdc27 and are functional in that they can rescue the phenotype of the cdc27L7123 mutant in vivo. However, when both of the Cdk1 phosphorylation sequence motifs were mutated, the resulting GFP::Cdc27P304A,P456A construct was not localised to the chromosomes during mitosis and was no longer functional, as it failed to rescue mutant phenotypes of the cdc27L7123 gene. High levels of cyclin B and cyclin A were detected in mutant third instar larvae brain samples compared with its wild-type control. These results show for the first time that the two potential Cdk1 phosphorylation sites on Drosophila Cdc27 are required for its chromosomal localisation during mitosis and imply that these localisations specific to Cdc27 are crucial for APC/C functions.
后期促进复合物或细胞周期体（APC/C）通过触发诸如分离酶和B型细胞周期蛋白等关键细胞周期调节因子的降解来控制中期到后期的转变以及有丝分裂的退出。然而，关于单个APC/C亚基的功能以及它们如何在空间和时间上调节APC/C活性，我们知之甚少。在这里，我们报告在有丝分裂期间，GFP::Cdc27的染色体定位需要两个潜在的Cdk1激酶磷酸化位点。Cdk1磷酸化共有序列基序中高度保守的脯氨酸残基中的一个或两个被突变为丙氨酸（Cdc27 P304A或P456A）。单突变融合蛋白GFP::Cdc27P304A和GFP::Cdc27P456A仍然可以以与野生型GFP::Cdc27相同的方式定位于有丝分裂染色体上，并且具有功能，因为它们可以在体内挽救cdc27L7123突变体的表型。然而，当两个Cdk1磷酸化序列基序都发生突变时，产生的GFP::Cdc27P304A,P456A构建体在有丝分裂期间不能定位于染色体上，并且不再具有功能，因为它无法挽救cdc27L7123基因的突变体表型。与野生型对照相比，在突变的三龄幼虫脑样本中检测到高水平的细胞周期蛋白B和细胞周期蛋白A。这些结果首次表明，果蝇Cdc27上的两个潜在Cdk1磷酸化位点在有丝分裂期间其染色体定位是必需的，并且暗示这些Cdc27特有的定位对于APC/C功能至关重要。
