Jallepalli P V, Brown G W, Muzi-Falconi M, Tien D, Kelly T J
Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 USA.
Genes Dev. 1997 Nov 1;11(21):2767-79. doi: 10.1101/gad.11.21.2767.
Cyclin-dependent kinases (CDKs) promote the initiation of DNA replication and prevent reinitiation before mitosis, presumably through phosphorylation of key substrates at origins of replication. In fission yeast, the p65cdc18 protein is required to initiate DNA replication and interacts with the origin recognition complex (ORC) and the p34cdc2 CDK. Here we report that p65cdc18 becomes highly phosphorylated as cells undergo the G1 --> S phase transition. This modification is dependent on p34cdc2 protein kinase activity, as well as six consensus CDK phosphorylation sites within the p65cdc18 polypeptide. Genetic interactions between cdc18+ and the S-phase cyclin cig2+ suggest that CDK-dependent phosphorylation antagonizes cdc18+ function in vivo. Using site-directed mutagenesis, we show that phosphorylation at CDK consensus sites directly targets p65cdc18 for rapid degradation and inhibits its replication activity, as strong expression of a constitutively hypophosphorylated mutant form of p65cdc18 results in large amounts of DNA over-replication in vivo. Furthermore, the over-replication phenotype produced by this mutant p65cdc18 is resistant to increased mitotic cyclin/CDK activity, a known inhibitor of over-replication. Therefore, p65cdc18 is the first example of a cellular initiation factor directly regulated in vivo by CDK-dependent phosphorylation and proteolysis. Regulation of p65cdc18 by CDK phosphorylation is likely to contribute to the CDK-driven "replication switch" that restricts initiation at eukaryotic origins to once per cell cycle.
细胞周期蛋白依赖性激酶（CDK）可促进DNA复制的起始，并在有丝分裂前防止重新起始，推测这是通过对复制起点处的关键底物进行磷酸化来实现的。在裂殖酵母中，p65cdc18蛋白是启动DNA复制所必需的，它与起始识别复合物（ORC）和p34cdc2 CDK相互作用。在此我们报告，当细胞经历G1期到S期的转变时，p65cdc18会发生高度磷酸化。这种修饰依赖于p34cdc2蛋白激酶的活性，以及p65cdc18多肽内的六个共有CDK磷酸化位点。cdc18 +与S期细胞周期蛋白cig2 +之间的遗传相互作用表明，CDK依赖性磷酸化在体内拮抗cdc18 +的功能。使用定点诱变，我们发现CDK共有位点的磷酸化直接靶向p65cdc18使其快速降解，并抑制其复制活性，因为组成型低磷酸化突变形式的p65cdc18的强表达会在体内导致大量DNA过度复制。此外，这种突变型p65cdc18产生的过度复制表型对有丝分裂细胞周期蛋白/CDK活性的增加具有抗性，而有丝分裂细胞周期蛋白/CDK活性是已知的过度复制抑制剂。因此，p65cdc18是体内第一个直接受CDK依赖性磷酸化和蛋白水解调控的细胞起始因子的例子。CDK磷酸化对p65cdc18的调控可能有助于CDK驱动的“复制开关”，该开关将真核生物复制起点的起始限制在每个细胞周期一次。
