Voitenleitner C, Rehfuess C, Hilmes M, O'Rear L, Liao P C, Gage D A, Ott R, Nasheuer H P, Fanning E
Department of Molecular Biology, Vanderbilt University, Nashville, Tennessee 37235, and Vanderbilt Cancer Center, Nashville, Tennessee 37232-6838, USA.
Mol Cell Biol. 1999 Jan;19(1):646-56. doi: 10.1128/MCB.19.1.646.
DNA polymerase alpha-primase is known to be phosphorylated in human and yeast cells in a cell cycle-dependent manner on the p180 and p68 subunits. Here we show that phosphorylation of purified human DNA polymerase alpha-primase by purified cyclin A/cdk2 in vitro reduced its ability to initiate simian virus 40 (SV40) DNA replication in vitro, while phosphorylation by cyclin E/cdk2 stimulated its initiation activity. Tryptic phosphopeptide mapping revealed a family of p68 peptides that was modified well by cyclin A/cdk2 and poorly by cyclin E/cdk2. The p180 phosphopeptides were identical with both kinases. By mass spectrometry, the p68 peptide family was identified as residues 141 to 160. Cyclin A/cdk2- and cyclin A/cdc2-modified p68 also displayed a phosphorylation-dependent shift to slower electrophoretic mobility. Mutation of the four putative phosphorylation sites within p68 peptide residues 141 to 160 prevented its phosphorylation by cyclin A/cdk2 and the inhibition of replication activity. Phosphopeptide maps of the p68 subunit of DNA polymerase alpha-primase from human cells, synchronized and labeled in G1/S and in G2, revealed a cyclin E/cdk2-like pattern in G1/S and a cyclin A/cdk2-like pattern in G2. The slower-electrophoretic-mobility form of p68 was absent in human cells in G1/S and appeared as the cells entered G2/M. Consistent with this, the ability of DNA polymerase alpha-primase isolated from synchronized human cells to initiate SV40 replication was maximal in G1/S, decreased as the cells completed S phase, and reached a minimum in G2/M. These results suggest that the replication activity of DNA polymerase alpha-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.
已知DNA聚合酶α-引发酶在人和酵母细胞中，其p180和p68亚基会以细胞周期依赖性方式发生磷酸化。在此我们表明，体外经纯化的细胞周期蛋白A/细胞周期蛋白依赖性激酶2（cyclin A/cdk2）对纯化的人DNA聚合酶α-引发酶进行磷酸化，会降低其体外启动猿猴病毒40（SV40）DNA复制的能力，而细胞周期蛋白E/细胞周期蛋白依赖性激酶2（cyclin E/cdk2）进行的磷酸化则会刺激其启动活性。胰蛋白酶磷酸肽图谱显示，有一族p68肽被细胞周期蛋白A/cdk2很好地修饰，而被细胞周期蛋白E/cdk2修饰得很差。p180磷酸肽与这两种激酶修饰的情况相同。通过质谱分析，p68肽族被鉴定为第141至160位氨基酸残基。细胞周期蛋白A/cdk2和细胞周期蛋白A/细胞周期蛋白依赖性激酶4（cyclin A/cdc2）修饰的p68在电泳迁移率上也表现出磷酸化依赖性的向较慢迁移率的转变。p68肽第141至160位氨基酸残基内四个假定的磷酸化位点发生突变，可阻止其被细胞周期蛋白A/cdk2磷酸化以及复制活性受到抑制。对来自人细胞、在G1/S期和G2期同步化并标记的DNA聚合酶α-引发酶的p68亚基进行磷酸肽图谱分析，结果显示在G1/S期呈现细胞周期蛋白E/cdk2样模式，在G2期呈现细胞周期蛋白A/cdk2样模式。在G1/S期的人细胞中不存在迁移率较慢的p68形式，而在细胞进入G2/M期时出现。与此一致的是，从同步化的人细胞中分离得到的DNA聚合酶α-引发酶启动SV40复制的能力在G1/S期最大，随着细胞完成S期而降低，并在G2/M期达到最低。这些结果表明，人细胞中DNA聚合酶α-引发酶的复制活性以细胞周期依赖性方式受到磷酸化的调控。
