Cell cycle-dependent regulation of human DNA polymerase alpha-primase activity by phosphorylation.
Cell cycle-dependent regulation of human DNA polymerase alpha-primase activity by phosphorylation.
复制标题
通过磷酸化对人类 DNA 聚合酶 α-引物酶活性进行细胞周期依赖性调节。
DOI：
10.1128/mcb.19.1.646
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发表时间：
1999
影响因子：
5.3
通讯作者：
Fanning,E
中科院分区：
文献类型：
--
作者：
Voitenleitner,C;Rehfuess,C;Hilmes,M;O'Rear,L;Liao,PC;Gage,DA;Ott,R;Nasheuer,HP;Fanning,E
已知DNA聚合酶α-引发酶在人和酵母细胞中以细胞周期依赖性方式在p180和p68亚基上磷酸化。我们发现，在体外，纯化的人DNA聚合酶α-引发酶被纯化的细胞周期蛋白A/cdk 2磷酸化，降低了其启动猿猴病毒40（SV 40）DNA复制的能力，而被细胞周期蛋白E/cdk 2磷酸化，刺激了其启动活性。胰蛋白酶磷酸肽图谱显示，一个家庭的p68肽，以及由细胞周期蛋白A/cdk 2和细胞周期蛋白E/cdk 2差的修改。p180磷酸肽与两种激酶相同。通过质谱法，p68肽家族被鉴定为残基141至160。细胞周期蛋白A/cdk 2-和细胞周期蛋白A/cdc 2-修饰的p68也显示磷酸化依赖的转变，以较慢的电泳迁移率。突变p68肽残基141至160内的四个假定的磷酸化位点，防止其被细胞周期蛋白A/cdk 2磷酸化和抑制复制活性。人细胞DNA聚合酶α-引发酶p68亚基的磷酸肽图谱，在G1/S和G2中同步和标记，显示G1/S中的细胞周期蛋白E/cdk 2样模式和G2中的细胞周期蛋白A/cdk 2样模式。在G1/S期的人细胞中不存在电泳迁移率较慢的p68，而在细胞进入G2/M期时出现。与此一致，从同步化的人细胞中分离的DNA聚合酶α-引发酶启动SV 40复制的能力在G1/S期最大，随着细胞完成S期而降低，在G2/M期达到最小。这些结果表明，DNA聚合酶α-引物酶在人类细胞中的复制活性是通过磷酸化以细胞周期依赖性方式调节的。
DNA polymerase α-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 α-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 α-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 α-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 α-primase in human cells is regulated by phosphorylation in a cell cycle-dependent manner.