Phosphorylation of serine-515 activates the mammalian maintenance methyltransferase Dnmt1
Phosphorylation of serine-515 activates the mammalian maintenance methyltransferase Dnmt1
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DOI：
10.4161/epi.2.3.4768
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发表时间：
2007-07-01
期刊：
影响因子：
3.7
通讯作者：
Jeltsch, Albert
中科院分区：
文献类型：
--
作者：
Goyal, Rachna;Rathert, Philipp;Jeltsch, Albert
DNA 甲基转移酶 1 将 DNA 复制过程中产生的半甲基化 CG 位点甲基化。该酶的丝氨酸 515 已被证明被磷酸化。为了探索 S515 磷酸化的重要性，我们生成了 Dnmt1 突变体，该突变体去除了磷酸化电位 (S515A) 或模拟磷酸丝氨酸 (S515E)，从昆虫细胞中纯化了蛋白质，并在体外分析了它们的 DNA 甲基化活性。发现S515E突变体具有活性，而与野生型蛋白相比，S515A突变体的活性严重丧失。 S515A 变体活性的丧失并不是由于 DNA 结合能力的丧失。此外，我们还发现，序列模拟 Ser515 ((EKIYISKIVVE)-K-P) 周围的磷酸化肽对野生型 Dnmt1 的活性的抑制是非磷酸化肽的十倍。该抑制对 Dnmt1 和特定肽序列具有特异性。我们的数据表明，Ser515 的磷酸化对于 Dnmt1 的 N 端结构域与其催化结构域之间的相互作用非常重要，而催化结构域是活性所必需的，并且这种相互作用会被磷酸化肽特异性破坏。我们得出结论，Dnmt1 Ser515 的磷酸化可能是细胞周期期间和增殖刺激后 Dnmt1 活性的重要调节因子。
DNA methyltransferase 1 methylates hemi-methylated CG sites generated during DNA replication. Serine 515 of this enzyme has been shown to be phosphorylated. To explore the importance of S515 phosphorylation, we generated mutants of Dnmt1 which removed the phosphorylation potential (S515A) or mimic phosphoserine (S515E), purified the proteins from insect cells and analyzed their DNA methylation activity in vitro. The S515E mutant was found to be active, while S515A mutant had severe loss in activity when compared to the wild type protein. The loss of activity of the S515A variant was not due to loss of DNA binding capacity. Furthermore, we show that a phosphorylated peptide whose sequence mimics the surrounding of Ser515 ((EKIYISKIVVE)-K-P) inhibited the activity of wild type Dnmt1 ten-fold more than the non-phosphorylated peptide. The inhibition was specific for Dnmt1 and for the particular peptide sequence. Our data suggest that phosphorylation of Ser515 is important for an interaction between the N-terminal domain of Dnmt1 and its catalytic domain that is necessary for activity and that this interaction is specifically disrupted by the phosphorylated peptide. We conclude that phosphorylation of Dnmt1 at Ser515 could be an important regulator of Dnmt1 activity during cell cycle and after proliferative stimuli.