Institut national de la recherche scientifique, INRS-Institut Armand-Frappier, Laval, QC, Canada.
BMC Biol. 2011 May 27;9:31. doi: 10.1186/1741-7007-9-31.
DNA methyltransferase 1 (DNMT1) has been shown to be phosphorylated on multiple serine and threonine residues, based on cell type and physiological conditions. Although recent studies have suggested that protein kinase C (PKC) may be involved, the individual contribution of PKC isoforms in their ability to phosphorylate DNMT1 remains unknown. The PKC family consists of at least 12 isoforms that possess distinct differences in structure, substrate requirement, expression and localization.
Here we show that PKCα, βI, βII, δ, γ, η, ζ and μ preferentially phosphorylate the N-terminal domain of human DNMT1. No such phosphorylation of DNMT1 was observed with PKCε. Using PKCζ as a prototype model, we also found that PKC physically interacts with and phosphorylates DNMT1. In vitro phosphorylation assays conducted with recombinant fragments of DNMT1 showed that PKCζ preferentially phosphorylated the N-terminal region of DNMT1. The interaction of PKCζ with DNMT1 was confirmed by GST pull-down and co-immunoprecipitation experiments. Co-localization experiments by fluorescent microscopy further showed that endogenous PKCζ and DNMT1 were present in the same molecular complex. Endogenous PKCζ activity was also detected when DNMT1 was immunoprecipitated from HEK-293 cells. Overexpression of both PKCζ and DNMT1 in HEK-293 cells, but not of either alone, reduced the methylation status of genes distributed across the genome. Moreover, in vitro phosphorylation of DNMT1 by PKCζ reduced its methytransferase activity.
Our results indicate that phosphorylation of human DNMT1 by PKC is isoform-specific and provides the first evidence of cooperation between PKCζ and DNMT1 in the control of the DNA methylation patterns of the genome.
根据细胞类型和生理条件，已证实 DNA 甲基转移酶 1（DNMT1）可在多个丝氨酸和苏氨酸残基上发生磷酸化。尽管最近的研究表明蛋白激酶 C（PKC）可能参与其中，但 PKC 同工型在其使 DNMT1 磷酸化的能力方面的个体贡献仍不清楚。PKC 家族至少由 12 种同工型组成，它们在结构、底物需求、表达和定位方面存在明显差异。
在这里，我们显示 PKCα、βI、βII、δ、γ、η、ζ 和 μ 优先磷酸化人 DNMT1 的 N 端结构域。未观察到 PKCε 对 DNMT1 的这种磷酸化。使用 PKCζ 作为原型模型，我们还发现 PKC 与 DNMT1 发生物理相互作用并使 DNMT1 磷酸化。用 DNMT1 的重组片段进行的体外磷酸化实验表明，PKCζ 优先磷酸化 DNMT1 的 N 端区域。GST 下拉和共免疫沉淀实验证实了 PKCζ 与 DNMT1 的相互作用。荧光显微镜的共定位实验进一步表明，内源性 PKCζ 和 DNMT1 存在于同一分子复合物中。当从 HEK-293 细胞中免疫沉淀 DNMT1 时，也检测到内源性 PKCζ 活性。在 HEK-293 细胞中过表达 PKCζ 和 DNMT1 两者，而不是单独过表达任何一种，都降低了分布在基因组中的基因的甲基化状态。此外，PKCζ 对 DNMT1 的体外磷酸化降低了其甲基转移酶活性。
我们的结果表明，PKC 对人 DNMT1 的磷酸化是同工型特异性的，并为 PKCζ 和 DNMT1 在控制基因组 DNA 甲基化模式方面的合作提供了首个证据。
