PKC isoforms interact with and phosphorylate DNMT1.

Geneviève Lavoie (1) , Pierre-Olivier Estève (2) , Nathalie Bibens Laulan (1) , Sriharsa Pradhan (2) , Yves St-Pierre (1)

1 INRS-IAF - Institut Armand Frappier
2 RNA Biology Division

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Domaines: Maladies infectieuses

Geneviève Lavoie, Pierre-Olivier Estève, Nathalie Bibens Laulan, Sriharsa Pradhan, Yves St-Pierre. PKC isoforms interact with and phosphorylate DNMT1.. BMC Biology, 2011, 9 (1), pp.31. ⟨10.1186/1741-7007-9-31⟩. ⟨pasteur-00723261⟩