Figure 2

PKC isoforms preferentially phosphorylate DNMT1 N-terminal domain. (A) Diagram of DNMT1 showing the corresponding regions of the GST fusion DNMT1 fragments used for phosphorylation assays. Methylation DNA-dependent allosteric activation (MDDAAD), bromo domain (BD), and nuclear localization sequences (NLS) of DNMT1 are indicated. (B) Coomassie-stained gel representing GST fusion DNMT1 proteins used for phosphorylation assays. Positions of the fusion fragments are marked with an asterisk. (C) Phosphorylation of GST fusion DNMT1 fragments following incubation with 20 nM of activated recombinant PKCα, βI, βII, γ, δ or η using (γ-³²P)ATP. Counts were obtained following subtraction of the negative control (GST alone). Data are representative of three independent experiments. (D) Phosphorylation of the GST fusion DNMT1 fragment 1 to 446 following incubation with 20 nM of activated recombinant PKCα, βI, βII, γ, δ, ε, η, μ or ζ using (γ-³²P)ATP. Counts were obtained following subtraction of the negative control (GST alone). Data represent the average of three independent experiments that gave similar results. Bars, S.D.