Figure 2

Phosphomimetic A3B mutants lose their CDA in vitro. (a) Schematic figure of phosphomimetic A3B mutants at Thr214. T214D CTD and H253R CTD are the purified protein constructs. (b) Phosphomimetic A3B mutants lose their CDA in vitro. FRET-based CDA assays were performed using lysates from HEK293T cells with exogenously-expressed A3B or its mutants. Each of the fluorescent intensity numerical values represent the average of three independent experiments, normalized to that of the WT sample. Bars indicate standard error (SE), and asterisks (*) show statistically significant difference (p < 0.05) (upper panel). The protein levels of the A3B mutants used in the experiment were comparable (lower panel). (c) Phosphomimetic A3B mutants lose CDA against the TCA sequence in the gel-based CDA assay. In this assay A3B CDA catalyzes substrate 43b oligo to 12b oligo transition. Lysates from cells transfected with EV, T214D, T214E or H253R have no CDA, and those with T214A have less CDA than those with WT. (d) FRET-based CDA assays using purified proteins. Purified C-terminal A3B T214D almost completely loses CDA in vitro. Each of the fluorescent intensity numerical values represent the average of three independent experiments. (e) Gel-based CDA assay using purified proteins. A3B T214D loses CDA, and T214A has weak CDA against the TCA sequence. (f) Phosphorylation of A3B reduces its cytidine deaminase activity. A3B-CTD and T214A-CTD were phosphorylated by PKACA in vitro, and subsequently performed in vitro CDA assays. Values represent the average of two independent experiments, and bars indicate SE.