Figure 5. PRMT-1 Binds to and Methylates DAF-16. (A) Expression pattern of
prmt-1 in C. elegans. Fluorescence micrograph of GFP proteins expressed under the control of the
prmt-1 promoter. Arrows represent GFP fluorescence in the intestine. Scale bar, 50 um. (B) PRMT-1 binds to DAF-16 in vitro. GST pull-down assays were performed with whole-worm extracts from zIs356[
daf-16::gfp] transgenic strain together with GST or GST-PRMT-1, followed by western blotting with anti-DAF-16 antibody. GST proteins are shown by CBB staining.(C) Physical interaction between PRMT-1 and DAF-16 in cells. Expression plasmids of FLAG-DAF-16 and/or HA-PRMT-1 were transfected into HEK293T cells, and whole-cell extracts were immunoprecipitated with anti-FLAG antibody, followed by western blotting with anti-FLAG and anti-HA antibodies. Two percent of total input is shown. (D) PRMT-1 methylates DAF-16. DAF-16 proteins were incubated with wild-type or G70A mutant of GST-PRMT-1 in the presence of [3H]SAM. Total amounts of DAF-16 are shown by CBB staining. An asterisk indicates GST-PRMT-1.(E) Determination of methylated region in DAF-16. In vitro methylation assay was performed using a series of GST-DAF-16 deletions as indicated. Forkhead (FH) domain is shown as blue boxes. An asterisk indicates GST-PRMT-1. (F) Comparison of the DAF-16 sequence with its orthologs of mouse FOXO1 and Drosophila dFOXO. Methylated arginines in mouse FOXO1 are shown in red. Single or dual consecutive RxR motifs are shown in blue.(G) Determination of methylated sites in DAF-16. GST-PRMT-1 proteins were incubated with wild-type or 3RK (R235K/R237K/R239K) mutant of GST-DAF-16 fragment (225-300 aa) in the presence of [3H]SAM.