Phosphorylation of MBP by mDYRK-3 and apparent autophosphorylation in 293 fibroblasts.

To initially assess catalytic activity, mDYRK-3 (and the type II subdomain mutant mDYRK-3–K202R) were expressed transiently as Myc epitope–tagged constructs in 293 cells. At 48 hours posttransfection, mAb 9E10 immunoprecipitates were prepared from whole-cell lysates and were used together with ³²P γATP plus MBP or histone 2B (H2B) in in vitro kinase assays. Shown in the upper panel are electrophoresed and autoradiographed ³²P products. Immunoprecipitates were also eluted, and epitope-tagged wild-type mDYRK-3 and mDYRK-3–K202R were assayed by Western blotting (lower panel). Indexed are 2 forms of mDYRK-3 (ie, molecular-weight 68 000 and 70 000 forms), and the positions of molecular-weight standards.