FIGURE 2.
Dyrk1A phosphorylates ASF in vitro at Ser-227, Ser-234, and Ser-238. a, schematic of ASF domain structures and the serine residues (227, 234, and 238) in three conserved motifs (gray box) of Dyrk1A phosphorylation sites that were identified in this study. b, autoradiography of ASF phosphorylation by Dyrk1A in vitro. Recombinant GST-ASF was incubated with various concentrations of Dyrk1A indicated above each lane for 30 min at 30 °C and separated by SDS-PAGE and visualized with Coomassie Blue staining (middle panel). The last lane is Dyrk1A alone, without GST-ASF. After drying the gel, the 32P incorporated into ASF was measured by using a phosphorimaging device (BAS-1500, Fuji) (upper panel). The level of 32P was normalized by the protein level detected by Coomassie Blue staining was plotted to Dyrk1A concentration (lower panel). c, inhibition of Dyrk1A by EGCG decreased the phosphorylation of ASF in cultured cells. COS7 cells were transfected with pCEP4-ASF-HA for 45 h and then treated with 10 μm Tg003 and 50 μm EGCG to inhibit Clk and Dyrk1A, respectively. At the same time, [32P]orthophosphate was added to label the phosphoproteins. After a 3-h treatment and phospho-labeling, the cells were harvested, and the cell lysates were subjected to immunoprecipitation with anti-HA. The immunoprecipitated ASF-HA was analyzed by autoradiography and Western blot with anti-HA. The 32P incorporated into ASF-HA was normalized by ASF-HA level detected with anti-HA. Con, control; Pi, phosphate group. d, mutation of Ser to Ala at Ser-227, Ser-234, and/or Ser-238 inhibited ASF phosphorylation by Dyrk1A. Mutants of GST-ASF at the Ser residue indicated above each lane were phosphorylated by Dyrk1A (10 μg/ml) in vitro for 30 min. 32P incorporation into GST-ASFs was measured by using phosphorimaging analysis after the phosphorylation products were separated by SDS-PAGE. The 32P incorporated was normalized by protein level detected with Coomassie Blue staining. WT, wild type.