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. 2016 Apr 22;7:11391. doi: 10.1038/ncomms11391

Figure 3. FINDY suppresses autophosphorylation of Ser97 in DYRK1A.

Figure 3

(a) The bicistronic expression of intact DYRK1A (WT) and the substituted mutants of Lys188 to Arg (K188R), Ser97 to Ala (S97A), Tyr111 to Phe (Y111F), Tyr319 to Phe (Y319F), Tyr321 to Phe (Y321F), Tyr319 and Tyr321 to Phe (Y319F/Y321F) and Ser529 to Ala (S529A) with EGFP in HEK293 cells. The expression vectors were stably integrated in the genome of the cells. Each protein was detected with the corresponding antibody against FLAG, EGFP or GAPDH. Representative data from the quadruplicate experiments are shown. (b) The alignment of Ser97 and its surrounding amino-acid sequences from DYRK1A of the indicated species. The conserved serine residues corresponding to Ser97 of Homo sapiens DYRK1A are highlighted by the red rectangular box. Underlined portions indicate the phospho-peptide used in the immunization. (c) The polyclonal antibody against phosphorylated Ser97 (p-Ser97) specifically detected FLAG-DYRK1A (WT), but not the kinase-dead (K188R) or S97A mutants. The cells were transiently transfected with the expression vectors of the indicated constructs and the proteins were overexpressed. Total cell lysates were subjected to SDS–PAGE followed by western blot analysis. Representative data from the triplicate experiments are shown. (d) FINDY suppressed Ser97 autophosphorylation. FLAG-DYRK1A was produced in HEK293 cells incubated with epoxomicin and FINDY for 5 h, then purified with anti-FLAG antibody-conjugated beads. Each protein was detected with the corresponding antibodies against p-Ser97, phospho-Tyr (p-Tyr) or FLAG. Representative data from the triplicate experiments are shown. (e) FINDY did not suppress Ser97 autophosphorylation in Tc 2. FLAG-DYRK1A was produced in HEK293 cells for 16 h in the presence of doxycycline, after which cells were treated with 10 μM of FINDY or RD0392 for 5 h. Total cell lysates were subjected to immunoprecipitation (IP) with anti-FLAG antibody-conjugated beads. Each protein was detected with the corresponding antibodies against p-Ser97 and FLAG. Representative data from the duplicate experiments are shown. Conc., concentration.