DYRK2 phosphorylated TBK1 at S527.
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Abstract
<p>(A and C) Schematics of human DYRK2 (A) and TBK1 (C) and their truncation mutants. (B) DYRK2 interacted with TBK1 via its kinase domain. The 293 cells (2×10<sup>6</sup>) were transfected with the indicated plasmids (5 μg each). Coimmunoprecipitations were performed with anti-Flag or IgG (control). The immunoprecipitates were analyzed by immunoblotting with an anti-HA antibody (top). The expressions of the transfected proteins were analyzed by immunoblotting with anti-HA (middle) or anti-Flag (bottom) antibodies. (D) DYRK2 bound to the coiled-coil domain of TBK1. The 293 cells (2×10<sup>6</sup>) were transfected with the indicated plasmids and then treated and analyzed as described in (B). (E) Wild-type DYRK2 but not mutant DYRK2 promoted TBK1 phosphorylation. The 293 cells (2×10<sup>6</sup>) were transfected with the indicated plasmids (5 μg each). Cell lysates were immunoprecipitated with an anti-Flag antibody, treated with or without calf intestine phosphatase (CIP), and analyzed by immunoblotting with an anti-Flag antibody (top). The expressions of the transfected proteins were analyzed by immunoblotting with an anti-HA antibody (bottom). (F) The effects of DYRK2 on the wild-type and mutant TBK1 as determined by ISRE activation. The 293 cells (1×10<sup>5</sup>) were transfected with an ISRE reporter plasmid (0.1 μg) and the indicated expression plasmids (0.1 μg each) for 20 h before the luciferase assays were performed. The graphical data are presented as the means ± the SDs (n = 3). (G) DYRK2 promoted the phosphorylation of wild-type TBK1 but not the TBK1 S527A mutant. The 293 cells (2×10<sup>6</sup>) were transfected with the indicated plasmids and then treated and analyzed as described in (E).</p