DYRK2 promoted TBK1 degradation via K48-linked ubiquitination.
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Abstract
<p>(A) Overexpression of DYRK2 induced TBK1 degradation in a dose-dependent manner. The 293T cells (2×10<sup>5</sup>) were transfected with the Flag-TBK1, HA-β-actin and HA-DYRK2 plasmids (0.1, 0.2 or 0.4 μg) and were treated with dimethyl sulfoxide (DMSO) or MG132. The cells were lysed, and the lysates were analyzed by immunoblotting with anti-Flag or anti-HA antibodies. (B) Overexpression of wild-type DYRK2 but not mutant DYRK2, promoted the ubiquitination of TBK1. The 293 cells (1×10<sup>7</sup>) were transfected with the indicated plasmids. Twenty-four hours after transfection, cell lysates were immunoprecipitated with an anti-TBK1 antibody. The immunoprecipitates were analyzed by immunoblotting with an anti-Myc antibody (upper). Protein expression was analyzed by immunoblotting with the indicated antibodies (lower). (C) Effects of DYRK2 RNAi on the SeV-induced ubiquitination of endogenous TBK1. The 293 cells (5×10<sup>7</sup>) were transfected with control or DYRK2 RNAi (#2) plasmids. Twenty hours after transfection, the cells were infected or not infected with SeV for 10 h. The cell lysates were immunoprecipitated with an anti-TBK1 antibody. The immunoprecipitates were analyzed by immunoblotting with an anti-ubiquitin antibody (top). The expressions of related proteins were examined by immunoblotting with the indicated antibodies (bottom). (D) DYRK2 promoted K48-linked but not K63-linked ubiquitination of TBK1. The 293 cells (2×10<sup>6</sup>) were transfected with HA-tagged Lys-48-only or Lys-63-only ubiquitin plasmids and the other indicated plasmids. Twenty-four hours after transfection, cell lysates were immunoprecipitated with an anti-TBK1 antibody and then analyzed by immunoblotting with an anti-HA antibody (upper panel). The expressions of related proteins were examined by immunoblotting with the indicated antibodies (lower panel).</p