Figure 6.

Phosphorylation of ZNF281 by GSK-3β is required for ZNF281 degradation (A1 and A2) Co-transfection approach was used to investigate co-IP of GSK-3β with ZNF281. (B) Endogenous GSK-3β and ZNF281 were immunoprecipitated from HCT116 cells. (C) The amino acid sequences of the conserved GSK-3β phosphorylation motif in ZNF281 (C1). GST-ZNF281 or its mutant protein was incubated with GST-GSK-3β, and the phosphorylation of ZNF281 was assayed with phosphor-Ser antibody (C1), and in vitro kinase assay was performed as described in Materials and Methods (C2). (D) HCT116 cells were cotransfected with indicated plasmids, and then were treated with MG132 alone or with LiCl for 10 h. Samples were then blotted with specific phospho-Ser antibody. (E) WT-ZNF281 or its mutant was cotransfected with indicated plamids into HCT116 cells. Samples were then analyzed by Western blotting with indicated antibodies. (F) Indicated plasmids were cotransfected into WT-MEF or GSK-3β knockout MEF cells. Western blotting was performed with indicated antibodies. (G) Indicated plasmids were transfected into HCT116 cells, which were then treated with CHX (20 mM) for the indicated times. The levels of WT-ZNF281 and its mutant were determined. (H) HCT116 cells were treated with CHX (20 μM) with or without LiCl (20 mM) for the indicated times. Endogenous expression of ZNF281 was determined.