FIGURE 2.

ZAP is phosphorylated by GSK3β. A and B, recombinant NZAP295 is phosphorylated by GSK3β in vitro. Recombinant proteins GST-NZAP295 and GST-NZAP295(S1–8A) were bacterially expressed, partially purified, and incubated with GSK3β, CDK1, or CK2 in the presence of [γ-³²P]ATP at 30 °C. The reactions were analyzed by SDS-PAGE and autoradiography (upper panel). That equal amounts of GST-NZAP295 and GST-NZAP295(S1–8A) were used was confirmed by Coomassie Blue staining (lower panel). A, GST-NZAP295 and GST-NZAP295(S1–8A) were incubated with the enzymes indicated for 30 min. The positions of the kinases are indicated by asterisks. −, no recombinant ZAP proteins were added to the reaction; M, GST-NZAP295(S1–8A); Z, GST-NZAP295. B, GST-NZAP295 and GST-NZAP295(S1–8A) were incubated with GSK3β for the lengths of time indicated. C, inhibition of GSK3β reduces ZAP phosphorylation. The plasmid expressing Myc-tagged NZAP332 was transfected into HEK293 cells. At 6 h post-transfection, cells were treated with SB216763 or mock-treated. The proteins were immunoprecipitated, incubated with alkaline phosphatase at 37 °C for 30 min, and analyzed by Western blotting. D, the plasmid expressing Myc-tagged NZAP332 was cotransfected with the plasmid expressing GSK3β. At 6 h post-transfection, the cells were mock-treated (−) or treated with SB216763 (+). Cells were lysed at 48 h post-transfection, and the lysates were analyzed by Western blotting with anti-Myc antibody to detect NZAP332 or with anti-FLAG antibody to detect GSK3β. E and F, a plasmid expressing the shRNA directed against GSK3β (Gi-3 or Gi-5) was transfected into HEK293 cells. At 24 h post-transfection, puromycin was added to a final concentration of 3 μg/ml to select for transfected cells. Three days later, a second transfection was done under the same conditions. At 24 h after the second transfection, a plasmid expressing Myc-tagged recombinant NZAP332 (rNZAP332) (E) or recombinant NZAP295 (rNZAP295) (F) was transfected. A plasmid expressing Myc-tagged GFP was included as a control for transfection efficiency and sample handling. At 24 h post-transfection, the cells were lysed, and the proteins were analyzed by Western blotting. Ctrl, control.