FIGURE 4.

Priming-dependent phosphorylation of p190A by GSK-3β. A, priming is required for p190A phosphorylation by GSK-3β. Bacterially purified p190A-50AA (pGEX-KG-50AA-WT) on beads was treated either with or without priming by COS-7 lysate. The beads were washed extensively and then subjected to in vitro phosphorylation by GSK-3β. p190A-50AA (pEBG-50AA-WT) expressed in transfected COS-7 cells was purified on glutathione beads, pretreated with either λ-phosphatase, λ-phosphatase plus phosphatase inhibitors (PPI), or phosphatase inhibitors alone. The beads were washed extensively and then subjected to in vitro phosphorylation by GSK-3β. Reaction products were resolved by SDS-PAGE and autoradiography. B, p38 or p42 MAPK, but not CKI, can prime the C-terminal domain of p190A for subsequent phosphorylation by GSK-3β in vitro. Bacterially purified p190A-50AA (pGEX-KG-50AA-WT) was first phosphorylated in vitro by purified recombinant p38MAPK, p42 MAPK, or CKI in the presence of cold ATP. Proteins were then subjected to GSK-3β kinase assays using [γ-³²P]ATP, and products were resolved by SDS-PAGE and visualized by autoradiography. Note the absolute requirement for priming by MAPKs.