FIGURE 5.

Mapping the p190A priming sites. A, mapping the sites of p38 and p42 MAPK phosphorylation on p190A. pEBG (vector) or pEBG-50AA-WT and the indicated phosphorylation site mutant proteins were captured from transfected COS-7 cells, and the proteins on beads were incubated with either p38MAPK or p42MAPK and [γ-³²P]ATP. The leftmost lane corresponds to the pEBG-WT protein with buffer only. Reaction products were analyzed by SDS-PAGE and autoradiography (upper panels) and Coomassie Blue staining (lower panels). B, mapping the priming sites on p190A in vivo. pEBG-50AA-WT and the various phosphorylation site mutant proteins were purified from transfected COS-7 cells as indicated. The proteins on beads were then either mock-treated or prephosphorylated in vitro by either p38 or p42 MAPK in the presence of cold ATP. They were then incubated with GSK-3β in the presence of [γ-³²P]ATP. Reaction products were analyzed by SDS-PAGE and autoradiography. C, inhibiting p38MAPK in the priming lysate disrupts priming activity. pEBG-50AA-WT proteins were purified from COS-7 cells as described under “Experimental Procedures.” For priming, following cell lysis the lysates were preincubated with either DMSO (vehicle control) or the p38 MAPK inhibitor SB203580. The lysates were then used to prime the pEBG-50AA-WT proteins on beads. The beads were then processed for GSK-3β kinase assays using [γ-³²P]ATP. Reaction products were analyzed by SDS-PAGE and autoradiography (upper panel) and Coomassie Blue staining (lower panel).