FIG. 4.

Phosphorylation by Pak2 inhibits the interaction of Myc with Max. (A) Analysis in vitro. Various forms of Myc were produced by translation in vitro. The ability of the Myc substrate to interact with Max after phosphorylation with activated Pak2 was evaluated as described in Materials and Methods. The relative amount of binding was determined by ImageQuant. Aliquots of cell lysates used in the binding assays were subjected directly to SDS-PAGE and immunoblotting for Myc. WT, wild type. (B) Analysis in vivo. The interaction between Myc and Max was evaluated by a two-hybrid assay with 293T cells, as described in Materials and Methods. Interaction was manifested as activation of a luciferase reporter. The expression of the Myc participant in the reaction was determined by immunoblotting. The data shown are the mean and standard deviation of three independent experiments (*, P < 0.005; **, P < 0.03). (C) Activation of Pak2 by stress blocks the interaction of Myc with Max in vivo. The stress conditions were applied to 293T cells as described in the legend to Fig. 3C. After stress treatment, endogenous Myc was immunoprecipitated from cellular extracts. The immunoprecipitates (IP) were examined for Myc and Max by immunoblotting (IB). Where required, the analyses were performed with cells expressing Pak2-AID that had been introduced by transfection. AraC, cytosine arabinoside; CisP, cisplatin.