FIG. 1.

Characterization of MKK7-JNK fusion proteins. (A) Plasmid expression vectors were constructed to express fusion proteins with an NH₂-terminal epitope tag (Flag). The structure of the fusion proteins is schematically illustrated. The MKK7-JNK fusion proteins contain residues 1 to 443 of MKK7β2 fused to JNK1α1, JNK2α2, and JNK3α2. The JNK1-MKK7 fusion protein contains residues 1 to 415 of JNK1α2 fused to MKK7β2. Point mutations were used to create kinase-negative MKK7 (Lys¹⁴⁹ replaced with Ala) and phosphorylation-negative JNK (Thr¹⁸⁰-Pro-Tyr¹⁸² replaced with Ala-Pro-Phe). (B) Measurement of JNK protein kinase activity. JNK1, JNK1 plus MKK7, and the MKK7-JNK fusion proteins were expressed in CHO cells and were detected by immunoblot (IB) analysis using an antibody to the Flag epitope tag (lower panel). Protein kinase assays were performed using an immune complex assay, with c-Jun and [γ-³²P]ATP as the substrates. The reaction products were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The c-Jun protein was detected by Coomassie staining (middle panel) and phosphorylated c-Jun was detected by autoradiography (upper panel). The relative kinase activity was quantitated by phosphorimager analysis. The numbers on the left correspond to the migration of molecular mass standards (in kilodaltons). (C) The activation state of JNK was examined by immunoblot analysis using an antibody to phospho-JNK. The expression of JNK1, MKK7, and the MKK7-JNK fusion proteins in CHO cell lysates was examined by immunoblot analysis using an antibody to the Flag epitope tag (lower panel) and to phospho-JNK (upper panel). (D) Comparison of the wild-type MKK7-JNK1 fusion protein with the catalytically inactive mutant proteins MKK7(K>A)-JNK1 and MKK7-JNK1(APF). Upper panel, the phosphorylation of c-Jun was examined in an in vitro Flag-immune complex protein kinase assay. c-Jun was detected by staining with Coomassie blue. Phosphorylated c-Jun was detected by autoradiography. Lower panel, the activation state of JNK was examined by immunoblot analysis by probing with antibodies to the epitope tag (Flag) and phospho-JNK.