Fig. 6. The G177/S179 residues in the JNK1 are crucial for its activity (A) SABLE analysis revealed a putative structural difference between JNK1 and JNK2 in the variable region. The arrows represent β-strands that are predicted in this region. An extra β-strand is predicted in JNK2, but not in JNK1. Asterisks identify the Thr and Tyr residues as JNK phosphate acceptor sites. The triangles indicate the G177/S179 residues in JNK1, which are divergent from C177/N179 in JNK2. (B) HEK293 cells were transiently transfected with wild-type HA-JNK1 and HA-JNK2, and mutant HA-JNK1 (CTN) and HA-JNK2 (GTS). The JNK proteins were isolated by immunoprecipitation using HA-conjugated beads and subjected to western blotting using anti-HA followed by quantification using chemiluminescence imaging analyses. The precipitates were also used for an in vitro kinase assay with an active MKK4 as the kinase in the presence of [-³²P]ATP. JNK phosphorylation was detected by exposure to X-ray film and phosphoimager analyses. The values of the relative ³²P-JNK/total JNK of the mutants were compared with that of their wild-type counterparts, which were designated as 1. (C,D) HEK293 cells were transiently co-transfected with active MEKK1, wild-type HA-JNK1 and HA-JNK2, and mutant HA-JNK1 (CTN) and HA-JNK2 (GTS), as indicated. The cell lysates were analyzed for MEKK1 expression by western blot analyses and were used for immunoprecipitation with anti-HA. The immunoprecipitates (C) were analyzed for the phospho- and total-JNK by western blotting and chemiluminescence imaging analyses, and (D) were subjected to in vitro kinase assay (KA) using GST-c-Jun as a substrate in the presence of [-³²P]ATP. The GST-c-Jun phosphorylation was detected by exposure to X-ray film and the GST-c-Jun and JNK proteins in the reaction were detected by western blot. The relative p-c-Jun/total c-Jun levels were calculated and are shown in the graph. Consistent results were obtained from at least three independent experiments.