Fig 3.

JNK phosphorylates 4E-T in vitro and in vivo. (A) Cells were cotransfected with myc-tagged 4E-T and Flag-tagged MKK7-JNK1 in constitutively activated (CA) or kinase-inactive (KD) forms. 4E-T phosphorylation was analyzed by mobility shift assay. (B) JNK activation was induced by anisomycin treatment (10 μM) for the indicated times, and 4E-T phosphorylation was analyzed as described for panel A. (C) HEK293T cells were transfected with Flag-tagged MKK7-JNK1 (CA or KD) or myc-tagged 4E-T. Immunoprecipitated myc-tagged 4E-T from serum-starved cells was incubated with immunopurified Flag-tagged MKK7-JNK1 in a kinase reaction with [γ-³²P]ATP. The resulting samples were subjected to SDS-PAGE, and the dried Coomassie-stained gel was autoradiographed. The histogram shows the n-fold increase of 4E-T phosphorylation normalized to the condition where JNK was not transfected.