Fig 6.

JNK phosphorylates 4E-T on six serine residues. (A) HEK293 cells were cotransfected with wt 4E-T or the S6A mutant (S301A, S374A, S513A, S587A, S693A, S752A) and activated JNK1. The phosphorylation of 4E-T was analyzed by mobility shift assay. (B) Cells were transfected with wt or S6A 4E-T and treated with arsenite (0.5 mM) for 45 min. 4E-T phosphorylation was assayed as described for panel A. (C) HEK293T cells were cotransfected with wt or S6A 4E-T and activated JNK1. Immunoprecipitated myc-tagged 4E-T from serum-starved cells was incubated with immunopurified Flag-tagged MKK7-JNK1 (CA) 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.