Figure 4. SGK1 phosphorylates MNK1 in vitro.

Kinase assay using [γ-³²P]ATP. (A) Immunoprecipitates of wild-type NDRG1 or the indicated mutants (see Methods for details) or (B) immunoprecipitated SGK1 were incubated with or without purified MNK1a at 30°C for 30 min. Incorporated radiolabel was assessed by phosphorimager (left). Protein levels were assessed by Coomassie blue staining (right). eIF4E was used as a positive control for MNK1a activity. * denotes a non-specific band in the SGK1 immunoprecipitate. (C) Kinase assays using [γ-³²P]ATP were performed with human MNK1a with or without SGK1′. and beads (with/without SGK1) on the left (phosphorimage), and protein levels were assessed by Coomassie blue staining (on the right). NDRG1 was used as a positive control for SGK1 activity. (D) Kinase assay using [γ-³²P]ATP was used to test the ability of SGK1 to phosphorylate purified MNK1a protein and the indicated mutants. NDRG1 was used as a positive control. In (C) and (D), +SGK1 indicates that SGK1was immunoprecipitated from cells transfected with a vector encoding SGK1. –SGK1 indicates mockimmunoprecipitaion from non-transfected cells.’ (E) The activity of activated MNK1a protein was assessed using non-radioactive ATP with eIF4E as substrate and samples were withdrawn at the indicated times. Samples were analysed by SDS-PAGE/immunoblot, using the indicated antibodies.