Fig. 2.

mTOR phosphorylates MNK2a on Ser74. a mTOR kinase assays were performed with mTOR IPed as in Fig. 1b, and, as substrates, GST-MNK2a (WT or mutants) that had been purified as in Fig. 1c. b Quantitation of a. c HEK293 cells were treated as in Fig. 1a, after SDS-PAGE, phosphorylated (P-) or total proteins were detected as indicated. d HEK293 cells were transfected with indicated GST-MNK2a constructs. Cells were starved of serum for 16 h and then kept in KRB in the presence or absence of AZD8055 for 30 min. GST-MNK2a proteins were then pulled down with glutathione beads and assayed for kinase activity against recombinant eIF4E as substrate. Quantification of data in panel B and D is presented as means ± SD, n = 3. *0.01 ≤ P < 0.05; ***P < 0.001 (one-way ANOVA); ^$P < 0.001 (two-way ANOVA)