Distinct signalling pathways mediate insulin and phorbol ester-stimulated eukaryotic initiation factor 4F assembly and protein synthesis in HEK 293 cells

Stimulation of serum-starved human, embryonic kidney (HEK) 293 cells with e ither the phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA), or ins ulin resulted in increases in the phosphorylation of 4E-BP1 and p70 S6 kina se, eIF4F assembly, and protein synthesis. All these effects were blocked b y rapamycin, a specific inhibitor of mTOR. Phosphatidylinositol 3-kinase an d protein kinase B were activated by insulin but not by TPA. Therefore TPA can induce eIF4F assembly, protein synthesis, and the phosphorylation of p7 0 Se kinase and 4E-BP1 independently of both phosphatidylinositol 3-kinase and protein kinase B. Using two structurally unrelated inhibitors of MEK (P D098059 and U0126), we provide evidence that Erk activation is important in TPA stimulation of eIF4F assembly and the phosphorylation of p70 S6 kinase and 4E-BP1 and that basal MEK activity is important for basal, insulin, an d TPA-stimulated protein synthesis. Transient transfection of constitutivel y active mitogen-activated protein kinase interacting kinase 1 (the eIF4E k inase) indicated that inhibition of protein synthesis and eIF4F assembly by PD098059 is not through inhibition of eIF4E phosphorylation but of other s ignals emanating from MEK. This report also provides evidence that increase d eIF4E phosphorylation alone does not affect the assembly of the eIF4F com plex or general protein synthesis.