Fig. 8.

Inhibition of Mnk1 with CGP57380 does not affect the activation of PKCα, Erk1/2, and phosphorylation of eIF4G(Ser1186). (A) Inhibition of eIF4E(Ser209) phosphorylation by CGP57380. Lysates from serum-starved, Tet-induced HEK293^eIF4G-e cells expressing the indicated forms of eIF4G-e were subjected to immunoblotting. The effects of pretreatment with 10 μM CGP57380 and stimulation with 0.5 nM STD are shown. (B) Treatments with CGP57380 and STD-mediated stimulation enhance Mnk1 binding to eIF4G. Serum-starved, Tet-induced HEK293^eIF4G-e cells or the corresponding eIF4G(Ser1186/1188Ala) or eIF4G(Ser1186/1188Glu) mutants were pretreated for 1 h with 10 μM CGP57380 and stimulated with 0.5 nM STD as indicated. Cell lysates were subjected to Flag-IP followed by immunoblotting. Bands were quantified as described in Materials and Methods, and the Mnk1 signal density was adjusted for the signal of the loading control (eIF4E). The Mnk1 signal in mock-induced, untreated cells was set at 1. Comparison of exogenous eIF4G-e variants reveals a potent increase in Mnk1 binding upon STD stimulation in cells pretreated with CGP57380.