FIG 8.
Regulation of eIF4A and Mnk1 interactions by mitotic phosphorylation of eIF4G(Ser1232) by Cdk1:cyclin B1. (A) Schematic representation of the eIF4G(1222-1600) fragment. Structural HEAT2 and -3 domains, the distal portion (aa 1222 to 1235) of the IDL, the position of p-Ser1232 (*), and the interactions with eIF3A/-4A/-4B and Mnk1 are shown. Dynamics of endogenous eIF4G(Ser1232) and H3(Ser10) phosphorylation (B) and eIF4G(1222-1600) interactions (C) during cell cycle progression are also shown. HEK293 cells transfected with wt or Ser1232Ala mutant myc-eIF4G(1220-1600)-Flag fragments were synchronized by Th block (4 mM, 9 h) at the G1/S boundary, followed by NOC (200 nM, 16 h) arrest in mitosis. Cells were released from NOC block into growth medium for 15 to 180 min. Lysates were subjected to immunoblotting (B) or Flag-IP followed by immunoblotting with the indicated antibodies. Endogenous eIF4G (B) was detected with an antibody against an N-terminal epitope; myc-eIF4G(1222-1600) was detected with anti-myc antibody (C). The assay was repeated three times, and a representative series is shown.