FIG. 4.

Human and mouse Mnk1 and Ad late 100K protein share a hydrophilically charged amino acid-rich sequence required for binding to eIF4G. (A) Alignment of homologous sequences found in human and mouse eIF4E kinase, Mnk1 proteins, and Ad 100K protein. (B) 293T cells were cotransfected with plasmids expressing HA-eIF4GI and GST or GST-100K plasmids. 100K peptides are indicated by amino acid number of retained fragments. 280-345 RRKA is a 100K mutant peptide containing AAA instead of the homologous RRK sequence. GST-Mnk1 is a wild-type clone. GST-Mnk1[RRKA] is a mouse Mnk1 mutant with AAA instead of RRK. At 36 h posttransfection cellular extracts were prepared and equal amounts of total protein were incubated with glutathione-Sepharose to recover GST fusion proteins. Complexes were resolved by SDS-10% PAGE, and the association of eIF4GI with GST proteins was detected by immunoblot analysis with HA antibody. Expression levels of input HA-eIF4GI and GST fusion proteins (first and third panels from left) were determined by immunoblotting. (C) 293T cells were transfected with plasmids expressing HA-eIF4GI and either Flag or Flag-Mnk1, or GST or GST-Mnk1. Cellular lysates were prepared with 50 mM NaF or 150 mM NaCl, respectively, at 36 h posttransfection. Flag-tagged or GST fusion proteins were isolated by immunoprecipitation with anti-Flag antibody or by glutathione-Sepharose 4B chromatography, respectively, and proteins were resolved by SDS-10% PAGE. Interaction of Flag-Mnk1 and GST-100K [280-345] with HA-eIF4GI was detected by immunoblotting with an antibody to the HA epitope. Typical results from at least three independent experiments are shown.