Characterization of 100K protein. (A) Predicted structural regions identified within the 100K protein are indicated by amino acid position: coiled-coil region (CCR 439-458), RRM 383-458, Rev-like NES 383-392, RGG box, and overlapping NLS 727-764. (B) 293T cells were transfected with plasmids expressing Flag, Flag-100K, or truncated versions of 100K retaining amino acid sequences as indicated: Flag-100K [1-726] (with RGG box) or Flag-100K [1-345] (without RRM and RRG box). Flag-tagged proteins were isolated by immunoprecipitation with anti-Flag antibody. Equal amounts of Flag immune complexes as shown by immunoblotting (right panel) were incubated with 4 ng of ³²P-labeled, in vitro-transcribed RNAs corresponding to the capped or uncapped-CR3 5′ UTR. (see Materials and Methods for more details). RNA-protein complexes were subjected to UV cross-linking, and complexes were analyzed by denaturing SDS-10% PAGE followed by autoradiography (left panel). PI indicates immunoprecipitation with preimmune serum. Quantification in all figures was obtained by densitometry of typical results from at least three independent experiments and is described in the text. (C) HeLa cells were transfected with plasmids expressing Flag-100K, Flag-100KΔRGG [1-726], or Flag-100KΔNES and grown on coverslips. Cells were fixed-permeabilized and incubated with anti-Flag antibody, followed by incubation with mouse secondary antibody coupled to fluorescein. Nuclei were stained with Hoechst 33258 dye. Cells were visualized and photographed using a Zeiss Axiophot microscope. IF, immunofluorescence.

Fine mapping of eIF4G-binding site within 100K protein. (A) 293T cells were cotransfected with plasmids expressing HA-eIF4GI (20) and either GST or GST-100K plasmids containing 100K peptides as shown. At 36 h posttransfection GST fusion proteins were recovered by glutathione-Sepharose chromatography and resolved by SDS-10% PAGE, and HA-eIF4GI-associated proteins were detected by immunoblot analysis. (B) 293T cells were cotransfected with plasmids as described above, and GST fusion proteins associated with HA-eIF4GI were analyzed. Typical results from at least three independent experiments are shown. CCR, coiled-coil region.

Analysis of Mnk1 protein displacement from eIF4G polypeptide by either wild-type or RRKA mutant 100K [280-345] peptides. 293T cells were transfected with plasmids expressing HA-eIF4GI, Flag or Flag-Mnk1, and either GST, GST-100K [280-345], or GST-100K [280-345 RRKA]. At 36 h posttransfection, cells were lysed and Flag-tagged proteins were immunoprecipitated by Ezview Red anti-Flag M2 affinity gel and resolved by SDS-10% PAGE. Proteins associated with Flag-tagged proteins (Flag or Flag-Mnk1) were detected by immunoblot analysis using antibodies specific to the HA epitope for HA-eIF4GI and anti-GST for GST-100K [280-345] peptides (right panels). GST proteins were recovered by glutathione-Sepharose beads and resolved by SDS-10% PAGE, and associated proteins were identified by immunoblot analysis as shown. Levels of ectopically expressed proteins are shown in the left panels. Immunoblots were quantified by densitometry, and standard deviations were calculated from three independent experiments.

RNA dependence for Mnk1 but not 100K interaction with eIF4F complexes. (A) 293T cells were transfected with plasmids expressing GST or GST-Mnk1 proteins, cellular lysates were prepared 36 h posttransfection, and GST fusion proteins were recovered by glutathione-Sepharose chromatography with or without prior digestion by RNase A (100 μg/ml for 2 h). Mnk1-associated proteins were resolved by SDS-10% PAGE and identified by immunoblot analysis with antibodies to eIF4G, eIF4E, eIF4A, and GST(-Mnk1) polypeptides. Original levels of proteins are shown in the right panels. (B) As in panel A but proteins were recovered by immunoprecipitation of eIF4G and subjected to immunoblot detection of eIF4G, GST(-Mnk1), eIF4A, and eIF4E. (C) Cells were transfected with Flag or Flag-100K protein and treated with RNase as described above, and immunoprecipitation of eIF4G was carried out. Associated proteins were resolved by SDS-PAGE and immunoblotted for eIF4G and Flag-100K protein. Autoradiograms were quantified by densitometry of three independent studies, and standard deviations were determined.

Mapping of eIF4G-binding site within 100K protein. 293T cells were transfected with plasmids as indicated, and then at 36 h posttransfection equal amounts of total protein lysates were incubated with eIF4G antiserum (eIF4G IP) or preimmune serum (Preim. IP). Immune complexes were resolved by SDS-10% PAGE, and associated proteins were detected by immunoblot analysis of endogenous eIF4G (anti-eIF4G) or Flag (anti-Flag). Typical results from at least three independent experiments are shown. IgG, immunoglobulin G; CCR, coiled-coil region.

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.

eIF4E phosphorylation and cap-dependent translation in cells expressing wild-type or mutant 100K peptides. (A) 293T cells were cotransfected with plasmids expressing HA-eIF4E and either GST, GST-100K [280-345], GST-100K [280-345 RRKA], mouse GST-Mnk1, or GST-Mnk1[RRKA] proteins. At 36 h posttransfection cellular extracts were prepared, HA-eIF4E was immunoprecipitated, and protein amounts were normalized by HA-eIF4E protein levels (middle panel), and then eIF4E phosphorylation was assessed by one-dimensional isoelectric focusing (IEF) gel electrophoresis (top panel). (B) 293T cells were transfected with plasmids expressing β-Gal (cap-dependent translation), GFP (EMCV IRES-dependent translation), and either GST, GST-100K [280-345], GST-100K [280-345 RRKA], GST-Mnk1, or GST-Mnk1[RRKA] proteins. At 36 h posttransfection cells were lysed, and after normalization by GFP, proteins were resolved by SDS-10% PAGE and subjected to immunoblot analysis for the indicated proteins. (C) 293T cells were transfected with plasmids expressing β-Gal (Ad tripartite leader-dependent translation), GFP (EMCV IRES-dependent translation), and either GST, GST-100K [280-345], GST-100K [280-345 RRKA], GST-Mnk1, or GST-Mnk1[RRKA]. At 36 h posttransfection, equal amounts of total protein were resolved by SDS-10% PAGE, transferred to a polyvinylidene difluoride membrane, and immunoblotted as shown. Autoradiograms were quantified by densitometry of three independent experiments, and standard deviations were determined.