Hyperphosphorylation and decreased binding of eIF4GII to eIF4E in mitosis. (A) Slower electrophoretic mobility of eIF4GII and decreased binding to eIF4E. Total extract from interphase (I) or nocodazole-arrested (M) HeLa cells was resolved by SDS-PAGE and analyzed by Western blotting with anti-eIF4GII antibody, or by far-Western with an ³²P-HMK–eIF4E probe. (B) Phosphatase treatment of eIF4GII. eIF4GII immunoprecipitated from interphase (I) or nocodazole-arrested (M) HeLa cells was untreated, treated with buffer alone, or with calf intestinal acid phosphatase (CIAP) as described in Materials and Methods. Immunoprecipitates resolved by SDS-PAGE were analyzed by Western blotting with anti-eIF4GII antibody.

Hyperphosphorylation and decreased binding of 4E-T to eIF4E in mitosis. (A) Slower electrophoretic mobility of 4E-T and decreased binding to eIF4E. Total extract from interphase (I) or nocodazole-arrested (M) HeLa cells was resolved by SDS-PAGE, and analyzed by Western blotting with anti-4E-T antibody (lanes 1,2) or by far-Western with an ³²P-HMK—eIF4E probe (lanes 3,4). (*) Cross-reacting polypeptide detected in Western blotting, but not following immunoprecipitation. (B) Phosphatase treatment of 4E-T. 4E-T immunoprecipitated from interphase (I) or nocodazole-arrested (M) HeLa cells were untreated, treated with buffer alone, or with CIAP as described in Materials and Methods. Immunoprecipitates were resolved by SDS-PAGE, and analyzed by Western blotting with anti-4E-T antibody.

Dephosphorylation and increased binding of 4E-BP1 to eIF4E in mitosis. (A) Hypophosphorylation of 4E-BP1. HeLa cell extract from interphase (I) or nocodazole-arrested (M) cells was resolved by SDS-PAGE, and analyzed by Western blotting with anti-actin (top), anti-eIF4E (middle), or anti 4E-BP1 (bottom). (B) Increased binding of 4E-BP1 to eIF4E. HeLa cell extract from interphase (I) and nocodazole-treated (M) cells was resolved by SDS-PAGE and analyzed by Western blotting with anti-4E-BP1 (top) or by far-Western with ³²P-HMK-eIF4E as a probe (bottom). (C) eIF4F complex disruption. Extract from interphase (I) or nocodazole-arrested (M) HeLa cells was incubated with an m⁷GDP resin. Input, flow-through, and bound material was resolved by SDS-PAGE and analyzed by Western blotting with the indicated antibodies. (D) Decreased eIF4E/Mnk1 interaction. Extracts from interphase (I) or nocodazole-arrested (M) HeLa cells transfected with Flag–Mnk1 and HA–eIF4E was immunoprecipitated with anti-Flag antibody. Immunoprecipitates were resolved by SDS-PAGE, and analyzed by Western blotting with anti-HA or anti-Flag antibody.

eIF4E/4E-BP1 expression and interaction throughout the cell cycle. (A) Flow cytometry analysis of HeLa cells following release from a double thymidine block. (x axis) Fluorescence intensity; (y axis) cell number. (B) Expression of eIF4E and 4E-BP1 through the cell cycle. HeLa extract from interphase cells (lane 1), cells arrested at G₁/S by double-thymidine block (lane 2), and from cells released from a double thymidine block (lanes 3–11) was resolved by SDS-PAGE and analyzed by Western blotting with the indicated antibodies. (C) eIF4E/4E-BP1 interaction through the cell cycle. The extract described in B was incubated with m⁷GDP resin. Bound proteins were resolved by SDS-PAGE and analyzed by Western blotting with the indicated antibodies. (D) eIF4F complex disruption in mitosis. HeLa extract from interphase cells (lane 1) or from cells collected by mitotic shake-off (lane 2) was incubated with m⁷GDP resin. Bound proteins were resolved by SDS-PAGE, and analyzed by Western blotting with the indicated antibodies. (E) Protein synthesis in mitotic versus interphase cells. [³⁵S]methionine incorporated into TCA precipitable material was measured and expressed in arbitrary units. Values represent the average of two separate experiments.

Decreased eIF4E phosphorylation, but increased Mnk1 activity in mitotic cells. (A) Specificity of anti-phospho-specific eIF4E antibody. Total HeLa extract from asynchronous cells was resolved by isoelectric focusing (as described in Materials and Methods), and analyzed by Western blotting with anti-phospho-specific eIF4E (lane 1), or anti-eIF4E (lane 2) antibodies. (B) eIF4E phosphorylation at different phases of the cell cycle. Total HeLa extract from interphase, aphidicolin-treated (G₁/S), mitotic (M shake-off), nocodazole-treated (M nocodazole), starved, or starved and TPA-treated cells was resolved by SDS-PAGE and analyzed by Western blotting with anti-phospho-specific eIF4E (top), or anti-eIF4E (bottom) antibody. (C) Mnk1 gel mobility. Total extract from cells transfected with Flag–Mnk1 and treated with aphidicolin (G₁/S), thymidine (S), nocodazole (M), or extract from starved cells (G₀) was resolved by SDS-PAGE, and analyzed by Western blotting with anti-Mnk1 (top) or anti-Flag (bottom) antibody. (D) Mnk1 activation during mitosis. Extract from interphase or mitotic (nocodazole-arrested) HeLa cells transfected with Flag–Mnk1 wild type or mutants was immunoprecipitated with anti-Flag antibody. Immunoprecipitates were used for immune complex kinase assay using [γ-³²P]ATP and recombinant mouse eIF4E as a substrate (as described in Materials and Methods). Samples were resolved by SDS-PAGE, and revealed by autoradiography (bottom) or by Western blotting using the indicated antibodies (top, middle).