Fig. 4.

4E-BP1 is hyperphosphorylated to the δ phosphoisoform during mitosis. (A) pH3^S10+ mitotic 293 cells have higher levels of p4E-BP1^T37/T46+ saturation than cells in other portions of the cell cycle. Dual flow cytometry staining for pH3^S10 and p4E-BP1^T37/T46 was performed on 293 cells synchronized by double-thymidine block and release, which have peak mitotic entry at 10 h postrelease (see also Fig. S6). Vertical bar represents the centroid for p4E-BP1^T37/T46+ fluorescence staining at time 0 h. To determine if 4E-BP1 phosphorylation depends on mTOR activity, cells were also treated 1 h before harvesting with 5 µM PP242. Mitotic cells formed an orthogonal pH3^S10+/p4E-BP1^T37/T46+ population having high levels of inactivated (phosphorylated) 4E-BP1 that were not dependent on mTOR activity. In contrast, interphase pH3^S10– cells were largely mTOR inhibition-sensitive. PP242 treatment increases mitotic entry at 8 h postrelease. (B) PP242-resistant δ-4E-BP1 is formed during peak mitotic entry. Protein lysates were collected from cells in A and immunoblotted for p4E-BP1 and pH3^S10. Representative results are shown from three independent experiments.