The study investigates the phosphorylation of the translation inhibitor 4E-BP1, which regulates translation initiation by binding to the eIF4E factor. The authors use mass spectrometry and two-dimensional isoelectric focusing/SDS-PAGE to identify the serum-responsive and rapamycin-sensitive phosphorylation sites as Ser 65 and Thr 70. They establish the order of 4E-BP1 phosphorylation in vivo, showing that Thr 37/Thr 46 is followed by Thr 70, and Ser 65 is phosphorylated last. Additionally, they demonstrate that phosphorylation of Ser 65 alone is insufficient to block binding to eIF4E, indicating that multiple phosphorylation events are necessary for the dissociation of 4E-BP1 from eIF4E. The findings provide insights into the regulatory mechanisms of 4E-BP1 and its role in controlling translation initiation.The study investigates the phosphorylation of the translation inhibitor 4E-BP1, which regulates translation initiation by binding to the eIF4E factor. The authors use mass spectrometry and two-dimensional isoelectric focusing/SDS-PAGE to identify the serum-responsive and rapamycin-sensitive phosphorylation sites as Ser 65 and Thr 70. They establish the order of 4E-BP1 phosphorylation in vivo, showing that Thr 37/Thr 46 is followed by Thr 70, and Ser 65 is phosphorylated last. Additionally, they demonstrate that phosphorylation of Ser 65 alone is insufficient to block binding to eIF4E, indicating that multiple phosphorylation events are necessary for the dissociation of 4E-BP1 from eIF4E. The findings provide insights into the regulatory mechanisms of 4E-BP1 and its role in controlling translation initiation.