The study investigates the hierarchical phosphorylation of 4E-BP1, a translation inhibitor that binds to eIF4E and prevents translation initiation. Phosphorylation of 4E-BP1 by kinases such as PI3K and mTOR leads to its dissociation from eIF4E, thereby promoting translation. The phosphorylation of 4E-BP1 is a sequential process, with Thr37 and Thr46 being phosphorylated first, followed by Thr70, and finally Ser65. These phosphorylation events are essential for the release of 4E-BP1 from eIF4P. However, phosphorylation of Ser65 and Thr70 alone is not sufficient to fully dissociate 4E-BP1 from eIF4E, indicating that multiple phosphorylation events are required for complete release. The study used mass spectrometry and phosphopeptide mapping to identify the phosphorylation sites and determine the order of phosphorylation events. The results show that the phosphorylation of 4E-BP1 is a tightly regulated process, with specific phosphorylation sites playing critical roles in the regulation of translation initiation. The findings highlight the importance of phosphorylation in the regulation of translation and provide insights into the molecular mechanisms underlying the control of protein synthesis.The study investigates the hierarchical phosphorylation of 4E-BP1, a translation inhibitor that binds to eIF4E and prevents translation initiation. Phosphorylation of 4E-BP1 by kinases such as PI3K and mTOR leads to its dissociation from eIF4E, thereby promoting translation. The phosphorylation of 4E-BP1 is a sequential process, with Thr37 and Thr46 being phosphorylated first, followed by Thr70, and finally Ser65. These phosphorylation events are essential for the release of 4E-BP1 from eIF4P. However, phosphorylation of Ser65 and Thr70 alone is not sufficient to fully dissociate 4E-BP1 from eIF4E, indicating that multiple phosphorylation events are required for complete release. The study used mass spectrometry and phosphopeptide mapping to identify the phosphorylation sites and determine the order of phosphorylation events. The results show that the phosphorylation of 4E-BP1 is a tightly regulated process, with specific phosphorylation sites playing critical roles in the regulation of translation initiation. The findings highlight the importance of phosphorylation in the regulation of translation and provide insights into the molecular mechanisms underlying the control of protein synthesis.