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00925njm 22002777a 4500 dc Ayuso, María I author Hernández Jiménez, Macarena author Martín, María E author Salinas, Matilde author Alcázar, Alberto author 2010 Eukaryotic initiation factor (eIF) 4E-binding protein 1 (4E-BP1) is a translational repressor that is characterized by its capacity to bind specifically to eIF4E and inhibit its interaction with eIF4G. Phosphorylation of 4E-BP1 regulates eIF4E availability, and therefore, cap-dependent translation, in cell stress. This study reports a physiological study of 4E-BP1 regulation by phosphorylation using control conditions and a stress-induced translational repression condition, ischemia-reperfusion (IR) stress, in brain tissue. In control conditions, 4E-BP1 was found in four phosphorylation states that were detected by two-dimensional gel electrophoresis and Western blotting, which corresponded to Thr(69)-phosphorylated alone, Thr(69)- and Thr(36)/Thr(45)-phosphorylated, all these plus Ser(64) phosphorylation, and dephosphorylation of the sites analyzed. In control or IR conditions, no Thr(36)/Thr(45) phosphorylation alone was detected without Thr(69) phosphorylation, and neither was Ser(64) phosphorylation without Thr(36)/Thr(45)/Thr(69) phosphorylation detected. Ischemic stress induced 4E-BP1 dephosphorylation at Thr(69), Thr(36)/Thr(45), and Ser(64) residues, with 4E-BP1 remaining phosphorylated at Thr(69) alone or dephosphorylated. In the subsequent reperfusion, 4E-BP1 phosphorylation was induced at Thr(36)/Thr(45) and Ser(64), in addition to Thr(69). Changes in 4E-BP1 phosphorylation after IR were according to those found for Akt and mammalian target of rapamycin (mTOR) kinases. These results demonstrate a new hierarchical phosphorylation for 4E-BP1 regulation in which Thr(69) is phosphorylated first followed by Thr(36)/Thr(45) phosphorylation, and Ser(64) is phosphorylated last. Thr(69) phosphorylation alone allows binding to eIF4E, and subsequent Thr(36)/Thr(45) phosphorylation was sufficient to dissociate 4E-BP1 from eIF4E, which led to eIF4E-4G interaction. These data help to elucidate the physiological role of 4E-BP1 phosphorylation in controlling protein synthesis. Ayuso MI, Hernández-Jiménez M, Martín ME, Salinas M, Alcázar A. New hierarchical phosphorylation pathway of the translational repressor eIF4E-binding protein 1 (4E-BP1) in ischemia-reperfusion stress. J Biol Chem. 2010 Nov 5;285(45):34355-63. doi: 10.1074/jbc.M110.135103. Epub 2010 Aug 24. PMID: 20736160; PMCID: PMC2966049. 0021-9258 10.1074/jbc.M110.135103 https://hdl.handle.net/20.500.14352/100067 1083-351X https://www.doi.org/10.1074/jbc.M110.135103 New hierarchical phosphorylation pathway of the translational repressor eIF4E-binding protein 1 (4E-BP1) in ischemia-reperfusion stress