title: New hierarchical phosphorylation pathway of the translational repressor eIF4E-binding protein 1 (4E-BP1) in ischemia-reperfusion stress.
abstract: 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.
authors.affiliation: Department of Investigation, Hospital Ramón y Cajal, 28034 Madrid, Spain.
authors.forename: María I
authors.initials: MI
authors.lastname: Ayuso
authors.forename: Macarena
authors.initials: M
authors.lastname: Hernández-Jiménez
authors.forename: María E
authors.initials: ME
authors.lastname: Martín
authors.forename: Matilde
authors.initials: M
authors.lastname: Salinas
authors.forename: Alberto
authors.initials: A
authors.lastname: Alcázar
mesh_terms: D000818:Animals; D001921:Brain / Q000378:metabolism* / Q000473:pathology; D002352:Carrier Proteins / Q000378:metabolism*; D039561:Eukaryotic Initiation Factor-4E / Q000378:metabolism; D039603:Eukaryotic Initiation Factor-4G / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D014176:Protein Biosynthesis; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D015427:Reperfusion Injury / Q000378:metabolism* / Q000473:pathology; D013312:Stress, Physiological; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; C499526:Eif4ebp1 protein, rat; D039561:Eukaryotic Initiation Factor-4E; D039603:Eukaryotic Initiation Factor-4G; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; C546845:mTOR protein, rat; D051057:Proto-Oncogene Proteins c-akt; D058570:TOR Serine-Threonine Kinases