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 Thr69-phosphorylated alone, Thr69- and Thr36/Thr45-phosphorylated, all these plus Ser64 phosphorylation, and dephosphorylation of the sites analyzed. In control or IR conditions, no Thr36/Thr45 phosphorylation alone was detected without Thr69 phosphorylation, and neither was Ser64 phosphorylation without Thr36/Thr45/Thr69 phosphorylation detected. Ischemic stress induced 4E-BP1 dephosphorylation at Thr69, Thr36/Thr45, and Ser64 residues, with 4E-BP1 remaining phosphorylated at Thr69 alone or dephosphorylated. In the subsequent reperfusion, 4E-BP1 phosphorylation was induced at Thr36/Thr45 and Ser64, in addition to Thr69. 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 Thr69 is phosphorylated first followed by Thr36/Thr45 phosphorylation, and Ser64 is phosphorylated last. Thr69 phosphorylation alone allows binding to eIF4E, and subsequent Thr36/Thr45 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.
Highlights
An important control point in the translation process in eukaryotic organisms is the recruitment of the 40 S ribosomal
A key step in this process is the assembly of eukaryotic initiation factor 4F complex, which contains: the initiation factor eIF4A, an ATP-dependent RNA helicase; eIF4E, which binds to the mRNA 5Ј cap structure m7GpppN (7-methylguanosine triphosphate and N is any nucleotide); and eIF4G, a scaffolding protein that provides docking sites for the aforementioned initiation factors and binds the poly(A)-binding protein (PABP), thereby circularizing the mRNA [1,2,3]. eIF4E recruits eIF4G and eIF4A to assemble the eIF4F complex and binds to the 5Ј cap [3]
Phosphorylation—we studied the phosphorylation of mammalian target of rapamycin (mTOR) at Ser2448, a residue that is correlated with kinase activity [24], and the activity of the upstream effector of mTOR complex 1 (mTORC1), the serine/threonine-protein kinase Akt, through phosphorylation at Thr308 and Ser473 [25]
Summary
4E-BP, eIF4E-binding protein; IR, ischemiareperfusion; SHC, sham control; mTOR, mammalian target of rapamycin; mTORC1, mTOR complex 1; TOS, TOR signaling; ROS, reactive oxygen species; PMS, postmitochondrial supernatant; PP2A, protein phosphatase 2A. New Hierarchical Phosphorylation of 4E-BP1 tion of 4E-BP1 at Thr, Thr, Ser, and Thr sites and the release of 4E-BP1 from eIF4E [6, 7]. The hierarchical phosphorylation of 4E-BP1 at these sites is stimulated by agents such as insulin, growth factors and serum, and in many cell types, this effect requires the presence of amino acids in the culture medium This compelling evidence has been reported in stimulated cultured cells, including cell lines and transfected cells, and the physiological roles of these phosphorylation reactions in the normal control of 4E-BP1 remain to be fully established [6]. We discovered a new hierarchical phosphorylation pathway for 4E-BP1 and suggest a new physiological outcome for the phosphorylation sites in 4E-BP1
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