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.

Oligodendrocyte precursor cells (OPCs) are extremely efficient at remyelination. These cells persist in the adult human central nervous system and can proliferate. However, the failure to remyelinate is a pathological characteristic of the human demyelinating disease multiple sclerosis (MS), which suggests that these cells are ineffective in this disorder. This paper reports that IgG antibodies in the cerebrospinal fluid (CSF) of MS patients specifically recognize an antigen on OPCs in culture. Control patients were found not to possess these antibodies. The antigen was immunoprecipitated in cell extracts from cultures with purified IgG from MS CSF. Peptide mass fingerprinting identified it as the β type of heat shock protein 90 (Hsp90). Two‐dimensional electrophoresis and immunoblot showed that this antigen in fact corresponds to two specific isoforms of Hsp90β. Several control assays using monoclonal and polyclonal anti‐Hsp90 antibodies confirmed the specific expression of Hsp90 on OPCs. Labeling OPCs in vivo with MS CSF and anti‐Hsp90 antibodies and subsequent immunofluorescence confocal microscopy located the antigen on the cell surface. The binding of CSF antibodies from MS patients to the OPC surface led to complement activation and significant extinction of the OPC population. These results suggest that OPCs may be a target of anti‐Hsp90 antibodies in MS patients and that this could prevent remyelination.