This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The eIF4E-binding proteins (4E-BPs) are a family of three conserved polypeptides that inhibit translation by binding to and sequestering the mRNA 5 cap binding protein, eIF4E. Binding of 4E-BPs to eIF4E is normally regulated by phosphorylation: hypophosphorylated 4E-BPs bind strongly to eIF4E, whereas hyperphosphorylated isoforms do not. The 4E-BPs are differentially expressed in mouse tissues such that 4E-BP2 is highly enriched in brain. Concordant with this, our laboratory has shown that 4E-BP2 plays an important role in learning and memory in mice. Two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis of a mixture of protein extracts from brain and cultured mouse embryonic fibroblasts (MEFs) revealed that the brain-derived 4E-BP2 isoforms diverge from those corresponding to multiple phosphorylation events in MEFs. Unlike 4E-BP2 from cultured cells, brain-derived 4E-BP2 is also resistant to phosphatase treatment. These findings suggest that 4E-BP2 in brain is subject to different or additional posttranslational modifications (PTMs) than phosphorylation. Furthermore, these modifications show developmental regulation: the slowest migrating of the three 4E-BP2 isoforms is not present in the brain until postnatal day 15 and subsequently persists into adulthood. It is therefore of biochemical, neurobiological, and developmental interest to identify these modifications. A top-down mass spectrometric approach employing 12T FTICR mass spectrometer equipped with electron capture dissociation capability will be used to determine both the identity of the modifications and the amino acid sites of covalent attachment. These novel PTMs of 4E-BP2 may influence its binding to 4E; consequently, protein synthesis in the brain will be subject to an altered control mechanism. Subsequent analyses will compare the PTMs of adult brain 4E-BP2 with that of early postnatal brains (<15 days) to understand the changes that this protein undergoes during mammalian development.
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