title: Capillary Isoelectric Focusing of Akt Isoforms Identifies Highly Dynamic Phosphorylation in Neuronal Cells and Brain Tissue.
abstract: The PI3K/PTEN/Akt pathway has been established as a core signaling pathway that is crucial for the integration of neurons into neuronal circuits and the maintenance of the architecture and function of neurons in the adult brain. Akt1-3 kinases are specifically activated by two phosphorylation events on residues Thr(308) and Ser(473) upon growth factor signaling, which subsequently phosphorylate a vast cohort of downstream targets. However, we still lack a clear understanding of the complexity and regulation of isoform specificity within the PI3K/PTEN/Akt pathway. We utilized a capillary-based isoelectric focusing method to study dynamics of Akt phosphorylation in neuronal cells and the developing brain and identify previously undescribed features of Akt phosphorylation and activation. First, we show that the accumulation of multiple phosphorylation events on Akt forms occur concurrently with Ser(473) and Thr(308) phosphorylation upon acute PI3K activation and provide evidence for uncoupling of Ser(473) and Thr(308) phosphorylation, as well as differential sensitivities of Akt1 forms upon PI3K inhibition. Second, we detect a transient shift in Akt isoform phosphorylation and activation pattern during early postnatal brain development, at stages corresponding to synapse development and maturation. Third, we show differential sensitivities of Ser(473)-Akt species to PTEN deletion in mature neurons, which suggests inherent differences in the Akt pools that are accessible to growth factors as compared with the pools that are controlled by PTEN. Our study demonstrates the presence of complex phosphorylation events of Akt in a time- and signal-dependent manner in neurons.
authors.affiliation: From the Charité-Universitätsmedizin Berlin, Cluster of Excellence NeuroCure and Institute of Biochemistry, Berlin 10117, Germany.
authors.forename: Sandra
authors.initials: S
authors.lastname: Schrötter
authors.affiliation: From the Charité-Universitätsmedizin Berlin, Cluster of Excellence NeuroCure and Institute of Biochemistry, Berlin 10117, Germany.
authors.forename: George
authors.initials: G
authors.lastname: Leondaritis
authors.affiliation: From the Charité-Universitätsmedizin Berlin, Cluster of Excellence NeuroCure and Institute of Biochemistry, Berlin 10117, Germany Britta.Eickholt@charite.de.
authors.forename: Britta J
authors.initials: BJ
authors.lastname: Eickholt
mesh_terms: D000328:Adult; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism*; D006801:Humans; D007525:Isoelectric Focusing / Q000379:methods*; D018345:Mice, Knockout; D009447:Neuroblastoma / Q000378:metabolism* / Q000473:pathology; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D051059:PTEN Phosphohydrolase / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D020033:Protein Isoforms; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: D020033:Protein Isoforms; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt; D051059:PTEN Phosphohydrolase
keywords: Akt PKB; brain; capillary isoelectric focusing, phosphorylation; cell signaling; growth factors; neurons; phosphatase and tensin homolog (PTEN); post-translational modification (PTM)