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 Thr308 and Ser473 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 Ser473 and Thr308 phosphorylation upon acute PI3K activation and provide evidence for uncoupling of Ser473 and Thr308 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 Ser473-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.

Inhibition of the amino‐acid transporter LAT1 demonstrates anti‐neoplastic activity in medulloblastoma

“…Primary murine neurons were isolated from E16.5 mouse embryos cortices dissociated with trypsin as previously described. 29 Briefly, neurons were plated onto 12-well plates and cul-…”

Gap junctions regulate vessel diameter in chick chorioallantoic membrane vasculature by both tone‐dependent and structural mechanisms

“…25 CAM tissue lysates were prepared by excision of 5 mm 2 of CAM tissue, which was washed two times with ice-cold PBS, lysed in ice-cold RIPA buffer supplemented with protease inhibitors (Roche cOmplete, Mini Protease Inhibitor Cocktail) and subsequently homogenized with a homogenizer (Minilys, Bertin Instruments). 25 CAM tissue lysates were prepared by excision of 5 mm 2 of CAM tissue, which was washed two times with ice-cold PBS, lysed in ice-cold RIPA buffer supplemented with protease inhibitors (Roche cOmplete, Mini Protease Inhibitor Cocktail) and subsequently homogenized with a homogenizer (Minilys, Bertin Instruments).…”

“…Western blotting was performed as described. 25 CAM tissue lysates were prepared by excision of 5 mm 2 of CAM tissue, which was washed two times with ice-cold PBS, lysed in ice-cold RIPA buffer supplemented with protease inhibitors (Roche cOmplete, Mini Protease Inhibitor Cocktail) and subsequently homogenized with a homogenizer (Minilys, Bertin Instruments). Homogenates were centrifuged at 20 000 g, and supernatant was collected for protein quantification analysis using BCA Thermo Scientific Pierce™ Protein Assay.…”

Inhibition of the amino-acid transporter LAT1 demonstrates anti-neoplastic activity in medulloblastoma

“…The murine cerebellar astrocyte cells C8-D1A were obtained from the American Tissue and HD-MB03 [27] (Creative Bioarray Cat.No.CSC-C6241X, passage 60) were obtained from Dr. C. Pouponnot (Institut Curie, France) and cultivated in Minimum Essential Medium (MEM, Invitrogen) supplemented with 10% iFCS and 90% RPMI-1640 supplemented with 10% iFCS respectively. Primary murine neurons were isolated from E16.5 mouse embryos cortices dissociated with trypsin as previously described [29]. Briefly, neurons were plated onto 12-wells plates and cultured in neurobasal medium (21103049 Gibco) supplemented with B27 (A3582801 Gibco) and glutamine.…”