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. 2017 Sep 7;140(10):2610–2622. doi: 10.1093/brain/awx203

Figure 3.

Figure 3

Analysis of AKT3 activity in vitro. (A) The primary structure of AKT3 showing the relative positions of the pleckstrin homology (PH) domain for lipid binding the catalytic kinase domain and C-terminal (C-ter) region. Mutations identified to date are shown along with the numbers of patients with these mutations in brackets. (B) Catalytic kinase domain and C-terminal localizing patient-derived AKT3 mutations are associated with elevated kinase activity. Ectopically expressed wild-type (WT) AKT, a kinase dead variant K177M, the E17K activating pleckstrin homology domain mutant and various patient mutants were assessed for kinase activity using a GSK3β peptide as a substrate in an ex vivo kinase assay. The upper panel shows immune detection of phosphorylated GSK3β peptide following western blotting with anti-phospho-GSK3β (Ser9/Ser21) antibody. The patient mutants all exhibit elevated phospho-activity compared to wild-type. The graph depicts quantitation of phospho-GSK3β (Ser9/Ser21) signal (a.u. = arbitrary units). Error bars represent mean ± SD (n = 4), P-values were determined using Student’s t-test. (C) Pleckstrin homology domain localizing patient mutations are associated with elevated kinase activity and altered phospholipid-binding profile. Left panels show western blot analysis of phospho-GSK3β (Ser9/Ser21) of ectopically expressed wild-type, K177M kinase dead and three pleckstrin homology domain patient mutants; E17K, N53K and F54Y. The graph depicts quantitation of phospho-GSK3β (Ser9/Ser21) signal. Error bars represent mean ± SD (n = 4), P-values were determined using Student’s t-test. The bottom panels depict PIP-membranes seeded with various lipids and phospholipids for dot blot binding analysis. Ectopically expressed FLAG-tagged wild-type and AKT3 pleckstrin homology domain mutants were incubated with the PIP Strips and bound protein detected by western blotting using anti-FLAG. All three pleckstrin homology domain mutants exhibit altered and elevated binding to specific phospholipids compared to wild-type. DMEG = dysplastic megalencephaly; HMEG = hemimegalencephaly; LPA = lysophophatidic acid; LPC = lysophosphocholine; MEG = megalencephaly; P = phosphate; PA = phosphatidic acid; PC = phosphatidylcholine; PE = phosphatidylethanolamine; PMG = polymicrogryria; PS = phosphatidylserine; PtdIns = phosphatidylinositol; S1P = sphingosine-1-phosphate.