Journal: Brain

Article Title: Mutations of AKT3 are associated with a wide spectrum of developmental disorders including extreme megalencephaly

doi: 10.1093/brain/awx203

Figure Lengend Snippet: Summary of the clinical and molecular findings of AKT3 mutation-positive patients ( n = 24) [AKT3: {"type":"entrez-nucleotide","attrs":{"text":"NM_005465.4","term_id":"332078467","term_text":"NM_005465.4"}} NM_005465.4 ]

Article Snippet: AKT3 expression vector was obtained from OriGene (RC221051) as pCMV6-FLAG-MYC tagged Human cDNA ORF Clone containing AKT3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_005465","term_id":"332078467","term_text":"NM_005465"}} NM_005465 ).

Techniques: Mutagenesis, Functional Assay

Journal: Brain

Article Title: Mutations of AKT3 are associated with a wide spectrum of developmental disorders including extreme megalencephaly

doi: 10.1093/brain/awx203

Figure Lengend Snippet: Brain MRIs of AKT3 mutation-positive children. (A and B) Brain MRI of Patient LR15-262 showing markedly enlarged and dysplastic right cerebral hemisphere with diffuse cortical dysplasia and dysmyelination consistent with hemimegalencephaly. The contralateral hemisphere is markedly decreased in size with areas of cortical dysplasia (hemimicroencephaly). (C and D) Images of Patient LR16-251 showing multifocal areas of dysplastic cortex in the perisylvian, frontal, temporal and occipital regions (arrows). (E and F) Images of Patient LR16-372 showing a thick and dysplastic corpus callosum and deeply infolded perisylvian regions. (G–I) Images of Patient LR16-301 showing striking megalencephaly, ventriculomegaly, stretched but thick corpus callosum, diffuse polymicrogyria with deep infolding in the right occipital lobe, and bilateral periventricular nodular heterotopia (arrowheads). (J) Image of Patient LP96-103 showing diffuse bilateral perisylvian polymicrogyria, ventriculomegaly, cavum septum pellucidum et vergae and diffuse periventricular nodular heterotopia (arrowheads). (K and L) Images of Patient LR13-041 showing a large cerebellum with cerebellar tonsillar ectopia, bilateral polymicrogyria predominantly in the perisylvian region (more severe on the right, arrows) with dysmyelination. (M and N) Images of Patient LR14-271 showing diffuse megalencephaly with a thick corpus callosum and deep infolding in the perisylvian region suggestive of polymicrogyria (arrows). (O and P) Images of Patient LR14-254 showing diffuse megalencephaly, thick corpus callosum and bilaterally diffuse infolding of the perisylvian region suggestive of polymicrogyria (arrows). (Q and R) Images of Patient LR14-025 showing megalencephaly, thick corpus callosum and bilateral diffuse polymicrogyria with increased extra-axial space. (S and T) Images of Patient LR12-470 showing megalencephaly and thick corpus callosum. This patient also had deep infolding in the right perisylvian region suspicious for polymicrogyria, with very limited involvement (arrows). (U and V) Images of Patient LR13-008 showing diffuse megalencephaly and possible area of cortical dysplasia in the right perisylvian region. (W and X) Images of Patient LR14-112 showing diffuse megalencephaly, bilateral perisylvian polymicrogyria and bilateral ventriculomegaly.

Article Snippet: AKT3 expression vector was obtained from OriGene (RC221051) as pCMV6-FLAG-MYC tagged Human cDNA ORF Clone containing AKT3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_005465","term_id":"332078467","term_text":"NM_005465"}} NM_005465 ).

Techniques: Mutagenesis

Journal: Brain

Article Title: Mutations of AKT3 are associated with a wide spectrum of developmental disorders including extreme megalencephaly

doi: 10.1093/brain/awx203

Figure Lengend Snippet: 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.

Article Snippet: AKT3 expression vector was obtained from OriGene (RC221051) as pCMV6-FLAG-MYC tagged Human cDNA ORF Clone containing AKT3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_005465","term_id":"332078467","term_text":"NM_005465"}} NM_005465 ).

Techniques: Activity Assay, In Vitro, Binding Assay, Derivative Assay, Variant Assay, Mutagenesis, Ex Vivo, Kinase Assay, Western Blot, Quantitation Assay, Dot Blot, Incubation

Journal: Brain

Article Title: Mutations of AKT3 are associated with a wide spectrum of developmental disorders including extreme megalencephaly

doi: 10.1093/brain/awx203

Figure Lengend Snippet: Diagram showing the spectrum of AKT3-associated phenotypes. Several groups of partially overlapping developmental brain disorders are associated with AKT3 mutations that include the following phenotypes (i) focal malformations of cortical development that are highly segmental [e.g. focal cortical dysplasia (FCD), hemimegalencephaly (HMEG), dysplastic megalencephaly DMEG; orange]; (ii) bilateral polymicrogyria (PMG) (purple) with or without ventriculomegaly or hydrocephalus (VMEG-HYD; light purple), and heterotopia (HET; blue); (iii) diffuse megalencephaly with intellectual disability (ID) and/or autistic features (AUT) with subtle or no cortical dysplasia (green). *Of note, MCAP and MPPH fit within the second group of AKT3-related disorders, from the brain phenotype perspective. MCAP can be further clinically distinguished by somatic findings (somatic overgrowth, vascular or lymphatic abnormalities), and MPPH by the occurrence of polydactyly in a subset of affected individuals. A general molecular diagnostic workflow for megalencephaly has been proposed (Supplementary Fig. 1). OFC = orbitofrontal cortex.

Article Snippet: AKT3 expression vector was obtained from OriGene (RC221051) as pCMV6-FLAG-MYC tagged Human cDNA ORF Clone containing AKT3 ( {"type":"entrez-nucleotide","attrs":{"text":"NM_005465","term_id":"332078467","term_text":"NM_005465"}} NM_005465 ).

Techniques: Diagnostic Assay

Journal: Structure (London, England : 1993)

Article Title: Crystal Structure of Bicc1 SAM Polymer and Mapping of Interactions between the Ciliopathy-Associated Proteins Bicc1, ANKS3, and ANKS6

doi: 10.1016/j.str.2017.12.002

Figure Lengend Snippet: Full-length (FL) human Bicc1, truncated KH (residues 1–414), IVS (415–872), and SAM-domain (873–974) fragments, and full-length ANKS3 and ANKS6 and their respective ANK (34–220 and 8–423, respectively) and SAM (425–488 and 773–836, respectively) domains were used as baits and preys in yeast two-hybrid (Y2H) assays. A growth control in non-selective medium without leucine (L) and tryptophan (T) is shown in the first column (L‒T‒). Interactions are revealed in triple selective medium (L‒T‒H‒) lacking histidine, supplemented with the indicated increasing amounts of 3-aminotriazole (3-AT).

Article Snippet: ANKS3 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_133450","term_id":"339275864","term_text":"NM_133450"}} NM_133450 ), expressed from pCMV6-Entry and fused to C-terminal Flag tag, was from OriGene (clone ID: RC223862).

Techniques:

Journal: Structure (London, England : 1993)

Article Title: Crystal Structure of Bicc1 SAM Polymer and Mapping of Interactions between the Ciliopathy-Associated Proteins Bicc1, ANKS3, and ANKS6

doi: 10.1016/j.str.2017.12.002

Figure Lengend Snippet: (A) HA-Bicc1 and v5-ANKS6 co-immunoprecipitation with ANKS3-FLAG in HEK293T cell extracts by anti-FLAG beads. Asterisk denotes a second ANKS6-specific band of reduced size.

Article Snippet: ANKS3 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_133450","term_id":"339275864","term_text":"NM_133450"}} NM_133450 ), expressed from pCMV6-Entry and fused to C-terminal Flag tag, was from OriGene (clone ID: RC223862).

Techniques: Immunoprecipitation

Journal: Structure (London, England : 1993)

Article Title: Crystal Structure of Bicc1 SAM Polymer and Mapping of Interactions between the Ciliopathy-Associated Proteins Bicc1, ANKS3, and ANKS6

doi: 10.1016/j.str.2017.12.002

Figure Lengend Snippet: (A) Combinations of HA-Bicc1, ANKS3-Flag, ANKS3ΔCter-Flag and v5-ANKS6 were transiently expressed in HEK293T cells. Cell extracts were fractionated on a continuous15%–60% sucrose gradient and analyzed by western blotting. The migration from the top to the bottom is indicated. γ-Tubulin was used as internal control.

Article Snippet: ANKS3 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_133450","term_id":"339275864","term_text":"NM_133450"}} NM_133450 ), expressed from pCMV6-Entry and fused to C-terminal Flag tag, was from OriGene (clone ID: RC223862).

Techniques: Western Blot, Migration

Journal: Structure (London, England : 1993)

Article Title: Crystal Structure of Bicc1 SAM Polymer and Mapping of Interactions between the Ciliopathy-Associated Proteins Bicc1, ANKS3, and ANKS6

doi: 10.1016/j.str.2017.12.002

Figure Lengend Snippet: (A) Immunofluorescent staining of individually transfected HA-Bicc1, ANKS3-Flag, ANKS3ΔCter-Flag or v5-ANKS6.

Article Snippet: ANKS3 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_133450","term_id":"339275864","term_text":"NM_133450"}} NM_133450 ), expressed from pCMV6-Entry and fused to C-terminal Flag tag, was from OriGene (clone ID: RC223862).

Techniques: Staining, Transfection

Journal: Structure (London, England : 1993)

Article Title: Crystal Structure of Bicc1 SAM Polymer and Mapping of Interactions between the Ciliopathy-Associated Proteins Bicc1, ANKS3, and ANKS6

doi: 10.1016/j.str.2017.12.002

Figure Lengend Snippet: (A) Silencing of AC6 3′UTR luciferase reporter by HA-Bicc1 in the absence or presence of ANKS3-Flag or v5-ANKS6 in transfected HEK293T cells. β-Galactosidase was co-transfected for signal normalization. Data represent mean ± SEM of 3 experiments. *p < 0.05, **p < 0.01.

Article Snippet: ANKS3 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_133450","term_id":"339275864","term_text":"NM_133450"}} NM_133450 ), expressed from pCMV6-Entry and fused to C-terminal Flag tag, was from OriGene (clone ID: RC223862).

Techniques: Luciferase, Transfection

Journal: Structure (London, England : 1993)

Article Title: Crystal Structure of Bicc1 SAM Polymer and Mapping of Interactions between the Ciliopathy-Associated Proteins Bicc1, ANKS3, and ANKS6

doi: 10.1016/j.str.2017.12.002

Figure Lengend Snippet: (A) Co-immunoprecipitation (IP) of ANKS3-Flag or ANKS37ΔCter-Flag and v5-ANKS6 with wild-type or bpk mutant HA-Bicc1 by anti-HA antibodies. Five percent of total HEK293T cell extracts was loaded as input. γ-Tubulin was used as negative control. Asterisk denotes an ANKS6-specific band of reduced size.

Article Snippet: ANKS3 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_133450","term_id":"339275864","term_text":"NM_133450"}} NM_133450 ), expressed from pCMV6-Entry and fused to C-terminal Flag tag, was from OriGene (clone ID: RC223862).

Techniques: Immunoprecipitation, Mutagenesis, Negative Control

Journal: Structure (London, England : 1993)

Article Title: Crystal Structure of Bicc1 SAM Polymer and Mapping of Interactions between the Ciliopathy-Associated Proteins Bicc1, ANKS3, and ANKS6

doi: 10.1016/j.str.2017.12.002

Figure Lengend Snippet: (A) Sequence alignment of the SAM domains of human Bicc1 (residues 877–938), ANKS3 (residues 429–490), and ANKS6 (residues 771–840). α Helices are numbered above. Residues encompassing ML and EH surfaces are shaded in blue and red, respectively.

Article Snippet: ANKS3 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_133450","term_id":"339275864","term_text":"NM_133450"}} NM_133450 ), expressed from pCMV6-Entry and fused to C-terminal Flag tag, was from OriGene (clone ID: RC223862).

Techniques: Sequencing

Journal: Structure (London, England : 1993)

Article Title: Crystal Structure of Bicc1 SAM Polymer and Mapping of Interactions between the Ciliopathy-Associated Proteins Bicc1, ANKS3, and ANKS6

doi: 10.1016/j.str.2017.12.002

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ANKS3 cDNA ( {"type":"entrez-nucleotide","attrs":{"text":"NM_133450","term_id":"339275864","term_text":"NM_133450"}} NM_133450 ), expressed from pCMV6-Entry and fused to C-terminal Flag tag, was from OriGene (clone ID: RC223862).

Techniques: Produced, Recombinant, Mutagenesis, Amplification, Software