Journal: Science signaling

Article Title: Neurodevelopmental disorder-associated mutations in TAOK1 reveal its function as a plasma membrane remodeling kinase

doi: 10.1126/scisignal.add3269

Figure Lengend Snippet: (A) Schematic representation of secondary structure of Thousand and one amino acid kinase 1 orthologs found in human (TAOK1), mouse (Taok1), fish (Taok1b), fly (dTao) and worm (kin-18) species. The N-terminal kinase domain (orange) and three predicted coiled-coils (green) are highly evolutionarily conserved at the sequence and structural level. (B) In situ hybridization data from the Allen Brain Atlas using antisense probe against Taok1 on sagittal and coronal sections of P56 C57BL6/J male mouse brain. (C) Primary hippocampal neurons obtained from E15 rat embryos were fixed at 16 days in vitro (DIV) and immunostained using antibodies against Taok1 (cyan) and dendritic protein Map2 (magenta). Nucleus is stained with DAPI (yellow). Grayscale image shows Taok1 immunostaining. Scale bar, 10µm. (D) Immunofluorescence intensity of Taok1 in neuronal cells (Map2 positive) compared to non-neuronal cells. Data are means ± SEM from n= 10 cells each, neuronal and non-neuronal. *P= 0.0005 by unpaired t-test with Welch’s correction. (E) DIV9 hippocampal neurons were transfected with sfGFP-TAOK1 (green) along with cytosolic tdTomato (magenta) and fixed at DIV12 to visualize TAOK1 localization and neuronal morphology. Scale bar, 20µm. Yellow-demarcated section magnified to assess localization of TAOK1 within dendrites and dendritic spines; scale bar, 2µm.

Article Snippet: Antibodies used are TAOK1 (Rabbit, Proteintech Cat# 26250–1-AP, RRID:AB_2880446), TAOK1 (Mouse, BD Biosciences Cat# 611368, RRID:AB_398890), GST (Mouse, Thermo Scientific Cat# MA4–004, RRID:AB_10979611), Map2 (Chicken, Abcam Cat# ab5392, RRID:AB_2138153), p-TAOK S181 (Rabbit, R and D Systems Cat# PPS037, RRID:AB_2255678) and GFP (Mouse, Sigma-Aldrich Cat# 11814460001, RRID:AB_390913).

Techniques: Sequencing, In Situ Hybridization, In Vitro, Staining, Immunostaining, Immunofluorescence, Transfection

Journal: Science signaling

Article Title: Neurodevelopmental disorder-associated mutations in TAOK1 reveal its function as a plasma membrane remodeling kinase

doi: 10.1126/scisignal.add3269

Figure Lengend Snippet: (A) Schematic depicts the secondary protein structure of TAOK1 and marks the position of the four NDD-associated mutations clustered within the N-terminal kinase domain of TAOK1. (B) Western blot shows in vitro kinase activity of immunoprecipitated GFP-tagged wildtype (WT) TAOK1 and the four mutants as indicated. Autophosphorylation at residue Ser181 is used as a readout for kinase activity and amount of total TAOK1 protein is indicated by GFP blot. (C) Kinase activity as measured by intensity of pSer181 signal normalized to the GFP signal is plotted in the bar graph. Mean ±SEM from n=3 experiments. P<0.0001 by one way ANOVA with Dunnett’s multiple comparison test. (D) Images show representative HEK293T cells expressing plasma membrane marker myr-tdTomato (red) along with either wild-type TAOK1 (WT) or NDD-associated TAOK1 mutant S111F (cyan). Single confocal plane is shown. Scale bar, 10µm. (E) Plot depicts the normalized fluorescence intensity distribution of GFP-TAOK1 wild-type and ASD-associated mutants S111F, L167R, A219V and R269Q across a 5-µm line across a single z plane of a confocal image, drawn such that 2.5µm is intracellular, 0µm refers to plasma membrane, and the remaining 2.5µm is extracellular. Normalized mean intensity values as a function of distance are plotted and error bars represent SEM from n=6 cells per condition. (F) DIV11 rat hippocampal neurons expressing WT-TAOK1 or S111F ASD-associated mutant TAOK1 (green) along with cytoplasmic td-tomato (magenta) to visualize neuronal morphology. Right column shows the magnified somatic region of the neuron to highlight the localization of WT TAOK1 (top) compared to S111F mutant. (G) Total number of primary dendrites and secondary or tertiary (others) dendrites in hippocampal neurons expression GFP-tagged WT TAOK1 or the mutants S111F, L167R, A219V and R269Q is plotted. Mean ±SEM from n>14 neurons per condition from 3 different experiments. ns= not significant, ** p<0.005, and **** p<0.0001 by fitting a generalized linear model with a Poisson link function. (H) Total length of primary dendrites and secondary or tertiary (others) dendrites in hippocampal neurons expression GFP-tagged WT TAOK1 or the mutants S111F, L167R, A219V and R269Q is plotted. Mean ±SEM from n>14 neurons per condition from 3 different experiments. ns= not significant, *p<0.05, **p<0.005, ***p<0.001, and **** p<0.0001 by 2-way ANOVA with Dunnett’s multiple comparisons.

Article Snippet: Antibodies used are TAOK1 (Rabbit, Proteintech Cat# 26250–1-AP, RRID:AB_2880446), TAOK1 (Mouse, BD Biosciences Cat# 611368, RRID:AB_398890), GST (Mouse, Thermo Scientific Cat# MA4–004, RRID:AB_10979611), Map2 (Chicken, Abcam Cat# ab5392, RRID:AB_2138153), p-TAOK S181 (Rabbit, R and D Systems Cat# PPS037, RRID:AB_2255678) and GFP (Mouse, Sigma-Aldrich Cat# 11814460001, RRID:AB_390913).

Techniques: Western Blot, In Vitro, Activity Assay, Immunoprecipitation, Expressing, Marker, Mutagenesis, Fluorescence

Journal: Science signaling

Article Title: Neurodevelopmental disorder-associated mutations in TAOK1 reveal its function as a plasma membrane remodeling kinase

doi: 10.1126/scisignal.add3269

Figure Lengend Snippet: (A) Schematics show the phosphorylation sites in TAOK1 identified by mass spectrometry using immunoprecipitated WT TAOK1. Kinase-deficient (L167R) TAOK1 was used as negative control. The fold-change increase in WT over control for the three phosphothreonine sites (red) identified is shown in parenthesis after the phosphosite. The blue, serine phosphorylation sites were only identified in WT TAOK1. Evolutionary conservation of sites was determined using ConSurf. (B) Position of the phospho-threonine sites within the predicted 3D structure of TAOK1. Kinase domain is shown in blue, helical bundle in green, and phospho-sites in red. (C) Representative confocal images of HEK293T cells transfected with WT TAOK1, double-phosphomutant TAOK1 (T440A+T443A), and phosphomutant TAOK1 (T474A). Scale bar, 10µm. (D) Normalized fluorescence intensity distribution of the constructs described in (C) across a 5-µm line across a single z plane of a confocal image, drawn such that 2.5µm is intracellular, 0µm refers to plasma membrane, and the remaining 2.5µm is extracellular. Normalized mean intensity values as a function of distance are plotted ± SEM, from n=6 cells each per condition. (E and F) As described in (C and D) for HEK293T cells transfected with GFP-tagged TAOK1-(T440A+T443A) along with either the active isolated kinase domain mCh-TAOK1-(1–320WT) or the inactive kinase domain mCh-TAOK1-(1–320 K57A), also from n=6 cells each per condition. Scale bar, 5µm. (G) Working model of TAOK1 as a membrane-sculpting kinase autoregulated by its kinase activity through phosphorylation at critical residues Thr440 and Thr443, which regulates it shuttling between its active cytosolic state (orange) and inactive membrane-bound state (green). NDD-associated mutations (S111F, L167R, A219V and R269Q) render TAOK1 catalytically dead and traps the kinase permanently in the membrane bound state, where it induces aberrant membrane extensions and dendritic defects.

Article Snippet: Antibodies used are TAOK1 (Rabbit, Proteintech Cat# 26250–1-AP, RRID:AB_2880446), TAOK1 (Mouse, BD Biosciences Cat# 611368, RRID:AB_398890), GST (Mouse, Thermo Scientific Cat# MA4–004, RRID:AB_10979611), Map2 (Chicken, Abcam Cat# ab5392, RRID:AB_2138153), p-TAOK S181 (Rabbit, R and D Systems Cat# PPS037, RRID:AB_2255678) and GFP (Mouse, Sigma-Aldrich Cat# 11814460001, RRID:AB_390913).

Techniques: Mass Spectrometry, Immunoprecipitation, Negative Control, Transfection, Fluorescence, Construct, Isolation, Activity Assay

Journal: Science signaling

Article Title: Neurodevelopmental disorder-associated mutations in TAOK1 reveal its function as a plasma membrane remodeling kinase

doi: 10.1126/scisignal.add3269

Figure Lengend Snippet: (A) Schematic depicts the different deletion constructs generated to identify the membrane binding domain within TAOK1. The red box indicates the region necessary and sufficient for TAOK1 plasma membrane association. (B) Plot depicts the normalized fluorescence intensity distribution of full-length GFP-TAOK1 (1–1001 amino acids), kinase domain (1–320 amino acids) and coiled coil domain (321–901 amino acid) across a 5-µm line across a single z plane of a confocal image of HEK293T cells, drawn such that 2.5µm is intracellular, 0µm refers to plasma membrane and the remaining 2.5µm is extracellular. Normalized mean intensity values are plotted as a function of distance ± SEM from n=10 cells per condition. (C) Western blotting for the localization of GFP-tagged TAOK1 kinase domain (1–320) and coiled-coil domain (321–901) in supernatant (S) and membrane pellet (P) fractions, respectively, of HEK293T cells. Fractions were obtained by differential centrifugation at 100,000g. TAOK1 domains were detected by immunoblotting using anti-GFP antibodies. ER protein Stim1 and α-tubulin were controls for the pellet and supernatant fractions, respectively. (D) Mean percentage of GFP tagged kinase domain and coiled-coil domain protein present in the pellet and supernatant fractions from (C) is plotted from n=3 experiments. P<0.0001 by 2 way-ANOVA. (E) Mean length of plasma membrane protrusions in HEK293T cells expressing either the kinase domain (1–320) or the coiled coil domain (321–901) is plotted ± SEM, from n=50 protrusions from 10 cells each. P<0.0001 by unpaired t-test with Welch’s correction. (F and G) DIV11 rat hippocampal neurons expressing GFP-tagged TAOK1 kinase domain (1–320) or coiled coil domain (321–901) (cyan) along with membrane marker myristoylated-tdTomato (magenta) to visualize neuronal morphology (F) and analyze colocalization (G). Yellow box in (F) demarcates the area magnified in (G). Scale bars, 10 and 3 µm, respectively. (H) Plot profile of GFP (cyan) and myrTdTomato (magenta) fluorescence shown in (G) as a function of distance in pixels. Data are representative of 3 experiments.

Article Snippet: Antibodies used are TAOK1 (Rabbit, Proteintech Cat# 26250–1-AP, RRID:AB_2880446), TAOK1 (Mouse, BD Biosciences Cat# 611368, RRID:AB_398890), GST (Mouse, Thermo Scientific Cat# MA4–004, RRID:AB_10979611), Map2 (Chicken, Abcam Cat# ab5392, RRID:AB_2138153), p-TAOK S181 (Rabbit, R and D Systems Cat# PPS037, RRID:AB_2255678) and GFP (Mouse, Sigma-Aldrich Cat# 11814460001, RRID:AB_390913).

Techniques: Construct, Generated, Binding Assay, Fluorescence, Western Blot, Centrifugation, Expressing, Marker

Journal: Science signaling

Article Title: Neurodevelopmental disorder-associated mutations in TAOK1 reveal its function as a plasma membrane remodeling kinase

doi: 10.1126/scisignal.add3269

Figure Lengend Snippet: (A) AlphaFold2.0 prediction of the structure of human TAOK1. The kinase domain is shown in green (residues 1–320) and the rest of protein is shown in gray (321–1001). The three coiled coil domain (444–901) are predicted to fold into a triple helix. Expected position error at each residue is plotted for the entire length of the protein (top right). The linker region between kinase domain and triple helix (321–443) has a low per-residue confidence score, pLDDT <50. (B) The isolated triple helix bundle is shown in neon green. The APBS (Adaptive Poisson-Boltzmann Solver) function of Python was used on the Alphafold2.0 predicted structure of TAOK1 triple helix to create a surface electrostatic representation. The convex face of the crescent shaped protein domain (side view, top view) is highly enriched in positive charges (blue), and the concave side (side view, bottom view) is enriched in negatively charged amino acids (red). The scale of charges -5 to +5 is shown as gradation in shades of red and blue. (C) Lipid overlay blots show the binding affinity of a GST-tagged TAOK1 triple helix (321–901) construct to different species of lipids dotted on the blot. Darker dots depict greater binding, detected through antibody against GST tag. (D) Quantification of binding affinity of GST-TAOK1(321–901) with different lipids. Mean ±SEM from n=3 experiments. (E) Images shows representative HEK293T cells transfected with the PI(4,5)P2 sensor GFP-C1-PLC∂-PH along with either control (top) or mCherry-TAOK1(321–901), assessing for protrusions from the plasma membrane. Scale bar, 5µm.

Article Snippet: Antibodies used are TAOK1 (Rabbit, Proteintech Cat# 26250–1-AP, RRID:AB_2880446), TAOK1 (Mouse, BD Biosciences Cat# 611368, RRID:AB_398890), GST (Mouse, Thermo Scientific Cat# MA4–004, RRID:AB_10979611), Map2 (Chicken, Abcam Cat# ab5392, RRID:AB_2138153), p-TAOK S181 (Rabbit, R and D Systems Cat# PPS037, RRID:AB_2255678) and GFP (Mouse, Sigma-Aldrich Cat# 11814460001, RRID:AB_390913).

Techniques: Isolation, Binding Assay, Construct, Transfection

Journal: Science signaling

Article Title: Neurodevelopmental disorder-associated mutations in TAOK1 reveal its function as a plasma membrane remodeling kinase

doi: 10.1126/scisignal.add3269

Figure Lengend Snippet: (A) HEK293T cells transfected with TAOK1 helical bundle domain GFP-TAOK1 (321–901) alone or with TAOK1 kinase domain mCherry-TAOK1 (1–321). Scale bar, 5µm. (B) Plot depicts the normalized fluorescence intensity distribution of the helical bundle domain as in (A), alone (blue) or with the kinase domain (red) across a 5-µm line across a single z plane of a confocal image, drawn such that 2.5µm is intracellular, 0µm refers to plasma membrane, and the remaining 2.5µm is extracellular. Normalized mean intensity values as a function of distance are plotted ±SEM from n=10 cells per condition. (C) Representative HEK293T cells expressing NDD-associated TAOK1 mutants S111F (magenta), L167R (red), A219V (cyan) and R269Q (green) are shown in top row, and representative HEK293T cells expressing these mutants along with active kinase domain of TAOK1 (1–320) are shown in the bottom row. Scale bar, 10µm. (D) Plot depicts the normalized fluorescence intensity distribution of TAOK1 mutants in (C), assessed and shown as described in (B), from n=6 cells each per condition.

Article Snippet: Antibodies used are TAOK1 (Rabbit, Proteintech Cat# 26250–1-AP, RRID:AB_2880446), TAOK1 (Mouse, BD Biosciences Cat# 611368, RRID:AB_398890), GST (Mouse, Thermo Scientific Cat# MA4–004, RRID:AB_10979611), Map2 (Chicken, Abcam Cat# ab5392, RRID:AB_2138153), p-TAOK S181 (Rabbit, R and D Systems Cat# PPS037, RRID:AB_2255678) and GFP (Mouse, Sigma-Aldrich Cat# 11814460001, RRID:AB_390913).

Techniques: Transfection, Fluorescence, Expressing