Fig. 4

De novo mutations in TAOK2 impair phosphorylation at Ser181, localization in cortical neurons, and dendritic spine motility. a Diagram of TAOK2α and TAOK2β isoforms and location of de novo (A135P, P1022* and 563 + 12_563 + 15), truncating mutations (T604Sfs*45, Q622*, L1030fs*3), and rare-inherited variants (A335V and H781R). Different protein domains are represented by colored boxes (kinase domain: red, MEK binding domain: black, regulatory domains: blue (α) and light blue (β)). TAOK2α has two phosphorylation sites (ser181 and thr475) and caspase-9 cleavage site (⁹¹⁶DPGD⁹¹⁹). TAOK2β has three known phosphorylation sites (ser181, thr475, and ser1031). b Western blot of HEK293 cell lysates 48 h post transfection with TAOK2β and β variants (A135P, P1022*, A335V, and H781R). c TAOK2β A135P shows reduced protein expression and ser181 phosphorylation, TAOK2β P1022* shows only reduced expression, and TAOK2β A335V and H781R have no effect compared with TAOK2β (n = 6–8 western blots; one-sample t-test; TAOK2 levels: A135P t(5) = 5.303, p = 0.0032; P1022* t(5) = 8.903, p = 0.0003; A335V t(5) = 1.342, p = 0.2373, H781R* t(5) = 0.6381, p = 5515; pTAOK2 levels: A135P t(7) = 10.93, p < 0.0001; P1022* t(7) = 0.4992, p = 0.6330; A335V t(7) = 0.7243, p = 0.4924; H781R t(7) = 0.3526, p = 0.7348). d Western blot of HEK293 cell lysates 48 h post transfection with JNK1a1 only or with TAOK2β, TAOK2β A135P and TAOK2β P1022*. e TAOK2β P1022* significantly increases phosphorylation of JNK1a1 in HEK293 cells compared with TAOK2β (n = 7 western blots; one-way ANOVA, post hoc Dunnett’s test; F_{2, 18} = 6.88, p = 0.0060; TAOK2β vs. P1022* p = 0.0342, TAOK2β vs. A135P p = 0.5087; JNK1a1 only (set to 100%) vs. TAOK2β: one-sample t-test, t(6) = 3.167, p = 0.0194). f Schematic showing impairment of ser181 auto-phosphorylation by the A135P mutation resulting in reduced kinase activity on downstream targets, whereas the P1022* mutation causes increased kinase activity of TAOK2. g Western blot of LCLs from the A135P proband and the unaffected father. h The A135P proband has reduced TAOK2 and pTAOK2 levels compared with the unaffected father (n = 5 western blots; one-sample t-test; TAOK2: t(4) = 4.557, p = 0.0104; pTAOK2: t(4) = 2.74, p = 0.0519). i Images of DIV14 cortical neuron cultures transfected with only GFP (control) or TAOK2β, TAOK2β A135P, and TAOK2β P1022* with GFP and immunostained against GFP (green), TAOK2 (red) and stained with DAPI (blue). Scale bars represent 10 μm. Boxes are shown magnified (right), with scale bars representing 3 μm. Arrowheads represent dendritic spines filled with exogenous TAOK2β, but not TAOK2β A135P and P1022*. j Percentage of total exogenous TAOK2β A135P and P1022* is increased in the soma (top) and decreased in the dendrite and spines (bottom) compared with TAOK2β (TAOK2β = 47, TAOK2β A135P = 35 and TAOK2β P1022* = 40 neurons from three separate cultures; one-way ANOVA post hoc Dunnett’s test; Soma F_{2, 119} = 28.01, p < 0.0001: TAOK2β vs. A135P p < 0.0001, TAOK2β vs. P1022* p < 0.0001; Dendrite: F_{2, 119}= 28.01: TAOK2β vs. A135P p < 0.0001, TAOK2β vs. P1022* p < 0.0001). k Snapshots of DIV14 cortical neurons transfected with TAOK2β and β variants (A135P and P1022*) at 0 s, 124 s and 302 s. Green arrowheads indicate extending filopodia spines and red arrowhead indicates retraction of a filopodia spine. l Left: increased spine motility in neurons transfected with Taok2 shRNA compared with neurons transfected with control shRNA (Control shRNA = 12 and Taok2 shRNA = 18 neurons from three different cultures; one-sample t-test; t(17) = 3.799, p = 0.0014). Right: TAOK2β A135P transfected neurons have increased spine motility compared TAOK2β transfected neurons (Control = 10, TAOK2β = 22, A135P = 12 and P1022* = 14 neurons from three different cultures; one-way ANOVA, post hoc Dunnett’s test; F_{3, 42}= 17.2, p < 0.0001; TAOK2β vs. A135P p = 0.0002).*p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. Values are mean ±  s.e.m.