Fig. 5. Removal of single master site and kinase p38α abolishes tau multi-site hyperphosphorylation.

(A) Mapping of tau site interdependence and differential levels of p38α-mediated tau phosphorylation. Levels (circular graph) and significance (bar chart) of differential tau phosphorylation in brains of p38α-deficient mice (p38α^Δneu) and of recombinant tau by p38α correlates with predicted interdependence for p38α. (B) Schematic of in vitro kinase reactions of recombinant tauWT, tauT50A, tauT69A, or tauT181A with p38α to delineate intrinsic basis of tau phosphorylation interdependence. (C) Immunoblots (n = 6) and (D) phospho-proteomic quantification (n = 3) of kinase reactions of p38α with indicated tau variants showing markedly lower phosphorylation of recombinant tau variants at multiple epitopes. (E to G) Combination of master sites T50 and T181 and master kinase p38α governs phosphorylation of human tau. (E) Schematic of stereotaxic delivery of AAVs expressing human tau (tauWT) or indicated tau variants in hippocampus of p38α^Δneu and control p38α^loxP/loxP mice. (F) Representative immunoblots for tauWT- and tauT181A-expressing mice probed with phospho-tau epitope, tau, and p38α antibodies. n = 10 to 12. (G) Immunoblots were quantified relative to total tau and tauWT. Note that the absence of master sites T69 and T181 in human tau ablates the majority of tau phosphorylation in p38α^Δneu compared with p38α^loxP/loxP controls. (H) Hypothetical model of tau multi-site hyperphosphorylation governed by site interdependence and mediated by p38α. Initial phosphorylation by p38α at master sites, T50, T69, or T181, promotes subsequent multi-site phosphorylation by p38α. Thus, interdependence between master sites and other phospho-sites may promote tau hyperphosphorylation and contribute to Aβ-induced cognitive deficits. MTBR, microtubule-binding region. Data are presented as means ± SEM unless indicated otherwise. ***P < 0.001; **P < 0.01; *P < 0.05 (ANOVA).