Site-specific effects of tau phosphorylation on its microtubule assembly activity and self-aggregation

Abstract

Microtubule-associated protein tau is abnormally hyperphosphorylated and aggregated into neurofibrillary tangles in brains with Alzheimer's disease. The phosphorylation sites of tau are mainly localized in the proline-rich (residues 172-251) and C-terminal tail (residues 368-441) regions, which flank the microtubule-binding repeats. Here, we investigated the effects of tau phosphorylation at these distinct sites/regions on its activity of stimulating microtubule assembly and its self-aggregation. We found that tau phosphorylation at the proline-rich region by dual-specificity tyrosine-phosphorylated and -regulated kinase 1A inhibited its microtubule assembly activity moderately and promoted its self-aggregation slightly. Tau phosphorylation at the C-terminal tail region by glycogen synthase kinase-3beta increased its activity and promoted its self-aggregation markedly. Tau phosphorylation at both regions plus the microtubule-binding region by cAMP-dependent protein kinase diminished its activity (approximately 70% inhibition) and disrupted microtubules. These studies reveal the differential regulation of tau's biological activity and self-aggregation by phosphorylation at various sites/regions.

Fig. 1

Site-specific phosphorylation of tau by dual-specificity tyrosine-phosphorylated and -regulated kinase 1A (Dyrk1A), glycogen synthase kinase-3β (GSK-3β) and cAMP-dependent protein kinase (PKA). (A) Schematic diagram of tau₄₄₁, showing the proline-rich region (P-region), microtubule (MT)-binding region and C-terminal tail region (C-region), where the majority of the serine and threonine residues are phosphorylated in Alzheimer’s disease brain. (B and C) Recombinant tau₄₄₁ was first phosphorylated in vitro to ~2.0 mol phosphates/mol tau by catalysis with Dyrk1A, GSK-3β or PKA. The phosphorylated tau and the control-treated tau (Con) were then subjected to western blots developed with antibodies recognizing total tau or tau phosphorylated at specific sites, as indicated under each blot.

Fig. 2

Differential effects of tau phosphorylation by different protein kinases on its ability to stimulate microtubule (MT) assembly. Real-time MT assembly was monitored by measuring the turbidity (O.D._{350 nm}) of tubulin (2.0 mg/mL) at 35 °C in the presence of 0.07 mg/mL control-treated tau₄₄₁ (Con-tau) or tau₄₄₁ phosphorylated by dual-specificity tyrosine-phosphorylated and -regulated kinase 1A (Dyrk1A) (A), glycogen synthase kinase-3β (GSK-3β) (B) or cAMP-dependent protein kinase (PKA) (C). Representative real-time turbidimetric changes of MT assembly of three independent experiments with the same results are shown.

Fig. 3

Kinetics of glycogen synthase kinase-3β (GSK-3β)-catalysed tau phosphorylation at individual sites and its impact on microtubule (MT) assembly activity. (A) Tau₄₄₁ was incubated with GSK-3β for various periods of time and the aliquots of the reaction mixture were examined for both the stoichiometry of total tau phosphorylation and tau phosphorylation at individual sites. The site-specific phosphorylation was then plotted against the total tau phosphorylation. (B–D) Real-time MT assembly was monitored by measuring the turbidity (O.D._{350 nm}) of tubulin (2.0 mg/mL) at 35 °C in the presence of 0.07 mg/mL control-treated tau₄₄₁ (Con-tau) or tau₄₄₁ phosphorylated by GSK-3β to a stoichiometry of 1.0 mol phosphates (Pi)/mol tau (B) or 1.9 mol Pi/mol tau (C), or first phosphorylated by dual-specificity tyrosine-phosphorylated and -regulated kinase 1A (Dyrk1A) to 2.0 mol Pi/mol tau and then further phosphorylated by GSK-3β to a total stoichiometry of 4.3 mol Pi/mol tau (D). Representative data of three independent experiments with the same results are shown.

Fig. 4

Effect of tau phosphorylation at various sites on rebuilding of the microtubule (MT) network in situ. Nocodazole-treated and Triton X-100-extracted 3T3 cells were incubated at 37 °C for 1 h with 15% fresh rat brain cytosol in MT assembly buffer containing 0.5 mg/mL control-treated tau (Con-tau) or tau phosphorylated with dual-specificity tyrosine-phosphorylated and -regulated kinase 1A (Dyrk1A), glycogen synthase kinase-3β (GSK-3β) or cAMP-dependent protein kinase (PKA). The cells were then doubly stained with monoclonal antibody DM1A against tubulin (green) and polyclonal antibody R134d against tau (red), and visualized using a confocal microscope. Bar, 25 μm.

Fig. 5

Effect of phosphorylation by dual-specificity tyrosine-phosphorylated and -regulated kinase 1A (Dyrk1A), glycogen synthase kinase-3β (GSK-3β) and cAMP-dependent protein kinase (PKA) on self-aggregation of tau. Tau₄₄₁ was phosphorylated by Dyrk1A, GSK-3β or PKA to a stoichiometry of ~2.0 mol phosphates/mol tau and then incubated at 37 °C for 12 h, followed by centrifugation at 30 000 g for 20 min. The equivalent amounts of the supernatants (Sup) and the samples before centrifugation (Total) were assayed for tau levels by both western blots (A) and immuno-dot-blots (B and C). In B, all samples were analysed in three different dilutions and in triplicate, and the quantification of the blots is shown in C. Tau aggregates in the preparations were also examined by negatively stained electron microscopy (D). Con, control; Con-tau, control-treated tau. *P < 0.05 as compared with control group.