Objective: To explore the association between the abnormal phosphorylation sites found in Alzheimer disease (AD) tau and the inhibition of its biological activity.
Methods: Ultracentrifugation, chromatography, manual Edman degradation and autosequence techniques were used to prepare and phosphorylate human recombinant tau, isolate and purify 32P tau peptides and determine phosphorylation sites.
Results: (1) Phosphorylation of tau by casein kinase-1 (CK-1), cyclic AMP-dependent protein kinase (PKA) and glycogen synthetase kinase-3 (GSK-3) differentially inhibited its biological activity, and the inhibition of this activity of tau by GSK-3 was significantly increased if tau was prephosphorylated by CK-1 or PKA. The most potent inhibition was seen by a combined phosphorylation of tau with PKA and GSK-3. (2) The treatment of tau by PKA and GSK-3 combination induced phosphorylation of tau at Ser-195, Ser-198, Ser-199, Ser-202, Thr-205, Thr-231, Ser-235, Ser-262, Ser-356, Ser-404, whereas only Thr-181, Ser-184, Ser-262, Ser-356 and Ser-400 were phosphorylated by GSK-3 alone under the same conditions. Among the above-mentioned phosphorylation sites, Ser-198, Ser-199, Ser-202, Thr-205, Thr-231, Ser-235, Ser-262, Ser-400 and Ser-404 were seen in Alzheimer tau. The phosphorylation of Ser-262 only slightly inhibited its biological activity, and Ser-198, Ser-199, Ser-202, Thr-231, Ser-235, Ser-400 and Ser-404 also presented in fetal tau which was highly active, suggesting that Thr-205 was the unique site which both caused the potent inhibition of biological activity and specifically presented in AD abnormally phosphorylated tau.
Conclusions: Phosphorylation of Thr-205 might play a key role in tau pathology in AD.