The binding and phosphorylation of Thr231 is critical for Tau’s hyperphosphorylation and functional regulation by glycogen synthase kinase 3β

Abstract

Neuropathological hallmarks of Alzheimer's disease are extracellular senile plaques and intracellular neurofibrillary lesions. The neurofibrillary lesions mainly consist of the hyperphosphorylated microtubule-associated protein Tau predominantly expressed in the axon of CNS neurons. Hyperphosphorylation of Tau negatively affects its binding to tubulin and decreases the capacity to promote microtubule assembly. Among a number of proline-directed kinases capable of phosphorylating paired helical filament-Tau, glycogen synthase kinase 3b (GSK3b) was first identified as a Tau protein kinase I and has been demonstrated to phosphorylate Tau both in vivo and in vitro. However, the phosphorylation mechanism of Tau by GSK3b remained unclear. In this study, we show that the T231 is the primary phosphorylation site for GSK3b and the Tau227-237 (AVVRTPPKSPS) derived from Tau containing T231P232 motif is identified as the GSK3b binding site with high affinity of a Kd value 0.82 ± 0.16 lmol/L. Our results suggest that direct binding and phosphorylation of T231P232 motif by GSK3b induces conformational change of Tau and consequentially alters the inhibitory activity of its N-terminus that allows the phosphorylation of C-terminus of Tau by GSK3b. Furthermore, hyperphosphorylation reduces Tau's ability to promote tubulin assembly and to form bundles in N18 cells. T231A mutant completely abolishes Tau phosphorylation by GSK3b and retains the ability to promote tubulin polymerization and bundle formation. Taken together, these results suggest that phosphorylation of T231 by GSK3b may play an important role in Tau's hyperphosphorylation and functional regulation. Keywords: Alzheimer's disease, glycogen synthase kinase 3b, hyperphosphorylation, Tau, Thr231. J. Neurochem. (2007) 103, 802-813. A diagnosis of Alzheimer's disease (AD) is made when a patient exhibits clinical evidence of progressive dementia and when a post-mortem examination of brain reveals the characteristic neuropathology consisting of extracellular senile plaques and intracellular neurofibrillary lesions (Goedert and Spillantini 2006). The neurofibrillary lesions mainly consist of the hyperphosphorylated microtubule (MT)-associated protein Tau (Avila 2006). Tau is a family of MTassociated proteins that are produced by alternative mRNA splicing of a single gene as six isoforms and express predominantly within neurites and axons in adult brain. The largest Tau found in the brain containing 441 amino acids contains two N-terminal inserts and four MT-binding repeats. However, the smallest isoform has no N-terminal insert and has three MT-binding repeats instead (Buee et al. 2000;Avila 2006). Interestingly, 4R-tau was easily phosphorylated by brain protein kinases and aggregated into filaments Received January 31, 2007; revised manuscript received April 25, 2007; accepted June 13, 2007. Address correspondence and reprint requests to Pei-Jung Lu, PhD, Department of Medical Education and Research, Kaohsiung Veterans General Hospital 386 Ta-Chung 1st Rd., Kaoh...

GSK3β is a negative regulator of platelet function and thrombosis

“…MAPtau contains many sites for phosphorylation on ser or thr residues, but phosphorylation of thr-231 has been reported to be GSK3 dependent. [40][41][42] Therefore, we evaluated phosphorylation of this residue in mouse and human platelets with and without agonist stimulation. Since phosphorylation of GSK3␤-ser9 is expected to reduce its kinase activity, phosphorylation of GSK3␤ substrates should decrease as GSK␤-ser9 phosphorylation increases.…”

Tau Monoclonal Antibody Generation Based on Humanized Yeast Models

“…Furthermore, although we found the ADx215-positive oligomers to be phosphorylated mainly in the N-terminal part and MTBR domain of Tau, the study with T22 nicely documented oligomerization to start once Tau is phosphorylated at Thr 231 (8). Phosphorylation of Thr 231 by Gsk-3␤ is known to prevent Tau from binding microtubules (4) and to relieve the inhibitory activity of the N terminus over the C terminus of Tau so as to allow kinases such as Gsk-3␤ to access and subsequently phosphorylate Tau at other epitopes (48). Indeed, monomeric Tau is thought to adopt a so-called "paperclip" conformation when in solution, where the C-terminal end of Tau folds over the MTBR domain and the N terminus folds back to come in close proximity to the C terminus (49,50).…”

Tau and Amyloid- Conformational Change to  -Sheet Structures as Effectors in the Development of Alzheimer's Disease

“…The majority of the phosphorylation sites are Serine or Threonine residues followed by Prolines (SP/TP motifs) that lie in the domains flanking the repeats and can be phosphorylated by several Proline-directed kinases and by neuronal kinases MARK (affecting the KXGS motifs within Tau's repeat domain) (Eckermann et al, 2007;Trinczek et al, 1995). Among the kinases responsible for Tau phosphorylation are Glycogen Synthase Kinase 3 (Lin et al, 2007), cyclin-dependent kinase 5 (Cdk5), MT-affinity regulatory kinase, cAMP-dependent protein kinase (Johnson &Stoothoff, 2004), dual-specificity tyrosinephosphorylated and regulated kinase 1A, Tau-tubulin kinase 1, and calmodulin-dependent protein kinase 2 (Meraz-Ríos et al, 2010)…”

“…For elucidated this issue is used conformational-specific antibodies. The Alz-50 and MC-1, two conformational sensitive antibodies, have been demonstrated to recognize a pathological conformation of Tau molecule in NFTs (Jicha et al, 1997;Lin et al, 2007). Either antibody recognizes the N-termini nearby C-termini, determinate by fluorescence lifetime imaging and other techniques (Hyman et al, 2005).…”

“…Moreover, a number of studies have proposed that the phosphorylation modification at S-P or T-P motifs in Tau might cause Tau conformational change and the cis-trans isomerisation, made by the prolyl-peptide isomerise Pin-1, might regulate Tau function. This association is dependent on a folding of the Tau molecule, this bend occurring locally in the vicinity of Thr231-P motif, and actually recognized by TG3 antibody Lin et al, 2007;Luna-Muñoz et al, 2005;Weaver et al, 2000). The antibody Tau-66 is another conformational-associated antibody, recognizing residues between 155-244 and 305-314.…”