The Endogenous and Cell Cycle-dependent Phosphorylation of tau Protein in Living Cells: Implications for Alzheimer’s Disease

Abstract

In Alzheimer's disease the neuronal microtubule-associated protein tau becomes highly phosphorylated, loses its binding properties, and aggregates into paired helical filaments. There is increasing evidence that the events leading to this hyperphosphorylation are related to mitotic mechanisms. Hence, we have analyzed the physiological phosphorylation of endogenous tau protein in metabolically labeled human neuroblastoma cells and in Chinese hamster ovary cells stably transfected with tau. In nonsynchronized cultures the phosphorylation pattern was remarkably similar in both cell lines, suggesting a similar balance of kinases and phosphatases with respect to tau. Using phosphopeptide mapping and sequencing we identified 17 phosphorylation sites comprising 80-90% of the total phosphate incorporated. Most of these are in SP or TP motifs, except S214 and S262. Since phosphorylation of microtubule-associated proteins increases during mitosis, concomitant with increased microtubule dynamics, we analyzed cells mitotically arrested with nocodazole. This revealed that S214 is a prominent phosphorylation site in metaphase, but not in interphase. Phosphorylation of this residue strongly decreases the tau-microtubule interaction in vitro, suppresses microtubule assembly, and may be a key factor in the observed detachment of tau from microtubules during mitosis. Since S214 is also phosphorylated in Alzheimer's disease tau, our results support the view that reactivation of the cell cycle machinery is involved in tau hyperphosphorylation.

Quantitative mapping of microtubule-associated protein 2c (MAP2c) phosphorylation and regulatory protein 14-3-3ζ-binding sites reveals key differences between MAP2c and its homolog Tau

“…PKA seems to be the key kinase in this mechanism. Phosphorylation of Tau by PKA or PKB, but not by GSK-3␤, CDK2, or CK1, enhances the interaction with 14-3-3 (25), whereas Tau phosphorylated by PKA binds to tubulin with 7-fold lower affinity (41,20).…”

“…In vivo MAPs are substrates of various protein kinases, such as cAMP-dependent protein kinase A (PKA), protein kinase C (PKC), cdc2 kinase, and the like (16,17,20,21). Phosphorylation of Tau weakens its interaction with microtubules and increases its affinity to regulatory 14-3-3 proteins (vide infra).…”

Tau-Based Treatment Strategies in Neurodegenerative Diseases

“…[77][78][79][80] PKA additionally phosphorylates other sites including S214, a phospho-epitope that is especially upregulated during mitosis. 81 In AD brain, tau is hyperphosphorylated at nearly all phosphorylation sites, with approximately nine phosphates per molecule, in contrast to the two to three phosphorylated residues observed in healthy control brains. 82 Various antibodies directed against these phosphoepitopes preferentially recognize AD brain-derived tau and are used as diagnostic tools.…”

Regulation and function of the interaction between the APC tumour suppressor protein and EB1

“…Alfa et al, 1990;Andreassen et al, 1994;see Cassimeris, 1999 for a recent review). Furthermore, the association of tau protein with microtubules is also modi®ed during mitosis by phosphorylation at Ser-Pro and PKA consensus motifs (Illenberger et al, 1998). Interestingly, the in vitro biochemical and tubulin-binding properties of the basic domain of APC have recently been shown to be similar to that of tau (Deka et al, 1998).…”