Abstract
In Alzheimer's disease, the microtubule-associated protein tau forms paired helical filaments (PHFs) that are the major structural component of neurofibrillary tangles. Although tau isolated from PHFs (PHF-tau) is abnormally phosphorylated, the role of this abnormal phosphorylation in PHF assembly is not known. Previously, neuronal cdc2-like protein kinase (NCLK) was shown to phosphorylate tau on sites that are abnormally phosphorylated in PHF-tau (Paudel, H. K., Lew, J., Ali, Z., and Wang, J. H. (1993) J. Biol. Chem. 268, 23512-23518). In this study, phosphorylation by NCLK was found to promote dimerization of recombinant human tau (R-tau) and brain tau (B-tau) purified from brain extract. Chemical cross-linking by disuccinimidyl suberate (DSS), a homobifunctional chemical cross-linker that specifically cross-linked R-tau dimers, and a Superose 12 gel filtration chromatography revealed that R-tau preparations contain mixtures of monomeric and dimeric R-tau species. When the structure of NCLK-phosphorylated R-tau was studied by a similar approach, DSS preferentially cross-linked the phosphorylated R-tau over the nonphosphorylated R-tau, and the phosphorylated R-tau eluted as a dimeric species from the gel filtration column. Phosphorylated R-tau became resistant to DSS upon dephosphorylation and was recovered as a monomeric species from the gel filtration column. In the presence of a low concentration of dithiothreitol (1.65 microM), R-tau formed disulfide cross-linked R-tau dimers. When compared, phosphorylated R-tau formed more disulfide cross-linked dimers than the nonphosphorylated R-tau. B-tau also was specifically cross-linked to dimers by DSS. When B-tau and NCLK-phosphorylated B-tau were treated with DSS, phosphorylated B-tau was preferentially cross-linked over nonphosphorylated counterpart. Taken together, these results suggest that phosphorylation by NCLK promotes dimerization and formation of disulfide cross-linked tau dimers, which is suggested to be the key step leading to PHF assembly (Schweers, O., Mandelkow, E.-M., Biernat, J., and Mandelkow, E. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 8463-8467).
Highlights
Senile plaques and neurofibrillary tangles are two characteristic pathological lesions found in the brains of patients suffering from Alzheimer’s disease (AD)1
As has been pointed out [11], juvenile tau, which shares many abnormal phosphorylation sites with paired helical filaments (PHFs)-tau, does not exist as PHFs [12]. These observations raised the question as to whether the abnormal phosphorylation observed in PHF-tau is required for normal tau molecules to be converted into PHFs or whether it is merely a secondary event occurring during PHF assembly
Since sites S-202, T-231, S-235, S-396, and S-404 are phosphorylated in PHF-tau [3], NCLK was suggested to be one of the kinases that participates in the abnormal phosphorylation of tau occurring in AD brain [13, 14]
Summary
Senile plaques and neurofibrillary tangles are two characteristic pathological lesions found in the brains of patients suffering from Alzheimer’s disease (AD) (for a review, see Ref. 1). There does not appear to be any AD-specific mutation in the tau gene, and studies indicate that tau mRNA level does not change in AD brain [4] It is puzzling how tau, a highly soluble axonal protein, becomes insoluble PHF in degenerating neurons during the ontogeny of AD. Recombinant tau fragments derived from microtubule binding repeats of tau spontaneously dimerize [5, 6] These dimers, when treated with buffers containing high salt and/or low pH, aggregate into PHF-like filaments [5,6,7]. PHF-tau (tau found in PHFs) is abnormally phosphorylated (i.e. contains more phosphate than normal tau [1, 2]) It has a reduced mobility on SDS-PAGE and is incapable of binding to microtubules and promoting microtubule assembly. It is reported that phosphorylation by NCLK promotes self-association of tau molecules, leading to dimerization
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