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Phosphorylation by neuronal cdc2-like protein kinase promotes dimerization of Tau protein in vitro
1Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital and Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec, H3T 1E2, Canada. MDHP@MUSICA.McGILL.CA
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).
Insights
Phosphorylation of tau protein by neuronal cdc2-like protein kinase (NCLK) promotes tau dimerization. This dimerization is a critical step in the formation of paired helical filaments (PHFs) implicated in Alzheimer's disease.
Area of Science:
- Neuroscience
- Biochemistry
- Cell Biology
Background:
- Alzheimer's disease is characterized by neurofibrillary tangles composed of paired helical filaments (PHFs).
- PHFs are primarily formed by the microtubule-associated protein tau, which is abnormally phosphorylated in PHF-tau.
- The precise role of abnormal tau phosphorylation in PHF assembly remains unclear.
Purpose of the Study:
- To investigate the effect of neuronal cdc2-like protein kinase (NCLK) phosphorylation on tau protein structure and assembly.
- To determine if NCLK-mediated phosphorylation promotes tau dimerization, a potential precursor to PHF formation.
Main Methods:
- Utilized recombinant human tau (R-tau) and brain tau (B-tau).
- Employed chemical cross-linking with disuccinimidyl suberate (DSS) to detect tau dimers.
- Applied Superose 12 gel filtration chromatography to analyze tau species (monomeric vs. dimeric).
- Investigated disulfide cross-linking in the presence of dithiothreitol.
Main Results:
- NCLK phosphorylation significantly promoted the dimerization of both R-tau and B-tau.
- Phosphorylated tau preferentially formed dimers, which eluted as dimeric species in gel filtration.
- Dephosphorylation of tau reversed the dimeric state, making it monomeric and resistant to cross-linking.
- Phosphorylated tau exhibited increased disulfide cross-linked dimer formation compared to non-phosphorylated tau.
Conclusions:
- NCLK-mediated phosphorylation of tau facilitates its dimerization.
- Tau dimerization, particularly the formation of disulfide cross-linked dimers, is a key initiating step in PHF assembly.
- These findings provide mechanistic insight into tau pathology in Alzheimer's disease.