PHOSPHORYLATION BY NEURONAL CDC2-LIKE PROTEIN-KINASE PROMOTES DIMERIZATION OF TAU-PROTEIN IN-VITRO

In Alzheimer's disease, the microtubule-associated protein tau forms p aired 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 phosphorylati on in PHF assembly is not known. Previously, neuronal cdc2-like protei n kinase (NCLK) was shown to phosphorylate tau on sites that are abnor mally phosphorylated in PHF-tau (Paudel, H. g., Lew, J., All, Z., and Wang, J. H. (1993) J. Biol. Chem. 268, 23512-23518). In this study, ph osphorylation 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 homobifunct ional chemical cross-linker that specifically cross-linked R-tau dimer s, 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 simi lar 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 mu M), R-tau formed di sulfide crosslinked 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, phosphoryla ted B-tau was preferentially cross-linked over nonphosphorylated count erpart. Taken together, these results suggest that phosphorylation by NCLK promotes dimerization and formation of disulfide crosslinked 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).