Phosphorylation by neuronal cdc2-like protein kinase promotes dimerization of Tau protein in vitro

J Biol Chem. 1997 Nov 7;272(45):28328-34. doi: 10.1074/jbc.272.45.28328.

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).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Brain Chemistry
  • Cattle
  • Chromatography, High Pressure Liquid
  • Cross-Linking Reagents / pharmacology
  • Cyclin-Dependent Kinase 5
  • Cyclin-Dependent Kinases*
  • Dimerization
  • Disulfides / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • In Vitro Techniques
  • Neurons / enzymology*
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism*
  • Recombinant Proteins / metabolism
  • Succinimides / pharmacology
  • tau Proteins / metabolism*

Substances

  • Cross-Linking Reagents
  • Disulfides
  • Recombinant Proteins
  • Succinimides
  • tau Proteins
  • Cyclin-Dependent Kinase 5
  • Protein-Serine-Threonine Kinases
  • CDK5 protein, human
  • Cyclin-Dependent Kinases
  • disuccinimidyl suberate