The microtubule-associated protein tau, in a hyperphosphorylated form, 
aggregates into insoluble paired-helical filaments (PHFs) in Alzheimer's disease 
(AD) and other tauopathies. In AD, there is approximately 8 mol of phosphate per 
mole of tau distributed among approximately 30 PHF phosphorylation sites as 
compared to 2-3 mol of phosphate per mole in normal brain. In AD, kinases such 
as glycogen synthase kinase-3beta (GSK-3beta) are believed to be involved in the 
generation of hyperphosphorylated tau. However, the functional consequences of 
hyperphosphorylation on the microtubule binding and polymerization of tau are 
not well understood. To address this question, we have generated 
pseudohyperphosphorylation mutants consisting of six and seven sites in the 
proline-rich region and carboxy terminus of tau by amino acid substitution. In 
addition, several single, double, and triple pseudophosphorylation mutants were 
also generated. Pseudophosphorylation of tau decreases its affinity for 
microtubules, and pseudohyperphosphorylated forms of tau do not have 
significantly decreased levels of microtubule binding as compared to single and 
double sites. Three pseudohyperphosphorylated forms of tau with altered sodium 
dodecyl sulfate-polyacrylamide gel electrophoresis migration have a greater 
effect on its inducer-mediated polymerization, slowing the rate of nucleation 
and elongation. On the basis of the observations that pseudohyperphosphorylated 
tau has decreased affinity for microtubules and reduced inducer-initiated rates 
of nucleation and polymerization, we propose that this combination could be the 
cause of the increased cytotoxicity of hyperphosphorylated tau in Alzheimer's 
disease and also explain the potentially beneficial role of tau polymerization 
and NFT formation.