Sequential phosphorylation of tau protein by cAMP-dependent protein kinase and SAPK4/p38δ or JNK2 in the presence of heparin generates the AT100 epitope

Microtubule-associated protein tau in a hyperphosphorylated state is the major component of the filamentous lesions that define a number of neurodegenerative diseases, including Alzheimer's disease, progressive supranuclear palsy, corticobasal degeneration, Pick's disease, argyrophilic grain disease and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). Previous work has established that the phosphorylation-dependent anti-tau antibody AT100 is a specific marker for filamentous tau in adult human brain. Here we have identified protein kinases that generate the AT100 epitope in vitro and have used them, in conjunction with site-directed mutagenesis of tau, to map the epitope. We show that the sequential phosphorylation of recombinant tau by cAMP-dependent protein kinase (PKA) and the stress-activated protein kinases SAPK4/p38 delta or JNK2 generated the AT100 epitope and that this required phosphorylation of T212, S214 and T217. Tau protein from newborn, but not adult, mouse brain was weakly labelled by AT100. Phosphorylation by PKA and SAPK4/p38 delta abolished the ability of tau to promote microtubule assembly, but failed to influence significantly the heparin-induced assembly of tau into filaments.