Kinase-specific phosphorylation of tau outside the amyloid core encodes fibril fold selection

Abstract

Tauopathies are defined by the accumulation of filamentous tau assemblies that adopt disease-specific molecular conformations, or tau strains. Although tau is extensively hyperphosphorylated in Alzheimer’s disease, how phosphorylation patterns influence tau folding and strain selection remains unclear. Here, we show that site-specific phosphorylation of 0N3R tau by extracellular signal-regulated kinase 2 (ERK2), occurring predominantly within the proline-rich region and largely excluding the microtubule-binding domain, is sufficient to direct tau assembly into a structurally homogeneous fibril conformation. Residue-resolved NMR spectroscopy identifies a defined and quantifiable ERK2 phosphorylation pattern in the proline-rich region of tau. Cryo-electron microscopy at 3.0 Å resolution reveals that ERK2-phosphorylated 3R tau assembles into filaments with an ordered cross-β fold adopting an Alzheimer’s disease PHF fold, despite the absence of phospho-sites within the fibril core. These filaments exhibit robust seeding activity in tau biosensor cells. Together, these results demonstrate that kinase-specific phosphorylation outside the amyloid core can be sufficient to bias tau toward a defined fibril structure, establishing a direct mechanistic link between kinase specificity, post-translational modification, and tau strain formation.