Characterization of Neuronal Tau Protein as a Target of Extracellular Signal-regulated Kinase

Haoling Qi,Sudhakaran Prabakaran,François-Xavier Cantrelle,Béatrice Chambraud,Jeremy Gunawardena,Guy Lippens,Isabelle Landrieu

doi:10.1074/jbc.m115.700914

Abstract

Tau neuronal protein has a central role in neurodegeneration and is implicated in Alzheimer disease development. Abnormal phosphorylation of Tau impairs its interaction with other proteins and is associated with its dysregulation in pathological conditions. Molecular mechanisms leading to hyperphosphorylation of Tau in pathological conditions are unknown. Here, we characterize phosphorylation of Tau by extracellular-regulated kinase (ERK2), a mitogen-activated kinase (MAPK) that responds to extracellular signals. Analysis ofin vitrophosphorylated Tau by activated recombinant ERK2 with nuclear magnetic resonance spectroscopy (NMR) reveals phosphorylation of 15 Ser/Thr sites.In vitrophosphorylation of Tau using rat brain extract and subsequent NMR analysis identifies the same sites. Phosphorylation with rat brain extract is known to transform Tau into an Alzheimer disease-like state. Our results indicate that phosphorylation of Tau by ERK2 alone is sufficient to produce the same characteristics. We further investigate the mechanism of ERK2 phosphorylation of Tau. Kinases are known to recognize their protein substrates not only by their specificity for a targeted Ser or Thr phosphorylation site but also by binding to linear-peptide motifs called docking sites. We identify two main ERK2 docking sites in Tau sequence using NMR. Our results suggest that ERK2 dysregulation in Alzheimer disease could lead to abnormal phosphorylation of Tau resulting in the pathology of the disease.

Highlights

Tau is an intrinsically disordered protein whose primary sequence is divided into several functional domains: an N-terminal region, a proline-rich domain (PRD),2 a microtubule binding domain (MTBD) constituted of partially repeated sequences R1 to R4, and a C-terminal region
A comparison with previous assignments of the nuclear magnetic resonance spectroscopy (NMR) amide cross-peaks corresponding to Tau proline-directed phosphorylation sites, generated by cyclin-dependent kinase 2/CycA3 or glycogen synthase kinase 3 (GSK3) [46, 47], further confirmed identification of the ERK2 phosphorylation sites located in the PRD and C-terminal region
Using NMR spectroscopy, we identified Tau phosphorylation sites modified in vitro by the activated ERK2 and rat brain extracts

Summary

Introduction

Tau is an intrinsically disordered protein whose primary sequence is divided into several functional domains: an N-terminal region, a proline-rich domain (PRD),2 a microtubule binding domain (MTBD) constituted of partially repeated sequences R1 to R4, and a C-terminal region (see Fig. 1A). On incubation with MEK-activated recombinant ERK2 (double-phosphorylated on Thr-183/Tyr-185), several additional resonances were observed in the 1H,15N two-dimensional spectrum for the resulting phospho-Tau compared with the spectrum of the unphosphorylated Tau (compare Fig. 1, B and C). Phosphorylation sites were identified using three-dimensional triple resonance NMR spectroscopy on a 13C,15N-labeled Tau sample phosphorylated in vitro by activated ERK2.

Results

Conclusion