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

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 of in vitro phosphorylated Tau by activated recombinant ERK2 with nuclear magnetic resonance spectroscopy (NMR) reveals phosphorylation of 15 Ser/Thr sites. In vitro phosphorylation 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.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). Both PRD and MTBD are involved in microtubule stabilizing activity of Tau (1, 2).Phosphorylation of Tau is an essential process to regulate its physiological function(s); however, abnormal phosphorylation of Tau has been linked to neuronal dysfunction (3-5). In Alzheimer disease (AD) Tau is hyperphosphorylated and aggregated (6). The longest Tau protein isoform (441 residues) has 80 threonine (Thr) or serine (Ser) residues. These residues are exposed as Tau is an intrinsically disordered protein subject to modification by numerous kinases (7). Mass spectrometry (MS) analyses have identified ϳ45 phosphorylated sites on Tau aggregates extracted from AD patients with a typical paired helical filament (PHF) morphology (8, 9) ERK2 belongs to the mitogen-activated protein kinase (MAPKs) family and is activated by dual phosphorylation on a Thr-Xaa-Tyr sequence in its activation loop by MAP kinase kinases (MKK) (21-23). Human ERK2 is phosphorylated on Thr-183 and Tyr-185 by MEK kinase. MAP kinases have a classic kinase structure with an N-terminal lobe folded as a ␤ sheet associated to a larger C-terminal domain constituted of ␣-helices (24). The activation loop is located at the hinge between these domains and shows a large conformational change upon phosphor...

Identification of the Tau phosphorylation pattern that drives its aggregation

“…2 B and C). In agreement with our previous results on full-length Tau (31), no ThT response could be detected during the phosphorylation of the TauF8 sample, and further analysis by electron microscopy of this sample showed very few structures after 20 h of incubation (Fig. 2D).…”

“…Additional phosphorylation at position 208 hence prevents the turn-like structure in a manner similar to the Gly-to-Val mutation at position 207. We have previously confirmed that ERK2, as a proline-directed kinase, does not phosphorylate the Ser208 residue (31). However, the kinase activity of a rat brain extract (RBE) in the presence of serine/threonine phosphatase inhibitor okadaic acid can lead to phosphorylation of the Ser208 site, but also phosphorylates Ser262 (31) As phosphorylation of Ser262 has been described as inhibitory for Tau aggregation (27), we mutated Ser262 to alanine in the TauF8 fragment and phosphorylated the resulting TauF8-S262A sample by the RBE.…”

“…One study phosphorylated Tau with a RBE to 12-15 phosphates per Tau molecule and obtained fibers with the macroscopic aspect of those isolated from AD patient brains (16). However, no precise definition of the phosphorylation pattern generated by the RBE was determined, and attempts to repeat this experiment did not yield any appreciable amount of fibers (31,39).…”

“…However, when previously using the RBE kinase activity with Tau441 as a substrate, we did not obtain significant amounts of protein aggregates (31). With hindsight, and suspecting the inhibitory role of Ser262 phosphorylation not only in a heparin-derived assay (27) but equally when phosphorylation is the driver, we produced both WT and the S262A mutant of Tau441 (Tau441-S262A).…”

Oxidative Stress, Inflammation, and Formation of Beta‐ Amyloid 1‐42 in Brain

“…This receptor usually binds APP to the plasmatic membrane to prevent the internalization of the -amyloid peptide, reducing its production [87]. The failure of these mechanisms by the efect of oxidative stress and the hyperphosphorylation of tau protein induced a disturbance in the formation of microtubules, producing neuroibrillary tangles [88]. Furthermore, tau protein associates with -amyloids that might be involved in the internalization of the extracellular protein into the neurons.…”