Parkinson Disease Mutant E46K Enhances α-Synuclein Phosphorylation in Mammalian Cell Lines, in Yeast, and in Vivo

Martial Kamdem Mbefo,Mohamed-Bilal Fares,Katerina Paleologou,Abid Oueslati,Guowei Yin,Sandra Tenreiro,Madalena Pinto,Tiago Outeiro,Markus Zweckstetter,Eliezer Masliah,Hilal A Lashuel

doi:10.1074/jbc.m114.610774

Abstract

Although α-synuclein (α-syn) phosphorylation has been considered as a hallmark of sporadic and familial Parkinson disease (PD), little is known about the effect of PD-linked mutations on α-syn phosphorylation. In this study, we investigated the effects of the A30P, E46K, and A53T PD-linked mutations on α-syn phosphorylation at residues Ser-87 and Ser-129. Although the A30P and A53T mutants slightly affected Ser(P)-129 levels compared with WT α-syn, the E46K mutation significantly enhanced Ser-129 phosphorylation in yeast and mammalian cell lines. This effect was not due to the E46K mutant being a better kinase substrate nor due to alterations in endogenous kinase levels, but was mostly linked with enhanced nuclear and endoplasmic reticulum accumulation. Importantly, lentivirus-mediated overexpression in mice also showed enhanced Ser-129 phosphorylation of the E46K mutant compared to WT α-syn, thus providing in vivo validation of our findings. Altogether, our findings suggest that the different PD-linked mutations may contribute to PD pathogenesis via different mechanisms.

Highlights

Little is known about the effect of Parkinson disease (PD)-linked mutations on ␣-synuclein (␣-syn) phosphorylation
We investigated the effects of the A30P, E46K, and A53T PD-linked mutations on ␣-syn phosphorylation at residues Ser-87 and Ser-129
To further explore the interplay between ␣-syn PD-linked mutations and pathological post-translational modifications, we investigated the effects of the A30P, E46K, and A53T mutations on ␣-syn phosphorylation at Ser-87 and Ser-129 by endogenous kinases in mammalian cell lines and in yeast

Summary

Background

Little is known about the effect of PD-linked mutations on ␣-synuclein (␣-syn) phosphorylation. The A30P and A53T mutants slightly affected Ser(P)-129 levels compared with WT ␣-syn, the E46K mutation significantly enhanced Ser-129 phosphorylation in yeast and mammalian cell lines This effect was not due to the E46K mutant being a better kinase substrate nor due to alterations in endogenous kinase levels, but was mostly linked with enhanced nuclear and endoplasmic reticulum accumulation. Lentivirus-mediated overexpression of E46K ␣-syn in the hippocampus of mice showed enhanced Ser(P)-129 phosphorylation Intrigued by this observation, we set to determine whether this effect is due to 1) the E46K mutant being a better kinase substrate in vitro and in cells, 2) alterations in endogenous kinase levels, 3) impairment in Ser(P)-129 or total ␣-syn degradation, or 4) differences in ␣-syn subcellular localization. This suggests that the different PD-linked mutations may contribute to the pathogenesis of PD via different mechanisms, and that improper localization of ␣-syn could alter its phosphorylation state at Ser-129

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION