Assembly of Lysine 63-linked Ubiquitin Conjugates by Phosphorylated α-Synuclein Implies Lewy Body Biogenesis

Chao Liu,Erkang Fei,Nali Jia,Hongfeng Wang,Ruisong Tao,Atsushi Iwata,Nobuyuki Nukina,Jiangning Zhou,Guanghui Wang

doi:10.1074/jbc.m700422200

Abstract

alpha-Synuclein (alpha-syn) and ubiquitin (Ub) are major protein components deposited in Lewy bodies (LBs) and Lewy neurites, which are pathologic hallmarks of idiopathic Parkinson disease (PD). Almost 90% of alpha-syn in LBs is phosphorylated at serine 129 (Ser(129)). However, the role of Ser(129)-phosphorylated alpha-syn in the biogenesis of LBs remains unclear. Here, we show that compared with coexpression of wild type (WT)alpha-syn and Ub, coexpression of phospho-mimic mutant alpha-syn (S129D) and Ub in neuro2a cells results in an increase of Ub-conjugates and the formation of ubiquitinated inclusions. Furthermore, S129D alpha-syn fails to increase the Ub-conjugates and form ubiquitinated inclusions in the presence of a K63R mutant Ub. In addition, as compared with WT alpha-syn, S129D alpha-syn increased cytoplasmic and neuritic aggregates of itself in neuro2a cells treated with H(2)O(2) and serum deprivation. These results suggest that the contribution of Ser(129)-phosphorylated alpha-syn to the Lys(63)-linked Ub-conjugates and aggregation of itself may be involved in the biogenesis of LBs in Parkinson disease and other related synucleinopathies.

Highlights

Three pea (Pisum sativum) loci controlling photoperiod sensitivity, HIGH RESPONSE (HR), DIE NEUTRALIS (DNE), and STERILE NODES (SN), have recently been shown to correspond to orthologs of Arabidopsis (Arabidopsis thaliana) circadian clock genes EARLY FLOWERING3 (ELF3), ELF4, and LUX ARRHYTHMO, respectively
This interaction has been extensively explored in Arabidopsis (Arabidopsis thaliana), where the circadian clock consists of a network of genes that form several interlocking feedback loops and influences flowering time through control of several direct and indirect regulators of the florigen gene FLOWERING LOCUS T (FT; Andrés and Coupland, 2012; Song et al, 2012; Millar, 2016)
As all three ppd mutations appeared similar in their phenotypic effects, we used the ppd-3 mutant to explore the roles of PPD, as it was generated in the same genetic background as other relevant mutants, including sn and dne

Summary

Introduction

Three pea (Pisum sativum) loci controlling photoperiod sensitivity, HIGH RESPONSE (HR), DIE NEUTRALIS (DNE), and STERILE NODES (SN), have recently been shown to correspond to orthologs of Arabidopsis (Arabidopsis thaliana) circadian clock genes EARLY FLOWERING3 (ELF3), ELF4, and LUX ARRHYTHMO, respectively. EC proteins are involved in regulation of a number of clock-associated genes, including TIMING OF CAB EXPRESSION1 (TOC1), GIGANTEA (GI), LUX, PSEUDO RESPONSE REGULATOR7 (PRR7), and PRR9, and act to repress expression of these genes during the night (Huang and Nusinow, 2016). In some cases, this appears to reflect direct binding of LUX to LUX binding site elements in the promoters of these genes (Helfer et al, 2011; Nusinow et al, 2011; Chow et al, 2012; Herrero et al, 2012; Mizuno et al, 2014). It is clear that participation in the EC is a key role for ELF3, ELF4, and LUX proteins, it seems likely that the individual functions of ELF3 and ELF4 are not limited to this, as they have been reported to interact independently with other circadian clock, photoperiod pathway, and light signaling components (Liu et al, 2001; Kim et al, 2013; Kaiserli et al, 2015; Nieto et al, 2015)

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