Phosphorylated alpha-synuclein at Ser-129 is targeted to the proteasome pathway in a ubiquitin-independent manner.
α-Synuclein (a-Syn) is a major component of fibrillar aggregates in Lewy bodies (LBs), a characteristic hallmark of Parkinson disease. Almost 90% of a-Syn deposited in LBs is phosphorylated at Ser-129. However, the role of Ser-129-phosphorylated a-Syn in the biogenesis of LBs remains unclear. Here, we investigated the metabolism of Ser-129-phosphorylated a-Syn. In SH-SY5Y cells, inhibition of protein phosphatase 2A/1 by okadaic acid, and inhibition of the proteasome pathway by MG132 or lactacystin accumulated Ser-129-phosphorylated a-Syn. However, these inhibitions did not alter the amounts of total a-Syn within the observation time. Inhibition of the autophagy-lysosome pathway by 3-methyladenine or chloroquine accumulated Ser-129-phosphorylated a-Syn in parallel to total a-Syn during longer incubations. Experiments using cycloheximide showed that Ser-129-phosphorylated a-Syn diminished rapidly (t(&frac12;) = 54.9 ± 6.4 min), in contrast to the stably expressed total a-Syn. The short half-life of Ser-129-phosphorylated a-Syn was blocked by MG132 to a greater extent than okadaic acid. In rat primary cortical neurons, either MG132, lactacystin, or okadaic acid accumulated Ser-129-phosphorylated a-Syn. Additionally, we did not find that phosphorylated a-Syn was ubiquitinated in the presence of proteasome inhibitors. These data show that Ser-129-phosphorylated a-Syn is targeted to the proteasome pathway in a ubiquitin-independent manner, in addition to undergoing dephosphorylation. The proteasome pathway may play a role in the biogenesis of Ser-129-phosphorylated a-Syn-rich LBs.
Department of Neurology, Hematology, Metabolism, Endocrinology, and Diabetology, Faculty of Medicine, Yamagata University, Yamagata 990-9585, Japan.
Youhei
Y
Machiya
Susumu
S
Hara
Shigeki
S
Arawaka
Shingo
S
Fukushima
Hiroyasu
H
Sato
Masahiro
M
Sakamoto
Shingo
S
Koyama
Takeo
T
Kato
D000111:Acetylcysteine / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D045744:Cell Line, Tumor; D002540:Cerebral Cortex / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D006801:Humans; D007976:Leupeptins / Q000494:pharmacology; D016631:Lewy Bodies / Q000235:genetics / Q000378:metabolism; D009474:Neurons / Q000378:metabolism*; D019319:Okadaic Acid / Q000494:pharmacology; D010300:Parkinson Disease / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects / Q000235:genetics; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism*; D061988:Proteasome Inhibitors; D054645:Protein Phosphatase 1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D054648:Protein Phosphatase 2 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D011500:Protein Synthesis Inhibitors; D011817:Rabbits; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism*; D051844:alpha-Synuclein / Q000235:genetics / Q000378:metabolism*
D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D061988:Proteasome Inhibitors; D011500:Protein Synthesis Inhibitors; D025801:Ubiquitin; D051844:alpha-Synuclein; C067713:lactacystin; D019319:Okadaic Acid; D003513:Cycloheximide; C522189:Ppp2r1b protein, rat; D054645:Protein Phosphatase 1; D054648:Protein Phosphatase 2; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D000111:Acetylcysteine