Glycogen synthase kinase 3beta modulates synphilin-1 ubiquitylation and cellular inclusion formation by SIAH: implications for proteasomal function and Lewy body formation.
alpha-Synuclein is known to play a major role in the pathogenesis of Parkinson disease. We previously identified synphilin-1 as an alpha-synuclein-interacting protein and more recently found that synphilin-1 also interacts with the E3 ubiquitin ligases SIAH-1 and SIAH-2. SIAH proteins ubiquitylate synphilin-1 and promote its degradation through the ubiquitin proteasome system. Inability of the proteasome to degrade synphilin-1 promotes the formation of ubiquitylated inclusion bodies. We now show that synphilin-1 is phosphorylated by GSK3beta within amino acids 550-659 and that this phosphorylation is significantly decreased by pharmacological inhibition of GSK3beta and suppression of GSK3beta expression by small interfering RNA duplex. Mutation analysis showed that Ser556 is a major GSK3beta phosphorylation site in synphilin-1. GSK3beta co-immunoprecipitated with synphilin-1, and protein 14-3-3, an activator of GSK3beta activity, increased synphilin-1 phosphorylation. GSK3beta decreased the in vitro and in vivo ubiquitylation of synphilin-1 as well as its degradation promoted by SIAH. Pharmacological inhibition and small interfering RNA suppression of GSK3beta greatly increased ubiquitylation and inclusion body formation by SIAH. Additionally, synphilin-1 S556A mutant, which is less phosphorylated by GSK3beta, formed more inclusion bodies than wild type synphilin-1. Inhibition of GSK3beta in primary neuronal cultures decreased the levels of endogenous synphilin-1, indicating that synphilin-1 is a physiologic substrate of GSK3beta. Using GFPu as a reporter to measure proteasome function in vivo, we found that synphilin-1 S556A is more efficient in inhibiting the proteasome than wild type synphilin-1, raising the possibility that the degree of synphilin-1 phosphorylation may regulate the proteasome function. Activation of GSK3beta during endoplasmic reticulum stress and the specific phosphorylation of synphilin-1 by GSK3beta place synphilin-1 as a possible mediator of endoplasmic reticulum stress and proteasomal dysfunction observed in Parkinson disease.
Avraham
Eyal
E
Department of Pharmacology, The B. Rappaport Institute of Medical Research, Technion-Israel Institute of Technology, Haifa 31096, Israel.
Szargel
Raymonde
R
Eyal
Allon
A
Rott
Ruth
R
Engelender
Simone
S
D048948:14-3-3 Proteins / Q000737:chemistry; D001665:Binding Sites; D015153:Blotting, Western; D002352:Carrier Proteins / Q000737:chemistry*; D002460:Cell Line; D003593:Cytoplasm / Q000378:metabolism; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D017930:Genes, Reporter; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism / Q000502:physiology*; D000071679:Glycogen Synthase Kinase 3 beta; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D016631:Lewy Bodies / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D009154:Mutation; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D009687:Nuclear Proteins / Q000737:chemistry*; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D034741:RNA, Small Interfering / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D013997:Time Factors; D014162:Transfection; D025801:Ubiquitin / Q000737:chemistry* / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry*; D000098563:Seven in Absentia Proteins
D048948:14-3-3 Proteins; D002352:Carrier Proteins; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; C118958:SNCAIP protein, human; D025801:Ubiquitin; D049452:Green Fluorescent Proteins; D012694:Serine; D044767:Ubiquitin-Protein Ligases; D000098563:Seven in Absentia Proteins; C000605506:GSK3B protein, human; D000071679:Glycogen Synthase Kinase 3 beta; D038362:Glycogen Synthase Kinase 3; D046988:Proteasome Endopeptidase Complex