Glycogen Synthase Kinase 3β Modulates Synphilin-1 Ubiquitylation and Cellular Inclusion Formation by SIAH

• Published in: The Journal of biological chemistry 2005-12, Vol.280 (52), p.42877-42886
• Main Authors: Avraham, Eyal, Szargel, Raymonde, Eyal, Allon, Rott, Ruth, Engelender, Simone
• Format: Article
• Language: English
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M505608200

α-Synuclein is known to play a major role in the pathogenesis of Parkinson disease. We previously identified synphilin-1 as an α-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 GSK3β within amino acids 550–659 and that this phosphorylation is significantly decreased by pharmacological inhibition of GSK3β and suppression of GSK3β expression by small interfering RNA duplex. Mutation analysis showed that Ser556 is a major GSK3β phosphorylation site in synphilin-1. GSK3β co-immunoprecipitated with synphilin-1, and protein 14-3-3, an activator of GSK3β activity, increased synphilin-1 phosphorylation. GSK3β 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 GSK3β greatly increased ubiquitylation and inclusion body formation by SIAH. Additionally, synphilin-1 S556A mutant, which is less phosphorylated by GSK3β, formed more inclusion bodies than wild type synphilin-1. Inhibition of GSK3β in primary neuronal cultures decreased the levels of endogenous synphilin-1, indicating that synphilin-1 is a physiologic substrate of GSK3β. 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 GSK3β during endoplasmic reticulum stress and the specific phosphorylation of synphilin-1 by GSK3β place synphilin-1 as a possible mediator of endoplasmic reticulum stress and proteasomal dysfunction observed in Parkinson disease.