Glycogen synthase kinase 3β 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 GSK3 beta within amino acids 550-659 and that this phosphorylation is significantly decreased by pharmacological inhibition of GSK3 beta and suppression of GSK3 beta expression by small interfering RNA duplex. Mutation analysis showed that Ser(556) is a major GSK3 beta phosphorylation site in synphilin-1. GSK3 beta co-immunoprecipitated with synphilin-1, and protein 14-3-3, an activator of GSK3 beta activity, increased synphilin-1 phosphorylation. GSK3 beta 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 GSK3 beta greatly increased ubiquitylation and inclusion body formation by SIAH. Additionally, synphilin-1 S556A mutant, which is less phosphorylated by GSK3 beta, formed more inclusion bodies than wild type synphilin-1. Inhibition of GSK3 beta in primary neuronal cultures decreased the levels of endogenous synphilin-1, indicating that synphilin-1 is a physiologic substrate of GSK3 beta. 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 GSK3 beta during endoplasmic reticulum stress and the specific phosphorylation of synphilin-1 by GSK3 beta place synphilin-1 as a possible mediator of endoplasmic reticulum stress and proteasomal dysfunction observed in Parkinson disease.