Casein Kinase II-mediated Phosphorylation Regulates α-Synuclein/Synphilin-1 Interaction and Inclusion Body Formation

Gwang Lee, Mikiei Tanaka, Kiho Park, Sang Seop Lee, Yong Man Kim, Eunsung Junn, Sang-Hyeon Lee, M. Maral Mouradian
2003 Journal of Biological Chemistry  
␣-Synuclein is a phosphoprotein that accumulates as a major component of Lewy bodies in the brains of patients with Parkinson disease. Synphilin-1, which is also present in Lewy bodies, binds with ␣-synuclein and forms cytoplasmic inclusions in transfected cells. Yet the molecular determinants of this protein-protein interaction are unknown. Here we report that casein kinase II (CKII) phosphorylates synphilin-1 and that the ␤ subunit of this enzyme complex binds to synphilin-1. Additionally,
more » ... 1. Additionally, both CKII ␣ and ␤ subunits are present within cytoplasmic inclusions in cells that overexpress synphilin-1. Notably, the interaction between synphilin-1 and ␣-synuclein is markedly dependent on phosphorylation. Inhibition of CKII activity by 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole blocks the binding between these two proteins and significantly reduces the percentage of cells that contain eosinophilic cytoplasmic inclusions. Mutation of the major CKII phosphorylation site in ␣-synuclein (S129A) has no significant impact on the binding between ␣-synuclein and synphilin-1 or on the formation of synphilin-1/␣-synuclein-positive inclusions. These data suggest that the CKII-mediated phosphorylation of synphilin-1 rather than that of ␣-synuclein is critical in modulating their tendency to aggregate into inclusions. These observations collectively indicate that a ubiquitous post-translational modification such as phosphorylation can regulate inclusion body formation in the context of ␣-synuclein and synphilin-1 interaction. Parkinson disease (PD) 1 is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta. Accumulating evidence suggests that aberrations in protein processing or folding leading to the aggregation of pathogenic proteins or their partners are a common theme in many degenerative disorders affecting the brain, including PD (1-3). Lewy bodies, classically considered as pathological hallmark features of PD, are proteinaceous cytoplasmic inclusions that consist of many components, among which ␣-synuclein is a major constituent (4, 5). The first indication for the pathogenic role of ␣-synuclein in PD came from the linkage of mutations in its gene with autosomal dominant forms of PD (6, 7). Another component of Lewy bodies is synphilin-1, which was discovered by screening for proteins that interact with ␣-synuclein (8). Co-expression of these two proteins results in the formation of cytoplasmic inclusions in a small percentage of cultured cells (8, 9) . But the molecular determinants that regulate this interaction and perhaps consequent inclusion body formation are unknown. Such factors would elucidate not only the genesis of these aggregates but could also provide clues about potential therapeutic targets if the inclusions or their precursors are pathogenic. Phosphorylation is a common post-translational modification that regulates the function and other properties of many proteins. ␣-Synuclein itself is phosphorylated both in vitro and in vivo by casein kinase (CK) I and II (10). Additionally, ␣-synuclein is extensively phosphorylated at serine 129 in synucleinopathy lesions and in overexpressing transgenic mice and flies (11-13). Yet, the physiological significance of this phosphorylation and its link to the pathogenesis of PD are unclear. Synphilin-1 also has consensus sequences for putative CKII recognition sites, but whether it is indeed phosphorylated has not been addressed. In this investigation we studied the phosphorylation of synphilin-1 by CKII and elucidated the critical role of this post-translational modification in the interaction between synphilin-1 and ␣-synuclein and in inclusion body formation. EXPERIMENTAL PROCEDURES Construction of Synphilin-1 and CKII Expression Vectors-pFLAGsynph expressing full-length synphilin-1 with an N-terminal FLAG tag was generated as described previously (14) . ␣-Synuclein cDNA was amplified by PCR from a human brain cDNA library (Stratagene) and inserted into pcDNA3.1 expression vector (Invitrogen) to generate pSyn-WT. To generate ␣-synuclein cDNA with a serine 129-alanine substitution, pSyn-WT was used as a template to introduce the T385G point mutation using the QuickChange site-directed mutagenesis kit (Stratagene). To generate an expression vector for myc-tagged casein kinase II ␤ subunit, the entire open reading frame was amplified by PCR from a human brain cDNA library (Invitrogen) using primers
doi:10.1074/jbc.m312760200 pmid:14645218 fatcat:fgfeafp2brgc7g5lhk32rrynji