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

Lee, Gwang; Tanaka, Mikiei; Park, Ki Ho; Lee, Sang Seop; Kim, Yong Man; Junn, Eunsung; Lee, Sang-Hyeon; Mouradian, M. Maral

doi:10.1074/jbc.m312760200

Cited by 92 publications

(90 citation statements)

References 39 publications

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“…Our data now suggest that the modulation of inclusion body formation also depends on GSK3␤ phosphorylation. Recently, Mouradian and co-workers found that casein kinase II phosphorylation at a still unidentified region of synphilin-1 is required for synphilin-1/␣-synuclein aggregation (29). Under our experimental conditions, casein kinase II inhibitor had no effect on synphilin-1 ubiquitylation or its ability to form cytosolic aggregates with SIAH-1.…”

Section: Discussionmentioning

confidence: 59%

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

Avraham

Szargel

Eyal

et al. 2005

Journal of Biological Chemistry

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␣-Synuclein is known to play a major role in the pathogenesis of Parkinson disease. We previously identified synphilin-1 as an ␣-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␤ within amino acids 550 -659 and that this phosphorylation is significantly decreased by pharmacological inhibition of GSK3␤ and suppression of GSK3␤ expression by small interfering RNA duplex. Mutation analysis showed that Ser 556 is a major GSK3␤ phosphorylation site in synphilin-1. GSK3␤ co-immunoprecipitated with synphilin-1, and protein 14-3-3, an activator of GSK3␤ activity, increased synphilin-1 phosphorylation. GSK3␤ 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␤ greatly increased ubiquitylation and inclusion body formation by SIAH. Additionally, synphilin-1 S556A mutant, which is less phosphorylated by GSK3␤, formed more inclusion bodies than wild type synphilin-1. Inhibition of GSK3␤ in primary neuronal cultures decreased the levels of endogenous synphilin-1, indicating that synphilin-1 is a physiologic substrate of GSK3␤. 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␤ during endoplasmic reticulum stress and the specific phosphorylation of synphilin-1 by GSK3␤ place synphilin-1 as a possible mediator of endoplasmic reticulum stress and proteasomal dysfunction observed in Parkinson disease. Parkinson disease (PD)2 is characterized by loss of dopaminergic neurons in the substantia nigra and the presence of cytoplasmic inclusions called Lewy bodies in surviving neurons (1). Hereditary PD can be caused by mutations in the ␣-synuclein gene (2-4) and in components of the ubiquitin-proteasome system, such as the E3 ubiquitin ligase parkin and UCH-L1 (5, 6).␣-Synuclein is a major component of Lewy bodies in sporadic PD (7). Overexpression of ␣-synuclein inhibits the proteasomal activity (8 -10) and causes cell death in a variety of cell and animal models (11). In agreement, proteasomal activity is decreased in substantia nigra of PD patients (12).We have shown that synphilin-1 is a presynaptic protein that interacts with ␣-synuclein in vivo (13,14). Synphilin-1 leads to the formation of inclusion bodies when co-transfected with the non-A␤ component portion of ␣-synuclein in cultured cells and is an intrinsic component of Lewy bodies in PD, suggesting that it may play a role in Lewy body formation (13,...

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Section: Discussionmentioning

confidence: 59%

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

Avraham

Szargel

Eyal

et al. 2005

Journal of Biological Chemistry

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“…We reported previously that synphilin-1, a protein that interacts with ␣-synuclein (Engelender et al, 1999), is highly enriched in Lewy bodies (Wakabayashi et al, 2000). Cotransfection of ␣-synuclein and synphilin-1 in human embryonic kidney 293 (HEK 293) cells yields cytoplasmic eosinophilic inclusions resembling Lewy bodies (Engelender et al, 1999;Lee et al, 2004). We also identified synphilin-1 as a substrate of parkin (a ubiquitin ligase, also present in Lewy bodies) (Chung et al, 2001a;Shimura et al, 2001;Schlossmacher et al, 2002).…”

Section: Introductionmentioning

confidence: 85%

-Synuclein Phosphorylation Enhances Eosinophilic Cytoplasmic Inclusion Formation in SH-SY5Y Cells

Smith

Margolis

et al. 2005

Journal of Neuroscience

247

229

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Parkinson's disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. Previous reports have shown that ␣-synuclein deposited in brain tissue from individuals with synucleinopathy is extensively phosphorylated at Ser-129. Here, we investigate the role of phosphorylation of ␣-synuclein in the formation of inclusions involving synphilin-1 and parkin using site-directed mutagenesis to change Ser-129 of ␣-synuclein to alanine (S129A) to abolish phosphorylation at this site. Coexpression of wild-type ␣-synuclein and synphilin-1 in human neuroblastoma SH-SY5Y cells yielded cytoplasmic eosinophilic inclusions with some features resembling Lewy bodies, whereas coexpression of S129A ␣-synuclein and synphlin-1 formed few or no inclusions. Moreover, coexpression of parkin with ␣-synuclein and synphilin-1 formed more ubiquitinated inclusions, but these inclusions decreased with expression of S129A ␣-synuclein instead of wild-type ␣-synuclein. Coimmunoprecipitation assays revealed a decreased interaction of S129A ␣-synuclein with synphilin-1 compared with wild-type ␣-synuclein. Expression of S129A ␣-synuclein instead of wild-type ␣-synuclein also decreased the association of synphilin-1 and parkin and subsequently reduced the parkin-mediated ubiquitination of synphilin-1 and the formation of ubiquitinated inclusions. Treatment of SH-SY5Y cells with H 2 O 2 increased ␣-synuclein phosphorylation and enhanced the formation of inclusions formed by coexpression of ␣-synuclein, synphilin-1, and parkin, whereas treatment with the casein kinase 2 inhibitor 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole had the opposite affect. These results indicate that phosphorylation of ␣-synuclein at S129 may be important for the formation of inclusions in PD and related ␣ synucleinopathies.

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“…Therefore, it is reasonable to predict that the identification of factors that regulate the physical and functional interactions between ␣-synuclein and synphilin-1 will help to uncover the molecular etiology of LB formation and PD pathogenesis. CKII has been recently reported as a potent kinase that phosphorylates both ␣-synuclein and synphilin-1 and regulates the binding between these two proteins (11,31). Significantly, the CKII inhibitor, DRB, abolishes this interaction and reduces inclusion body formation in cell cultures (31).…”

Section: Discussionmentioning

confidence: 99%

“…Pin1 Promotes the Interaction of ␣-Synuclein with Synphilin-1-We next addressed whether Pin1 expression affects the interaction between ␣-synuclein and synphilin-1, because this binding is crucial for the formation of cytoplasmic inclusions (9,31). 293T cells were transfected with HA-␣-synuclein and Xpress-synphilin-1, in the presence or absence of exogenous Pin1 co-expression, and lysates were subjected to immunoprecipitation with anti-Xpress antibodies.…”

Section: Pin1 Accumulates In the Lewy Bodies Of Parkinson Disease-mentioning

confidence: 99%

Prolyl-isomerase Pin1 Accumulates in Lewy Bodies of Parkinson Disease and Facilitates Formation of α-Synuclein Inclusions

Ryo

Togo

Nakai

et al. 2006

Journal of Biological Chemistry

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Parkinson disease (PD) is a relatively common neurodegenerative disorder that is characterized by the loss of dopaminergic neurons and by the formation of Lewy bodies (LBs), which are cytoplasmic inclusions containing aggregates of ␣-synuclein. Although certain post-translational modifications of ␣-synuclein and its related proteins are implicated in the genesis of LBs, the specific molecular mechanisms that both regulate these processes and initiate subsequent inclusion body formation are not yet well understood. We demonstrate in our current study, however, that the prolyl-isomerase Pin1 localizes to the LBs in PD brain tissue and thereby enhances the formation of ␣-synuclein immunoreactive inclusions. Immunohistochemical analysis of brain tissue from PD patients revealed that Pin1 localizes to 50 -60% of the LBs that show an intense halo pattern resembling that of ␣-synuclein. By utilizing a cellular model of ␣-synuclein aggregation, we also demonstrate that, whereas Pin1 overexpression facilitates the formation of ␣-synuclein inclusions, dominant-negative Pin1 expression significantly suppresses this process. Consistent with these observations, Pin1 overexpression enhances the protein half-life and insolubility of ␣-synuclein. Finally, we show that Pin1 binds synphilin-1, an ␣-synuclein partner, via its Ser-211-Pro and Ser-215-Pro motifs, and enhances its interaction with ␣-synuclein, thus likely facilitating the formation of ␣-synuclein inclusions. These results indicate that Pin1-mediated prolyl-isomerization plays a pivotal role in a post-translational modification pathway for ␣-synuclein aggregation and in the resultant Lewy body formations in PD. Parkinson disease (PD)2 is one of the most common neurodegenerative disorders and is characterized by the loss of dopaminergic neurons in the substantia nigra and by the presence of cytoplasmic inclusions known as Lewy bodies (LBs) in surviving neurons (1, 2). LBs have classically been considered as a pathological hallmark of PD, consisting of many components, including ␣-synuclein, which is one of the major constituents (3, 4). The first indication of a pathogenic role for ␣-synuclein in PD came from the results of linkage analysis of mutations in its gene in autosomal dominant forms of the disease (5, 6). ␣-Synuclein is an unfolded protein in its native state, but in a pathological state it can be induced to form either ␣-helical or ␤-sheet structures that result in the formation of insoluble ␣-synuclein aggregates (7,8). The aggregation of ␣-synuclein can be modified by a range of factors, both in vitro and in vivo, including environmental regulators of pH, temperature, ionic strength, and oxidative stress and by intrinsic intracellular pathways (8). The latter of these regulatory networks includes several ␣-synuclein-binding proteins such as synphilin-1 (9) and posttranslational modifications of related molecules, such as phosphorylation and ubiquitination (10 -12). Synphilin-1 was identified as a protein that interacts with ␣-synuclein and has been shown t...

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

Cited by 92 publications

References 39 publications

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

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

-Synuclein Phosphorylation Enhances Eosinophilic Cytoplasmic Inclusion Formation in SH-SY5Y Cells

Prolyl-isomerase Pin1 Accumulates in Lewy Bodies of Parkinson Disease and Facilitates Formation of α-Synuclein Inclusions

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