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• W2037819682 abstract "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. 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. Parkinson disease (PD) 2The abbreviations used are: PDParkinson diseaseDRB5,6-dibromo-1-β-d-ribofuranosylbenzimidazoleGSK3βglycogen synthase kinase 3βsiRNAsmall interfering RNAHAhemagglutininSph-1synphilin-1.2The abbreviations used are: PDParkinson diseaseDRB5,6-dibromo-1-β-d-ribofuranosylbenzimidazoleGSK3βglycogen synthase kinase 3βsiRNAsmall interfering RNAHAhemagglutininSph-1synphilin-1. is characterized by loss of dopaminergic neurons in the substantia nigra and the presence of cytoplasmic inclusions called Lewy bodies in surviving neurons (1Dunnett S.B. Bjorklund A. Nature. 1999; 399: A32-A39Crossref PubMed Scopus (511) Google Scholar). Hereditary PD can be caused by mutations in the α-synuclein gene (2Polymeropoulos M.H. Lavedan C. Leroy E. Ide S.E. Dehejia A. Dutra A. Pike B. Root H. Rubenstein J. Boyer R. Stenroos E.S. Chandrasekharappa S. Athanassiadou A. Papapetropoulos T. Johnson W.G. Lazzarini A.M. Duvoisin R.C. Di Iorio G. Golbe L.I. Nussbaum R.L. Science. 1997; 276: 2045-2047Crossref PubMed Scopus (6510) Google Scholar, 3Krüger R. Kuhn W. Muller T. Woitalla D. Graeber M. Kosel S. Przuntek H. Epplen J.T. Schols L. Riess O. Nat. Genet. 1998; 18: 106-108Crossref PubMed Scopus (3263) Google Scholar, 4Zarranz J.J. Alegre J. Gomez-Esteban J.C. Lezcano E. Ros R. Ampuero I. Vidal L. Hoenicka J. Rodriguez O. Atares B. Llorens V. Gomez Tortosa E. del Ser T. Munoz D.G. de Yebenes J.G. Ann. Neurol. 2004; 55: 164-173Crossref PubMed Scopus (2118) Google Scholar) and in components of the ubiquitin-proteasome system, such as the E3 ubiquitin ligase parkin and UCH-L1 (5Kitada T. Asakawa S. Hattori N. Matsumine H. Yamamura Y. Minoshima S. Yokochi M. Mizuno Y. Shimizu N. Nature. 1998; 392: 605-608Crossref PubMed Scopus (4123) Google Scholar, 6Leroy E. Boyer R. Auburger G. Leube B. Ulm G. Mezey E. Harta G. Brownstein M.J. Jonnalagada S. Chernova T. Dehejia A. Lavedan C. Gasser T. Steinbach P.J. Wilkinson K.D. Polymeropoulos M.H. Nature. 1998; 395: 451-452Crossref PubMed Scopus (1372) Google Scholar).α-Synuclein is a major component of Lewy bodies in sporadic PD (7Spillantini M.G. Schmidt M.L. Lee V.M. Trojanowski J.Q. Jakes R. Goedert M. Nature. 1997; 388: 839-840Crossref PubMed Scopus (5964) Google Scholar). Overexpression of α-synuclein inhibits the proteasomal activity (8Tanaka Y. Engelender S. Igarashi S. Rao R.K. Wanner T. Tanzi R.E. Sawa A. Dawson V.L. Dawson T.M. Ross C. A. Hum. Mol. Genet. 2001; 10: 919-926Crossref PubMed Google Scholar, 9Snyder H. Mensah K. Theisler C. Lee J. Matouschek A. Wolozin B. J. Biol. Chem. 2003; 278: 11753-11759Abstract Full Text Full Text PDF PubMed Scopus (341) Google Scholar, 10Lindersson E. Beedholm R. Hojrup P. Moos T. Gai W. Hendil K.B. Jensen P.H. J. Biol. Chem. 2004; 279: 12924-12934Abstract Full Text Full Text PDF PubMed Scopus (306) Google Scholar) and causes cell death in a variety of cell and animal models (11Dawson T.M. Dawson V.L. Science. 2003; 302: 819-822Crossref PubMed Scopus (1377) Google Scholar). In agreement, proteasomal activity is decreased in substantia nigra of PD patients (12McNaught K.S. Jenner P. Neurosci. Lett. 2001; 297: 191-194Crossref PubMed Scopus (544) Google Scholar).We have shown that synphilin-1 is a presynaptic protein that interacts with α-synuclein in vivo (13Engelender S. Kaminsky Z. Guo X. Sharp A.H. Amaravi R.K. Kleiderlein J.J. Margolis R.L. Troncoso J.C. Lanahan A.A. Worley P.F. Dawson V.L. Dawson T.M. Ross C.A. Nat. Genet. 1999; 22: 110-114Crossref PubMed Scopus (435) Google Scholar, 14Ribeiro C.S. Carneiro K. Ross C.A. Menezes J.R. Engelender S. J. Biol. Chem. 2002; 277: 23927-23933Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Synphilin-1 leads to the formation of inclusion bodies when co-transfected with the non-Aβ component portion of α-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 (13Engelender S. Kaminsky Z. Guo X. Sharp A.H. Amaravi R.K. Kleiderlein J.J. Margolis R.L. Troncoso J.C. Lanahan A.A. Worley P.F. Dawson V.L. Dawson T.M. Ross C.A. Nat. Genet. 1999; 22: 110-114Crossref PubMed Scopus (435) Google Scholar, 15Wakabayashi K. Engelender S. Yoshimoto M. Tsuji S. Ross C.A. Takahashi H. Ann. Neurol. 2000; 47: 521-523Crossref PubMed Scopus (240) Google Scholar). Synphilin-1 seems to have a dual role in cell survival. Synphilin-1 is toxic to cells and inhibits proteasomal activity, raising the possibility that synphilin-1 might contribute to the death of dopaminergic neurons in PD (16Lee G. Junn E. Tanaka M. Kim Y.M. Lee S.S. Mouradian M.M. J. Neurochem. 2002; 83: 346-352Crossref PubMed Scopus (34) Google Scholar, 17Ihara M. Tomimoto H. Kitayama H. Morioka Y. Akiguchi I. Shibasaki H. Noda M. Kinoshita M. J. Biol. Chem. 2003; 278: 24095-24102Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar, 18Kalia S.K. Lee S. Smith P.D. Liu L. Crocker S.J. Thorarinsdottir T.E. Glover J.R. Fon E.A. Park D.S. Lozano A.M. Neuron. 2004; 44: 931-945Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). On the other hand, cells containing synphilin-1 inclusions are more resistant to death, indicating that inclusions might be neuroprotective (19Liani E. Eyal A. Avraham E. Shemer R. Szargel R. Berg D. Bornemann A. Riess O. Ross C.A. Rott R. Engelender S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5500-5505Crossref PubMed Scopus (158) Google Scholar, 20Marx F.P. Holzmann C. Strauss K.M. Li L. Eberhardt O. Gerhardt E. Cookson M.R. Hernandez D. Farrer M.J. Kachergus J. Engelender S. Ross C.A. Berger K. Schols L. Schulz J.B. Riess O. Kruger R. Hum. Mol. Genet. 2003; 12: 1223-1231Crossref PubMed Scopus (122) Google Scholar, 21Tanaka M. Kim Y.M. Lee G. Junn E. Iwatsubo T. Mouradian M.M. J. Biol. Chem. 2004; 279: 4625-4631Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). Additional evidence that synphilin-1 may be involved in PD comes from identification of a missense mutation in its gene in two patients that share a rare haplotype (20Marx F.P. Holzmann C. Strauss K.M. Li L. Eberhardt O. Gerhardt E. Cookson M.R. Hernandez D. Farrer M.J. Kachergus J. Engelender S. Ross C.A. Berger K. Schols L. Schulz J.B. Riess O. Kruger R. Hum. Mol. Genet. 2003; 12: 1223-1231Crossref PubMed Scopus (122) Google Scholar).It has been shown that Lewy bodies are ubiquitylated, and understanding the ubiquitylation mechanism of Lewy body proteins may be relevant for clarifying ubiquitin's role in Lewy body formation. We have recently reported that the E3 ubiquitin ligases SIAH-1 and SIAH-2 ubiquitylate and target synphilin-1 for degradation by the proteasome system (19Liani E. Eyal A. Avraham E. Shemer R. Szargel R. Berg D. Bornemann A. Riess O. Ross C.A. Rott R. Engelender S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5500-5505Crossref PubMed Scopus (158) Google Scholar). The inability of the proteasome to degrade the synphilin-1-SIAH complex leads to a robust formation of ubiquitylated cytosolic inclusions containing synphilin-1, SIAH, and α-synuclein (19Liani E. Eyal A. Avraham E. Shemer R. Szargel R. Berg D. Bornemann A. Riess O. Ross C.A. Rott R. Engelender S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5500-5505Crossref PubMed Scopus (158) Google Scholar). Ubiquitylation is required for inclusion body formation, since a catalytically inactive mutant of SIAH-1 that binds to synphilin-1 fails to promote inclusions (19Liani E. Eyal A. Avraham E. Shemer R. Szargel R. Berg D. Bornemann A. Riess O. Ross C.A. Rott R. Engelender S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5500-5505Crossref PubMed Scopus (158) Google Scholar). Additionally, both SIAH and synphilin-1 are present in Lewy bodies of PD patients, implying a role in inclusion formation.In an attempt to better understand the role of synphilin-1 in PD and Lewy body formation, we now sought to investigate mechanisms that regulate synphilin-1 ubiquitylation and inclusion formation. We present data indicating that synphilin-1 is phosphorylated in vivo by GSK3β, which regulates ubiquitin-dependent degradation of synphilin-1 and inclusion body formation mediated by SIAH. Selective inhibition of GSK3β or mutation of a GSK3β phosphorylation site greatly increased synphilin-1 aggregation into cytosolic inclusions, suggesting a role of phosphorylation in modulating synphilin-1 aggregation. GSK3β inhibitor also enhanced the degradation of endogenous synphilin-1 in neurons, indicating that synphilin-1 is a physiologic substrate of GSK3β. We also present data indicating that inhibition of proteasome function by synphilin-1 is modulated by its phosphorylation status. Our results shed light on the mechanism regulating synphilin-1 ubiquitylation and aggregation, with implications for inclusion body formation and possibly cell death in PD.EXPERIMENTAL PROCEDURESMaterials—Ubiquitin aldehyde and purified ubiquitin-activating enzyme were purchased Boston from teine, 32 Biochem. [35S]Methionine/cys-[P]orthophosphate, and [32P]ATP were purchased from Amersham Biosciences. 5,6-Dibromo-1-β-d-ribofuranosylbenzimidazole (DRB), SB415286, kenpaullone, and other protein kinase inhibitors were purchased from Sigma.Cell Culture and Transfections—HEK 293 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum in a5%CO2 atmosphere. Cells were transiently transfected with N-terminally tagged pRK5 and pFLAG-CMV-2 plasmids utilizing Lipofectamine 2000 (Invitrogen) and processed after 36 h.For experiments using siRNA, HEK 293 cells were transfected with 50 nm siRNAs using Lipofectamine 2000. After 48 h, cells were transfected with HA-synphilin-1 constructs and additional 50 nm siRNAs and processed after 36 h. Silencer-validated siRNA to GSK3β (sense siRNA strand, 5′-GGACAAGAGAUUUAAGAAUTT-3′; antisense siRNA strand, 5′-AUUCUUAAAUCUCUUGUCCTG-3′) and negative control siRNA 1 (sense siRNA strand, 5′-AGUACUGCUUACGAUACGGTT-3′; antisense siRNA strand, 5′-CCGUAUCGUAAGCAGUACUTT-3′) were obtained from Ambion.Western Blot Analysis—Total protein extracts were done by homogenizing HEK 293 cells and neurons in buffer containing 50 mm Tris (pH 7.4), 140 mm NaCl, 1% Triton X-100, 0.1% SDS, 30 μm MG132, and protease inhibitor mixture (Complete; Roche Applied Science). Blots were probed with antibodies anti-synphilin-1 (13Engelender S. Kaminsky Z. Guo X. Sharp A.H. Amaravi R.K. Kleiderlein J.J. Margolis R.L. Troncoso J.C. Lanahan A.A. Worley P.F. Dawson V.L. Dawson T.M. Ross C.A. Nat. Genet. 1999; 22: 110-114Crossref PubMed Scopus (435) Google Scholar), anti-α-synuclein (BD Biosciences), mouse anti-HA (Covance), rabbit anti-HA (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), mouse anti-Myc (Oncogene), rabbit anti-Myc (Santa Cruz Biotechnology), rabbit anti-FLAG (Sigma), mouse anti-GSK3β (Sigma), and mouse anti-actin (Santa Cruz Biotechnology).Co-immunoprecipitation Assays—Transfected HEK 293 cells were lysed in buffer containing 50 mm Tris (pH 7.4), 140 mm NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 30 μm MG132, and protease inhibitor mixture (Complete; Roche Applied Science). Cell extracts were clarified, and supernatant was incubated with anti-HA or anti-Myc as described (19Liani E. Eyal A. Avraham E. Shemer R. Szargel R. Berg D. Bornemann A. Riess O. Ross C.A. Rott R. Engelender S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5500-5505Crossref PubMed Scopus (158) Google Scholar). Immunoprecipitates were washed with lysis buffer containing 500 mm NaCl and detected by Western blot.In Vitro Kinase Assays—HEK 293 cells were transfected with HA-synphilin-1 cDNAs. After 36 h of transfection, cells were lysed as in the co-immunoprecipitation experiments. HA-synphilin-1 was immunoprecipitated with anti-HA antibody and washed in lysis buffer containing 500 mm NaCl. Immunoprecipitated synphilin-1 was incubated with recombinant GSK3β (New England Biolabs) at 37 °C for 1 h in buffer containing 40 mm Tris, pH 7.6, 2 mm dithiothreitol, 5 mm MgCl2, 2 μg/ml soybean trypsin inhibitor, 0.5 mm unlabeled ATP, 0.25 mCi/ml [γ-32P]ATP. Reactions were ended with sample buffer and analyzed by SDS-PAGE using 8% gel. The amount of 32P-labeled-synphilin-1 was quantified by PhosphorImager analysis. Equal loading of immunoprecipitated HA-synphilin-1 was determined by Western blot or Coomassie Blue staining.In Vivo Phosphorylation Assays—After overnight starvation in serum- and phosphate-free medium, transfected HEK 293 cells were incubated for 3–6 h at 37 °C with serum-free/phosphate-free medium containing 200–400 μCi/ml [32P]orthophosphate. Cells were harvested and lysed in buffer containing 50 mm Tris-HCl (pH 7.4), 140 mm NaCl, 1% Triton X-100, 0.1% SDS, 20 mm NaF, 2 mm Na3VO4, 30 μm MG132, and protease inhibitor mixture (Complete; Roche Applied Science). Immunoprecipitation of HA-synphilin-1 was carried out with anti-HA antibody for 4 h at 4°C. Beads were washed with lysis buffer supplemented with 500 mm NaCl and analyzed by 8% SDS-PAGE. Densitometric quantification of radiolabeled HA-synphilin-1 was carried out by PhosphorImager analysis. Equal loading of immunoprecipitated HA-synphilin-1 was determined by Western blot or Coomassie Blue staining.In Vitro Ubiquitylation Assays—Synphilin-1 was translated using TNT wheat germ in vitro translation kit from Promega using [35S]methionine (Amersham Biosciences). In vitro translated proteins were incubated in reaction medium containing 40 mm Tris (pH 7.6), 5 mm MgCl2, 2 mm dithiothreitol, 1 mm ATP, 10 mm phosphocreatine, 0.1 mg/ml creatine kinase, 7.5 μg of ubiquitin, 1 μg of ubiquitin aldehyde, 100 ng of ubiquitin-activating enzyme, and 200 ng of UbcH5b. Reactions were incubated at 37 °C for 1 h and analyzed by SDS-PAGE. 35S-Labeled synphilin-1 was determined by PhosphorImager analysis.For the in vitro ubiquitylation assays of prephosphorylated synphilin-1, HA-synphilin-1 was co-transfected with Myc-GSK3β into HEK 293 cells. After 36 h, cells were lysed by sonication in buffer containing 50 mm Tris-HCl (pH 7.4), 140 mm NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 20 mm NaF, 2 mm Na3VO4, 30 μm MG132, and protease inhibitor mixture (Complete; Roche Applied Science). Immunoprecipitation of HA-synphilin-1 was carried out with anti-HA antibody for 4 h at 4°C. Beads were washed with lysis buffer supplemented with 500 mm NaCl. Immunoprecipitated HA-synphilin-1 was incubated with the same ubiquitylation reaction medium used for the in vitro translated synphilin-1. Reactions were incubated at 37 °C for 1 h, and ubiquitylated synphilin-1 was detected by Western blot. Very harsh lysis conditions were used in order to prevent co-immunoprecipitation of GSK3β with synphilin-1.In Vivo Ubiquitylation Assays—Transfected HEK 293 cells were incubated with 10 μm lactacystin for 12 h and then lysed in buffer containing 50 mm Tris (pH 7.4), 140 mm NaCl, 1% Triton X-100, 1% deoxycholate, 0.1% SDS, 20 mm NaF, 2 mm Na3VO4, 10 μm lactacystin, and protease inhibitor mixture (Complete; Roche Applied Science). Immunoprecipitates were washed with lysis buffer containing 500 mm NaCl, and ubiquitylated synphilin-1 was detected by Western blot.Pulse-Chase Experiments—Transfected HEK 293 cells were washed, incubated with methionine/cysteine-free medium for 1 h, pulsed with methionine/cysteine-free medium containing 200 ml/μCi [35S]methionine/cysteine (PerkinElmer Life Sciences) for 3 h, and subsequently chased in normal medium for the times specified. Cells were harvested, and HA-synphilin-1 immunoprecipitation was carried out as described above for the in vivo ubiquitylation assays. Immunoprecipitates were resolved on 8% SDS-polyacrylamide gels, and the amount of 35S-labeled synphilin-1 was quantified by PhosphorImager analysis.Immunocytochemistry Assays—Transfected HEK 293 cells were treated for 8 h with 10 μm lactacystin, fixed with 4% paraformaldehyde for 15 min, and blocked in phosphate-buffered saline containing 0.2% Triton X-100 and 5% normal goat serum. Cells were stained with anti-HA (Covance) and anti-Myc (Santa Cruz) as described (19Liani E. Eyal A. Avraham E. Shemer R. Szargel R. Berg D. Bornemann A. Riess O. Ross C.A. Rott R. Engelender S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5500-5505Crossref PubMed Scopus (158) Google Scholar). Immunolabeling was detected using fluorescein isothiocyanate- and Cy3-labeled secondary antibodies (Jackson Laboratories). The percentage of cells containing cytosolic inclusions was counted by an investigator unaware of the treatment groups. Statistics of the number of inclusion-containing cells was analyzed by analysis of variance followed by Tukey's post-test and by paired t test, when appropriate.Primary Cortical Neuronal Cultures—Primary neuronal cultures were prepared from cerebral cortex of E18 Sprague-Dawley rats as described (22Brewer G.J. Torricelli J.R. Evege E.K. Price P.J. J. Neurosci. Res. 1993; 35: 567-576Crossref PubMed Scopus (1895) Google Scholar). Cells were cultured in 12-well plates coated with poly-d-lysine in neurobasal medium plus B27 for 2 weeks before use.RESULTSPhosphorylation by GSK3β—We found that synphilin-1 has 22 putative GSK3β phosphorylation sites according to the motif X(S/T)XXXS. Thus, we sought to investigate whether synphilin-1 is a target of GSK3β in vivo. Phosphorylation of full-length synphilin-1 in HEK 293 cells was significantly inhibited by two selective GSK3β inhibitors, SB415286 and kenpaullone (23Meijer L. Flajolet M. Greengard P. Trends Pharmacol. Sci. 2004; 25: 471-480Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar) (Fig. 1A). Lithium chloride, another GSK3β inhibitor, also inhibited the phosphorylation of full-length synphilin-1 (data not shown).In order to further verify the specificity of synphilin-1 phosphorylation by GSK3β, we carried out in vivo phosphorylation experiments using siRNA to suppress GSK3β expression. HEK 293 cells were transfected with full-length HA-synphilin-1 and siRNA to GSK3β or negative control siRNA. The siRNA to GSK3β, but not the control siRNA, was effective in decreasing the expression of GSK3β by at least 90% (Fig. 1B). We found that siRNA to GSK3 significantly decreased the phosphorylation of full-length synphilin-1 (Fig. 1B).To confirm that synphilin-1 is phosphorylated by GSK3β, we carried out in vivo phosphorylation assays in which HEK 293 cells were co-transfected with full-length HA-synphilin-1 and increasing amounts of Myc-GSK3β. Accordingly, GSK3β increased synphilin-1 phosphorylation in a dose-dependent manner (Fig. 1C). In addition, the constitutively active GSK3β S9A mutant (24Jope R.S. Johnson G.V. Trends Biochem. Sci. 2004; 29: 95-102Abstract Full Text Full Text PDF PubMed Scopus (1317) Google Scholar) phosphorylated synphilin-1 more efficiently than wild-type GSK3β (Fig. 1D). In vitro phosphorylation experiments with immunoprecipitated full-length synphilin-1 and recombinant GSK3β confirmed that synphilin-1 is phosphorylated by GSK3β, indicating that the GSK3β effect is direct (Fig. 1E).The ability of GSK3β to interact with synphilin-1 was also investigated by co-immunoprecipitation experiments using HEK 293 cells co-transfected with HA-synphilin-1 and Myc-GSK3β. When anti-HA was used to immunoprecipitate HA-synphilin-1, Myc-GSK3β was found associated with HA-synphilin-1 (Fig. 2). The interaction of GSK3β with synphilin-1 is specific, since the control Myc-FKBP12 did not co-immunoprecipitate with HA-synphilin-1 (Fig. 2). To map the GSK3β-binding domain of synphilin-1, we co-transfected different HA-tagged synphilin-1 fragments into HEK 293 cells with Myc-GSK3β and carried out co-immunoprecipitation experiments. Different regions of synphilin-1 interacted with GSK3β, and a higher co-immunoprecipitation of GSK3β was observed with the synphilin-1 region containing amino acids 550–769 (Fig. 2).FIGURE 2GSK3β co-immunoprecipitates with synphilin-1. HEK 293 cells were co-transfected with HA-synphilin-1 (full-length or different fragments) and Myc-GSK3β or Myc-FKBP12 as control. HA-synphilin-1 was immunoprecipitated (IP HA) using an anti-HA antibody, and samples were subjected to Western blot analysis using an anti-Myc antibody to check for Myc-GSK3β co-immunoprecipitation (middle panel). The upper panel shows equal expression of Myc-GSK3β and Myc-FKBP12 in the HEK 293 cells determined by Western blot using an anti-Myc antibody. The lower panel shows the amount of immunoprecipitated HA-synphilin-1 (full-length or different fragments) by Western blot using an anti-HA antibody.View Large Image Figure ViewerDownload Hi-res image Download (PPT)GSK3β activity is strongly stimulated by 14-3-3 protein, which also interacts with α-synuclein and accumulates in Lewy bodies (25Yuan Z. Agarwal-Mawal A. Paudel H.K. J. Biol. Chem. 2004; 279: 26105-26114Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 26Ostrerova N. Petrucelli L. Farrer M. Mehta N. Choi P. Hardy J. Wolozin B. J. Neurosci. 1999; 19: 5782-5791Crossref PubMed Google Scholar, 27Kawamoto Y. Akiguchi I. Nakamura S. Honjyo Y. Shibasaki H. Budka H. J. Neuropathol. Exp. Neurol. 2002; 61: 245-253Crossref PubMed Scopus (126) Google Scholar). We now identified 14-3-3 as an accessory protein for synphilin-1 phosphorylation. Protein 14-3-3 co-immunoprecipitated with synphilin-1 from HEK 293 cells (Fig. 3A). Co-transfection of Myc-14-3-3 did not increase the amount of Myc-GSK3β that co-immunoprecipitated with HA-synphilin-1 (Fig. 3A). However, protein 14-3-3 significantly enhanced the in vivo phosphorylation of synphilin-1 by GSK3β, suggesting the formation of a ternary protein complex (14-3-3, synphilin-1, GSK3β) (Fig. 3B).FIGURE 3Protein 14-3-3 co-immunoprecipitates with synphilin-1 and enhances synphilin-1 phosphorylation by GSK3β. A, HEK 293 cells were co-transfected with HA-synphilin-1, Myc-GSK3β, Myc-14-3-3, or Myc-FKBP12 as control. HA-synphilin-1 was immunoprecipitated (HA IP) using an anti-HA antibody, and samples were subjected to Western blot analysis using an anti-Myc antibody to check for Myc-GSK3β and Myc-14-3-3 co-immunoprecipitations (middle panel). The upper panel shows total expression of Myc-GSK3β, Myc-14-3-3, and Myc-FKBP12 in the HEK 293 cells determined by Western blot using an anti-Myc antibody. The lower panel shows the amount of immunoprecipitated full-length HA-synphilin-1 by Western blot using an anti-HA antibody. B, HEK 293 cells were co-transfected with HA-synphilin-1, Myc-GSK3β, and Myc-14-3-3. Cells were incubated with [32P]orthophosphate, HA-synphilin-1 was immunoprecipitated with an anti-HA antibody, and phosphorylated synphilin-1 was detected by autoradiography. The lower panel shows equal immunoprecipitation of HA-synphilin-1 by Coomassie Blue staining.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Synphilin-1 Ubiquitylation—SIAH-1 and SIAH-2 are the major E3 ubiquitin ligases that regulate ubiquitin-dependent degradation of synphilin-1 (19Liani E. Eyal A. Avraham E. Shemer R. Szargel R. Berg D. Bornemann A. Riess O. Ross C.A. Rott R. Engelender S. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 5500-5505Crossref PubMed Scopus (158) Google Scholar, 28Nagano Y. Yamashita H. Takahashi T. Kishida S. Nakamura T. Iseki E. Hattori N. Mizuno Y. Kikuchi A. Matsumoto M. J. Biol. Chem. 2003; 278: 51504-51514Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). To investigate whether phosphorylation of synphilin-1 by GSK3β regulates ubiquitylation of synphilin-1, we carried out in vitro and in vivo ubiquitylation assays. Ubiquitylation of in vitro translated synphilin-1 promoted by SIAH-2 was significantly inhibited by the addition of GSK3β (Fig. 4A). By contrast, the addition of casein kinase II, previously shown to phosphorylate synphilin-1 (29Lee G Tanaka M. Park K. Lee S.S. Kim Y.M. Junn E. Lee S.H. Mouradian M.M. J. Biol. Chem. 2004; 279: 6834-6839Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar), did not affect synphilin-1 ubiquitylation promoted by SIAH-2 (Fig. 4A). The same results with GSK3β and casein kinase II were obtained when SIAH-1 was replaced for SIAH-2 (data not shown).FIGURE 4Effect of GSK3β in the ubiquitylation of synphilin-1 by SIAH. A, GSK3β decreases in vitro synphilin-1 ubiquitylation by SIAH. In vitro translated synphilin-1 was incubated with the indicated components of the ubiquitin system, in the presence of glutathione S-transferase (GST)-SIAH-2, GSK3β, and CKII, and ubiquitylated synphilin-1 was visualized by autoradiography. E1, ubiquitin-activating enzyme. B, autoubiquitylation of SIAH-1 is not affected by GSK3β. In vitro translated SIAH-1 was incubated with ubiquitin system components in the presence or absence of GSK3β, and ubiquitylated SIAH-1 was visualized by autoradiography. C, GSK3β decreases in vitro ubiquitylation of prephosphorylated synphilin-1. HEK 293 cells were transfected with HA-synphilin-1 and Myc-GSK3β or the control Myc-FKBP12. HA-immunoprecipitates (IP HA) were incubated with the indicated components of the ubiquitin system and glutathione S-transferase-SIAH-2, and ubiquitylation of HA-synphilin-1 was determined by Western blot using purified anti-synphilin-1 antibody (middle panel). The upper panel shows the expression of Myc-GSK3β in the transfected HEK 293 cells determined by Western blot using an anti-Myc antibody. The lower panel shows the lack of residual Myc-GSK3β in the synphilin-1 immunoprecipitate due to the harsh immunoprecipitation conditions, as described under “Experimental Procedures.” D, GSK3β decreases the in vivo ubiq" @default.
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• W2037819682 title "Glycogen Synthase Kinase 3β Modulates Synphilin-1 Ubiquitylation and Cellular Inclusion Formation by SIAH" @default.
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• W2037819682 cites W1527671059 @default.
• W2037819682 cites W1530609782 @default.
• W2037819682 cites W1552448470 @default.
• W2037819682 cites W1618846043 @default.
• W2037819682 cites W1877873625 @default.
• W2037819682 cites W1965778124 @default.
• W2037819682 cites W1968957266 @default.
• W2037819682 cites W1969571310 @default.
• W2037819682 cites W1971389338 @default.
• W2037819682 cites W197448336 @default.
• W2037819682 cites W1991231925 @default.
• W2037819682 cites W1993878844 @default.
• W2037819682 cites W1997369314 @default.
• W2037819682 cites W1998180291 @default.
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• W2037819682 cites W2014300038 @default.
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• W2037819682 cites W2019978095 @default.
• W2037819682 cites W2034595626 @default.
• W2037819682 cites W2046197575 @default.
• W2037819682 cites W2053680640 @default.
• W2037819682 cites W2054427773 @default.
• W2037819682 cites W2056865885 @default.
• W2037819682 cites W2059737503 @default.
• W2037819682 cites W2062918974 @default.
• W2037819682 cites W2064913654 @default.
• W2037819682 cites W2091265794 @default.
• W2037819682 cites W2091693910 @default.
• W2037819682 cites W2104912043 @default.
• W2037819682 cites W2116598203 @default.
• W2037819682 cites W2134573238 @default.
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• W2037819682 cites W2150656456 @default.
• W2037819682 cites W2152693222 @default.
• W2037819682 cites W2153244109 @default.
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