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Phosphorylation of Synucleins by Members of the Polo-like Kinase Family

2009; Elsevier BV; Volume: 285; Issue: 4 Linguagem: Inglês

10.1074/jbc.m109.081950

1083-351X

Martial Mbefo, Katerina E. Paleologou, Ahmed Boucharaba, Abid Oueslati, Heinrich Schell, Margot Fournier, Diana Olschewski, Guowei Yin, Markus Zweckstetter, Eliezer Masliah, Philipp J. Kahle, Harald Hirling, Hilal A. Lashuel,

Genetic Neurodegenerative Diseases

Phosphorylation of α-synuclein (α-syn) at Ser-129 is a hallmark of Parkinson disease and related synucleinopathies. However, the identity of the natural kinases and phosphatases responsible for regulating α-syn phosphorylation remain unknown. Here we demonstrate that three closely related members of the human Polo-like kinase (PLK) family (PLK1, PLK2, and PLK3) phosphorylate α-syn and β-syn specifically at Ser-129 and Ser-118, respectively. Unlike other kinases reported to partially phosphorylate α-syn at Ser-129 in vitro, phosphorylation by PLK2 and PLK3 is quantitative (>95% conversion). Only PLK1 and PLK3 phosphorylate β-syn at Ser-118, whereas no phosphorylation of γ-syn was detected by any of the four PLKs (PLK1 to -4). PLK-mediated phosphorylation was greatly reduced in an isolated C-terminal fragment (residues 103–140) of α-syn, suggesting substrate recognition via the N-terminal repeats and/or the non-amyloid component domain of α-syn. PLKs specifically co-localized with phosphorylated Ser-129 (Ser(P)-129) α-syn in various subcellular compartments (cytoplasm, nucleus, and membranes) of mammalian cell lines and primary neurons as well as in α-syn transgenic mice, especially cortical brain areas involved in synaptic plasticity. Furthermore, we report that the levels of PLK2 are significantly increased in brains of Alzheimer disease and Lewy body disease patients. Taken together, these results provide biochemical and in vivo evidence of α-syn and β-syn phosphorylation by specific PLKs. Our results suggest a need for further studies to elucidate the potential role of PLK-syn interactions in the normal biology of these proteins as well as their involvement in the pathogenesis of Parkinson disease and other synucleinopathies. Phosphorylation of α-synuclein (α-syn) at Ser-129 is a hallmark of Parkinson disease and related synucleinopathies. However, the identity of the natural kinases and phosphatases responsible for regulating α-syn phosphorylation remain unknown. Here we demonstrate that three closely related members of the human Polo-like kinase (PLK) family (PLK1, PLK2, and PLK3) phosphorylate α-syn and β-syn specifically at Ser-129 and Ser-118, respectively. Unlike other kinases reported to partially phosphorylate α-syn at Ser-129 in vitro, phosphorylation by PLK2 and PLK3 is quantitative (>95% conversion). Only PLK1 and PLK3 phosphorylate β-syn at Ser-118, whereas no phosphorylation of γ-syn was detected by any of the four PLKs (PLK1 to -4). PLK-mediated phosphorylation was greatly reduced in an isolated C-terminal fragment (residues 103–140) of α-syn, suggesting substrate recognition via the N-terminal repeats and/or the non-amyloid component domain of α-syn. PLKs specifically co-localized with phosphorylated Ser-129 (Ser(P)-129) α-syn in various subcellular compartments (cytoplasm, nucleus, and membranes) of mammalian cell lines and primary neurons as well as in α-syn transgenic mice, especially cortical brain areas involved in synaptic plasticity. Furthermore, we report that the levels of PLK2 are significantly increased in brains of Alzheimer disease and Lewy body disease patients. Taken together, these results provide biochemical and in vivo evidence of α-syn and β-syn phosphorylation by specific PLKs. Our results suggest a need for further studies to elucidate the potential role of PLK-syn interactions in the normal biology of these proteins as well as their involvement in the pathogenesis of Parkinson disease and other synucleinopathies. IntroductionIncreasing evidence suggests that phosphorylation may play an important role in the oligomerization and fibrillogenesis (1Fujiwara H. Hasegawa M. Dohmae N. Kawashima A. Masliah E. Goldberg M.S. Shen J. Takio K. Iwatsubo T. Nat. Cell Biol. 2002; 4: 160-164Crossref PubMed Scopus (157) Google Scholar), Lewy body formation (1Fujiwara H. Hasegawa M. Dohmae N. Kawashima A. Masliah E. Goldberg M.S. Shen J. Takio K. Iwatsubo T. Nat. Cell Biol. 2002; 4: 160-164Crossref PubMed Scopus (157) Google Scholar, 2Anderson J.P. Walker D.E. Goldstein J.M. de Laat R. Banducci K. Caccavello R.J. Barbour R. Huang J. Kling K. Lee M. Diep L. Keim P.S. Shen X. Chataway T. Schlossmacher M.G. Seubert P. Schenk D. Sinha S. Gai W.P. Chilcote T.J. J. Biol. Chem. 2006; 281: 29739-29752Abstract Full Text Full Text PDF PubMed Scopus (866) Google Scholar) and neurotoxicity of α-synuclein (α-syn) 5The abbreviations used are: α-β- and γ-syn, α-, β-, and γ-synuclein, respectivelyLBLewy bodyPDParkinson diseasePLKPolo-like kinasePBDPolo box domainADAlzheimer diseaseLBDLewy body diseaseWTwild typeMALDImatrix-assisted laser desorption ionizationTOFtime-of-flightHSQCheteronuclear single quantum coherencesiRNAsmall interference RNAMOPS3-(N-morpholino)propanesulfonic acidTBSTris-buffered salineNACnon-amyloid component. in vivo (3Chen L. Feany M.B. Nat. Neurosci. 2005; 8: 657-663Crossref PubMed Scopus (513) Google Scholar). The majority of α-syn within Lewy bodies (LBs) in diseased human brains and animal models of Parkinson disease (PD) and related synucleinopathies is phosphorylated at Ser-129 (Ser(P)-129) (1Fujiwara H. Hasegawa M. Dohmae N. Kawashima A. Masliah E. Goldberg M.S. Shen J. Takio K. Iwatsubo T. Nat. Cell Biol. 2002; 4: 160-164Crossref PubMed Scopus (157) Google Scholar, 2Anderson J.P. Walker D.E. Goldstein J.M. de Laat R. Banducci K. Caccavello R.J. Barbour R. Huang J. Kling K. Lee M. Diep L. Keim P.S. Shen X. Chataway T. Schlossmacher M.G. Seubert P. Schenk D. Sinha S. Gai W.P. Chilcote T.J. J. Biol. Chem. 2006; 281: 29739-29752Abstract Full Text Full Text PDF PubMed Scopus (866) Google Scholar, 4Kahle P.J. Neumann M. Ozmen L. Haass C. Ann. N.Y. Acad. Sci. 2000; 920: 33-41Crossref PubMed Scopus (82) Google Scholar, 5Neumann M. Kahle P.J. Giasson B.I. Ozmen L. Borroni E. Spooren W. Müller V. Odoy S. Fujiwara H. Hasegawa M. Iwatsubo T. Trojanowski J.Q. Kretzschmar H.A. Haass C. J. Clin. Invest. 2002; 110: 1429-1439Crossref PubMed Scopus (292) Google Scholar, 6Takahashi M. Kanuka H. Fujiwara H. Koyama A. Hasegawa M. Miura M. Iwatsubo T. Neurosci. Lett. 2003; 336: 155-158Crossref PubMed Scopus (112) Google Scholar, 7Hasegawa M. Fujiwara H. Nonaka T. Wakabayashi K. Takahashi H. Lee V.M. Trojanowski J.Q. Mann D. Iwatsubo T. J. Biol. Chem. 2002; 277: 49071-49076Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar). Although recent studies support the notion that phosphorylation at Ser-129 is related to pathology and blocks α-syn fibrillization in vitro (8Paleologou K.E. Schmid A.W. Rospigliosi C.C. Kim H.Y. Lamberto G.R. Fredenburg R.A. Lansbury Jr., P.T. Fernandez C.O. Eliezer D. Zweckstetter M. Lashuel H.A. J. Biol. Chem. 2008; 283: 16895-16905Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar, 9Waxman E.A. Giasson B.I. J. Neuropathol. Exp. Neurol. 2008; (in press)PubMed Google Scholar), the exact mechanisms by which phosphorylation at Ser-129 modulates α-syn aggregation and toxicity in vivo remain elusive. Unraveling the role of phosphorylation in modulating the physiological and pathogenic activities of α-syn requires identification of the kinases and phosphatases involved in regulating its phosphorylation in vivo.Several kinases that phosphorylate α-syn at serine and tyrosine residues, primarily in its C-terminal region, have been identified using in vitro kinase assays and co-transfection studies. Casein kinase I and II, G-protein-coupled receptor kinases (GRK1, GRK2, GRK5, and GRK6), and calmodulin-dependent kinase II (10Pronin A.N. Morris A.J. Surguchov A. Benovic J.L. J. Biol. Chem. 2000; 275: 26515-26522Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 11Arawaka S. Wada M. Goto S. Karube H. Sakamoto M. Ren C.H. Koyama S. Nagasawa H. Kimura H. Kawanami T. Kurita K. Tajima K. Daimon M. Baba M. Kido T. Saino S. Goto K. Asao H. Kitanaka C. Takashita E. Hongo S. Nakamura T. Kayama T. Suzuki Y. Kobayashi K. Katagiri T. Kurokawa K. Kurimura M. Toyoshima I. Niizato K. 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Tyrosine phosphorylation has also been reported at Tyr-125 by Fyn (14Nakamura T. Yamashita H. Takahashi T. Nakamura S. Biochem. Biophys. Res. Commun. 2001; 280: 1085-1092Crossref PubMed Scopus (108) Google Scholar), Syk (15Negro A. Brunati A.M. Donella-Deana A. Massimino M.L. Pinna L.A. FASEB J. 2002; 16: 210-212Crossref PubMed Google Scholar), Lyn (15Negro A. Brunati A.M. Donella-Deana A. Massimino M.L. Pinna L.A. FASEB J. 2002; 16: 210-212Crossref PubMed Google Scholar), c-Frg (15Negro A. Brunati A.M. Donella-Deana A. Massimino M.L. Pinna L.A. FASEB J. 2002; 16: 210-212Crossref PubMed Google Scholar), and Src (16Ellis C.E. Schwartzberg P.L. Grider T.L. Fink D.W. Nussbaum R.L. J. Biol. Chem. 2001; 276: 3879-3884Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) tyrosine kinases, with Syk (15Negro A. Brunati A.M. Donella-Deana A. Massimino M.L. Pinna L.A. FASEB J. 2002; 16: 210-212Crossref PubMed Google Scholar) also phosphorylating at Tyr-133 and Tyr-136.The Polo-like kinases (PLKs) comprise a family of conserved Ser/Thr protein kinases that play key roles in cell cycle regulation, cellular response to stress, and carcinogenesis. In mammalian cells, the PLK family consists of three closely related kinases PLK1, PLK2/Snk (serum-inducible kinase), PLK3/Fnk (fibroblast growth factor-inducible kinase) also designated Prk (proliferation-related kinase), and a distant member PLK4/Sak (Snk akin kinase) (17Clay F.J. McEwen S.J. Bertoncello I. Wilks A.F. Dunn A.R. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 4882-4886Crossref PubMed Scopus (134) Google Scholar, 18Hamanaka R. Maloid S. Smith M.R. O'Connell C.D. Longo D.L. Ferris D.K. Cell Growth Differ. 1994; 5: 249-257PubMed Google Scholar, 19Golsteyn R.M. Schultz S.J. Bartek J. Ziemiecki A. Ried T. Nigg E.A. J. Cell Sci. 1994; 107: 1509-1517Crossref PubMed Google Scholar, 20Fode C. Motro B. Yousefi S. Heffernan M. Dennis J.W. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 6388-6392Crossref PubMed Scopus (87) Google Scholar, 21Li B. Ouyang B. Pan H. Reissmann P.T. Slamon D.J. Arceci R. Lu L. Dai W. J. Biol. Chem. 1996; 271: 19402-19408Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). The four PLKs share a conserved sequence motif characterized by two regions: a highly conserved N-terminal serine/threonine catalytic domain and a C-terminal non-catalytic termed the Polo box domain (PBD) (Fig. 1) (22Barr F.A. Silljé H.H. Nigg E.A. Nat. Rev. Mol. Cell Biol. 2004; 5: 429-440Crossref PubMed Scopus (893) Google Scholar, 23Xie S. Xie B. Lee M.Y. Dai W. Oncogene. 2005; 24: 277-286Crossref PubMed Scopus (94) Google Scholar). The PBD plays important roles in regulating substrate interactions, targeting, subcellular localization, and autoinhibition of the PLKs (24Cheng K.Y. Lowe E.D. Sinclair J. Nigg E.A. Johnson L.N. EMBO J. 2003; 22: 5757-5768Crossref PubMed Scopus (191) Google Scholar). The PBD of PLK1, PLK2, and PLK3 contain two tandem Polo boxes (∼80 residues in length) that associate to form a phosphopeptide binding site, whereas PLK4 possesses only a single PB, which mediates the dimerization of PLK4, resulting in a structure that resembles the PBD of the other PLKs (24Cheng K.Y. Lowe E.D. Sinclair J. Nigg E.A. Johnson L.N. EMBO J. 2003; 22: 5757-5768Crossref PubMed Scopus (191) Google Scholar, 25Dai W. Oncogene. 2005; 24: 214-216Crossref PubMed Scopus (37) Google Scholar, 26Leung G.C. Hudson J.W. Kozarova A. Davidson A. Dennis J.W. Sicheri F. Nat. Struct. Biol. 2002; 9: 719-724Crossref PubMed Scopus (121) Google Scholar).Recent reports demonstrate that α-syn is phosphorylated by PLK2 (27Inglis K.J. Chereau D. Brigham E.F. Chiou S.S. Schöbel S. Frigon N.L. Yu M. Caccavello R.J. Nelson S. Motter R. Wright S. Chian D. Santiago P. Soriano F. Ramos C. Powell K. Goldstein J.M. Babcock M. Yednock T. Bard F. Basi G.S. Sham H. Chilcote T.J. McConlogue L. Griswold-Prenner I. Anderson J.P. J. Biol. Chem. 2009; 284: 2598-2602Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 28Mbefo M.K. Paleologou K.E. Boucharaba A. Oueslati A. Olschewski D. Hirling H. Lashuel H. Synuclein in Health and Disease, Lausanne, Switzerland, September 24–26. 2008; (Abstr): 21Google Scholar). In this report, we confirm these findings and demonstrate for the first time, using in vitro kinase assays, co-transfection, and small interference RNA (siRNA)-mediated knockdown of PLKs, that α- and β-, but not γ-syn are phosphorylated by specific members of the PLK family. PLK phosphorylation of synucleins occurs specifically at Ser-129 in α-syn and Ser-118 in β-syn and appears to be mediated by specific interactions between the PLKs and the N-terminal region (residues 1–95) of syn. These findings were validated by colocalization of α-syn and PLKs in different subcellular compartments and co-transfection studies as well as siRNA-mediated knockdown of PLKs in mammalian cells and primary neurons. PLK2 and PLK3 partly co-localized with Ser(P)-129 α-syn in primary hippocampal neurons as well as in cortical brain areas of α-syn transgenic mice. Furthermore, we demonstrate that the level of neuronal PLK2 is elevated in the Alzheimer disease (AD) and Lewy body disease (LBD) brains and correlates with the increased levels of Ser(P)-129 in these brains compared with healthy controls. Together, our findings point to PLK2 and PLK3 as the primary PLKs responsible for α-syn phosphorylation and highlight the importance of further studies to elucidate the potential role of the interactions between the PLKs and α-syn and their implications for α-syn aggregation and toxicity in PD and other synucleinopathies.RESULTSTo determine whether α-syn is a substrate for members of the PLK family of kinases (PLK1, PLK2, PLK3, and PLK4), we performed in vitro kinase assays using purified recombinant α-syn together with each of the four PLKs, and co-transfection experiments in mammalian cells (human embryonic kidney HEK 293T, HeLa and SH-SY5Y neuroblastoma cells).PLK1 to -3 Phosphorylate α-Syn Specifically at Ser-129 in VitroTo assess the phosphorylation of α-syn by the PLKs, α-syn (100 μm) was incubated with either of the four kinases at 1 μg of PLK, 144 μg of α-syn. The reactions were incubated at 30 °C, and the extent of phosphorylation was monitored by MALDI-TOF mass spectrometry. Upon incubation with PLK1, PLK2, and PLK3, we observed a shift in the molecular mass of α-syn by 80 Da (from 14,462 to 14,542 Da) for PLK1, -2, and -3, which corresponds to the addition of one phosphate group (Fig. 2). A closer examination of Fig. 2A demonstrates that PLK2, and PLK3 phosphorylate α-syn quantitatively, whereas ∼60–70% conversion was observed for PLK1 (Fig. 2A). No other kinase has been observed (data not shown) or reported to phosphorylate α-syn with the same efficiency. The MALDI-TOF results suggest that PLK1 to -3 phosphorylate α-syn at a single site. To determine if PLK1 to -3-mediated phosphorylation occurs at Ser-129, the phosphorylation reactions were analyzed by Western blotting using an antibody against Ser(P)-129 α-syn and subjected to trypsin digestion and peptide mapping by MALDI-TOF and liquid chromatography-electrospray ionization MS/MS. α-Syn samples incubated with the PLK1 to -3 showed a band at 14 kDa that was detectable with both anti-α-syn and anti-Ser(P)-129 antibodies (Fig. 2B). Comparative analysis of the tryptic fragments of these bands revealed that phosphorylation of α-syn occurs only within peptide fragments that encompass Ser-129. Trypsin digestion resulted in a peptide with a molecular mass of 4353.0 Da (M + H)+ corresponding to the monophosphorylated C-terminal fragment 103–140 containing Ser-129 (calculated mass 4272.4 Da (M) (NEEGAPQ EGILEDMPVDPDNEAYEMPpSEEGYQDYEPEA, where pS represents phosphoserine) (data not shown). This peptide fragment does not contain other serine or threonine residues, suggesting that phosphorylation indeed occurs at Ser-129. To further confirm these results, in vitro phosphorylation was also performed with a mutant that cannot be phosphorylated at position 129, S129A. As predicted, none of the PLKs could phosphorylate S129A mutant α-syn (data not shown).FIGURE 2In vitro phosphorylation of synucleins by PLK1 to -4. A, MALDI-TOF analysis of the WT α-syn after phosphorylation by PLK1 to -4. For PLK1 to -3, there is an 80-Da increase in the molecular mass of WT α-syn (14,461 + 80 = 14,541), corresponding to one phosphorylation. B, Western blot analysis of the same samples in A. The anti-Ser(P)-129 antibody detected a band, suggesting that the phosphorylation detected by mass spectrometry is at position 129. C, comparison of two-dimensional 1H,15N HSQC spectra of unphosphorylated WT (green) and α-syn phosphorylated by PLK3 (red). A dashed rectangle marks glutamine (Q) and asparagine (N) side chain resonances. D, kinetics of in vitro phosphorylation of Ser-129 in α-syn by PLK3 (black), PLK2 (red), and PLK1 (blue) as monitored by real-time NMR spectroscopy. NMR samples contained ∼0.1 mm 15N-labeled α-syn in 200 mm HEPES, 10 mm MgCl2, 2 mm dithiothreitol, and 1.09 mm ATP, pH 6.9. The real-time assay was started by the addition of kinase into the NMR sample using a protein/kinase ratio of 100:0.5 mg. The error bars were determined based on the signal/noise ratio observed in the NMR spectra.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Real-time Spectroscopy of the Phosphorylation ReactionHetereonuclear NMR spectroscopy on 15N-labeled protein modified by PLK allows identification of all phosphorylation sites, measures the level of integration, and yields kinetic data for the enzymatic modification of the individual sites. Although the reaction mixture is complex with enzyme, ATP and α-syn, filtering through the 15N label allows the monitoring of the kinase activity in the NMR tube without any further sample purification. To obtain single-residue resolution and identify all potential phosphorylation sites, the enzymatic reaction was followed by two-dimensional 1H-15N heteronuclear correlation spectra. Phosphorylated serine and threonine residues are readily detected because phosphorylation shifts their amide proton resonance downfield 8.8 ppm, to an empty region of the 15N-α-syn HSQC spectrum (Fig. 2C). The real-time NMR assay was applied to PLK1, PLK2, and PLK3. In the case of PLK3, the peak of phosphorylated Ser-129 was clearly observed already after 45 min (Fig. 2D). With increasing incubation time, the intensity of the NMR signal of phosphorylated Ser-129 increased. Quantitative analysis of the increase of the NMR signal of phosphorylated Ser-129 indicated that PLK3 fully phosphorylates α-syn within 2.5 h under the conditions of the assay (Fig. 2D). Importantly, no additional NMR signals of phosphorylated residues appeared, indicating that PLK3 exclusively phosphorylated α-syn at Ser-129. In the case of PLK1, no phosphorylation of α-syn could be detected during the time course of the experiment. The significantly reduced PLK1 phosphorylation of α-syn in the NMR experiments, compared with the in vitro kinase assay, is most likely due to the fact that the NMR phosphorylation experiments were performed at lower temperature (15 °C versus 30 °C for in vitro kinase assays) to reduce the impact of signal broadening due to amide proton exchange. Real-time spectroscopy of α-syn phosphorylation by PLK2 showed that PLK2 phosphorylates α-syn only at Ser-129. However, the kinetics of the enzymatic reaction is significantly slower for PLK2 than for PLK3, and α-syn was not fully phosphorylated at the end of the assay.α- and β-Syn but Not γ-Syn Are Phosphorylated by PLK1 to -3To evaluate the specificity of PLKs for α-syn, we also performed in vitro phosphorylation employing purified β- and γ-syn. β-Syn is a 134-amino acid-long protein sharing 61% sequence homology with α-syn. The sequence homology of the two synucleins is more prominent in their N-terminal regions (∼90% sequence similarity) (33Jakes R. Spillantini M.G. Goedert M. FEBS Lett. 1994; 345: 27-32Crossref PubMed Scopus (887) Google Scholar). γ-Syn is 127 amino acids long and shares 55.9 and 54.3% sequence homology with α-syn and β-syn, respectively (34Lavedan C. Genome Res. 1998; 8: 871-880Crossref PubMed Scopus (267) Google Scholar) (Fig. 1B). Unlike α-syn, both β- and γ-syn are not found in LBs (35Spillantini M.G. Crowther R.A. Jakes R. Hasegawa M. Goedert M. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 6469-6473Crossref PubMed Scopus (2345) Google Scholar). As illustrated in Fig. 3, incubation with PLK1 and PLK3 resulted in quantitative conversion of β-syn into its monophosphorylated form, as evidenced by the shift of the β-syn molecular mass, m/z, from 14,291 to 14,369.7 Da. A minor ( 94%) sequence homology with human WT α-syn, and their sequence differences are mostly located in the NAC and C-terminal regions (residues 83, 87, 100, 103, 107, 121, and 122), including a serine → asparagine mutation at position 87 (S87N). These mutations appear to reduce PLK1-mediated phosphorylation of α-syn without influencing phosphorylation by PLK2 and PLK3 (supplemental Fig. 3). The effect of mutations at the C terminus of α-syn was also assessed. Mutating Ser-129 to alanine blocked the phosphorylation by PLK1 to -3. In addition, mutations near Ser-129 alter the specificity of the three kinases, because only PLK2 was observed to phosphorylate E126A (supplemental Fig. 3C).In Vitro Phosphorylation of Fibrillar α-Syn by PLK1 to -4Whether phosphorylation at Ser-129 occurs prior to or after α-syn fibrillization and LB formation in vivo remains unclear. Recent studies by Waxman and Giasson (36Waxman E.A. Giasson B.I. J. Neuropathol. Exp. Neurol. 2008; 67: 402-416Crossref PubMed Scopus (161) Google Scholar) and our laboratory demonstrated that fibrillar α-syn is a better substrate for casein kinase I phosphorylation. To determine whether α-syn fibrils are good substrates for phosphorylation by PLKs, we generated fibrils of WT α-syn and subjected them to in vitro phosphorylation with PLK1 to -4. Briefly, WT α-syn was aggregated at a concentration of 200–400 μm at 37 °C with continuous shaking for 24–72 h. The fibrils formed were harvested by centrifugation, sonicated, and phosphorylated at a concentration of 100 μm as described above. As shown in Fig. 5, the efficiency of in vitro phosphorylation of α-syn fibril was as follows: PLK3 > PLK2 > PLK1. As is the case with monomeric α-syn, PLK1 exhibited decreased phosphorylation activity toward fibrillar α-syn relative to PLK2 and PLK3.FIGURE 5In vitro phosphorylation of α-syn fibrils by PLK1 to -4. Top, preformed α- fibrils were incubated with recombinant PLKs in the appropriate reaction buffers, and phosphorylation at Ser-129 was assessed by anti-Ser(P)-129 antibodies. Bottom, quantification of the level of Ser(P)-129 signal normalized against α-syn and β-actin (pS118/(α-syn + β actin)), n = 3.View Large Image Figure ViewerDownload Hi-res image Download (PPT)PLK Phosphorylation of α-Syn in Mammalian Cell Lines and Primary NeuronsHaving established that PLK1 to -3 phosphorylate α-syn at Ser-129