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Open Access 01-12-2016 | Research article

LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate

Authors: Elisa Belluzzi, Adriano Gonnelli, Maria-Daniela Cirnaru, Antonella Marte, Nicoletta Plotegher, Isabella Russo, Laura Civiero, Susanna Cogo, Maria Perèz Carrion, Cinzia Franchin, Giorgio Arrigoni, Mariano Beltramini, Luigi Bubacco, Franco Onofri, Giovanni Piccoli, Elisa Greggio

Published in: Molecular Neurodegeneration | Issue 1/2016

Abstract

Background

Lrrk2, a gene linked to Parkinson’s disease, encodes a large scaffolding protein with kinase and GTPase activities implicated in vesicle and cytoskeletal-related processes. At the presynaptic site, LRRK2 associates with synaptic vesicles through interaction with a panel of presynaptic proteins.

Results

Here, we show that LRRK2 kinase activity influences the dynamics of synaptic vesicle fusion. We therefore investigated whether LRRK2 phosphorylates component(s) of the exo/endocytosis machinery. We have previously observed that LRRK2 interacts with NSF, a hexameric AAA+ ATPase that couples ATP hydrolysis to the disassembling of SNARE proteins allowing them to enter another fusion cycle during synaptic exocytosis. Here, we demonstrate that NSF is a substrate of LRRK2 kinase activity. LRRK2 phosphorylates full-length NSF at threonine 645 in the ATP binding pocket of D2 domain. Functionally, NSF phosphorylated by LRRK2 displays enhanced ATPase activity and increased rate of SNARE complex disassembling. Substitution of threonine 645 with alanine abrogates LRRK2-mediated increased ATPase activity.

Conclusions

Given that the most common Parkinson’s disease LRRK2 G2019S mutation displays increased kinase activity, our results suggest that mutant LRRK2 may impair synaptic vesicle dynamics via aberrant phosphorylation of NSF.

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Gasper R, Meyer S, Gotthardt K, Sirajuddin M, Wittinghofer A. It takes two to tango: regulation of G proteins by dimerization. Nat Rev Mol Cell Biol. 2009;10(6):423–9. doi:10.1038/nrm2689.CrossRefPubMed

Taymans JM. The GTPase function of LRRK2. Biochem Soc Trans. 2012;40(5):1063–9. doi:10.1042/BST20120133.CrossRefPubMed

Greggio E. Role of LRRK2 kinase activity in the pathogenesis of Parkinson’s disease. Biochem Soc Trans. 2012;40(5):1058–62. doi:10.1042/BST20120054.CrossRefPubMed

Paisan-Ruiz C, Jain S, Evans EW, Gilks WP, Simon J, van der Brug M, et al. Cloning of the gene containing mutations that cause PARK8-linked Parkinson’s disease. Neuron. 2004;44(4):595–600. doi:10.1016/j.neuron.2004.10.023.CrossRefPubMed

Zimprich A, Biskup S, Leitner P, Lichtner P, Farrer M, Lincoln S, et al. Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology. Neuron. 2004;44(4):601–7. doi:10.1016/j.neuron.2004.11.005.CrossRefPubMed

Satake W, Nakabayashi Y, Mizuta I, Hirota Y, Ito C, Kubo M, et al. Genome-wide association study identifies common variants at four loci as genetic risk factors for Parkinson’s disease. Nat Genet. 2009;41(12):1303–7. doi:10.1038/ng.485.CrossRefPubMed

Simon-Sanchez J, Schulte C, Bras JM, Sharma M, Gibbs JR, Berg D, et al. Genome-wide association study reveals genetic risk underlying Parkinson’s disease. Nat Genet. 2009;41(12):1308–12. doi:10.1038/ng.487.CrossRefPubMedPubMedCentral

Rudenko IN, Cookson MR. Heterogeneity of leucine-rich repeat kinase 2 mutations: genetics, mechanisms and therapeutic implications. Neurotherapeutics. 2014;11(4):738–50. doi:10.1007/s13311-014-0284-z.CrossRefPubMedPubMedCentral

Reynolds A, Doggett EA, Riddle SM, Lebakken CS, Nichols RJ. LRRK2 kinase activity and biology are not uniformly predicted by its autophosphorylation and cellular phosphorylation site status. Front Mol Neurosci. 2014;7:54. doi:10.3389/fnmol.2014.00054.CrossRefPubMedPubMedCentral

10.

Alegre-Abarrategui J, Christian H, Lufino MM, Mutihac R, Venda LL, Ansorge O, et al. LRRK2 regulates autophagic activity and localizes to specific membrane microdomains in a novel human genomic reporter cellular model. Hum Mol Genet. 2009;18(21):4022–34. doi:10.1093/hmg/ddp346.CrossRefPubMedPubMedCentral

11.

Beilina A, Rudenko IN, Kaganovich A, Civiero L, Chau H, Kalia SK, et al. Unbiased screen for interactors of leucine-rich repeat kinase 2 supports a common pathway for sporadic and familial Parkinson disease. Proc Natl Acad Sci. 2014;111(7):2626–31. doi:10.1073/pnas.1318306111.CrossRefPubMedPubMedCentral

12.

Berger Z, Smith KA, Lavoie MJ. Membrane localization of LRRK2 is associated with increased formation of the highly active LRRK2 dimer and changes in its phosphorylation. Biochemistry. 2010;49(26):5511–23. doi:10.1021/bi100157u.CrossRefPubMedPubMedCentral

13.

Biskup S, Moore DJ, Celsi F, Higashi S, West AB, Andrabi SA, et al. Localization of LRRK2 to membranous and vesicular structures in mammalian brain. Ann Neurol. 2006;60(5):557–69. doi:10.1002/ana.21019.CrossRefPubMed

14.

Hatano T, S-i K, Imai S, Maeda M, Ishikawa K, Mizuno Y, et al. Leucine-rich repeat kinase 2 associates with lipid rafts. Hum Mol Genet. 2007;16(6):678–90. doi:10.1093/hmg/ddm013.CrossRefPubMed

15.

Shin N, Jeong H, Kwon J, Heo HY, Kwon JJ, Yun HJ, et al. LRRK2 regulates synaptic vesicle endocytosis. Exp Cell Res. 2008;314(10):2055–65. doi:10.1016/j.yexcr.2008.02.015.CrossRefPubMed

16.

Beccano-Kelly DA, Kuhlmann N, Tatarnikov I, Volta M, Munsie LN, Chou P, et al. Synaptic function is modulated by LRRK2 and glutamate release is increased in cortical neurons of G2019S LRRK2 knock-in mice. Front Cell Neurosci. 2014;8:301. doi:10.3389/fncel.2014.00301.CrossRefPubMedPubMedCentral

17.

Li X, Patel JC, Wang J, Avshalumov MV, Nicholson C, Buxbaum JD, et al. Enhanced striatal dopamine transmission and motor performance with LRRK2 overexpression in mice is eliminated by familial Parkinson’s disease mutation G2019S. J Neurosci. 2010;30(5):1788–97. doi:10.1523/JNEUROSCI.5604-09.2010.CrossRefPubMedPubMedCentral

18.

Li Y, Liu W, Oo TF, Wang L, Tang Y, Jackson-Lewis V, et al. Mutant LRRK2(R1441G) BAC transgenic mice recapitulate cardinal features of Parkinson’s disease. Nat Neurosci. 2009;12(7):826–8. doi:10.1038/nn.2349.CrossRefPubMedPubMedCentral

19.

Tong Y, Pisani A, Martella G, Karouani M, Yamaguchi H, Pothos EN, et al. R1441C mutation in LRRK2 impairs dopaminergic neurotransmission in mice. Proc Natl Acad Sci U S A. 2009;106(34):14622–7. doi:10.1073/pnas.0906334106.CrossRefPubMedPubMedCentral

20.

Piccoli G, Condliffe SB, Bauer M, Giesert F, Boldt K, De Astis S, et al. LRRK2 controls synaptic vesicle storage and mobilization within the recycling pool. J Neurosci. 2011;31(6):2225–37. doi:10.1523/JNEUROSCI.3730-10.2011.CrossRefPubMed

21.

Piccoli G, Onofri F, Cirnaru MD, Kaiser CJ, Jagtap P, Kastenmuller A, et al. LRRK2 binds to neuronal vesicles through protein interactions mediated by its C-terminal WD40 domain. Mol Cell Biol. 2014;34:2147–61. doi:10.1128/MCB.00914-13.CrossRefPubMedPubMedCentral

22.

Cirnaru MD, Marte A, Belluzzi E, Russo I, Gabrielli M, Longo F, et al. LRRK2 kinase activity regulates synaptic vesicle trafficking and neurotransmitter release through modulation of LRRK2 macro-molecular complex. Front Mol Neurosci. 2014;7:49. doi:10.3389/fnmol.2014.00049.CrossRefPubMedPubMedCentral

23.

Sudhof TC, Rizo J (2011) Synaptic vesicle exocytosis. Cold Spring Harb Perspect Biol 3 (12). doi: 10.1101/cshperspect.a005637

24.

Huynh H, Bottini N, Williams S, Cherepanov V, Musumeci L, Saito K, et al. Control of vesicle fusion by a tyrosine phosphatase. Nat Cell Biol. 2004;6(9):831–9. doi:10.1038/ncb1164.CrossRefPubMed

25.

Liu Y, Cheng K, Gong K, Fu AK, Ip NY. Pctaire1 phosphorylates N-ethylmaleimide-sensitive fusion protein: implications in the regulation of its hexamerization and exocytosis. J Biol Chem. 2006;281(15):9852–8. doi:10.1074/jbc.M513496200.CrossRefPubMed

26.

Matveeva EA, Whiteheart SW, Vanaman TC, Slevin JT. Phosphorylation of the N-ethylmaleimide-sensitive factor is associated with depolarization-dependent neurotransmitter release from synaptosomes. J Biol Chem. 2001;276(15):12174–81. doi:10.1074/jbc.M007394200.CrossRefPubMed

27.

Reith AD, Bamborough P, Jandu K, Andreotti D, Mensah L, Dossang P, et al. GSK2578215A; a potent and highly selective 2-arylmethyloxy-5-substitutent-N-arylbenzamide LRRK2 kinase inhibitor. Bioorg Med Chem Lett. 2012;22(17):5625–9. doi:10.1016/j.bmcl.2012.06.104.CrossRefPubMedPubMedCentral

28.

Melrose HL, Dachsel JC, Behrouz B, Lincoln SJ, Yue M, Hinkle KM, et al. Impaired dopaminergic neurotransmission and microtubule-associated protein tau alterations in human LRRK2 transgenic mice. Neurobiol Dis. 2010;40(3):503–17. doi:10.1016/j.nbd.2010.07.010.CrossRefPubMedPubMedCentral

29.

Granseth B, Odermatt B, Royle SJ, Lagnado L. Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses. Neuron. 2006;51(6):773–86. doi:10.1016/j.neuron.2006.08.029.CrossRefPubMed

30.

Matteoli M, Takei K, Perin MS, Sudhof TC, De Camilli P. Exo-endocytotic recycling of synaptic vesicles in developing processes of cultured hippocampal neurons. J Cell Biol. 1992;117(4):849–61.CrossRefPubMed

31.

Civiero L, Vancraenenbroeck R, Belluzzi E, Beilina A, Lobbestael E, Reyniers L, et al. Biochemical characterization of highly purified leucine-rich repeat kinases 1 and 2 demonstrates formation of homodimers. PLoS One. 2012;7(8):e43472. doi:10.1371/journal.pone.0043472.CrossRefPubMedPubMedCentral

32.

Webber PJ, Smith AD, Sen S, Renfrow MB, Mobley JA, West AB. Autophosphorylation in the leucine-rich repeat kinase 2 (LRRK2) GTPase domain modifies kinase and GTP-binding activities. J Mol Biol. 2011;412(1):94–110. doi:10.1016/j.jmb.2011.07.033.CrossRefPubMedPubMedCentral

33.

Zhao C, Slevin JT, Whiteheart SW. Cellular functions of NSF: not just SNAPs and SNAREs. FEBS Lett. 2007;581(11):2140–9. doi:10.1016/j.febslet.2007.03.032.CrossRefPubMedPubMedCentral

34.

Cipriano DJ, Jung J, Vivona S, Fenn TD, Brunger AT, Bryant Z. Processive ATP-driven Substrate Disassembly by the N-Ethylmaleimide-sensitive Factor (NSF) Molecular Machine. J Biol Chem. 2013;288(32):23436–45. doi:10.1074/jbc.M113.476705.CrossRefPubMedPubMedCentral

35.

Vivona S, Cipriano DJ, O’Leary S, Li YH, Fenn TD, Brunger AT. Disassembly of all SNARE complexes by N-ethylmaleimide-sensitive factor (NSF) is initiated by a conserved 1:1 interaction between alpha-soluble NSF attachment protein (SNAP) and SNARE complex. J Biol Chem. 2013;288(34):24984–91. doi:10.1074/jbc.M113.489807.CrossRefPubMedPubMedCentral

36.

Yun HJ, Park J, Ho DH, Kim H, Kim C-H, Oh H, et al. LRRK2 phosphorylates Snapin and inhibits interaction of Snapin with SNAP-25. Exp Mol Med. 2013;45:e36. doi:10.1038/emm.2013.68.CrossRefPubMedPubMedCentral

37.

Arranz AM, Delbroek L, Van Kolen K, Guimaraes MR, Mandemakers W, Daneels G, et al. LRRK2 functions in synaptic vesicle endocytosis through a kinase-dependent mechanism. J Cell Sci. 2015;128(3):541–52. doi:10.1242/jcs.158196.CrossRefPubMed

38.

Matta S, Van Kolen K, da Cunha R, van den Bogaart G, Mandemakers W, Miskiewicz K, et al. LRRK2 Controls an EndoA Phosphorylation Cycle in Synaptic Endocytosis. Neuron. 2012;75(6):1008–21. http://dx.doi.org/10.1016/j.neuron.2012.08.022.CrossRefPubMed

39.

Martin I, Kim JW, Lee BD, Kang HC, Xu JC, Jia H, et al. Ribosomal protein s15 phosphorylation mediates LRRK2 neurodegeneration in Parkinson’s disease. Cell. 2014;157(2):472–85. doi:10.1016/j.cell.2014.01.064.CrossRefPubMedPubMedCentral

40.

Matveeva EA, He P, Whiteheart SW. N-Ethylmaleimide-sensitive fusion protein contains high and low affinity ATP-binding sites that are functionally distinct. J Biol Chem. 1997;272(42):26413–8.CrossRefPubMed

41.

Yu RC, Hanson PI, Jahn R, Brunger AT. Structure of the ATP-dependent oligomerization domain of N-ethylmaleimide sensitive factor complexed with ATP. Nat Struct Biol. 1998;5(9):803–11. doi:10.1038/1843.CrossRefPubMed

42.

Liu G, Sgobio C, Gu X, Sun L, Lin X, Yu J, Parisiadou L, Xie C, Sastry N, Ding J, Lohr KM, Miller GW, Mateo Y, Lovinger DM, Cai H (2015) Selective Expression of Parkinson’s disease-related Leucine-rich Repeat Kinase 2 G2019S Missense Mutation in Midbrain Dopaminergic Neurons Impairs Dopamine Release and Dopaminergic Gene Expression. Hum Mol Genet. doi: 10.1093/hmg/ddv249

43.

Fuji RN, Flagella M, Baca M, Baptista MA, Brodbeck J, Chan BK, et al. Effect of selective LRRK2 kinase inhibition on nonhuman primate lung. Sci Transl Med. 2015;7(273):273ra215. doi:10.1126/scitranslmed.aaa3634.CrossRef

44.

Piccoli G, Verpelli C, Tonna N, Romorini S, Alessio M, Nairn AC, et al. Proteomic analysis of activity-dependent synaptic plasticity in hippocampal neurons. J Proteome Res. 2007;6(8):3203–15. doi:10.1021/pr0701308.CrossRefPubMed

45.

Nakamura Y, Iga K, Shibata T, Shudo M, Kataoka K. Glial plasmalemmal vesicles: a subcellular fraction from rat hippocampal homogenate distinct from synaptosomes. Glia. 1993;9(1):48–56. doi:10.1002/glia.440090107.CrossRefPubMed

46.

Sankaranarayanan S, Ryan TA. Real-time measurements of vesicle-SNARE recycling in synapses of the central nervous system. Nat Cell Biol. 2000;2(4):197–204. doi:10.1038/35008615.CrossRefPubMed

47.

Verderio C, Coco S, Bacci A, Rossetto O, De Camilli P, Montecucco C, et al. Tetanus toxin blocks the exocytosis of synaptic vesicles clustered at synapses but not of synaptic vesicles in isolated axons. J Neurosci. 1999;19(16):6723–32.PubMed

48.

Salvi M, Trashi E, Cozza G, Franchin C, Arrigoni G, Pinna LA. Investigation on PLK2 and PLK3 substrate recognition. Biochim Biophys Acta. 2012;1824(12):1366–73. doi:10.1016/j.bbapap.2012.07.003.CrossRefPubMed

49.

Venerando A, Franchin C, Cant N, Cozza G, Pagano MA, Tosoni K, et al. Detection of phospho-sites generated by protein kinase CK2 in CFTR: mechanistic aspects of Thr1471 phosphorylation. PLoS One. 2013;8(9):e74232. doi:10.1371/journal.pone.0074232.CrossRefPubMedPubMedCentral

50.

Lanzetta PA, Alvarez LJ, Reinach PS, Candia OA. An improved assay for nanomole amounts of inorganic phosphate. Anal Biochem. 1979;100(1):95–7.CrossRefPubMed

Title: LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate
Authors: Elisa Belluzzi
Adriano Gonnelli
Maria-Daniela Cirnaru
Antonella Marte
Nicoletta Plotegher
Isabella Russo
Laura Civiero
Susanna Cogo
Maria Perèz Carrion
Cinzia Franchin
Giorgio Arrigoni
Mariano Beltramini
Luigi Bubacco
Franco Onofri
Giovanni Piccoli
Elisa Greggio
Publication date: 01-12-2016
Publisher: BioMed Central
Published in: Molecular Neurodegeneration / Issue 1/2016
Electronic ISSN: 1750-1326
DOI: https://doi.org/10.1186/s13024-015-0066-z

2024 ASCO Annual Meeting

Springer Medicine

LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate

Abstract

Background

Results

Conclusions

2024 ASCO Annual Meeting

Springer Medicine

Abstract

Background

Results

Conclusions

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