title: LRRK2 phosphorylates pre-synaptic N-ethylmaleimide sensitive fusion (NSF) protein enhancing its ATPase activity and SNARE complex disassembling rate.
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.
authors.affiliation: Department of Biology, University of Padova, via Ugo Bassi 58/B, 35131, Padova, Italy. elisa.belluzzi@gmail.com.|Present Address: Rheumatology Unit, Department of Medicine - DIMED, University Hospital of Padova, Padova, Italy. elisa.belluzzi@gmail.com.
authors.forename: Elisa
authors.initials: E
authors.lastname: Belluzzi
authors.affiliation: Department of Biology, University of Padova, via Ugo Bassi 58/B, 35131, Padova, Italy. adriano.gonnelli@bio.unipd.it.
authors.forename: Adriano
authors.initials: A
authors.lastname: Gonnelli
authors.affiliation: San Raffaele Scientific Park & University Vita-Salute San Raffaele, Milan, Italy. Cirnaru.mariadaniela@hsr.it.
authors.forename: Maria-Daniela
authors.initials: MD
authors.lastname: Cirnaru
authors.affiliation: Department of Experimental Medicine, University of Genova, Genova, Italy. antonella.marte@unige.it.
authors.forename: Antonella
authors.initials: A
authors.lastname: Marte
authors.affiliation: Department of Biology, University of Padova, via Ugo Bassi 58/B, 35131, Padova, Italy. n.plotegher@ucl.ac.uk.|Present Address: Department of Cell and Developmental Biology, University College London, London, WC1E 6BT, UK. n.plotegher@ucl.ac.uk.
authors.forename: Nicoletta
authors.initials: N
authors.lastname: Plotegher
authors.affiliation: Department of Biology, University of Padova, via Ugo Bassi 58/B, 35131, Padova, Italy. isabella.russo@unipd.it.
authors.forename: Isabella
authors.initials: I
authors.lastname: Russo
authors.affiliation: Department of Biology, University of Padova, via Ugo Bassi 58/B, 35131, Padova, Italy. laura.civiero@unipd.it.
authors.forename: Laura
authors.initials: L
authors.lastname: Civiero
authors.affiliation: Department of Biology, University of Padova, via Ugo Bassi 58/B, 35131, Padova, Italy. susanna.cogo@studenti.unipd.it.
authors.forename: Susanna
authors.initials: S
authors.lastname: Cogo
authors.affiliation: San Raffaele Scientific Park & University Vita-Salute San Raffaele, Milan, Italy. mdolores.perez.carrion@gmail.com.
authors.forename: Maria Perèz
authors.initials: MP
authors.lastname: Carrion
authors.affiliation: Department of Biomedical Sciences, University of Padova, Padova, Italy. cinzia.franchin@unipd.it.|Proteomics Center of Padova University, Padova, Italy. cinzia.franchin@unipd.it.
authors.forename: Cinzia
authors.initials: C
authors.lastname: Franchin
authors.affiliation: Department of Biomedical Sciences, University of Padova, Padova, Italy. giorgio.arrigoni@unipd.it.|Proteomics Center of Padova University, Padova, Italy. giorgio.arrigoni@unipd.it.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Arrigoni
authors.affiliation: Department of Biology, University of Padova, via Ugo Bassi 58/B, 35131, Padova, Italy. mariano.beltramini@unipd.it.
authors.forename: Mariano
authors.initials: M
authors.lastname: Beltramini
authors.affiliation: Department of Biology, University of Padova, via Ugo Bassi 58/B, 35131, Padova, Italy. luigi.bubacco@unipd.it.
authors.forename: Luigi
authors.initials: L
authors.lastname: Bubacco
authors.affiliation: Department of Experimental Medicine, University of Genova, Genova, Italy. franco.onofri@unige.it.
authors.forename: Franco
authors.initials: F
authors.lastname: Onofri
authors.affiliation: San Raffaele Scientific Park & University Vita-Salute San Raffaele, Milan, Italy. piccoli.giovanni@hsr.it.|IN-CNR Milano, Milano, Italy. piccoli.giovanni@hsr.it.
authors.forename: Giovanni
authors.initials: G
authors.lastname: Piccoli
authors.affiliation: Department of Biology, University of Padova, via Ugo Bassi 58/B, 35131, Padova, Italy. elisa.greggio@unipd.it.
authors.forename: Elisa
authors.initials: E
authors.lastname: Greggio
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D005033:Ethylmaleimide / Q000378:metabolism; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation / Q000235:genetics*; D052066:N-Ethylmaleimide-Sensitive Proteins / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D050600:SNARE Proteins / Q000378:metabolism*; D033921:Vesicular Transport Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D002352:Carrier Proteins; D050600:SNARE Proteins; D033921:Vesicular Transport Proteins; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; C495279:Lrrk2 protein, mouse; D017346:Protein Serine-Threonine Kinases; D000251:Adenosine Triphosphatases; D052066:N-Ethylmaleimide-Sensitive Proteins; C498733:Nsf protein, mouse; D005033:Ethylmaleimide