LRRK2 phosphorylates Snapin and inhibits interaction of Snapin with SNAP-25.
Leucine-rich repeat kinase 2 (LRRK2) is a gene that, upon mutation, causes autosomal-dominant familial Parkinson's disease (PD). Yeast two-hybrid screening revealed that Snapin, a SNAP-25 (synaptosomal-associated protein-25) interacting protein, interacts with LRRK2. An in vitro kinase assay exhibited that Snapin is phosphorylated by LRRK2. A glutathione-S-transferase (GST) pull-down assay showed that LRRK2 may interact with Snapin via its Ras-of-complex (ROC) and N-terminal domains, with no significant difference on interaction of Snapin with LRRK2 wild type (WT) or its pathogenic mutants. Further analysis by mutation study revealed that Threonine 117 of Snapin is one of the sites phosphorylated by LRRK2. Furthermore, a Snapin T117D phosphomimetic mutant decreased its interaction with SNAP-25 in the GST pull-down assay. SNAP-25 is a component of the SNARE (Soluble NSF Attachment protein REceptor) complex and is critical for the exocytosis of synaptic vesicles. Incubation of rat brain lysate with recombinant Snapin T117D, but not WT, protein caused decreased interaction of synaptotagmin with the SNARE complex based on a co-immunoprecipitation assay. We further found that LRRK2-dependent phosphorylation of Snapin in the hippocampal neurons resulted in a decrease in the number of readily releasable vesicles and the extent of exocytotic release. Combined, these data suggest that LRRK2 may regulate neurotransmitter release via control of Snapin function by inhibitory phosphorylation.
Institute for Brain Science and Technology, Inje University, Gaegumdong, South Korea.
Hye Jin
HJ
Yun
Joohyun
J
Park
Dong Hwan
DH
Ho
Heyjung
H
Kim
Cy-Hyun
CH
Kim
Hakjin
H
Oh
Inhwa
I
Ga
Hyemyung
H
Seo
Sunghoe
S
Chang
Ilhong
I
Son
Wongi
W
Seol
D000595:Amino Acid Sequence; D000818:Animals; D005089:Exocytosis; D005260:Female; D057809:HEK293 Cells; D006801:Humans; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; D051379:Mice; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D050765:Qa-SNARE Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D050825:Synaptosomal-Associated Protein 25 / Q000378:metabolism*; D050857:Synaptotagmins / Q000378:metabolism; D050988:Vesicle-Associated Membrane Protein 2 / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000378:metabolism*
D050505:Mutant Proteins; D050765:Qa-SNARE Proteins; C118519:SNAPIN protein, human; D050825:Synaptosomal-Associated Protein 25; D050988:Vesicle-Associated Membrane Protein 2; D033921:Vesicular Transport Proteins; D010769:Phosphothreonine; D050857:Synaptotagmins; D000071158:Leucine-Rich Repeat Serine-Threonine Protein Kinase-2; C495279:Lrrk2 protein, mouse; D017346:Protein Serine-Threonine Kinases