LRRK2 phosphorylates Snapin and inhibits interaction of Snapin with SNAP-25

Abstract

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. Researchers in Korea have found that a protein linked with Parkinson's disease (PD) may play a role in regulating neuronal signaling. Mutations in the gene encoding the leucine-rich repeat kinase 2 (LRRK2) protein, a kinase whose enhanced enzyme activity is related to PD pathogenesis, are hereditary risk factors for this neurodegenerative disorder. Researchers led by Wongi Seol of Wonkwang University searched for substrates for LRRK2 kinase that might explain this enzyme's involvement in PD. They determined that LRRK2 phosphorylates Snapin, a protein that ensures proper localization of neurotransmitter reserves and LRRK2-mediated phosphorylation inhibits Snapin, thus reducing the size of these reserves and the amount of neurotransmitter release. The extent to which LRRK2-Snapin interaction contributes to Parkinson's remains unclear, but this pathway nevertheless seems to play an important role in proper brain function.