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.

Figure 1

Snapin phosphorylation by leucine-rich repeat kinase 2 (LRRK2). (a) Immunoprecipitated LRRK2 phosphorylates Snapin. Recombinant Snapin protein was expressed in E. coli as His-tagged fusion protein and isolated by affinity chromatography with nickel resin. Indicated LRRK2 wild-type (WT) or mutant proteins were expressed in HEK293T cells as Myc-tagged forms and immunoprecipitated by anti-myc antibody. The immunoprecipitates (IP) were incubated with Snapin for an in vitro kinase assay. Autoradiograms of phosphorylated LRRK2 (i) and phosphorylated Snapin (iii) are shown. In addition, the amount of immunoprecipitated LRRK2 (ii) and recombinant Snapin (iv) proteins were detected by anti-myc antibody and Coomassie blue staining, respectively. The empty vector was also transfected to HEK293T cells and the cell lysates were used as a control (vector). (b) Recombinant GST-LRRK2 (Invitrogen) proteins also phosphorylate Snapin, but not ARF1. The purchased LRRK2 WT (WT), G2019S (G) or R1441C (R) proteins were used for an in vitro kinase assay. Autoradiograms showed that LRRK2 and Snapin (i), but not ARF1, proteins (ii) were phosphorylated by LRRK2 (ii). (iii) Positions of both Arf1 and Snapin proteins are indicated by arrows. (c) The amino-acid sequence of human Snapin. The conserved phosphorylation candidate sites at the 20th and 117th threonine residues are indicated as bold letters. (d) LRRK2 phosphorylates Snapin at Thr-117. GST-LRRK2 WT or G2019S (Invitrogen) were subjected to an in vitro kinase assay with recombinant Snapin WT, T20A or T117A protein. Autoradiograms of autophosphorylated GST-LRRK2 (i) and phosphorylated Snapin (ii) are shown. For each kinase assay, use of equal amounts of substrates was confirmed by SDS–PAGE and Coomassie blue staining (aiv, biii, diii).

Figure 2

Leucine-rich repeat kinase 2 (LRRK2) interacts with Snapin. (a) Lysates of 293T cells transfected with myc-LRRK2 (LRRK2 WT) or HA-murine CAR (CAR) were co-incubated with purified GST-Snapin (S) or GST proteins and subjected to a GST pull-down assay and western blot analysis using anti-myc or anti-HA antibody. (b) LRRK2 WT, G2019S, R1441C or I2020T was expressed in HEK293T cells and the cell lysates were used for a GST pull-down assay as (a) Input (I) of each sample is 2.5% of the total proteins. The amount of GST-Snapin or GST proteins used was detected by Coomassie staining, and it was confirmed that similar amounts of GST proteins were used (a, b). (c) Co-immunoprecipitation of flag-LRRK2 with myc-Snapin. The HEK293T cells were induced by doxycycline to stably express flag-LRRK2 and transfected with plasmids expressing myc-Snapin. Cell lysates were immunoprecipitated with flag antibody-agarose. The immunoprecipitate (IP) was washed and prepared for western analysis with anti-flag or anti-myc. 10% of cell lysates were shown as input (Input). * indicates a non-specific band. (d) SH-SY5Y cells differentiated by retinoic acid (10 μℳ for 5 days) were immunoprecipitated by LRRK2 (MJFF2, Epitomics) antibody and the immunoprecipitates were analyzed by antibody against LRRK2 (1E11) or Snapin (SYSY). An input of 2% was not enough to detect endogenous LRRK2, but sufficient for Snapin detection. WB, western blot.

Figure 3

Mapping of leucine-rich repeat kinase 2 (LRRK2) domain interacting with Snapin. (a) A scheme of several LRRK2 deletion mutant constructs. LRR: Leucine-rich repeats; Roc, Ras of complex proteins; CoR, C-terminal of Roc domain. (b) GST pull-down assay of LRRK2. The indicated LRRK2-mutant proteins were expressed in 293T cells as a Myc-tagged form by transient transfection and total cell lysates were subjected to a GST pull-down assay using GST (-) or GST-Snapin (S). Input (I) of each sample was 2.5% of the total proteins. The LRRK2 proteins were detected with anti-myc antibody, except for the kinase domain, which was detected by a previously reported LRRK2 antibody, 1E11. Because expressions of the ΔApaI, Roc and kinase domains were weaker those of the other domains, exposure times of these domains were extended. * indicates nonspecific protein band.

Figure 4

Phosphorylation of Snapin by leucine-rich repeat kinase 2 (LRRK2) causes a decrease of interaction with SNAP-25. (a) Snapin T117D-mutant mimicking phosphorylation by LRRK2 interacts with SNAP-25 more weakly than the wild type (WT). Recombinant Flag-tagged Snapin WT and indicated mutant proteins were subjected to a GST pull-down assay using GST-SNAP-25 and detected by western blot analysis using anti-Flag antibody. Coomassie blue staining of GST and GST-SNAP-25 showed that similar amounts of each protein were used. (b) Snapin WT protein phosphorylated by LRRK2 exhibited weaker interaction with SNAP-25 than the untreated and presumably unphosphorylated Snapin (control). The myc-tagged LRRK2 G2019S was transiently expressed in HEK293T cells and immunoprecipitated by anti-myc antibody. The immunoprecipitated beads were co-incubated with recombinant flag tagged Snapin WT protein with ATP under the in vitro kinase assay conditions. The supernatant was co-incubated with purified GST-SNAP-25 and subjected to a GST pull-down assay and SDS–PAGE. The bound Snapin was detected by anti-flag antibody (Sigma) and the amount was compared to the control transfected with an empty vector (i). The presence of LRRK2 in immunoprecipitates was confirmed by anti-myc antibody (ii). Coomassie blue staining (iii) confirmed that equal amounts of GST-SANP25 protein were used for both incubations (control and G2019S). (c) Interaction of SNAP25 with Snapin was weaker in G2019S transgenic (TG) than in a control (C) mouse brain lysate. Whole brain lysates were prepared from both G2019S TG and normal control mice and immunoprecipitated with SNAP25 antibody. The amount of Snapin in each brain lysates (20% input) was shown with Snapin antibody (Abnova, 1:500 dilution, Snapin-low). The immunoprecipitates, which showed no signal under the diluted condition above, were subjected to western blot analysis with more concentrated Snapin antibody (Abnova, 1:50 dilution, Snapin-high). The amount of specific immunoprecipitated Snapin was calculated as the amount of immunoprecipitated Snapin divided by the amount of immunoprecipitated SNAP25. An arrow or * indicated Snapin or nonspecific protein band, respectively.

Figure 5

T117D Snapin, a phosphomimetic mutant, decreased interaction of synaptotagmin with VAMP2-syntaxin-SNAP 25 complex. Rat brain lysate was incubated with none (−), recombinant Snapin wild type (WT) or T117D overnight and immunoprecipitated with syntaxin1 antibody or control rabbit immunoglobulin G antibody. The immunoprecipitate was subjected to SDS–PAGE for western blot analysis. Co-precipitated synaptotagmin-1 was detected by synaptotagmin-1 antibody and each component of the VAMP2-syntaxin-SNAP-25 SNARE complex was also detected by specific antibodies. The experiment was repeated three times and then band densities were analyzed. Data are presented as means±s.e.m. *P⩽0.01 (analysis of variance and Turkey's honestly significant difference post hoc test). A representative image (a) and the resulting graph are shown (b). Five percent of the total brain lysate (a) is shown as input. Use of similar amounts of the recombinant Snapin proteins was confirmed (c). Snapin was detected by the His tag, which was used for purification of the recombinant protein. *P⩽0.05 (analysis of variance and Turkey's honestly significant difference post hoc test).

Figure 6

Leucine-rich repeat kinase 2 (LRRK2)-dependent phosphorylation of Snapin decreased the size of RRP and the extent of exocytotic release. (a) Rat hippocampal neurons were co-transfected with wild-type Snapin alone, T117D alone, G2019S and wild-type Snapin, or D1994A and wild-type Snapin with vGpH. To measure RRP size, neurons were stimulated with 40 APs at 20 Hz. They were then stimulated with 1800 APs at 20 Hz in the presence of bafilomycin (baf). Subtraction of RRP fluorescence from the fluorescence plateau reflected the reserve pool (RP). All remaining acidic vesicles were alkalized by NH₄Cl treatment, revealing the size of the resting pool. Fluorescence intensity was normalized to the maximum fluorescence change upon NH₄Cl treatment. (b) (left) Average fraction of RRP in wild-type Snapin alone, T117D alone, G2019S and wild-type Snapin, or D1994A and wild-type Snapin (20.7%±2.8% for wild-type Snapin; 16.6%±2.2% for T117D; 12.4%±1.3% for G2019S and wild-type Snapin; 29.2%±3.6% for D1994A and wild-type Snapin). (middle) Average fraction of reserve pool (29.6%±3.0% for wild-type Snapin; 32.8%±3.1% for T117D; 41.6%±2.9% for G2019S and wild-type Snapin; 37.4%±6.4% for D1994A and wild-type Snapin). (right) Average fraction of resting pool (49.7%±3.0% for wild-type Snapin; 50.5%±1.4% for T117D; 46.0%±2.8% for G2019S and wild-type Snapin; 33.8%±6.6% for D1994A and wild-type Snapin). (c) The extent of exocytotic release. Rat hippocampal neurons were co-transfected with wild-type Snapin alone, T117D alone, G2019S and wild-type Snapin, or D1994A and wild-type Snapin with vGpH. The neurons were stimulated with 100 APs at 10 Hz, and then treated with NH₄Cl to obtain the total synaptic vesicle pool size. Fluorescence intensity was normalized to the maximum fluorescence change upon NH₄Cl treatment. Data are presented as means±s.e.m. *P⩽0.01 (analysis of variance and Turkey's honestly significant difference post hoc test).