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. 2016 Jan 1;25(1):109-22.
doi: 10.1093/hmg/ddv453. Epub 2015 Nov 5.

14-3-3 Proteins regulate mutant LRRK2 kinase activity and neurite shortening

Nicholas J Lavalley  1 , Sunny R Slone  1 , Huiping Ding  1 , Andrew B West  1 , Talene A Yacoubian  2
Affiliations

14-3-3 Proteins regulate mutant LRRK2 kinase activity and neurite shortening

Nicholas J Lavalley et al. Hum Mol Genet. .

Abstract

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common known cause of inherited Parkinson's disease (PD), and LRRK2 is a risk factor for idiopathic PD. How LRRK2 function is regulated is not well understood. Recently, the highly conserved 14-3-3 proteins, which play a key role in many cellular functions including cell death, have been shown to interact with LRRK2. In this study, we investigated whether 14-3-3s can regulate mutant LRRK2-induced neurite shortening and kinase activity. In the presence of 14-3-3θ overexpression, neurite length of primary neurons from BAC transgenic G2019S-LRRK2 mice returned back to wild-type levels. Similarly, 14-3-3θ overexpression reversed neurite shortening in neuronal cultures from BAC transgenic R1441G-LRRK2 mice. Conversely, inhibition of 14-3-3s by the pan-14-3-3 inhibitor difopein or dominant-negative 14-3-3θ further reduced neurite length in G2019S-LRRK2 cultures. Since G2019S-LRRK2 toxicity is likely mediated through increased kinase activity, we examined 14-3-3θ's effects on LRRK2 kinase activity. 14-3-3θ overexpression reduced the kinase activity of G2019S-LRRK2, while difopein promoted the kinase activity of G2019S-LRRK2. The ability of 14-3-3θ to reduce LRRK2 kinase activity required direct binding of 14-3-3θ with LRRK2. The potentiation of neurite shortening by difopein in G2019S-LRRK2 neurons was reversed by LRRK2 kinase inhibitors. Taken together, we conclude that 14-3-3θ can regulate LRRK2 and reduce the toxicity of mutant LRRK2 through a reduction of kinase activity.

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Figures

Figure 1.
Figure 1.
14-3-3s regulate LRRK2 phosphorylation at serine 910 and 935. (A) Western blots from co-immunoprecipitation experiments of endogenous 14-3-3 interaction with HA-tagged LRRK2 protein. Difopein-eYFP migrates slightly higher than mutant difopein-eYFP since difopein has two R18 peptide sequences while the mutant difopein peptide has one copy of the mutated R18 peptide sequence (38). (B) Lysates from HEK 293T cells transfected with difopein and either wild type or G2019S-LRRK2 were analyzed for LRRK2 phosphorylation at S910 and S935 and total LRRK2 by western blot. Representative western blots are shown. The ratio of phosphorylated LRRK2 to total LRRK2 is quantified from three independent experiments. ***P < 0.001 (unpaired t-test). (C) Quantification of S935 phosphorylation in hippocampal lysates from wild-type and difopein-eYFP transgenic mice. n = 4 mice per group, **P < 0.01 (unpaired t-test). (D) Lysates from HEK 293T cells transfected with GFP or 14-3-3θ and either wild type or G2019S-LRRK2 were analyzed for LRRK2 phosphorylation at S910 and S935 and total LRRK2 by western blot. n = 3 independent rounds, **P < 0.01, ***P < 0.001 (unpaired t-test). (E) Representative western blots and quantification of S935 phosphorylation in cortical lysates generated from transgenic mice overexpressing 14-3-3θ. n = 4 mice per group, *P < 0.05 (unpaired t-test).
Figure 2.
Figure 2.
14-3-3θ overexpression reverses G2019S-LRRK2-mediated neurite shortening in primary hippocampal cultures. (A) Western blots against HA-tagged 14-3-3θ and LRRK2 from lysates of dissected hippocampi from 8-day-old transgenic mice. (B and C) Immunohistochemistry for HA-tagged 14-3-3θ in coronal brain section through the hippocampus from a 14-3-3θ mouse. Scale bar = 200 µm. (D and E) Western blot of lysates from DIV8 primary hippocampal cultures prepared from nontransgenic (ntg), G2019S-LRRK2, 14-3-3θ and G2019S-LRRK2/14-3-3θ mice to measure S935 LRRK2 phosphorylation. n = 4 mice per group, **P < 0.01 (unpaired t-test). (F–I) Representative images of neurons stained for MAP2 from each of the four genotypes represented in the cross. Scale bar = 50 µm. (J) Total neurite length analysis of neurons from nontransgenic, G2019S-LRRK2, 14-3-3θ and G2019S-LRRK2/14-3-3θ mouse cultures. n = 52 neurons for ntg, n = 52 for 14-3-3θ, n = 65 for G2019S-LRRK2 and n = 44 for G2019S-LRRK2/14-3-3θ combined from three independent rounds. *P < 0.05, **P < 0.01 (Tukey's multiple comparison test).
Figure 3.
Figure 3.
14-3-3 inhibition with difopein promotes neurite shortening induced by G2019S-LRRK2. (A) Primary hippocampal cultures from nontransgenic (ntg) and G2019S-LRRK2 littermates were transduced with a doxycycline-inducible lentivirus expressing either difopein-eYFP (dif) or mutant difopein-eYFP (mut dif) and then treated with doxycycline (2 µg/ml). Total neurite length was measured for neurons expressing eYFP. n = 22 neurons for ntg/mut dif, n = 39 for ntg/dif, n = 36 for G2019S-LRRK2/mut dif and n = 66 for G2019S-LRRK2/dif combined from three independent rounds. *P < 0.05, ***P < 0.001 (Tukey's multiple comparison test). (B) Total neurite length analysis of neurons from nontransgenic, G2019S-LRRK2, difopein and G2019S-LRRK2/difopein cultures. n = 34 neurons for ntg, n = 17 for dif, n = 34 for G2019S-LRRK2 and n = 22 for G2019S-LRRK2/dif combined from three independent rounds. *P < 0.05, **P < 0.01, ***P < 0.001 (Tukey's multiple comparison test). (C) Western blot for eYFP-tagged difopein and LRRK2 of lysates from dissected hippocampi from 8-day-old transgenic mice. (D) Western blot for S935 phosphorylation of lysates from DIV8 primary hippocampal cultures from transgenic mice. n = 3 mice per group, **P < 0.01 (unpaired t-test). (E) Immunohistochemistry for difopein-eYFP in coronal sections through the hippocampus of difopein mice and nontransgenic mice. Scale bar = 500 µm. (F) Representative tracings of neurons analyzed in each resulting genotype in the cross. Scale bar = 50 µm.
Figure 4.
Figure 4.
Dominant-negative 14-3-3θ expression also promotes G2019S-LRRK2-induced neurite shortening. (A) Immunohistochemistry for HA-tagged DN 14-3-3θ in coronal sections through the hippocampus of DN 14-3-3θ and nontransgenic mice. Scale bar = 500 µm. (B) Western blot for HA-tagged 14-3-3θ and LRRK2 from lysates from DIV8 primary hippocampal cultures from transgenic mice. (C) Total neurite length analysis of neurons from nontransgenic, G2019S-LRRK2, DN 14-3-3θ and G2019S-LRRK2/DN 14-3-3θ cultures. n = 25 neurons for ntg, n = 50 for DN 14-3-3θ, n = 75 for G2019S-LRRK2 and n = 35 for G2019S-LRRK2/DN 14-3-3θ combined from three independent rounds. *P < 0.05, **P < 0.01, ***P < 0.001 (Tukey's multiple comparison test).
Figure 5.
Figure 5.
14-3-3θ overexpression ameliorates R1441G-LRRK2-induced neurite shortening. (A) Cell lysates from HEK 293T cells transfected with myc-tagged wild-type, G2019S, or R1441G-LRRK2 were immunoprecipitated with a monoclonal antibody against myc, and resulting immunoprecipitants were analyzed by western blot with a polyclonal rabbit antibody against endogenous 14-3-3s (pan). The ratio of pan 14-3-3 to myc LRRK2 was quantified for three independent rounds. **P < 0.01 (Tukey's multiple comparison test). (B) Hippocampal lysates from 8-day-old BAC wild-type, G2019S, or R1441G-LRRK2 transgenic mice were immunoprecipitated for LRRK2 and the resulting immunoprecipitates were probed for endogenous 14-3-3s by western blot. (C) Lysates from HEK 293T cells transfected with V5-tagged 14-3-3θ and either wild type or R1441G-LRRK2 were analyzed for LRRK2 phosphorylation at S910 and S935 and total LRRK2 by western blot. The ratio of phosphorylated LRRK2 to total LRRK2 was quantified for three independent rounds. ***P < 0.001, ns = not significant (Tukey's multiple comparison test). (D) Total neurite length analysis of neurons from nontransgenic, R1441G-LRRK2, 14-3-3θ and R1441G-LRRK2/DN 14-3-3θ cultures. n = 29 neurons for ntg, n = 22 for 14-3-3θ, n = 48 for R1441G-LRRK2 and n = 55 for R1441G-LRRK2/14-3-3θ combined from three independent rounds. ***P < 0.001 (Tukey's multiple comparison test).
Figure 6.
Figure 6.
Difopein and DN 14-3-3θ cause neurite shortening in the absence of LRRK2 expression. (A) Total neurite length analysis of neurons from difopein-eYFP crossed with LRRK2 −/− cultures. n = 22 neurons for wild type (wt), n = 16 for LRRK2 −/−, n = 16 for dif and n = 22 for LRRK2 −/− + dif combined from four independent rounds. *P < 0.05, ***P < 0.001, ns = nonsignificant (Tukey's multiple comparison test). (B) Total neurite length analysis of neurons from DN 14-3-3θ crossed with LRRK2 −/− cultures. n = 23 neurons for wt, n = 25 for LRRK2 −/−, n = 44 for DN 14-3-3θ and n = 39 for LRRK2 −/− + DN 14-3-3θ combined from four independent rounds. **P < 0.01, ***P < 0.001, ns = nonsignificant (Tukey's multiple comparison test).
Figure 7.
Figure 7.
14-3-3s regulates LRRK2 autophosphorylation and trans-phosphorylation activities. (A) Lysates from HEK293T cells transfected with wt or G2019S-LRRK2 together with increasing amounts of V5-tagged 14-3-3θ plasmid were generated. LRRK2 was immunoprecipitated with a HA-specific antibody to isolate LRRK2 for in vitro kinase reactions. Following kinase reaction, immunoprecipitates were then analyzed for the LRRK2 autophosphorylation residue phospho-T1503 and total HA-tagged LRRK2 by western blot. n = 3 independent rounds. **P < 0.01, ***P < 0.001, ****P < 0.0001 (Tukey's multiple comparison test). (B) Lysates from HEK293T cells transfected with wt or G2019S-LRRK2 together with difopein-eYFP or mutant difopein-eYFP were immunoprecipitated with a HA-specific antibody prior to the kinase assay. After the kinase reaction, immunoprecipitates were probed for phospho-T1503 and total HA-tagged LRRK2 by western blot. n = 3 independent rounds. *P < 0.05, **P < 0.01 (Tukey's multiple comparison test). (C) Lysates from HEK 293T cells transfected with wt or G2019S-LRRK2 along with increasing amounts of V5-tagged 14-3-3θ plasmid were analyzed for LRRK2 autophosphorylation at S1292 and total LRRK2 by western blot. n = 4 independent rounds. ***P < 0.001, ****P < 0.0001 (Tukey's multiple comparison test). (D) Lysates from HEK 293T cells transfected with wt or G2019S-LRRK2 along with difopein-eYFP or mutant difopein-eYFP were analyzed for LRRK2 autophosphorylation at S1292 and total LRRK2 by western blot. n = 3 independent rounds. **P < 0.01, ***P < 0.001 (Tukey's multiple comparison test). (E) Hippocampal lysates from 6-week-old G2019S-LRRK2 or G2019S-LRRK2/14-3-3θ littermates were analyzed for LRRK2 autophosphorylation at S1292 and total LRRK2 by western blot. n = 3 mice per group, *P < 0.05 (unpaired t-test). (F) Hippocampal lysates from 6-week-old G2019S-LRRK2 or G2019S-LRRK2/difopein-eYFP littermates were analyzed for LRRK2 autophosphorylation at S1292 and total LRRK2 by western blot. n = 3 mice per group, ***P < 0.001 (unpaired t-test). (G) Lysates from HEK293T cells transfected with wt or G2019S-LRRK2 with and without V5-tagged 14-3-3θ were immunoprecipitated with an HA-specific antibody prior to performing the kinase assay. Kinase assays were performed with immunoprecipitates together with recombinant GST-tagged ArfGAP1. Following incubation in kinase buffer, immunoprecipitates were then probed for phospho-threonine and GST by western blot. n = 3 independent rounds. **P < 0.01 (Tukey's multiple comparison test).
Figure 8.
Figure 8.
Binding to LRRK2 is required for 14-3-3θ regulation of LRRK2 kinase activity. HEK 293T cells were cotransfected with either FLAG-tagged wt or G2019S-LRRK2 with and without the S935A mutation and V5-tagged 14-3-3θ. Lysates were immunoprecipitated with FLAG antibodies. Kinase assays were performed with immunoprecipitates. Resultant immunoprecipitants were analyzed by western blot for phospho-T1503, and for LRRK2 and V5 to verify that 14-3-3 co-immunoprecipitation was disrupted by the S935A mutation. n = 4 independent rounds. *P < 0.05, ***P < 0.001, n.s. = not significant (Tukey's multiple comparison test).
Figure 9.
Figure 9.
The LRRK2 kinase inhibitor HG-10-102-01 reverses the enhanced neurite shortening observed in the G2019S-LRRK2/difopein double transgenic mice. (A) Total neurite length analysis of primary hippocampal neurons from nontransgenic, G2019S-LRRK2, difopein-eYFP and G2019S-LRRK2/difopein-eYFP mouse cultures treated with HG-10-102-01 (1 µm) for 48 h prior to fixing and staining. n = 45 neurons for ntg, n = 21 for G2019S-LRRK2, n = 25 for G2019S-LRRK2 + HG-10-102, n = 24 for dif and n = 21 for dif + HG-10-102, n = 23 for G2019S-LRRK2/dif, n = 24 for G2019S-LRRK2/dif + HG-10-102 combined from four independent rounds. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (Tukey's multiple comparison test). (B) Total neurite length analysis of neurons from nontransgenic, G2019S-LRRK2, 14-3-3θ and G2019S-LRRK2/14-3-3θ mouse cultures treated with HG-10-102-01 (1 µm) for 48 h prior to fixing and staining. n = 27 neurons for ntg, n = 44 for G2019S-LRRK2 and n = 20 for G2019S-LRRK2/14-3-3θ, n = 11 for G2019S-LRRK2/14-3-3θ + HG-10-102 combined from three independent rounds. *P < 0.05, ***P < 0.001 (Tukey's multiple comparison test).
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