Fig 1: Model of how Rab29 activates and recruits LRRK2 to the trans‐Golgi network greatly stimulating its kinase activityOur data suggest that Rab29 binds to the LRRK2 ankyrin domain and that GTP binding to the ROC domain of LRRK2 promotes Rab29‐mediated activation. This explains why pathogenic LRRK2 R1441G/C and Y1699C mutants that promote GTP binding are more readily recruited to the Golgi and activated by Rab29 than wild‐type LRRK2. Recruitment of LRRK2 to Rab29 at the Golgi also promotes phosphorylation of a cluster of highly studied biomarker phosphorylation sites (Ser910, Ser935, Ser955, and Ser973). More work is needed to define whether these biomarker residues are phosphorylated by autophosphorylation or by a Golgi‐resident upstream kinase. Finally, our data suggest that LRRK2‐mediated phosphorylation of Rab29 might act as a negative feedback loop and prevent Rab29 from activating LRRK2.
Fig 2: Exposure to low doses of LRRK2 kinase inhibitor also restored mtDNA damage to basal levels. Healthy control or LRRK2 G2019S patient derived LCLs were treated with either RA334 or RA283 with doses ranging from 1 to 100 nM for 24 h. (A) Treatment with RA334 reversed mtDNA damage in LRRK2 G2019S-patient derived LCLs, with (B) no effect on mtDNA copy number. (C) Similarly, exposure to RA283 reduced LRRK2 G2019S-induced mtDNA damage to healthy control levels. (D) Treatment with RA283 did not change mtDNA copy number. The PCR-based assay was performed in technical triplicate for each biological replicate. (*p < 0.001, determined by one-way ANOVA with a Tukey’s post-hoc comparison). n = 3 biological replicates (6 cell lines total), each performed in technical replicate. Data are presented as mean ± SEM.
Fig 3: Acute LRRK2 kinase inhibition. (A) Representative western blots of healthy control and LRRK2 G2019S patient-derived LCLs treated for 1.5 h with RA334. (B) Quantification of western blots demonstrated that RA334 decreased LRRK2 pSer935 levels at all doses tested. (C) Representative western blots of healthy control and LRRK2 G2019S patient-derived LCLs treated for 1.5 h with RA283. (D) Quantification of western blots demonstrate that RA283 decreased LRRK2 pSer935 levels at all doses tested. All experiments were performed with at least three biological replicates (6 cell lines total), each performed in technical replicate. Data are presented as mean ± SEM. (*p < 0.001, determined by one-way ANOVA with a Tukey’s post-hoc comparison). Full blots available in Supplemental Figs. S14 and S15.
Fig 4: Inhibitor-induced dephosphorylation of kinase-inactive LRRK2.HEK293 cells transfected with wild-type, K1906A, K1906M, D1994A, D1994N, D2017A, S2032A or T2035A LRRK2 were treated with (A) 3 µM LRRK2-IN-1 or the solvent (0.1% DMSO) for 30 min, (B) 5 µM sunitinib or the solvent (0.1% DMSO) for 90 min, or (C) 30 µM H-1152 or the solvent (1% sterilized distilled water) for 90 min, and the phosphorylation of LRRK2 at Ser910, Ser935, or Ser955 was examined by immunoblotting. The levels of the phosphorylation were quantified and normalized by the expression levels of LRRK2 determined by immunoblotting with the anti-LRRK2 antibody (bottom panel). The data are given as the percentage of those observed in solvent-treated WT LRRK2 (n = 3, mean ± standard error). *p<0.05, **p<0.01, and ***p<0.001 (Two-way ANOVA test followed by Bonferroni’s test).
Fig 5: Ankyrin domain residues permit activation of LRRK2 by Rab29 AHEK293 cells were transfected with the indicated wild‐type human full length (FL) or a fragment lacking the N‐terminal 969 residues (LRRK2[residues 970‐end]) pathogenic LRRK2 variants with either HA‐empty vector (−) or HA‐tagged Rab29 (+). 24 h post‐transfection, cells were lysed and analyzed by immunoblotting with the indicated antibodies. WT is wild‐type and D2017A corresponds to the kinase‐inactive LRRK2 mutant. Similar results were obtained in two independent experiments, each performed in duplicate.BUpper panel: Schematic representation of how Rab29 might interact with the ankyrin domain (ANK) of LRRK2 by analogy with how Rab32 binds VARP. Lower panel: Sequence alignments of the three Leu‐rich regions in the ankyrin domain of LRRK2 in the indicated species.C, DAs in (A) except that HEK293 cells were transfected with the wild type and indicated LRRK2 ankyrin domain mutations with either HA‐empty vector (−) or HA‐tagged Rab29 (+). KD is the Kinase Dead LRRK2[D2017A] mutant. Similar results were obtained in two experiments.EAs in (C) except that the indicated forms of LRRK2 were immunoprecipitated from cell extracts and then subjected to an LRRK2 kinase activity by measuring phosphorylation of the Nictide peptide substrate in the presence of 0.2 mM 32PγATP and in the absence (−) or presence (+) of 1 μM MLi‐2 LRRK2 in a 30‐min kinase reaction. After the kinase assay, phosphorylation of Nictide was quantified by Cherenkov counts and data presented as average ± SEM for three independent experiments each undertaken in triplicate. Cherenkov counts recorded for no LRRK2 (‐) controls were subtracted from all values. There was a statistically significant difference between groups (P < 0.0001, one‐way ANOVA, F(9, 20) = 95.87) ***P < 0.001 by one‐way ANOVA with Dunnett's multiple comparison with mean difference 95% confidence intervals of groups compared to WT: WT MLI‐2 0.8668–1.126; C727D 0.7428–1.002; C727D MLI‐2 0.8731–1.132; L728D 0.7602–1.019; L728D MLI‐2 0.8559–1.115; L729D 0.7570–1.016, L729D MLI‐2 0.8708–1.130, L728D+L729D 0.7466–1.006; L728D+L729D MLI‐2 0.8655–1.124. Assay mixtures were subjected to immunoblot analysis with the indicated antibodies.FAs in (E) except phosphorylation of Rab8A by LRRK2 was assessed using a phospho‐specific antibody. Similar results were obtained in two experiments each undertaken in duplicate. Source data are available online for this figure.
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