Fig 1: Identification of a substrate trapping mutant of PPM1H that forms a stable interaction with endogenous LRRK2 phosphorylated Rab8A and Rab10.(A) HEK293 cells were transiently transfected with constructs expressing Flag-LRRK2[R1441G] and either wild-type HA-PPM1H or the PPM1H[D288A] mutant, as well as the corresponding mutants of the closely related PPM1J[D279A] and PPM1M[D235A]. 24 hr post-transfection cells were lysed and subjected to a HA-immunoprecipitation and analyzed by immunoblotting with the indicated antibodies (1 µg/ml). Membranes were developed using Odyssey CLx Western Blot imaging. Each lane represents cell extract obtained from a different dish of cells (three replicates per condition). (B) HEK293 cells were transiently transfected with constructs expressing Flag-LRRK2[R1441G] and either wild-type HA-PPM1H or the HA-PPM1H[D288A] mutant, lysed and subjected to a HA-immunoprecipitation and total immunoprecipitates were subjected to ‘on-bead’ digestion using trypsin following multiplexed TMT labeling the samples were pooled and fractionated into four fractions and analyzed on an Orbitrap Fusion Lumos Tribrid mass spectrometer in MS3 mode. The raw data was processed using MaxQuant pipeline and protein groups were further processed using Perseus software suite. The x-axis of the volcano plot represents the differential enrichment between HA-PPM1H[D288A]+LRRK2[R1441G] and wild-type HA-PPM1H+LRRK2[R1441G] and the y-axis represents the permutation-based false discovery rate corrected significance of a two tailed t-test. The differential enriched protein groups are highlighted in red filled circles along with the protein groups that are not changing are highlighted in blue with their gene names. The list of protein groups is included in Supplementary file 3.
Fig 2: PPM1H dephosphorylates Rab8A in vitro.(A) The indicated amounts of recombinant wild-type and mutant PPM1H (with a His-Sumo N-terminal tag, expressed in E. coli) were incubated in vitro with 2.5 µg pT72 phosphorylated Rab8A[1–181, Q67L-GTP bound conformation], 2.5 µg of Rab8A[1–181 wild-type, GDP-bound] or 2.5 µg of Rab8A[1–181 wild-type, GTP-bound] for 30 min in the presence of 10 mM MgCl2 in 40 mM HEPES pH 7.0 buffer. Reactions were terminated by addition of SDS Sample Buffer and analyzed by Phos-tag gel electrophoresis that separates phosphorylated and dephosphorylated Rab8A. The gel was stained with Instant Blue Coomassie. Bands corresponding to phosphorylated and non-phosphorylated Rab10 are marked with open (?) and closed (?) circles, respectively. (B) As in (A) except that a time-course assay was performed using 2.5 µg pT72 phosphorylated Rab8A[1–181, Q67L-GTP bound conformation], 2.5 µg of Rab8A[1–181 wild-type, GDP-bound] or 2.5 µg of Rab8A[1–181 wild-type, GTP-bound] and 40 ng wild-type or mutant PPM1H for the indicated times. (C) As in (A) except that PPM1J was assessed. (D) As in (A) except and PPM1M was assessed.
Fig 3: Effect of LRRK2 kinase inhibitor on the phosphorylation of Lrrk2, Rab8a and Rab10 in adipocyte. 3T3‐L1 cells treated with or without CZC25146 (1 and 2 μm) and MLi‐2 (0.1 and 0.2 μm) after serum starved and then stimulated with insulin for 30 min. The cells were harvested and analyzed by western blotting using antibodies against phosphorylated or total protein such as (A) Lrrk2, (B) phospho‐Lrrk2 (Ser935), (C) phospho‐Rab8a (Thr72), and (D) phospho‐Rab10 (Thr73). The expression level of phosphorylated protein was normalized against the total expression level of the target protein. Data are presented as means ± SD (n = 6). The data were analyzed by one‐way ANOVA combined with Tukey's post hoc test. *P < 0.05; ***P < 0.001; ****P < 0.0001.
Fig 4: Comparison of protein expression and phosphorylation of Lrrk2, Rab8a and Rab10 in epididymal adipose tissue of ND‐ or HFD‐fed WT and Lrrk2‐KO mice. The expression of Lrrk2 (A), phospho‐Lrrk2 (Ser935) (B), phospho‐Rab8a (Thr72) (C), phospho‐Rab10 (Thr73) (D), phospho‐AS160 (Thr642) (E), GLUT4 (F), phospho‐Akt (Thr308) (G), total Akt (H), phospho‐Ampk (Thr172) (I), and total Ampk (J) was determined by western blot analysis. All proteins and GAPDH (as a loading control) were visualized by chemiluminescence and using an Odyssey Fc Dual‐Mode Imaging System (LI‐COR Biosciences, USA). The band intensity of Lrrk2, GLUT4, Akt, and Ampk were normalized by GAPDH. The intensity of phospho‐Lrrk2, phospho‐Rab8a and phospho‐Rab10, phospho‐AS160, phospho‐Akt, and phospho‐Ampk was normalized by their total protein band intensity. (K) Quantification of GLUT4 in the plasma membrane (PM) fraction obtained from adipose tissue of each mouse by western blotting as described in the Materials and methods. GLUT4 contents in PM fraction were determined by western blotting with an anti‐GLUT4 antibody. The intensity of Glut4 bands was normalized to E‐cadherin. Data are presented as mean ± SD (n = 6). The data were analyzed by one‐way ANOVA followed by Tukey's post hoc test. **P < 0.01; ***P < 0.001; ****P < 0.0001.
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