Fig 1: Mutational Analyses Reveal Hotspots of pRab8a:RILPL2 Interactions(A) HEK293 cells were transiently transfected with constructs expressing Flag-LRRK2[R1441G], HA-Rab8a and WT or mutant RILPL2-GFP. At 48 h post transfection, cells were treated with ±500 nM MLi-2 for 90 min and then lysed. Upper panel, labeled IP:GFP: RILPL2-GFP was immunoprecipitated using GFP binder Sepharose and immunoprecipitates evaluated by immunoblotting with the indicated antibodies. Immunoblots were developed using the LI-COR Odyssey CLx western blot imaging system with the indicated antibodies at 0.5–1 μg/mL concentration. Lower panel, labeled input: 10 μg whole-cell lysate was subjected to LI-COR immunoblot analysis. Each lane represents cell extract obtained from a different dish of cells. Similar results were obtained in two separate experiments. (B) Same as A, but HEK293 cells were transiently transfected with WT or mutant HA-Rab8a as well as Flag-LRRK2[R1441G] and RILPL2-GFP WT. At 48 h post transfection, cells were treated with ±500 nM MLi-2 for 90 min and then lysed. RILPL2-GFP was immunoprecipitated using GFP binder Sepharose and as in (A), immunoprecipitates and input were evaluated by immunoblotting with the indicated antibodies. Each lane represents cell extract obtained from a different dish of cells. Similar results were obtained in two separate experiments.(C) Sequence alignment of the first α helix (α1) of the RILP family RH2 domains. Residues corresponding to the second α helix (α2) of RILP are not shown. Red circles are hotspots for the interactions where mutations severely reduce affinity between pRab8a and RILPL2. Blue circles indicate residues that are tolerant to mutations. The α-helical secondary structure above the alignment corresponds to RILPL2.
Fig 2: Evidence that JIP3 and JIP4 Bind to LRRK2-Phosphorylated Rab10 in Cells(A) HEK293 cells were transiently transfected with constructs expressing the indicated components. At 24 h post transfection, cells were treated with ±100 nM MLi-2 for 90 min and then lysed. Upper panel, labeled IP:GFP: RILPL2-GFP, JIP3-GFP, JIP4-GFP were immunoprecipitated using GFP binder Sepharose and immunoprecipitates evaluated by immunoblotting with the indicated antibodies. Immunoblots were developed using the LI-COR Odyssey CLx western blot imaging system with the indicated antibodies at 0.5–1 μg/mL concentration. Lower panel, labeled input: 10 μg whole-cell lysate was subjected to LI-COR immunoblot analysis. Each lane represents cell extract obtained from a different dish of cells. Similar results were obtained in two separate experiments.(B) Domain organization of JIP3 and JIP4. Sequence numbers correspond to JIP3, the interacting partners are shown above the cartoon, and p150G refers to p150Glued. The figure is adapted from the recent structure of the RH1-LZI domain of JIP3 (Vilela et al., 2019). KHC, kinesin heavy chain; KLC, kinesin light chain; DLIC, dynein light intermediate chain.
Fig 3: Model for the Control of Rab8a Functions by LRRK2Rab29 recruits LRRK2 to membranes and Rab8a is subsequently phosphorylated by LRRK2. RILPL2 is then recruited to membranes by pRab8a via the X-cap. RILPL2 is an adaptor that links pRab8a to the GTD of MyoVa. The structure of the mouse complex of RILPL2 with myosin was used to generate this figure (PDB: 4kp3; Wei et al., 2013).
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