Effect of 4E-BP and eIF4E complex on phosphorylation of 4E-BP by LRRK2 <i>in vitro</i>.

<p>(A) Time-course assays were performed for LRRK2 wild-type (upper panel) by measuring the incorporation of [γ³³P] ATP at different time points (0, 5, 15, 30 and 60 mins) and at 3 µM concentrations of 4E-BP (upper left panel), 3 µM concentrations of eIF4E (upper middle panel) and 3 µM concentrations of 4E-BP and 3 µM concentrations of eIF4E (upper right panel), respectively, followed by SDS-PAGE and autoradiography. (B) Phosphorylation of 4E-BP by LRRK2-wild type were plotted as a function of time.</p

V_max and K_m for LRRK2 and MAPK14/P38α phosphorylation of 4E-BP <i>in vitro</i>.

<p>Each data point is calculated V_max and K_m values (±SEM) for assays carried out in triplicate.</p

4E-BP phosphorylation in inducible HEK 293FT cells.

<p>(A) 4E-BP phosphorylation before and after induction of LRRK2 expression. The panels represent phospho-4E-BP (T37/46), phospho-4E-BP (S65), phospho-4E-BP (T70), total 4E-BP and V5-LRRK2, respectively. (B) Quantification analysis of 4E-BP phosphorylation of n = 14 independent experiments. (C) Autophosphorylation assays of LRRK2 immunoprecipitated from HEK 293FT cells. Lanes 1 and 2 are immunoprecipitated V5-tagged LRRK2 proteins transiently transfected and lanes 3–7 represent V5-LRRK2 immunoprecipitated from inducible cells.</p

Stoichiometry of 4E-BP phosphorylation and LRRK2 autophosphorylation.

<p>Ratio between the number of inorganic phosphates (Pi) incorporated by LRRK2 or 4E-BP and the total number of LRRK2 or 4E-BP molecules at different time points. Each figure is the mean +/− for assays carried out in triplicate.</p

Phosphorylation of 4E-BP by LRRK2 <i>in vitro</i>.

<p>(A) Time-course catalytic assays were performed for GST-LRRK2 wild-type (upper panel) or GST-LRRK2-G2019S (lower panels) by measuring the incorporation of [γ³³P] ATP at different time points (0, 5, 15, 30 and 60 mins) and at different concentrations of 4E-BP (1.5, 3, 6 and 12 µM), respectively, followed by SDS-PAGE and autoradiography. (B) Autophosphorylation of LRRK2-wild type (left graph) and G2019S (right graph) were plotted as a function of time. (C) Phosphorylation of 4E-BP by LRRK2-wild type (left graph) and G2019S (right graph) were plotted as a function of time. (D) Results of the catalytic assays were derived by calculating the reaction rate of each assay at different 4E-BP concentrations (1.5, 3, 6 and 12 µM). For all experiments in A–D, each point is the average of three independent experiments and error bars indicate the SEM.</p

4E-BP phosphorylation in transiently transfected HEK 293FT cells.

<p>4E-BP phosphorylation in HEK 293FT cell after transient transfection of LRRK2-wild type, LRRK2-R1441C, LRRK2-G2019S and MAPK14/P38α constructs. The panels represent myc-LRRK2 and myc-MAPK14/P38α, phospho-4E-BP (T37/46), phospho-4E-BP (S65), phospho-4E-BP (T70), total 4E-BP and β-actin, respectively. (B) Quantification of 4E-BP phosphorylation at T37/46, normalized to total 4E-BP from n = 3 samples representative of two independent experiments.</p

Comparative kinase activities of LRRK1 and LRRK2.

<p>(A) Autophosphorylation assays of 3xFlag-LRRK1 wild-type, LRRK1 kinase dead, LRRK2 wild-type and LRRK2 kinase dead. End-point reactions (60 minutes) were resolved on a 4–20% SDS-PAGE and transferred onto PVDF membranes. Upper panel is autoradiography and lower panel western blot to correct activity for total loading (with anti-Flag antibody). The experiment is representative of n = 3 replicates. Markers to the right of the blots are in kilodaltons. (B) Quantitation of ³³P signal by densitometry normalized to total loading. (C–D) LRRKtide (C) and (D) Nictide phosphorylation assessed by P81 filter binding assay reveals that both peptides are specific substrates for LRRK2. (E) Rate of P³³ incorporation as a function of LRRK2 protein content (from 10 to 550 ng) measured by LRRKtide phosphorylation assays. (F) Kinetic constants of wild-type and G2019S LRRK2 for LRRKtide were determined by incubating 25 nM LRRK2 with varying concentrations of LRRKtide in the presence of 100 µM ATP and by fitting the data to a hyperbolic function. K_m was 171±20 µM for wild-type and 257±63 µM for G2019S. V_max were 1.92±0.06 pmol/min/µg for wild-type and 7.71±0.95 pmol/min/µg for G2019S. (G) ATP binding was tested for both LRRK1 and LRRK2 by affinity binding of the proteins to 4 different forms of ATP-bound agarose beads (ie ATP is coupled to the beads in different conformations) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0043472#s4" target="_blank">materials and methods</a>. Both LRRK1 and LRRK2 bound to the beads when ATP is coupled via the adenine moiety (6-AH-ATP-A or 8-AH-ATP-A). Binding was negligible for ATP coupled via the gamma-phosphate (AP-ATP-A) or ribose group (EDA-ATP-A). The position of the 250 kilodalton M_w marker is shown.</p

Purification of soluble full-length 3xFlag-LRRK1 and 3xFlag-LRRK2.

<p>(A) Representative silver staining of purified 3xFlag-LRRK1 and LRRK2 purification indicates highly pure protein fractions. Markers are in kilodaltons (B) Circular dichroism analysis of purified 3xFlag LRRK1 and LRRK2. Representative spectra are reported as mean residue molar ellipticity (deg cm² dmol⁻¹). (C) Representative fluorescence spectra of purified LRRK1 (right) and LRRK2 (left) before (solid line) and after (dashed line) addition of 6M GdHCl using an excitation wavelength of 280 nm. Fluorescence intensity was normalized to the highest peak.</p

Analysis of LRRK1 and LRRK2 structure by transmission electron microscopy (TEM).

<p>Distributions of gold particle distances of double-gold labeled LRRK1 (A) and LRRK2 (B) particles and representative images. Particles were stained with uranyl acetate and subsequently labeled with primary anti-Flag (M2) antibody and secondary 5 nm gold-labeled secondary anti-mouse antibody. (C and D) Distributions of particles diameters of purified LRRK1 and LRRK2 negative stained with uranyl acetate and representative images of protein shapes. (E) Scatter plots of gold distances measured for double-gold labeled LRRK1 and LRRK2. Distributions are significantly different as assessed by t-test (**, <i>P</i> = 0.001). (F) Scatter plots of particle size for LRRK1 and LRRK2. Distribution of LRRK1 and LRRK2 are significantly different by t-test (***, <i>P</i><0.0001).</p

Characterization of HEK293T cell line stably expressing 3xFlag-LRRK1 and LRRK2.

<p>(A) Schematic alignment of LRRK1 and LRRK2. Predicted functional domains are drawn to scale at the relative location within the full protein sequence. For domains containing repeat sequences, predicted individual repeat units are depicted. The sequence identity and similarity for the LRR, ROC, COR and Kinase domains are given below the schematic. Also given are detailed alignments of LRRK1 and LRRK2 at the level of common LRRK2 clinical mutations. Abbreviations for the domains: ARM, armadillo repeat domain; ANK, ankyrin repeat domain; LRR, leucine rich repeat domain; ROC, Ras of comple proteins domain; COR, C-terminal of ROC domain; Kin, kinase domain; WD40, WD40 repeat domain. (B) Representative western blot analysis of HEK293T cells stably expressing (from lane 1 to 7) 3xFlag-tagged LRRK2 wild-type, T1348N GTP deficient binding mutant, K1906M kinase dead, G2019S pathogenic mutant and LRRK1 wild-type K650A GTP deficient binding mutant, K1269M kinase dead. Upper panel shows membranes probed with Flag (M2) antibody (note that LRRK2 and LRRK1 have different exposure time due to the very low expression of T1348N mutant). Lower panel shows β-tubulin loading control. (C) Representative confocal images of stable HEK293T cells expressing LRRK1 and LRRK2 wild-type and mutants. Scale bar 10 µm.</p