Journal: PLoS Genetics
Article Title: RIT1 controls actin dynamics via complex formation with RAC1/CDC42 and PAK1
Figure Lengend Snippet: Expression of RIT1 mutants enhances phosphorylation of ERK1/2. (A) Schematic representation of RIT1 with selected NS-associated amino acid substitutions. RIT1 isoform 2 (protein RefSeq NP_008843.1) comprises 219 amino acids and has five conserved GDP/GTP binding motifs (G1 to G5, light blue). Motifs representing the P-loop and switch I and II regions are shown in dark blue. The P-loop binds γ-phosphate of GTP and GDP, the switch regions are critical for GDP/GTP binding and for interaction with upstream and downstream partners. RIT1 amino acid substitutions identified in patients with NS and selected for functional studies in this work are given in the one-letter code above the scheme. (B) HEK293T cells were transfected with empty vector (EV) or constructs expressing HA-RIT wildtype (WT), HA-RIT1 p.A57G, p.F82L, or p.G95A as indicated. Cells were serum-starved (0.1% serum) and subsequently stimulated with 20% serum for 5, 15, or 30 min or left untreated (0 min). Cell extracts were analyzed by immunoblotting using anti-phospho-ERK1/2 (pERK1/2) and anti-ERK1/2 (ERK1/2) antibodies. Expression of HA-tagged RIT1 protein variants was monitored by immunoblotting using anti-HA antibody, and anti-GAPDH antibody was used to control for equal loading. Data shown are representative of three independent experiments. Autoradiographic signals were quantified by scanning densitometry. Levels of phosphorylated ERK1/2 were normalized relative to amounts of total ERK1/2. To conserve the relative variance of the samples, values for RIT1 wildtype and mutants were divided by the mean of the wildtype samples [ 79 ]. Graphs show phosphorylation levels upon serum starvation (0 min) and after 5, 15, and 30 min serum stimulation in cells expressing RIT1 wildtype (WT), RIT1 p.A57G, p.F82L or p.G95A (arbitrary units). The mean of three independent experiments ± SD is given. Unpaired t -tests were used to determine statistical significance (*, P
Article Snippet: Depending on the experimental design, recombinant His-RIT1WT/A57G/F82L , GST-CDC42 and GST-RAC1 were coupled to GDP (G7127, Sigma-Aldrich) or non-hydrolyzable GTPγS (G8634, Sigma-Aldrich) for 30 min by incubation with 20 mM Tris-HCl, 100 mM NaCl, 0.1% Triton X-100, 1 mM DTT, 2.5 mM EDTA and 1 mM of GDP or GTPγS at room temperature.
Techniques: Expressing, Binding Assay, Functional Assay, Transfection, Plasmid Preparation, Construct