NS-associated RIT1 amino acid changes enhance binding of RIT1 to PAK1.

<p>(A) HEK293T cells were transfected with empty vector (EV) and RIT1 expression constructs (WT, p.K23N, p.G31R, p.A57G, p.F82L, p.M90V, and p.G95A) as indicated and cultured under serum-starved conditions (0.1% serum). HA-tagged RIT1 protein variants were precipitated from cell extracts using GST-PAK[CRIB] fusion proteins (PD, pull down). Precipitated HA-RIT1 (PD) and HA-RIT1 in the total cell lysates (TCL) were detected by immunoblotting using anti-HA antibody. Anti-GAPDH antibody was used to control for equal loading (TCL, total cell lysate). Data shown are representative of three independent experiments. Autoradiographic signals were quantified by scanning densitometry. The amount of co-precipitated HA-RIT1 was normalized relative to the amount of total HA-RIT1. To conserve the relative variance of the samples, values for RIT1 wildtype and mutants were divided by the mean of the wildtype samples [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007370#pgen.1007370.ref079" target="_blank">79</a>]. The graphs show the relative amounts of co-precipitated RIT1 protein variants (arbitrary units). Data represent the mean of three independent experiments ± SD. One-way ANOVA between groups: <i>P</i> < 0.01. <i>Post hoc P</i> values were calculated by <i>t</i>-tests and Bonferroni correction; *, <i>P</i> < 0.05; **, <i>P</i> < 0.01. (B) HEK293T cells transfected with empty vector (EV) or expressing wild-type RIT1 (WT) or RIT1 p.G95A were either serum-deprived (0.1%) or kept under full serum (10%). Endogenous PAK1 was immunoprecipitated from cell extracts using an anti-PAK1 antibody [IP: PAK1 (#1)]. As IP control an irrelevant isotype-matched antibody (anti-pSMAD2 antibody) was used (IP ctrl). Co-precipitated HA-RIT1 and expression of HA-RIT1 in total cell lysates (TCL) was detected by immunoblotting using anti-HA antibody. Expression of endogenous PAK1 in TCL is shown below. Data shown are representative of three independent experiments. Autoradiographic signals were quantified by scanning densitometry. Levels of co-IPed HA-RIT1 were double-normalized relative to amounts of immunoprecipitated PAK1 and HA-RIT1 in total cell lysates. To conserve the relative variance of the samples, values for RIT1 wildtype and p.G95A were divided by the mean of the wildtype samples [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007370#pgen.1007370.ref079" target="_blank">79</a>]. The graphs show the relative amount of co-precipitated HA-RIT1 in cells expressing RIT1 WT and RIT1 p.G95A (arbitrary units) and cultivated in 0.1% or 10% serum. The mean of three independent experiments ± SD is given. Unpaired <i>t</i>-tests were used to determine statistical significance. ns, not significant. (C) HEK293T cells were transfected with empty vector (EV) and RIT1 expression constructs as indicated and cultured under serum deprivation (0.1% serum). Endogenous PAK1 was immunoprecipitated with an anti-PAK1 antibody [IP: PAK1 (#1)], and co-precipitated HA-RIT1 was detected using an anti-HA antibody. Enrichment of PAK1 in the precipitates was demonstrated with an anti-PAK1 antibody. The amount of HA-RIT1 and PAK1 in TCL is shown. Data shown are representative of two independent experiments. Autoradiographic signals were quantified by scanning densitometry. Levels of co-IPed HA-RIT1 were double-normalized relative to amounts of immunoprecipitated PAK1 and HA-RIT1 in total cell lysates. To conserve the relative variance of the samples, values for RIT1 wildtype and RIT1 mutants were divided by the mean of the wildtype samples [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007370#pgen.1007370.ref079" target="_blank">79</a>]. The graphs show the relative amount (arbitrary units) of co-precipitated RIT1 protein variants. The mean of two independent experiments ± SD is given. Unpaired <i>t</i>-tests were used to determine statistical significance (*, <i>P</i> <0.05).</p

RIT1 enhances migration and invasive capabilities of cells by regulating actin dynamics.

<p>(A) Transwell assay of HEK293T cells transiently transfected with the indicated constructs and kept under serum starvation overnight. Cells were seeded in serum-free medium into an upper compartment of a transwell chamber. Cells passed a growth factor-reduced matrigel as a barrier towards a lower compartment with medium containing 10% serum as a chemoattractant (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007370#sec013" target="_blank">materials and methods</a> for details). After 48 hours, only cells located in the lower compartment were detached and counted by flow cytometry; a constant number of counting beads ensured comparability. The graphs show the mean ± SD of cell counts from three independent experiments. One-way ANOVA between groups: <i>P</i> < 0.001. <i>Post hoc P</i> values calculated by pairwise <i>t</i>-tests and Bonferroni correction were not significant. (B) Model depicting RIT1’s function in the regulation of cytoskeletal dynamics. Extracellular stimuli induce the formation of various parallel signaling hubs, such as PI3K-AKT, MEK-ERK and RAC1/CDC42-PAK1 modules. RIT1 can stimulate the activation of AKT and MEK-ERK signaling cascades. On the other hand, RIT1 interacts with PAK1 and RAC1/CDC42 to regulate actin-dependent structures, such as stress fibers and focal adhesions that may positively influence cell migration and adhesion.</p

Expression of RIT1 mutants enhances phosphorylation of ERK1/2.

<p>(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 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007370#pgen.1007370.ref079" target="_blank">79</a>]. 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 <i>t</i>-tests were used to determine statistical significance (*, <i>P</i> < 0.05; **, <i>P</i> < 0.01). (C) HEK293T cells were transfected with empty vector (EV) and RIT1 expression constructs (WT, p.K23N, p.G31R, p.A57G, p.F82L, p.M90V, and p.G95A) as indicated and cultured under steady-state condition (10% serum). Total cell lysates were analyzed as described in (B). 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 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007370#pgen.1007370.ref079" target="_blank">79</a>]. The graph on the left shows relative phosphorylation levels in cells expressing RIT1 wildtype (WT), RIT1 p.A57G, p.F82L or p.G95A; the graph on the right shows relative phosphorylation levels in cells expressing RIT1 wildtype (WT), RIT1 p.K23N, p.G31R or p.M90V (arbitrary units). The mean of three independent experiments ± SD is given. Statistical significance was assessed by one-way ANOVA: not significant and <i>P</i> < 0.01 for RIT1 variants shown in the left and right graph, respectively. <i>Post hoc P</i> values were calculated by <i>t</i>-tests and Bonferroni correction; *, <i>P</i> < 0.05.</p

Serum and EGF stimulate the association of RIT1 with PIK3CA and PAK1.

<p>(A) HEK293T cells expressing empty vector (EV) or wild-type RIT1 (WT) were either serum-deprived (0.1%) and subsequently stimulated with 10 ng/ml EGF for 20 min (0.1%; +) or kept under full serum (10%). GTP-bound HA-tagged RIT1 was precipitated from cell extracts using GST-RALGDS[RA], GST-PLCE1[RA], GST-PIK3CA[RBD], and GST-PAK[CRIB] fusion proteins (PD, pull down). Precipitated HA-RIT1 (PD) and HA-RIT1 in the total cell lysates (TCL) were detected by immunoblotting (IB) using anti-HA antibody. Cellular extracts were probed with anti-GAPDH antibody to control for equal loading. For control purpose cell extracts were incubated with GST-coupled agarose beads. Data shown are representative of three independent experiments. (B) The graph shows the relative amount (arbitrary units) of co-precipitated RIT1 protein. The mean of four independent experiments ± SD is given. One-way ANOVA between groups: <i>P</i> < 0.01. <i>Post hoc P</i> values were calculated by <i>t</i>-tests and Bonferroni correction; **, <i>P</i> < 0.01; ***, <i>P</i> < 0.001.</p

Dissolution of actin stress fibers in cells expressing RIT1 and NS-associated RIT1 mutants.

<p>(A) COS7 cells were plated on collagen-coated glass slides, transiently transfected with the indicated constructs and kept under serum starvation overnight. HA-tagged RIT1 was stained by rabbit anti-HA antibody followed by anti-rabbit Alexa Fluor488-conjugated antibody. Polymerized F-actin was visualized using Texas Red-X Phalloidin, and nuclear DNA was labeled by DAPI. Cells were imaged by epifluorescence microscopy. White boxes indicate magnified parts of specimen shown on the very right-hand side. Scale bars, 10 μm. (B) Cells were divided into the two indicated categories: (I) normal and (II) reduced or absent actin stress fibers (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007370#pgen.1007370.s009" target="_blank">S8B Fig</a> shows exemplary images). A minimum of 50 cells per dataset were analyzed.</p

Co-expression of kinase-dead PAK1^K299A prevents loss of actin stress fibers in cells expressing RIT1.

<p>COS7 cells were plated on collagen-coated glass slides, transiently transfected with GFP-PAK1^K299A expression construct together with HA-RIT^WT construct or empty vector (EV) and serum-starved overnight. HA-tagged RIT1 was stained by rabbit anti-HA antibody followed by anti-rabbit Alexa Fluor647-conjugated antibody. F-actin was visualized using Texas Red-X Phalloidin, and nuclear DNA was labeled by DAPI. White boxes indicate magnified parts of specimen shown on the very right-hand side. Scale bar, 10 μm.</p

CDC42 and RAC1 interact with RIT1, and NS-associated RIT1 amino acid substitutions increase complex formation between RIT1 and RAC1/CDC42.

<p>(A and C) HEK293T cells transfected with empty vector (EV) or expressing wild-type RIT1 (WT) or RIT1 p.G95A were either serum-deprived (0.1% serum) or kept under full serum (10% serum). Endogenous CDC42 and RAC1 were immunoprecipitated with an anti-CDC42 antibody ([CDC42 (#1)] (A) and an anti-RAC1 antibody (C), respectively (IP). Co-precipitated HA-RIT1 and HA-RIT in total cell lysates (TCL) was detected by an anti-HA antibody. Enrichment of CDC42 and RAC1 in the precipitates and amount of endogenous CDC42 and RAC1 in TCL was demonstrated with an anti-CDC42 antibody ([CDC42 (#2)] (A) and an anti-RAC1/2/3 antibody (C), respectively. As a control, immunoprecipitation with an anti-V5 antibody was carried out (IP ctrl). Data shown are representative of three independent experiments. (B and D) HEK293T cells were transfected with empty vector (EV) and RIT1 expression constructs as indicated and cultured under serum deprivation (0.1% serum). Cell lysates were processed as described in (A) and (C). Data shown for CDC42 and RAC1 are representative of seven (B) and four (D) independent experiments, respectively. Autoradiographic signals were quantified by scanning densitometry. The amount of co-precipitated HA-RIT1 was double-normalized relative to amounts of immunoprecipitated CDC42/RAC1 and HA-RIT1 in total cell lysates. To conserve the relative variance of the samples, values for RIT1 wildtype and RIT1 mutants were divided by the mean of the wildtype samples [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007370#pgen.1007370.ref079" target="_blank">79</a>]. The graphs show the relative amount (in arbitrary units) of co-precipitated RIT1 protein variants. The mean of five (B) and four (D) independent experiments ± SD is given. One-way ANOVA between groups: <i>P</i> < 0.05 (B and D). <i>Post hoc P</i> values were calculated by <i>t</i>-tests and Bonferroni correction; *, <i>P</i> < 0.05; **, <i>P</i> < 0.01 (B and D).</p

The mammalian Arg/N-end rule pathway and missense mutations in human <i>UBR1</i> that underlie specific cases of the Johanson-Blizzard syndrome (JBS).

<p>(A) The mammalian N-end rule pathway. N-terminal residues are indicated by single-letter abbreviations for amino acids. Yellow ovals denote the rest of a protein substrate. ‘Primary’, ‘secondary’ and ‘tertiary’ denote mechanistically distinct subsets of destabilizing N-terminal residues (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#s1" target="_blank">Introduction</a>). C* denotes oxidized Cys, either Cys-sulfinate or Cys-sulfonate. MetAPs, Met-aminopeptidases. (B) Single-residue mutations in the UBR1 proteins of JBS patients #1 and #2. The positions of mutant residues are indicated both for the original mutations in human UBR1 and for their mimics in <i>S. cerevisiae</i>. (C) Same as in B but the mutation in UBR1 of patient #3 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#s2" target="_blank">Results</a>).</p

Clinical features in JBS patients.

a<p>Ref. 1;</p>b<p>Ref. 6 and unpublished data.</p

Functional activity of yeast Ubr1 mimics of missense JBS-UBR1 mutants.

<p>(A) Relative enzymatic activity of βgal in extracts from <i>S. cerevisiae</i> JD55 (<i>ubr1Δ</i>) that expressed His-βgal or Tyr-βgal, and also carried an empty vector, or an otherwise identical plasmid expressing wild-type <i>S. cerevisiae</i> Ubr1, or (separately) its three missense mutants Ubr1^V146L, Ubr1^H160R, or Ubr1^Q1224E. The activity of βgal was measured in triplicates, with standard deviations shown. (B) Relative levels of induction of the peptide transporter Ptr2 were assayed by measuring the activity of a plasmid-borne <i>lacZ</i> (βgal-encoding) reporter that was expressed from the P<i>_PTR2</i> promoter in <i>ubr1Δ S. cerevisiae</i> that carried either an empty vector or otherwise identical plasmids that expressed either wild-type Ubr1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Hwang1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Xia1" target="_blank">[28]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Turner1" target="_blank">[52]</a> or its indicated mutants. Cells were grown to A₆₀₀ of ∼0.8 in SC(-Ura, -Leu) medium at 30°C, followed by measurements, in triplicate, of βgal activity in cell extracts, with standard deviations shown. (C) The lysine-requiring JD55 (<i>ubr1Δ</i>) <i>S. cerevisiae</i> strain was grown on plates containing 110 µM lysine (Lys) or 66 µM Lys-Ala dipeptide as the sole source of Lys in the medium <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Hwang1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Hwang3" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Turner1" target="_blank">[52]</a>. JD52 (<i>ubr1Δ</i>) cells carried a vector plasmid or otherwise identical plasmids expressing wild-type Ubr1 or its missense mutants Ubr1^H160R, Ubr1^V146L and Ubr1^Q1224E. Cells were grown to A₆₀₀ of ∼1 in SC(-Leu) medium at 30°C, washed in sterile water, serially diluted 5-fold, spotted on SC(-Leu, -Lys) plates containing 110 µM Lys or 66 µM Lys-Ala, and incubated at 30°C for 3 days. (D) Cell extracts (equal total protein levels) from experiments described in panels A and B were subjected to SDS-PAGE, followed by immunoblotting with affinity-purified anti-Ubr1 antibody (upper panel) and anti-tubulin antibody (a loading control; lower panel). Asterisk indicates a protein that crossreacts with anti-Ubr1 antibody. (E) Extracts from human lymphocytes (equal amounts of total protein) were subjected to SDS-PAGE, followed by immunoblotting with antibody to human UBR1 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#s4" target="_blank">Materials and Methods</a>). Lane 1, wild-type lymphocytes. Lane 2, same as lane 1 but from lymphocytes of patient #2 (see the main text and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g001" target="_blank">Figs. 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone-0024925-g002" target="_blank">2</a>). Lane 3, same as lane 1 but with lymphocytes from patient #3. Lane 4, same as lane 1, but with lymphocytes from a JBS patient with a homozygous nonsense mutation in <i>UBR1</i>, previously shown to have no detectable UBR1 (null UBR1 control) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024925#pone.0024925-Varshavsky3" target="_blank">[17]</a>. Lane 5, same as a lane 1.</p