αPIX binds to the E3 ubiquitin ligase c-Cbl.

<p>A. Lysates from CHO-K1 cells were subjected to co-immunoprecipitation, either using rabbit IgG or rabbit anti-Cbl (S.C., Santa Cruz) antibodies. Total cell lysates (tcl) and immunoprecipitates (p) were resolved on an SDS-polyacrylamide gel and analyzed by immunoblotting using the indicated antibodies. B. Modular architecture of αPIX and c-Cbl. The protein domains of αPIX (CH, calponin homology; SH3, src-homology 3; DH, Dbl homology; PH, pleckstrin homology; GBD, GIT-binding domain; CC, coiled-coil domain) and c-Cbl (TKB, tyrosine-kinase-binding region; RING, RING finger domain; PRD, proline-rich domain; PKPFPR binding motif; UBA, ubiquitin associated domain) are schematically shown. Amino acid substitutions that are functionally important for this study are indicated. The total number of amino acids (aa) for αPIX and c-Cbl is given. C. Trp¹⁹⁷ in αPIX and Arg⁸²⁹ in c-Cbl are essential for the αPIX::c-Cbl interaction. COS-7 cells were transfected with the indicated expression constructs. HA-tagged αPIX was immunoprecipitated from cell extracts by using anti-HA-conjugated agarose beads. After SDS-PAGE and western blotting, immunoprecipitates (IP) and total cell lysates (tcl) were probed with anti-HA and anti-Cbl antibodies. The HA-membrane was re-probed using anti-GAPDH antibodies to control for equal loading.</p

αPIX::c-Cbl complex formation and degradation.

<p>A. EGF regulates complex formation of αPIX and c-Cbl. COS-7 cells transiently co-expressing HA-αPIX^WT and c-Cbl^WT were serum-starved or cultured under basal growth conditions (+S). Starved cells were stimulated with 5 ng/ml EGF for 5, 10, 30 or 60 min at 37°C (t_EGF) or left untreated (0 min). αPIX was immunoprecipitated from cell extracts by using anti-HA antibodies and protein levels of HA-αPIX, c-Cbl and GAPDH were determined in cell lysates (tcl) and precipitates (IP) by immunoblotting. Based on densitometric quantification of autoradiographic signals derived from immunoblots, the graphs show relative amounts of c-Cbl co-precipitated with HA-αPIX in unstimulated cells and at 30 min upon EGF induction. Amounts of c-Cbl in the precipitates were normalized to total c-Cbl and considered as 1 for unstimulated cells (0 min). Data represent the mean of six (n = 6) independent experiments ± sd. <i>P</i> value was calculated by paired Student’s t-test. B. Downregulation of αPIX and c-Cbl depends on complex formation of these proteins. COS-7 cells transiently expressing various HA-αPIX and c-Cbl protein variants were cultivated under basal growth conditions (+S), serum starved (0 min) or serum-starved and stimulated with 5 ng/ml EGF for 15 or 30 min. Cells were harvested and protein levels of HA-αPIX, c-Cbl, and GAPDH were determined by immunoblotting. Based on densitometric quantification of autoradiographic signals derived from immunoblots, the graphs show relative amounts of HA-αPIX^WT and c-Cbl^WT in the total lysates from cells overexpressing HA-αPIX^WT and c-Cbl^WT. Measurements were normalized to GAPDH and considered as 1 for unstimulated cells (0 min t_EGF). Data represent the mean of five (n = 5) independent experiments ± sd. <i>P</i> values were calculated by paired Student’s t-test. C. Both proteasomal and lysosomal inhibitors prevent EGF-induced αPIX and c-Cbl degradation. Serum-starved COS-7 cells transiently co-expressing HA-αPIX^WT and c-Cbl^WT were incubated with 20 μM MG132 or 50 μM chloroquine for 6h or left untreated (vehicle). Upon stimulation with 25 ng/ml EGF for the indicated times, cell extracts were subjected to SDS-PAGE and immunoblotting using anti-HA and anti-Cbl antibodies. Blots were reprobed with anti-GAPDH antibody to test for loading equality. Based on densitometric quantification of autoradiographic signals derived from immunoblots, the graphs show relative amounts of HA-αPIX and Cbl in the cell lysates. Measurements were normalized to GAPDH and considered as 1 for unstimulated cells (0 min t_EGF). Data represent the mean of four (n = 4) independent experiments ± sd. <i>P</i> values were calculated by unpaired Student’s t-test.</p

Model depicting the balancing effect of αPIX on EGFR trafficking to maintain EGFR signaling homeostasis.

<p>(For details see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132737#sec010" target="_blank">discussion</a>.)</p

Stimulation of EGFR recycling is the dominant αPIX function during EGFR trafficking.

<p>A. CHO cells stably expressing αPIX^WT or CAT (control) were transfected with EGFR expression constructs. Following serum starvation overnight, surface proteins were biotinylated on ice and cells were stimulated with 25 ng/ml EGF for 15, 30 or 60 min at 37°C to induce EGF receptor trafficking. A parallel culture was left unstimulated (0 min). Cells were transferred to 4°C, surface proteins were de-biotinylated and intracellular biotinylated proteins were precipitated from cell extracts. Representative autoradiographs show EGFR levels in total cell lysates (tcl) and precipitates (p) upon SDS-PAGE and immunoblotting. GAPDH served as a loading control. Based on densitometric quantification of autoradiographic signals the graphs show relative amounts of intracellular EGFR. Amounts of precipitated EGFR were normalized to total EGFR levels and considered as 100% in control cells after 60 min EGF stimulation (note: standard deviation for control cells at 60 min t_EGF was calculated subsequent to normalization to total EGFR levels). Data represent the mean of three independent experiments ± sd. For <i>P</i> value was calculated by paired Student’s t-test. B. Immunocytochemical analysis of EGFR distribution. Stable αPIX^WT and control (CAT) CHO cells were transfected with EGFR constructs and serum-starved overnight. Cells were stimulated with 25 ng/ml EGF for 15 or 60 min at 37°C to induce EGF receptor trafficking. After fixation, EGFR was visualized by anti-EGFR antibodies followed by Alexa Fluor488-conjugated antibodies and the nucleus was detected by staining with DAPI. Note the enrichment of EGFR at the plasma membrane in αPIX^WT overexpressing cells upon 60 min EGF stimulation (arrowheads, lower panel). 25 cells each [stably expressing CAT (control) and αPIX^WT cells] derived from three independent experiments have been analyzed, representative cells are shown. Scale bars, 20 μm. C. Serum-starved COS-7 cells transiently expressing HA-αPIX^WT were stimulated with 25 ng/ml Alexa Fluor 488-conjugated EGF (EGF488) for 15 or 60 min. Subsequently, extracellular receptor-bound EGF was removed and cells were fixed. HA-tagged αPIX was visualized by staining with anti-HA antibodies followed by Alexa Flour 546-conjugated secondary antibodies and the nucleus was detected by staining with DAPI. Dotted lines indicate αPIX-expressing cells, dashed lines indicate untransfected cells. 50 cells each (non-transfected cells and HA-αPIX^WT overexpressing cells) derived from three independent specimen have been examined, representative cells are shown. Scale bars, 20 μm.</p

αPIX interferes with EGFR ubiquitination and degradation.

<p>A. CHO cells stably expressing the indicated αPIX protein variants or CAT (control) were transfected with an EGFR expression construct. The experimental procedure was essentially the same as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132737#pone.0132737.g003" target="_blank">Fig 3A</a>, except for the medium that was supplemented with 0.3 mM of the recycling inhibitor primaquine to block EGFR recycling. Cells were harvested after various times of rewarming followed by surface de-biotinylation (5, 15, 30 min) or before rewarming (0 min). Intracellular biotinylated proteins were precipitated from cell extracts and cell lysates (tcl) and precipitates (p) were subjected to immunoblotting using anti-EGFR antibodies. B. Graphs show relative amounts of intracellular EGFR derived from densitometric quantification of autoradiographic signals obtained from EGFR degradation assays (A). Precipitated (intracellular) EGFR fractions were normalized to total EGFR levels and considered as 100% in cultures that haven’t been rewarmed (0 min). Data represent the mean of three (n = 3) independent experiments ± sd. <i>P</i> values were calculated by unpaired Student’s t-test. C. c-Cbl co-expression rescues αPIX^WT-induced inhibition of EGFR degradation. CHO cells stably expressing αPIX^WT were co-transfected with EGFR and c-Cbl expression constructs and subsequently treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0132737#pone.0132737.g003" target="_blank">Fig 3A</a>. After immunoblotting cell lysates (tcl) were probed with anti c-Cbl and anti-EGFR antibodies, and precipitates (p) were probed with anti-EGFR antibodies. D. αPIX^WT sequesters c-Cbl from EGF receptors. COS-7 cells were transfected with expression constructs as indicated. Endogenous EGFR was immunoprecipitated from cell extracts by using anti-EGFR antibodies. Upon SDS-PAGE and western blotting, precipitates (IP) and total cell lysates (tcl) were probed with anti-EGFR and anti-Cbl antibodies. Expression of αPIX^WT was demonstrated by immunodetection with anti-HA antibodies. E. αPIX^WT reduces c-Cbl-mediated EGFR ubiquitination. COS-7 cells were transiently co-transfected with <i>c-Cbl</i> and FLAG-tagged <i>αPIX</i> expression constructs (as indicated) together with HA-tagged ubiquitin and EGFR expression constructs. For control purpose cells were transfected with empty FLAG-vector. Subsequent to incubation under serum-free culture conditions overnight, cells were stimulated with 20 ng/ml EGF for 30 min and harvested. EGFR was immunoprecipitated with anti-EGFR antibodies and protein A-agarose and samples were subjected to SDS-PAGE and immunoblotting. Levels of ubiquitinated EGFR in precipitates (IP) were monitored by using anti-HA antibodies. EGFR levels in total cell lysates (tcl) and precipitates (IP) were determined by using anti-EGFR antibodies and expression of c-Cbl and FLAG-αPIX protein variants in total cell lysates was shown by using anti-c-Cbl and anti-FLAG antibodies, respectively. Tubulin served as a loading control. Representative blots from one out of three independent experiments are shown. Based on densitometric quantification of autoradiographic signals derived from immunoblots, the graph shows relative amounts of ubiquitinated EGFR. Amounts of HA-ubiquitinated EGFR in the precipitates were normalized to total EGFR and considered as 1 for cells expressing FLAG-αPIX^WT. Data represent the mean of three (n = 3) independent experiments ± sd. <i>P</i> value was calculated by paired Student’s t-test.</p

αPIX regulates EGFR trafficking.

<p>A. Stable αPIX^WT and control (CAT) CHO cell lines were transiently transfected with EGFR expression constructs. Following serum starvation, surface proteins were biotinylated and cells were stimulated with 25 ng/ml EGF for 30 min at 37°C (pulse) to induce EGF receptor trafficking. Then, cells were transferred to 4°C, residual surface biotin was removed and cells were rewarmed to 37°C for the indicated times (chase). Recycled surface proteins were de-biotinylated and intracellular biotinylated proteins were precipitated from cell extracts. Parallel cultures were harvested before rewarming (0 min). Representative autoradiographs show EGFR levels in precipitates (p) and total cell lysates (tcl). Equal loading was verified by reprobing membranes with anti-GAPDH antibodies. B. Based on densitometric quantification of autoradiographic signals derived from EGFR trafficking assays (A), the graphs show relative amounts of intracellular EGFR. Precipitated (intracellular) EGFR fractions were normalized to total EGFR levels and considered as 100% in cultures that haven’t been rewarmed (0 min). Data represent the mean of four (n = 4) independent experiments ± sd. <i>P</i> values were calculated by unpaired Student’s t-test. C. Serum-starved COS-7 cells transiently expressing HA-αPIX^WT were stimulated with 25 ng/ml EGF for 30 min (pulse). Subsequently, cells were either immediately fixed (0 min) or incubated in starvation medium for further 30 min (chase) and then fixed. HA-tagged αPIX was visualized by staining with anti-HA followed by Alexa Fluor 488-conjugated secondary antibodies. The early endosomal compartment was visualized by anti-EEA1 antibodies followed by Alexa Fluor546-conjugated antibodies and the nucleus was detected by staining with DAPI. Note the enlarged morphology (arrowheads, upper panel) and the reduced number (outlined cell, lower panel) of EEA1-positive vesicles in αPIX overexpressing cells compared to surrounding non-transfected cells (asterisks) at 0 min and 30 min chase, respectively. Specific details are shown enlarged at the right hand side of the images. 50 cells each (non-transfected cells and HA-αPIX^WT overexpressing cells) derived from three independent specimen have been examined, representative cells are shown. Scale bar, 20 μm.</p

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

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