Research ArticleCell Biology

Contact inhibitory Eph signaling suppresses EGF-promoted cell migration by decoupling EGFR activity from vesicular recycling

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Science Signaling  31 Jul 2018:
Vol. 11, Issue 541, eaat0114
DOI: 10.1126/scisignal.aat0114
  • Fig. 1 Eph receptor activation regulates Akt-dependent EGFR trafficking.

    (A and B) Representative immunofluorescence images (A) and quantification (B) of endogenous PM EGFR in Cos-7 cells (n = 16 to 29 cells per condition) after stimulation with A1 (2 μg/ml) for the indicated times (means ± SD). (C) Quantification of Akt activation by In-Cell Western (ICW) in Cos-7 cells after A1 stimulation (left, 2 μg/ml; right, 15 min) (means ± SEM from at least three independent experiments). (D) Quantification of endogenous PM EGFR abundance by On-Cell Western (OCW) and Akt activation in Cos-7 cells by ICW after treatment with the Akt inhibitor AktVIII (10 μM) for the times indicated (means ± SEM from at least three independent experiments). (E) EGF–Alexa Fluor 647 (Alexa647) binding (200 ng/ml, 2 min) to endogenous EGFR in Cos-7 cells as a measure of PM EGFR abundance after 1-hour pretreatment with A1 (2 μg/ml), the PIKfyve inhibitor YM201636 (YM; 200 nM), or both (n = 33 to 59 cells per condition; means ± SD). (F) Representative confocal images of Cos-7 cells expressing EGFR-mCherry before (top left) and after (top right) treatment with AktVIII (10 μM, 1 hour) and quantification of the increase in endosomal EGFR-mCherry during AktVIII treatment (bottom, n = 6 cells, means ± SD). (G) Representative time-lapse confocal images of Cos-7 cells expressing EGFR-mCherry and EphA2-mCitrine after A1 stimulation (2 μg/ml). (H) Quantification of endosomal EGFR-mCherry and EphA2-mCitrine from time-lapse confocal imaging (G) during A1 stimulation (n = 7 cells, means ± SD). (I) Quantification of PM EGFR-mCherry and EphA2-mCitrine abundance by OCW during A1 stimulation (2 μg/ml; means ± SEM from at least three independent experiments). (J) Immunofluorescence measurements of EGFR-mCherry intensity in Rab5-, Rab5/Rab7-, and Rab7-positive endosomal compartments in control, A1-pretreated (2 μg/ml, 1 hour), and AktVIII-pretreated (10 μM, 1 hour) Cos-7 cells before (left) and after EGF stimulation (right, 100 ng/ml, 1 hour; n = 6 to 11 cells per condition). Data are represented by Tukey boxplots, with the mean denoted as a cross and the median denoted as a line. (K) Immunofluorescence measurements of endogenous EGFR intensity in Rab5-positive endosomes after A1 stimulation (2 μg/ml; n = 83 to 98 cells per condition, means ± SD). (L) Immunofluorescence measurements of PM/total EGFR in control and A1-pretreated cells (2 μg/ml, 1 hour) before (Pre), after EGF stimulation (10 ng/ml, 15 min), and 15 min after EGF washout (n = 34 to 40 cells per condition, means ± SD). Statistical significance was determined in (B), (E), and (J) to (L) using one-way analysis of variance (ANOVA) with Sidak’s post hoc test (***P < 0.001). Data in (K) were log-transformed before statistical analysis to compare normally distributed populations. Scale bars, 20 μm. a.u., arbitrary units; NS, not significant.

  • Fig. 2 Eph receptor activation changes the spatial distribution of EGFR activity.

    (A and B) Average spatiotemporal maps (A) of endogenous EGFR abundance (top) and Tyr845 phosphorylation (p) (bottom) in radially segmented Cos-7 cells [PM→nuclear membrane (NM)] before and during EGF stimulation (20 ng/ml for 5, 30, and 60 min) in control, A1-pretreated (2 μg/ml, 1 hour), AktVIII-pretreated (10 μM, 1 hour), and YM201636-pretreated (200 nM, 1 hour) cells (n = 50 to 90 cells per condition). (B) Single-cell measurements of EGFR pTyr845 phosphorylation in the PM segment during EGF stimulation (means ± SD). (C) Phosphorylated fraction of EGFR-mCitrine as detected by FLIM-FRET (α) and representative images of EGFR-mCitrine and EGF–Alexa Fluor 647 fluorescence in control and A1-pretreated (2 μg/ml, 1 hour), AktVIII-pretreated (10 μM, 1 hour), and YM201636-pretreated (200 nM, 1 hour) Cos-7 cells after 60 min of EGF–Alexa Fluor 647 stimulation (20 ng/ml). (D to F) Quantification of the phosphorylated fraction of EGFR-mCitrine (α) at the PM and endosomes (D) and the PM/endosome ratio for EGFR-mCitrine (E) and EGF–Alexa Fluor 647 fluorescence intensity (F) (n = 10 to 14 cells per condition; means ± SD). Statistical significance was determined in (B) and (D) to (F) using one-way ANOVA with Sidak’s post hoc test (***P < 0.001, **P < 0.01, *P < 0.05). Scale bar, 20 μm.

  • Fig. 3 Eph receptor activation at cell-cell contacts alters the EGFR signaling response.

    (A) Quantification of Akt (top) and ERK (bottom) activation by ICW in control and A1-pretreated (2 μg/ml, 1 hour) Cos-7 cells after EGF stimulation (1 ng/ml; means ± SEM from at least three independent experiments). (B) Quantification of Akt activation by ICW in control or A1-pretreated (2 μg/ml, 1 hour) HEK293 cells, followed by 30-min treatment with the dynamin inhibitor dynole 34-2 (100 μM, top) or its negative control analog dynole 31-2 (100 μM, bottom), and then stimulated with EGF (1 ng/ml) for the times indicated (means ± SEM from at least three independent experiments). (C) Quantification of EGF-promoted Akt activation by ICW in Cos-7 cells after pretreatment with increasing concentrations of A1 (0.02, 0.2, and 2 μg/ml, 1 hour; means ± SEM from at least three independent experiments). (D) Representative images of a FRET-based sensor of EphA2 activity (LIFEA2) (24), whereby a decrease in fluorescence lifetime (τ, ns) represents an increase in EphA2 activity, and fluorescence intensity measurements of LIFEA2-mCitrine and SH2-mCherry in Cos-7 cells (n = 20 cells). Scale bars, 20 μm. (E and F) Single-cell measurements of Akt (E) and ERK (F) activation by ICW compared to cell-cell contact in 2D cultures (% cell circumference) in unstimulated and EGF-stimulated (20 ng/ml, 1 hour) Cos-7 cells from three independent experiments. A sum-of-squares F test was used to determine significance: Akt, unstimulated: F = 16.0, P = 0.001, r 2 = 0.432; Akt, EGF: F = 21.4, P < 0.001, r 2 = 0.322; ERK, unstimulated: F = 0.180, P = 0.673, r 2 = 0.003; ERK, EGF: F = 0.321, P = 0.575, r 2 = 0.009.

  • Fig. 4 Coupling EGFR activity to vesicular recycling generates positive feedback.

    (A) Quantification of endogenous PM EGFR abundance and Akt activation in Cos-7 cells by OCW and ICW, respectively, after treatment with the PP2A inhibitor okadaic acid (OA; 1 μM, 2 hours) (means ± SEM from at least three independent experiments). DMSO, dimethyl sulfoxide. (B) Quantification of PM EGF–Alexa Fluor 647 binding (200 ng/ml, 2 min, left) and Akt(Ser473) phosphorylation (p) by immunofluorescence in Cos-7 cells ectopically expressing the constitutively active mutant Akt1D323A/D325A-EGFP (n = 75 cells) compared to nontransfected control cells (n = 100 cells). (C) Representative images and quantification of EGFR-paGFP recycling to the PM after endosomal photoactivation in Cos-7 cells (top) in control, EGF-pretreated (20 ng/ml, 15 min), AktVIII-pretreated (10 μM, 1 hour), and AktVIII-pretreated, EGF-stimulated cells (bottom, n = 6 to 10 cells per condition; means ± SEM). Scale bar, 20 μm. (D) Spatial network topology showing positive feedback generated by coupling PM EGFR activity and Akt-dependent vesicular recycling. PIKfyve inhibition by YM201636 decouples Akt activation from its effect on EGFR recycling. (E and F) Single-cell measurements of Akt phosphorylation by flow cytometry in control (top), YM201636-pretreated (200 nM, 1 hour, middle), and A1-pretreated (2 μg/ml, 1 hour, bottom) Cos-7 cells after stimulation with the EGF concentrations indicated (ng/ml, 1 hour). (E) Solid lines represent the sum of two Gaussian fits for data accumulated from at least 10,000 cells per condition in three to four independent experiments. (F) Mean Akt activation in populations of cells showing low (gray) and high (red) Akt activity derived from the Gaussian distributions for each EGF concentration in (E). Circle sizes represent the relative proportions of cells exhibiting low and high Akt activity at each EGF concentration, as determined by the relative amplitudes of each population (means ± SEM from at least three independent experiments). Statistical significance was determined in (A) and (B) using two-tailed Student’s t test.

  • Fig. 5 Eph activation at cell-cell contacts suppresses the EGF-promoted transition to a migratory state.

    (A and B) Representative immunofluorescence images (A) and quantification of endogenous PM EGFR abundance (B) in MEFs after A1 (2 μg/ml) stimulation (means ± SD). Statistical significance was determined using one-way ANOVA with Sidak’s post hoc test (***P < 0.001). (C) Percent of MEFs initiating a migratory response (top, means ± SEM) and the distance traveled by migrating cells (bottom, means ± SD) after EGF stimulation. MEFs were pretreated with vehicle (control, blue), YM201636 (200 nM, 1 hour, purple), or A1 (2 μg/ml, 1 hour, green; or 0.02, 0.2, and 2 μg/ml, red) followed by EGF stimulation (0 to 100 ng/ml as indicated) for 16 hours. (D) Percent of migrating MEFs when seeded at low or high density after pretreatment with vehicle or A1 (2 μg/ml, 1 hour) and stimulated with EGF (20 ng/ml) for 16 hours (means ± SEM). Data in (C) and (D) were obtained from at least three independent experiments, consisting of at least two replicates per experiment (n = 581 to 1483 cells per condition), and statistical significance was determined using ordinary one-way ANOVA with Holm-Sidak’s multiple corrections post hoc test. (E to G) Measurements of transwell migration of wild-type (WT) MEFs (E), Pten−/− MEFs (F), and MDA-MB-231 cells (G) toward EGF (blue, 20 ng/ml) or serum-free medium (gray, control). Cells were pretreated for 1 hour with serum-free medium, A1 (green, 2 μg/ml), or YM201636 (magenta, 200 nM) before seeding (means ± SEM from three independent experiments). (H) Quantification of retinoblastoma (Rb) phosphorylation by ICW for vehicle-pretreated (control), A1-pretreated (2 μg/ml, 1 hour), and YM201636-pretreated (200 nM, 1 hour) WT MEFs after 24-hour EGF stimulation at the concentrations indicated [means ± SEM from three independent experiments; data points from EGF (100 ng/ml) conditions were excluded from curve fitting].

  • Fig. 6 Kiss1 receptor activation regulates Akt-dependent EGFR trafficking and signaling.

    (A) Quantification of Akt activation (pink) and PM EGFR abundance (purple) in HEK293 cells by ICW and OCW, respectively, after stimulation with Kp-10 (100 nM; means ± SEM from at least three independent experiments). (B and C) Quantification of Akt and ERK activation by ICW in HEK293 cells for control (gray) and Kp-10–pretreated (100 nM, 1 hour, purple) cells after EGF (1 ng/ml) stimulation (means ± SEM from at least three independent experiments). (D to F) Measurements of transwell migration of WT MEFs (D), Pten−/− MEFs (E), and MDA-MB-231 cells (F) toward EGF (20 ng/ml, blue) or serum-free medium (gray, control). Cells were pretreated for 1 hour with serum-free medium or Kp-10 (purple, 100 nM) before seeding (means ± SEM from three independent experiments). Control and EGF data were previously presented in Fig. 5 (E to G).

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/541/eaat0114/DC1

    Fig. S1. Eph receptor activation reduces PM EGFR abundance.

    Fig. S2. Akt and its effector PIKfyve regulate EGFR vesicular trafficking.

    Fig. S3. Akt is preferentially activated at the PM after EGFR stimulation.

    Fig. S4. A1 pretreatment inhibits EGFR-promoted Akt activation.

    Fig. S5. Distributions of cell migration distances.

    Table S1. Primer list for RT-qPCR.

    Movie S1. Akt inhibition induces EGFR endosomal accumulation.

    Movie S2. A1 stimulation induces EGFR endosomal accumulation.

    Movie S3. A1-EphA2 interactions at cell-cell contact induce EGFR endosomal accumulation.

    Movie S4. EGF-promoted migration in MEFs.

  • The PDF file includes:

    • Fig. S1. Eph receptor activation reduces PM EGFR abundance.
    • Fig. S2. Akt and its effector PIKfyve regulate EGFR vesicular trafficking.
    • Fig. S3. Akt is preferentially activated at the PM after EGFR stimulation.
    • Fig. S4. A1 pretreatment inhibits EGFR-promoted Akt activation.
    • Fig. S5. Distributions of cell migration distances.
    • Table S1. Primer list for RT-qPCR.
    • Legends for movies S1 to S4

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Akt inhibition induces EGFR endosomal accumulation.
    • Movie S2 (.avi format). A1 stimulation induces EGFR endosomal accumulation.
    • Movie S3 (.mp4 format). A1-EphA2 interactions at cell-cell contact induce EGFR endosomal accumulation.
    • Movie S4 (.mp4 format). EGF-promoted migration in MEFs.

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