Epidermal growth factor-stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis

Abstract

Epidermal growth factor (EGF) binding to its receptor (EGFR) activates several signaling intermediates, including Akt, leading to control of cell survival and metabolism. Concomitantly, ligand-bound EGFR is incorporated into clathrin-coated pits--membrane structures containing clathrin and other proteins--eventually leading to receptor internalization. Whether clathrin might regulate EGFR signaling at the plasma membrane before vesicle scission is poorly understood. We compared the effect of clathrin perturbation (preventing formation of, or receptor recruitment to, clathrin structures) to that of dynamin2 (allowing formation of clathrin structures but preventing EGFR internalization) under conditions in which EGFR endocytosis is clathrin dependent. Clathrin perturbation by siRNA gene silencing, with the clathrin inhibitor pitstop2, or knocksideways silencing inhibited EGF-simulated Gab1 and Akt phosphorylation in ARPE-19 cells. In contrast, perturbation of dynamin2 with inhibitors or by siRNA gene silencing did not affect EGF-stimulated Gab1 or Akt phosphorylation. EGF stimulation enriched Gab1 and phospho-Gab1 within clathrin structures. ARPE-19 cells have low ErbB2 expression, and overexpression and knockdown experiments revealed that robust ErbB2 expression bypassed the requirement for clathrin for EGF-stimulated Akt phosphorylation. Thus clathrin scaffolds may represent unique plasma membrane signaling microdomains required for signaling by certain receptors, a function that can be separated from vesicle formation.

FIGURE 1:

SiRNA gene silencing of clathrin heavy chain but not of dynamin2 inhibits EGF-stimulated Akt phosphoryla­tion in ARPE-19 cells. ARPE-19 cells were transfected with siRNA targeting clathrin heavy chain sequence 1 (clathrin siRNA 1), clathrin heavy chain sequence 2 (clathrin siRNA 2), dynamin2, or nontargeting siRNA (control). (A) EGF internalization was measured using 5 ng/ml EGF in cells transfected as indicated; mean ± SE of EGF internalized (n = 3); *p < 0.05 relative to the corresponding control condition. (B–E) After transfection, cells were stimulated with 5 ng/ml EGF or left unstimulated (basal), and whole-cell lysates were prepared and resolved by immunoblotting and probed with anti–phospho-Akt (pS473), anti–total pan-Akt, or anti–pan-actin antibodies. (B) Immunoblots representative of at least five independent experiments. (C–E) Means ± SE of pS473-Akt values; n = 12 (C), 7 (D), 7 (E); *p < 0.05 relative to control, EGF-stimulated condition. (F, G) After siRNA transfection, intact cells were subjected to immunofluorescence microscopy with antibodies that selectively recognize the EGFR ectodomain. (F) Representative fluorescence microscopy micrographs of cell surface EGFR staining. Scale, 20 μm. (G) Fluorescence intensity of cell-surface EGFR was quantified; mean cell-surface EGFR levels (n = 4).

FIGURE 2:

Treatment of ARPE-19 cells with the clathrin inhibitor pitstop2 but not the dynamin inhibitor dynasore or dyngo4A inhibits EGF localization to clathrin structures and EGF-stimulated Akt phosphorylation. ARPE-19 cells were treated with 10 μM pitstop2, 30 μM dyngo4A, 80 μM dynasore, or vehicle control (0.1% [vol/vol] DMSO) for 30 min. (A) After drug treatment, EGF internalization was measured using 5 ng/ml EGF in cells transfected as indicated; mean ± SE of EGF internalized (n = 4), *p < 0.05 relative to the corresponding control condition. (B–D) After drug treatment as indicated in ARPE-19 cells stably expressing GFP fused to clathrin light chain (GFP-CLC), cells were stimulated with A555-EGF (5 ng/ml) for 3 min at 37°C, followed immediately by fixation and then imaging using TIRFM. (B, C) Representative micrographs showing regions of larger images depicting whole cells, which are shown in Supplemental Figure S2, A and B. Scale bar, 5 μm. (D) Micrographs obtained by TIRFM were subjected to automated detection of clathrin structures as in Aguet et al. (2013; see Materials and Methods), followed by quantification of A555-EGF fluorescence intensity within each detected object. Mean A555-EGF fluorescence intensity measurements within clathrin structures in each image (>10,000 clathrin structures from >50 cells from three independent experiments; *p < 0.05). (E, F) After drug treatment, cells were stimulated with 5 ng/ml EGF for an additional 5 min in the continued presence of the inhibitors or left unstimulated (basal). Subsequently whole-cell lysates were immediately prepared and resolved by immunoblotting and probed with anti–phospho-Akt (S473) or total pan-Akt antibodies. (E) Immunoblots representative of at least five independent experiments. (F) Mean ± SE of pS473-Akt values (n = 5); *p < 0.05, relative to control, EGF-stimulated condition.

FIGURE 3:

Knocksideways silencing of clathrin light chain reduces EGF-stimulated Akt phosphorylation in ARPE-19 cells. ARPE-19 cells were cotransfected with cDNA encoding MitoTrap (pMito-mCherry-FRB) or GFP-FKBP-CLCa, after which cells were treated with 1 μM rapamycin for 10 min or left untreated (Supplemental Figure S3) and then stimulated with 5 ng/ml EGF for an additional 5 min in the continued presence of rapamycin. After this, cells were fixed and processed for immunofluorescence staining using anti–phospho-Akt (pS473)-specific antibodies. (A) Representative micrographs; scale bar, 20 μm. (B) Mean fluorescence intensity of phospho-Akt (pS473) staining was quantified in cells expressing both MitoTrap and GFP-FKBP-CLCa (KS transfected +) or only one of these exogenous fluorescent proteins (KS transfected –). Mean ± SE of EGF-stimulated gain in phospho-Akt (pS473) levels (n = 3).

FIGURE 4:

Perturbation of clathrin by pitstop2 treatment or siRNA gene silencing does not alter EGF-stimulated EGFR phosphorylation in ARPE-19 cells. (A, B) ARPE-19 cells were treated with 10 μM pitstop2, 30 μM dyngo4A, or vehicle control (0.1% [vol/vol] DMSO) for 30 min. After drug treatment, cells were stimulated with 5 ng/ml EGF for an additional 5 min, in the continued presence of the inhibitors, or left unstimulated (basal). Whole-cell lysates were then immediately prepared, resolved by immunoblotting, and probed with anti–phospho-EGFR or anti-actin antibodies. (A) A sample immunoblot probed with anti–phospho-EGFR (pY1068) and anti-actin antibodies, representative of three independent experiments. (B) Sample immunoblots probed with anti–phospho-EGFR (recognizing pY1068, pY1086, pY845, pY1173, or pY1045 motifs), representative of three independent experiments. (C) ARPE-19 cells were transfected with siRNA targeting clathrin heavy chain sequence 1(CHC seq. 1) or sequence 2 (CHC seq. 2) or nontargeting siRNA (control). After transfection, cells were stimulated with 5 ng/ml EGF or left unstimulated (basal) and then fixed and processed for immuno­fluorescence staining with anti–phospho-EGFR (pY1068) antibodies. Mean ± SE of the EGF-stimulated gain in phospho-EGFR (pY1068; n = 3, minimum 20 cells per condition per experiment). Representative micrographs are shown in Supplemental Figure S4.

FIGURE 5:

Perturbation of clathrin by pitstop2 treatment or siRNA gene silencing inhibits EGF-stimulated Gab1 phosphorylation in ARPE-19 cells. (A, B) ARPE-19 cells were treated with 10 μM pitstop2 or vehicle control (0.1% [vol/vol] DMSO) for 30 min. After drug treatment, cells were stimulated with 5 ng/ml EGF for 5 min in the continued presence of the inhibitors or left unstimulated (basal). Whole-cell lysates were then immediately prepared and resolved by immunoblotting and probed with anti–phospho-Gab1 (pY627) or anti-actin antibodies. (A) Immunoblots representative of three independent experiments. (B) Mean ± SE of pY627-Gab1 values (n = 4); *p < 0.05 (C) ARPE-19 cells were transfected with cDNA encoding the p85 subunit of class I PI3K fused to GFP (p85-GFP) and then treated with 10 μM pitstop2 or vehicle control (0.1% [vol/vol] DMSO) for 30 min. After drug treatment, cells were stimulated with 5 ng/ml EGF for an additional 5 min in the continued presence of the inhibitors or left unstimulated (basal), followed by fixation and fluorescence microscopy. Left, representative fluorescence micrographs. Scale, 10 μm. Right, density of GFP-p85 puncta detected by automated image analysis in individual cells (diamonds) and the means ± SE of these measurements in each condition (n = 4); *p < 0.05. (D) ARPE-19 cells were transfected with siRNA targeting clathrin heavy chain sequence 1 (CHC seq. 1), dynamin2 (dyn2), or nontargeting siRNA (control). After transfection, cells were stimulated with 5 ng/ml for 5 min EGF or left unstimulated (basal), and whole-cell lysates were prepared and resolved by immunoblotting and probed with anti–phospho-Gab1 (pY627) or anti-actin antibodies.

FIGURE 6:

Gab1 is enriched within plasma membrane clathrin structures upon EGF stimulation in ARPE-19 cells. ARPE-19 cells stably expressing Tag-RFP-T-CLC (RFP-CLC) were transfected with GFP fused to Gab1 (GFP-Gab1). Cells were then stimulated with 5 ng/ml EGF for 3 min or left unstimulated (basal) and then fixed and imaged using TIRFM). (A) Representative micrographs; scale bar, 5 μm. (B) Micrographs obtained by TIRFM were subjected to automated detection of clathrin structures as in Aguet et al. (2013; see Materials and Methods, followed by quantification of GFP-Gab1 and RFP-CLC in each detected object. (B) Mean GFP-Gab1 fluorescence intensity detected within all clathrin structures in each image (>10,000 structures from >45 cells from four independent experiments; *p < 0.05). (C) The 2D histogram of the difference in GFP-Gab1 and RFP-CLC intensity frequencies between basal and EGF-stimulated cells; also see Materials and Methods. Bins shown in green indicate RFP-CLC and GFP-Gab1 fluorescence intensity ranges within detected clathrin structures that are more frequently detected in EGF-stimulated cells, and bins shown in red indicate RFP-CLC and GFP-Gab1 fluorescence intensity ranges more frequently detected in basal cells.

FIGURE 7:

Phosphorylated Gab1 is enriched within plasma membrane clathrin structures upon EGF stimulation in ARPE-19 cells. ARPE-19 cells stably expressing Tag-RFP-T-CLC (RFP-CLC) were stimulated with 5 ng/ml EGF for 3 min or left unstimulated (basal), fixed, and labeled with antibodies that specifically recognize Gab1 phosphorylated on Y627 (pGab1), followed by imaging using TIRFM. (A) Representative micrographs; scale bar, 5 μm. (B) Micrographs obtained by TIRFM were subjected to automated detection of clathrin structures as in Aguet et al. (2013; see Materials and Methods), followed by quantification of pGab1 and RFP-CLC in each detected object. (B) Mean GFP-Gab1 fluorescence intensity detected within all clathrin structures in each image (actual image), as well as in images in which the pGab1 signal was randomized (see Materials and Methods) to determine the amount of pGab1 that occurs as a result of random overlap of clathrin and pGab1 puncta (>10,000 clathrin structures from >45 cells from four independent experiments; *p < 0.05). (C) The 2D histogram of the difference in pGab1 and RFP-CLC intensity frequencies between basal and EGF-stimulated cells; also see Materials and Methods. Bins shown in green indicate RFP-CLC and pGab1 fluorescence intensity ranges within detected clathrin structures that are more frequently detected in EGF-stimulated cells, and bins shown in red indicate RFP-CLC and pGab1 fluorescence intensity ranges more frequently detected in basal cells.

FIGURE 8:

Expression of ErbB2 renders EGF-stimulated Akt phosphorylation insensitive to clathrin perturbation. (A–D) MDA-MB-231 cells were treated with 10 μM pitstop2, 80 μM dynasore, or vehicle control (0.1% [vol/vol] DMSO) for 20 min (A, B) or transfected with siRNA targeting clathrin heavy chain sequence 1 (clathrin siRNA 1), or nontargeting siRNA (control; C, D). After siRNA or drug treatment, MDA-MB-231 cells were stimulated with 5 ng/ml EGF for an additional 5 min in the continued presence of the inhibitors or left unstimulated (basal). Subsequently whole-cell lysates were immediately prepared and resolved by immunoblotting and probed with anti–phospho-Akt (S473), total pan-Akt antibodies, anti-actin, or anti-CHC antibodies. (A, C) Immunoblots representative of at least three independent experiments. (B, D) Mean ± SE of pS473-Akt values (n > 3); *p < 0.05 relative to vehicle control (B) or control siRNA, EGF-stimulated condition (D). (E, F) ARPE-19 cells stably expressing ErbB2 (RPE-ErbB2) or parental ARPE-19 cells (RPE-WT), which do not express detectable levels of ErbB2, were treated with 10 μM pitstop2 or vehicle control (0.1% [vol/vol] DMSO) for 30 min. After drug treatment, cells were stimulated with 5 ng/ml EGF for an additional 5 min in the continued presence or absence of the inhibitors or left unstimulated (basal). Whole-cell lysates were then immediately prepared, resolved by immunoblotting, and probed with anti–phospho-Akt (S473), total pan-Akt, or anti-ErbB2 antibodies. (E) Immunoblots representative of at least four independent experiments. (F) Mean ± SE of the EGF-stimulated gain in pS473-Akt values (n = 4). (G–I) HeLa cells were transfected with siRNA targeting ErbB2 or nontargeting (control) siRNA. (G) Western blot of HeLa whole-cell lysates probed with either anti-ErbB2 or anti-Akt (total, as a loading control) antibodies. (H, I) After siRNA transfection, HeLa cells were treated with 10 μM pitstop2 or vehicle control (0.1% [vol/vol] DMSO) for 20 min and then stimulated with 5 ng/ml EGF for an additional 5 min in the continued presence of the inhibitors or left unstimulated (basal). (H) Immunoblots representative of four independent experiments. (I) Mean ± SE of the EGF-stimulated gain in pS473-Akt values (n > 3); p < 0.05 relative to the control siRNA, pitstop2-treated condition.