Endosomal accumulation of the activated epidermal growth factor receptor (EGFR) induces apoptosis

Abstract

Endocytosis positively and negatively regulates cell surface receptor signaling by temporally and spatially controlling interactions with downstream effectors. This process controls receptor-effector communication. However, the relationship between receptor endocytic trafficking and cell physiology is unclear. In MDA-MB-468 cells, cell surface EGF receptors (EGFRs) promote cell growth, whereas intracellular EGFRs induce apoptosis, making these cells an excellent model for studying the endocytic regulation of EGFR signaling. In addition, MDA-MB-468 cells have limited EGFR degradation following stimulation. Here, we report that in MDA-MB-468 cells the phosphorylated EGFR accumulates on the limiting membrane of the endosome with its carboxyl terminus oriented to the cytoplasm. To determine whether perturbation of EGFR trafficking is sufficient to cause apoptosis, we used pharmacological and biochemical strategies to disrupt EGFR endocytic trafficking in HeLa cells, which do not undergo EGF-dependent apoptosis. Manipulation of HeLa cells so that active EGF·EGFRs accumulate on the limiting membrane of endosomes reveals that receptor phosphorylation is sustained and leads to apoptosis. When EGF·EGFR complexes accumulated in the intraluminal vesicles of the late endosome, phosphorylation of the receptor was not sustained, nor did the cells undergo apoptosis. These data demonstrate that EGFR-mediated apoptosis is initiated by the activated EGFR from the limiting membrane of the endosome.

FIGURE 1.

EGF treatment of MDA-MB-468 cells causes the receptors to accumulate in the early endosome. MDA-MB-468 cells were treated with 10 ng/ml EGF for 15 min (A), 2 h (B), 16 h (C), or 24 h (D). Postnuclear supernatants were prepared, run on isotonic 17% Percoll gradients, and fractionated into ∼30 fractions (∼330 ml each). Every other fraction was resolved by 7.5% SDS-PAGE, transferred to nitrocellulose, and probed with antibodies against the phosphorylated EGFR, the transferrin receptor (TfnR), and lysosomal-associate membrane protein 2 (LAMP2). Densitometric measurements of the immunoblot analyses were performed using National Institutes of Health ImageJ software, and the intensities were normalized to the maximum value and plotted for each fraction as indicated by the legend. Data shown are from representative experiments repeated for each time point in triplicate.

FIGURE 2.

EGF stimulated EGFRs in MDA-MB-468 cells accumulate on the limiting membrane of endosomes with the carboxyl terminus oriented toward the cytoplasm. A, serum-starved MDA-MB-468 cells were treated with 10 ng/ml EGF for the indicated times, fixed, and treated with or without 0.015% digitonin to permeabilize the cell membrane while leaving the limiting membranes of intracellular organelles intact. Cells were fixed and processed for double indirect immunofluorescence with an EGFR amino terminal antibody (Ab-1, red) and an EGFR carboxyl-terminal antibody (SC-03, green) as described under “Experimental Procedures.” Labeling for the amino terminus of the EGFR is present on the cell membrane at all time points but is absent from endosomal compartment in the cytoplasm. In contrast, labeling for the carboxyl terminus is present in both the cell membrane and in the endosome with longer EGR treatments, indicating that the C terminus of the internalized receptor is oriented toward the cytoplasm. Shown are representative single optical sections from an experiment performed three times. Scale bar = 20 μm. B, magnified images of data collected as described in A. Included is a positive control of cells permeabilized with 0.1% saponin to demonstrate endosomal staining with the amino-terminal specific EGFR antibody (Ab-1). Scale bar = 5 μm.

FIGURE 3.

Endosomal accumulation of the EGFR by monensin treatment is sufficient to promote EGF-dependent apoptosis. A, HeLa cells grown on coverslips were pretreated with or without 5 μm monensin for 30 min, followed by treatment with 10 ng/ml EGF in the presence or absence of 5 μm monensin. At the indicated time points, the cells on the coverslip were fixed and processed for indirect immunofluorescence (IF) using an EGFR antibody (Ab-1) (green). Nuclei were stained with DAPI (blue). Scale bar = 10 μm. B, HeLa cells were pretreated with 5 μm monensin for 30 min, followed by treatment with 10 ng/ml EGF as indicated. Cell lysates were prepared, resolved by 7.5% SDS-PAGE, and immunoblotted (IB) with the indicated antibodies. C, HeLa cells were plated in 96-well dish, pretreated with 5 μm monensin for 30 min, and then stimulated with the indicated concentrations of EGF in DMEM or 5% serum in DMEM for 48 h in the absence or presence of 5 μm monensin. The number of viable cells was quantified by MTT assay. Data are plotted as the relative change in the number of cells as compared with cells maintained in DMEM. Data are presented as the average ± S.E. (n = 4). *, p < 0.05 calculated by a paired Student's t test.

FIGURE 4.

Knockdown of either RAB7 or TSG101 results in attenuated kinetics of degradation of ¹²⁵I-EGF. HeLa cells were transfected with either control siRNA (siCon) or siRNA targeting either RAB7 or TSG101. A, representative immunoblot (IB) analysis indicating the extent of RAB7 and TSG101 knockdown 72 h post-transfection. Serial dilutions of each cell lysate (25 mg, 12.5 mg, and 6.25 mg) were resolved by 12% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibodies against either TSG101 (Genetex) or RAB7 (Sigma). B and C, 72 h post-transfection, cells were incubated at 37 °C with ¹²⁵I-EGF for 7 min and, following washing to remove unbound radioligand, were incubated in radioligand-free media for the indicated times. At each time point the medium was collected to determine the levels of secreted ¹²⁵I. In addition, cell lysates were harvested, and intact radioligand was precipitated with trichloroacetic acid. Data are plotted as the percentage of intact ¹²⁵I-EGF for each time point (B) or the percentage of ¹²⁵I-EGF that was secreted into the media at each time point (C) (mean ± S.E., n = 4). *, p < 0.05 **; p < 0.10; calculated by a paired Student's t test.

FIGURE 5.

RAB7 knockdown causes ¹²⁵I-EGF to accumulate in high density endosomes and TSG101 knockdown causes ¹²⁵I-EGF to accumulate in endosomes of a range of densities. The endocytic accumulation of the ¹²⁵I-EGF·EGFR complex was assessed by pulse-labeling cells with ¹²⁵I-EGF. Following 120 min of chase with radioligand-free media, postnuclear supernatant was prepared from HeLa cells transfected with either control siRNA (siCON) (A), siRNA targeting either RAB7 (B), or TSG101 (C). Data were plotted as the relative distribution of ¹²⁵I in each fraction and were normalized to the total radioactivity. Shown are representative graphs from four independent experiments. D, plot of the relative total radioactivity in transfected HeLa cells following incubation for 120 min at 37 °C. Circles along the x axis indicate the distribution of density beads (from lowest to highest Rf: 1.040 g/ml, 1.055 g/ml, 1.069 g/ml, and 1.109 g/ml). Early endosomes migrate at 1.035–1.042 g/ml. The heavier late endosomes sediment with a density of 1.048–1.060 g/ml.

FIGURE 6.

Knockdown of TSG101, but not RAB7, prolongs EGFR signaling. HeLa cells were transfected with either control siRNA (siCon) or siRNA targeting either RAB7 or TSG101. Following recovery from transfection (72 h), cells were serum-starved for 2 h and then treated with 10 ng/ml EGF for the indicated amounts of time. A, cell lysates were prepared, and equivalent amounts of protein were resolved by 7.5% SDS-PAGE, transferred to nitrocellulose, and immunoblotted (IB) with antibodies for either phosphorylated EGFR (pY1068 EGFR), EGFR, or α-tubulin. Shown is a representative blot of an experiment repeated four times. B and C, densitometric readings from four independent experiments were quantified using ImageJ software and normalized to the intensity of the 15-min time point in siCon cells for phosphorylated EGFR or to the 0-min time point for total EGFR. Data are presented as the mean ± S.E. *, p < 0.10; **, p < 0.05; calculated by a paired Student's t test.

FIGURE 7.

Retention of the EGF·EGFR complex on the limiting membrane of the endosome induces apoptosis. A, siRNA-treated cells were grown in either growth media, DMEM alone, or DMEM with 10 ng/ml EGF for 48 h. Shown are representative phase contrast images of cell morphology collected using a ×40 objective on a Nikon Eclipse TE-2000U microscope. Scale bar = 10 μm. Shown are representative images from an experiment performed three times. B, 24 h post-transfection, cells were replated in a 96-well dish and treated with varying concentrations of EGF (0 ng/ml, 0.1 ng/ml, 1.0 ng/ml, 10 ng/ml) or media containing 5% FBS for 24 or 48 h. The number of viable cells were assessed using an MTT assay as described under “Experimental Procedures.” Data are plotted as fold change in cell number relative to cells maintained in DMEM without ligand. *, p value < 0.05 as measured by a Student's t test (n = 4). C, cell lysates from the cells in B were prepared, resolved by 7.5% SDS-PAGE, and immunoblotted for PARP. Full-length and cleaved PARP are indicated by the upper and lower arrows. Shown is a representative image from an experiment performed three times.