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WASH and Tsg101/ALIX-dependent diversion of stress-internalized EGFR from the canonical endocytic pathway.

Tomas A, Vaughan SO, Burgoyne T, Sorkin A, Hartley JA, Hochhauser D, Futter CE - Nat Commun (2015)

Bottom Line: Stress-internalized EGFR is retained intracellularly by continued p38 activity in a mechanism involving ubiquitin-independent, ESCRT/ALIX-dependent incorporation onto intraluminal vesicles (ILVs) of MVBs.In contrast to the internalization-independent EGF-stimulated activation, UVC/cisplatin-triggered EGFR activation depends on EGFR internalization and intracellular retention.EGFR signalling from this MVB subpopulation delays apoptosis and might contribute to chemoresistance.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Cell Biology, UCL Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, UK [2].

ABSTRACT
Stress exposure triggers ligand-independent EGF receptor (EGFR) endocytosis, but its post-endocytic fate and role in regulating signalling are unclear. We show that the p38 MAP kinase-dependent, EGFR tyrosine kinase (TK)-independent EGFR internalization induced by ultraviolet light C (UVC) or the cancer therapeutic cisplatin, is followed by diversion from the canonical endocytic pathway. Instead of lysosomal degradation or plasma membrane recycling, EGFR accumulates in a subset of LBPA-rich perinuclear multivesicular bodies (MVBs) distinct from those carrying EGF-stimulated EGFR. Stress-internalized EGFR co-segregates with exogenously expressed pre-melanosomal markers OA1 and fibrillar PMEL, following early endosomal sorting by the actin polymerization-promoting WASH complex. Stress-internalized EGFR is retained intracellularly by continued p38 activity in a mechanism involving ubiquitin-independent, ESCRT/ALIX-dependent incorporation onto intraluminal vesicles (ILVs) of MVBs. In contrast to the internalization-independent EGF-stimulated activation, UVC/cisplatin-triggered EGFR activation depends on EGFR internalization and intracellular retention. EGFR signalling from this MVB subpopulation delays apoptosis and might contribute to chemoresistance.

No MeSH data available.


Related in: MedlinePlus

Dual role of p38 in ligand-independent stress-triggered EGFR trafficking in HeLa cells.(a) EGFR immunolocalization in untreated versus UVC-exposed cells 1 h post UVC exposure (left), and quantification of surface downregulation in cells exposed to UVC or treated with EGF for the indicated times (right). Arrows indicate perinuclear accumulation of EGFR (green) after UVC exposure. Data are mean±s.e.m. of three independent experiments. (b) Untreated or UVC-exposed HeLa cells were fixed after 1 h, or further incubated with the p38 inhibitor SB202190 (SB) for 1 h (left). Untreated or EGF-treated cells were fixed after 30 min, or further incubated with SB for 1 h in the continuous presence of EGF (right). p38 inhibition causes EGFR (green) redistribution to the plasma membrane following UVC, but not EGF exposure. (c) Pre-treatment for 30 min with PITSTOPII prevents UVC-induced EGFR (green) internalization (left) but PITSTOPII addition 1 h after UVC-induced EGFR internalization does not affect perinuclear EGFR accumulation or the recycling induced by simultaneous p38 inhibition (right). (d) Cells transfected with control or Rab11 siRNA were immunoblotted after 72 h for Rab11 and tubulin to assess knockdown efficiency (top). Rab11 knockdown did not prevent UVC-induced EGFR (green) internalization but prevented EGFR recycling after subsequent SB treatment (bottom). (e) Cells transfected with EGFR-GFP were fixed 1 h after UVC exposure and ultrathin cryosections were immuno-labelled for EGFR with 8 nm gold. EGFR-GFP (arrows) is on the limiting membrane and ILVs of MVBs. (f) Immunofluorescence analysis of UVC and EGF sequentially exposed HeLa cells (see Methods for experimental details). Red arrows show endosomes containing EGFR (green) and EGF (red). White arrows show EGFR+ve, EGF-ve endosomes, indicating a separate subset of MVBs containing stress-internalized but not EGF-bound EGFR. Scale bar, 5 μm. (g) Cells transfected with constitutively active Rab5-Q79L-DsRed were exposed to UVC and incubated for 1 h before treatment with EGF-AlexaFluor 647 (red) for 3 h. Red and white arrows show EGFR+ve/EGF+ve and EGFR+ve/EGF-ve endosomes, respectively. Scale bars, 10 μm for confocal and 100 nm for EM, unless otherwise indicated; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.
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f1: Dual role of p38 in ligand-independent stress-triggered EGFR trafficking in HeLa cells.(a) EGFR immunolocalization in untreated versus UVC-exposed cells 1 h post UVC exposure (left), and quantification of surface downregulation in cells exposed to UVC or treated with EGF for the indicated times (right). Arrows indicate perinuclear accumulation of EGFR (green) after UVC exposure. Data are mean±s.e.m. of three independent experiments. (b) Untreated or UVC-exposed HeLa cells were fixed after 1 h, or further incubated with the p38 inhibitor SB202190 (SB) for 1 h (left). Untreated or EGF-treated cells were fixed after 30 min, or further incubated with SB for 1 h in the continuous presence of EGF (right). p38 inhibition causes EGFR (green) redistribution to the plasma membrane following UVC, but not EGF exposure. (c) Pre-treatment for 30 min with PITSTOPII prevents UVC-induced EGFR (green) internalization (left) but PITSTOPII addition 1 h after UVC-induced EGFR internalization does not affect perinuclear EGFR accumulation or the recycling induced by simultaneous p38 inhibition (right). (d) Cells transfected with control or Rab11 siRNA were immunoblotted after 72 h for Rab11 and tubulin to assess knockdown efficiency (top). Rab11 knockdown did not prevent UVC-induced EGFR (green) internalization but prevented EGFR recycling after subsequent SB treatment (bottom). (e) Cells transfected with EGFR-GFP were fixed 1 h after UVC exposure and ultrathin cryosections were immuno-labelled for EGFR with 8 nm gold. EGFR-GFP (arrows) is on the limiting membrane and ILVs of MVBs. (f) Immunofluorescence analysis of UVC and EGF sequentially exposed HeLa cells (see Methods for experimental details). Red arrows show endosomes containing EGFR (green) and EGF (red). White arrows show EGFR+ve, EGF-ve endosomes, indicating a separate subset of MVBs containing stress-internalized but not EGF-bound EGFR. Scale bar, 5 μm. (g) Cells transfected with constitutively active Rab5-Q79L-DsRed were exposed to UVC and incubated for 1 h before treatment with EGF-AlexaFluor 647 (red) for 3 h. Red and white arrows show EGFR+ve/EGF+ve and EGFR+ve/EGF-ve endosomes, respectively. Scale bars, 10 μm for confocal and 100 nm for EM, unless otherwise indicated; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.

Mentions: Immunofluorescence staining of HeLa cells showed that both UVC and cisplatin elicit a striking perinuclear accumulation of internalized EGFR (Fig. 1a and Supplementary Fig. 1a), but western blotting cell lysates showed that, unlike EGF stimulation, they do not induce detectable EGFR degradation (Supplementary Fig. 1b). Quantitative analysis of EGFR surface downregulation showed that the perinuclear intracellular pool of EGFR is maintained for >2 h and accounts for ∼50% of total EGFR (Fig. 1a). In keeping with previous studies, UVC-elicited p38 activity was required to promote initial EGFR internalization (Supplementary Fig. 1c). Interestingly, p38 inhibition after UVC-induced perinuclear EGFR accumulation resulted in redistribution of perinuclear EGFR to the plasma membrane (Fig. 1b), revealing a novel role for p38 in EGFR retention in the perinuclear compartment distinct from its role in EGFR internalization. This is in marked contrast to the p38-independent post-endocytic fate of ligand-stimulated EGFR, which was degraded. Treatment with the clathrin inhibitor PITSTOPII blocked UVC-induced EGFR internalization but when PITSTOPII was added after UVC-induced EGFR perinuclear accumulation to prevent re-internalization of any recycled receptor, the EGFR was retained intracellularly (Fig. 1c). This indicates that UVC-exposed EGFR does not undergo continuous internalization and recycling. However, if p38 is inhibited after UVC-induced EGFR perinuclear accumulation, EGFR recycles to the plasma membrane in a manner that siRNA-mediated depletion shows is dependent on Rab11 (Fig. 1d). In contrast, endocytosis of Transferrin Receptor (TfR) did not require p38 activity, and TfR recycling was unaffected by UVC exposure, indicating EGFR-specific p38-dependent internalization and retention (Supplementary Fig. 1c,d).


WASH and Tsg101/ALIX-dependent diversion of stress-internalized EGFR from the canonical endocytic pathway.

Tomas A, Vaughan SO, Burgoyne T, Sorkin A, Hartley JA, Hochhauser D, Futter CE - Nat Commun (2015)

Dual role of p38 in ligand-independent stress-triggered EGFR trafficking in HeLa cells.(a) EGFR immunolocalization in untreated versus UVC-exposed cells 1 h post UVC exposure (left), and quantification of surface downregulation in cells exposed to UVC or treated with EGF for the indicated times (right). Arrows indicate perinuclear accumulation of EGFR (green) after UVC exposure. Data are mean±s.e.m. of three independent experiments. (b) Untreated or UVC-exposed HeLa cells were fixed after 1 h, or further incubated with the p38 inhibitor SB202190 (SB) for 1 h (left). Untreated or EGF-treated cells were fixed after 30 min, or further incubated with SB for 1 h in the continuous presence of EGF (right). p38 inhibition causes EGFR (green) redistribution to the plasma membrane following UVC, but not EGF exposure. (c) Pre-treatment for 30 min with PITSTOPII prevents UVC-induced EGFR (green) internalization (left) but PITSTOPII addition 1 h after UVC-induced EGFR internalization does not affect perinuclear EGFR accumulation or the recycling induced by simultaneous p38 inhibition (right). (d) Cells transfected with control or Rab11 siRNA were immunoblotted after 72 h for Rab11 and tubulin to assess knockdown efficiency (top). Rab11 knockdown did not prevent UVC-induced EGFR (green) internalization but prevented EGFR recycling after subsequent SB treatment (bottom). (e) Cells transfected with EGFR-GFP were fixed 1 h after UVC exposure and ultrathin cryosections were immuno-labelled for EGFR with 8 nm gold. EGFR-GFP (arrows) is on the limiting membrane and ILVs of MVBs. (f) Immunofluorescence analysis of UVC and EGF sequentially exposed HeLa cells (see Methods for experimental details). Red arrows show endosomes containing EGFR (green) and EGF (red). White arrows show EGFR+ve, EGF-ve endosomes, indicating a separate subset of MVBs containing stress-internalized but not EGF-bound EGFR. Scale bar, 5 μm. (g) Cells transfected with constitutively active Rab5-Q79L-DsRed were exposed to UVC and incubated for 1 h before treatment with EGF-AlexaFluor 647 (red) for 3 h. Red and white arrows show EGFR+ve/EGF+ve and EGFR+ve/EGF-ve endosomes, respectively. Scale bars, 10 μm for confocal and 100 nm for EM, unless otherwise indicated; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.
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f1: Dual role of p38 in ligand-independent stress-triggered EGFR trafficking in HeLa cells.(a) EGFR immunolocalization in untreated versus UVC-exposed cells 1 h post UVC exposure (left), and quantification of surface downregulation in cells exposed to UVC or treated with EGF for the indicated times (right). Arrows indicate perinuclear accumulation of EGFR (green) after UVC exposure. Data are mean±s.e.m. of three independent experiments. (b) Untreated or UVC-exposed HeLa cells were fixed after 1 h, or further incubated with the p38 inhibitor SB202190 (SB) for 1 h (left). Untreated or EGF-treated cells were fixed after 30 min, or further incubated with SB for 1 h in the continuous presence of EGF (right). p38 inhibition causes EGFR (green) redistribution to the plasma membrane following UVC, but not EGF exposure. (c) Pre-treatment for 30 min with PITSTOPII prevents UVC-induced EGFR (green) internalization (left) but PITSTOPII addition 1 h after UVC-induced EGFR internalization does not affect perinuclear EGFR accumulation or the recycling induced by simultaneous p38 inhibition (right). (d) Cells transfected with control or Rab11 siRNA were immunoblotted after 72 h for Rab11 and tubulin to assess knockdown efficiency (top). Rab11 knockdown did not prevent UVC-induced EGFR (green) internalization but prevented EGFR recycling after subsequent SB treatment (bottom). (e) Cells transfected with EGFR-GFP were fixed 1 h after UVC exposure and ultrathin cryosections were immuno-labelled for EGFR with 8 nm gold. EGFR-GFP (arrows) is on the limiting membrane and ILVs of MVBs. (f) Immunofluorescence analysis of UVC and EGF sequentially exposed HeLa cells (see Methods for experimental details). Red arrows show endosomes containing EGFR (green) and EGF (red). White arrows show EGFR+ve, EGF-ve endosomes, indicating a separate subset of MVBs containing stress-internalized but not EGF-bound EGFR. Scale bar, 5 μm. (g) Cells transfected with constitutively active Rab5-Q79L-DsRed were exposed to UVC and incubated for 1 h before treatment with EGF-AlexaFluor 647 (red) for 3 h. Red and white arrows show EGFR+ve/EGF+ve and EGFR+ve/EGF-ve endosomes, respectively. Scale bars, 10 μm for confocal and 100 nm for EM, unless otherwise indicated; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.
Mentions: Immunofluorescence staining of HeLa cells showed that both UVC and cisplatin elicit a striking perinuclear accumulation of internalized EGFR (Fig. 1a and Supplementary Fig. 1a), but western blotting cell lysates showed that, unlike EGF stimulation, they do not induce detectable EGFR degradation (Supplementary Fig. 1b). Quantitative analysis of EGFR surface downregulation showed that the perinuclear intracellular pool of EGFR is maintained for >2 h and accounts for ∼50% of total EGFR (Fig. 1a). In keeping with previous studies, UVC-elicited p38 activity was required to promote initial EGFR internalization (Supplementary Fig. 1c). Interestingly, p38 inhibition after UVC-induced perinuclear EGFR accumulation resulted in redistribution of perinuclear EGFR to the plasma membrane (Fig. 1b), revealing a novel role for p38 in EGFR retention in the perinuclear compartment distinct from its role in EGFR internalization. This is in marked contrast to the p38-independent post-endocytic fate of ligand-stimulated EGFR, which was degraded. Treatment with the clathrin inhibitor PITSTOPII blocked UVC-induced EGFR internalization but when PITSTOPII was added after UVC-induced EGFR perinuclear accumulation to prevent re-internalization of any recycled receptor, the EGFR was retained intracellularly (Fig. 1c). This indicates that UVC-exposed EGFR does not undergo continuous internalization and recycling. However, if p38 is inhibited after UVC-induced EGFR perinuclear accumulation, EGFR recycles to the plasma membrane in a manner that siRNA-mediated depletion shows is dependent on Rab11 (Fig. 1d). In contrast, endocytosis of Transferrin Receptor (TfR) did not require p38 activity, and TfR recycling was unaffected by UVC exposure, indicating EGFR-specific p38-dependent internalization and retention (Supplementary Fig. 1c,d).

Bottom Line: Stress-internalized EGFR is retained intracellularly by continued p38 activity in a mechanism involving ubiquitin-independent, ESCRT/ALIX-dependent incorporation onto intraluminal vesicles (ILVs) of MVBs.In contrast to the internalization-independent EGF-stimulated activation, UVC/cisplatin-triggered EGFR activation depends on EGFR internalization and intracellular retention.EGFR signalling from this MVB subpopulation delays apoptosis and might contribute to chemoresistance.

View Article: PubMed Central - PubMed

Affiliation: 1] Department of Cell Biology, UCL Institute of Ophthalmology, University College London, 11-43 Bath Street, London EC1V 9EL, UK [2].

ABSTRACT
Stress exposure triggers ligand-independent EGF receptor (EGFR) endocytosis, but its post-endocytic fate and role in regulating signalling are unclear. We show that the p38 MAP kinase-dependent, EGFR tyrosine kinase (TK)-independent EGFR internalization induced by ultraviolet light C (UVC) or the cancer therapeutic cisplatin, is followed by diversion from the canonical endocytic pathway. Instead of lysosomal degradation or plasma membrane recycling, EGFR accumulates in a subset of LBPA-rich perinuclear multivesicular bodies (MVBs) distinct from those carrying EGF-stimulated EGFR. Stress-internalized EGFR co-segregates with exogenously expressed pre-melanosomal markers OA1 and fibrillar PMEL, following early endosomal sorting by the actin polymerization-promoting WASH complex. Stress-internalized EGFR is retained intracellularly by continued p38 activity in a mechanism involving ubiquitin-independent, ESCRT/ALIX-dependent incorporation onto intraluminal vesicles (ILVs) of MVBs. In contrast to the internalization-independent EGF-stimulated activation, UVC/cisplatin-triggered EGFR activation depends on EGFR internalization and intracellular retention. EGFR signalling from this MVB subpopulation delays apoptosis and might contribute to chemoresistance.

No MeSH data available.


Related in: MedlinePlus