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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

Ubiquitin-independent, ESCRT-dependent sorting of stress-internalized EGFR onto ILVs of MVBs.(a) Immunoprecipitation (IP) of EGFR from HeLa cell lysates and immunoblotting for ubiquitin and EGFR showed robust EGFR ubiquitination after EGF stimulation but not after exposure to UVC or cisplatin. (b) Treatment of HeLa cells with EGF 1 h post-UVC exposure induced strong EGFR ubiquitination, measured as in a, that was reduced compared with EGF alone, most likely because only 50% of EGFR was available for EGF stimulation after UVC exposure. (c) Stable PAE cell sublines expressing EGFR-wt or a ubiquitination-defective EGFR (EGFR-15KR) were exposed to UVC and incubated for 1 h with anti-EGFR-gold. Cells were fixed and processed for EM. Representative images of ultrathin sections with gold in ILVs (black arrows) and on the limiting membrane of MVBs (white arrows) from both sublines are shown. (d) Immunofluorescence shows perinuclear EGFR (green) accumulation 1 h after UVC exposure followed by recycling to the plasma membrane on subsequent p38 inhibition in both PAE EGFR-wt and -15KR cells, consistent with no role for ubiquitination in stress-induced EGFR traffic. (e) PAE EGFR-wt and -15KR cells were transfected with OA1-myc and either treated with EGF for 30 min or exposed to UVC and incubated for 1 h. Ubiquitination-deficient EGFR-15KR (green) showed increased co-staining with OA1-myc (red) following EGF stimulation compared with EGFR-wt, to a similar level to that shown by UVC-internalized EGFR (-wt or -15KR). Data are mean±s.e.m. of three independent experiments, ***P<0.001 (Student's t-test). (f) Lysates from HeLa cells transfected with Hrs or Tsg101 siRNA were immunoblotted after 72 h for Hrs, Tsg101 and tubulin to assess knockdown efficiency (left). RNAi-treated cells were exposed to UVC, incubated for 1 h with anti-EGFR-gold (arrows) and processed for EM. Ultrathin sections (right) show enlarged MVBs containing reduced numbers of EGFR-positive ILVs in Hrs and Tsg101 siRNA-treated compared with control RNAi cells. Scale bars, 10 μm for confocal and 100 nm for EM images; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.
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f4: Ubiquitin-independent, ESCRT-dependent sorting of stress-internalized EGFR onto ILVs of MVBs.(a) Immunoprecipitation (IP) of EGFR from HeLa cell lysates and immunoblotting for ubiquitin and EGFR showed robust EGFR ubiquitination after EGF stimulation but not after exposure to UVC or cisplatin. (b) Treatment of HeLa cells with EGF 1 h post-UVC exposure induced strong EGFR ubiquitination, measured as in a, that was reduced compared with EGF alone, most likely because only 50% of EGFR was available for EGF stimulation after UVC exposure. (c) Stable PAE cell sublines expressing EGFR-wt or a ubiquitination-defective EGFR (EGFR-15KR) were exposed to UVC and incubated for 1 h with anti-EGFR-gold. Cells were fixed and processed for EM. Representative images of ultrathin sections with gold in ILVs (black arrows) and on the limiting membrane of MVBs (white arrows) from both sublines are shown. (d) Immunofluorescence shows perinuclear EGFR (green) accumulation 1 h after UVC exposure followed by recycling to the plasma membrane on subsequent p38 inhibition in both PAE EGFR-wt and -15KR cells, consistent with no role for ubiquitination in stress-induced EGFR traffic. (e) PAE EGFR-wt and -15KR cells were transfected with OA1-myc and either treated with EGF for 30 min or exposed to UVC and incubated for 1 h. Ubiquitination-deficient EGFR-15KR (green) showed increased co-staining with OA1-myc (red) following EGF stimulation compared with EGFR-wt, to a similar level to that shown by UVC-internalized EGFR (-wt or -15KR). Data are mean±s.e.m. of three independent experiments, ***P<0.001 (Student's t-test). (f) Lysates from HeLa cells transfected with Hrs or Tsg101 siRNA were immunoblotted after 72 h for Hrs, Tsg101 and tubulin to assess knockdown efficiency (left). RNAi-treated cells were exposed to UVC, incubated for 1 h with anti-EGFR-gold (arrows) and processed for EM. Ultrathin sections (right) show enlarged MVBs containing reduced numbers of EGFR-positive ILVs in Hrs and Tsg101 siRNA-treated compared with control RNAi cells. Scale bars, 10 μm for confocal and 100 nm for EM images; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.

Mentions: The best characterized MVB sorting machinery is the ESCRT complex system24, which recognizes ubiquitinated cargoes such as ligand-stimulated EGFR and sorts them onto intraluminal vesicles (ILVs) of MVBs that subsequently fuse with lysosomes for degradation. We found that, in contrast to EGF stimulation, UVC or cisplatin exposure did not trigger EGFR ubiquitination (Fig. 4a). Ultraviolet exposure may downregulate the cellular ubiquitination machinery25, but EGF-dependent EGFR ubiquitination was still present, although slightly reduced, in UVC-pre-treated cells, showing that the ubiquitination capacity was only marginally abridged (Fig. 4b). Consistent with the absence of a role for ubiquitination in stress-induced EGFR trafficking, ubiquitination-deficient mutant EGFR-15KR26 accumulated on both MVB-limiting membranes and ILVs following UVC exposure (Fig. 4c), and recycled to the plasma membrane following p38 inhibition (Fig. 4d), just as control EGFR-wt, when expressed in porcine aortic endothelial (PAE) cells. Moreover, EGFR-15KR co-segregated with transfected OA1 following both EGF and UVC exposure, whereas, as reported above in HeLa and flox/flox MEFs, EGFR-wt only co-localized with OA1 following UVC exposure (Fig. 4e), suggesting receptor ubiquitination as critical for its retention on the degradative pathway.


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)

Ubiquitin-independent, ESCRT-dependent sorting of stress-internalized EGFR onto ILVs of MVBs.(a) Immunoprecipitation (IP) of EGFR from HeLa cell lysates and immunoblotting for ubiquitin and EGFR showed robust EGFR ubiquitination after EGF stimulation but not after exposure to UVC or cisplatin. (b) Treatment of HeLa cells with EGF 1 h post-UVC exposure induced strong EGFR ubiquitination, measured as in a, that was reduced compared with EGF alone, most likely because only 50% of EGFR was available for EGF stimulation after UVC exposure. (c) Stable PAE cell sublines expressing EGFR-wt or a ubiquitination-defective EGFR (EGFR-15KR) were exposed to UVC and incubated for 1 h with anti-EGFR-gold. Cells were fixed and processed for EM. Representative images of ultrathin sections with gold in ILVs (black arrows) and on the limiting membrane of MVBs (white arrows) from both sublines are shown. (d) Immunofluorescence shows perinuclear EGFR (green) accumulation 1 h after UVC exposure followed by recycling to the plasma membrane on subsequent p38 inhibition in both PAE EGFR-wt and -15KR cells, consistent with no role for ubiquitination in stress-induced EGFR traffic. (e) PAE EGFR-wt and -15KR cells were transfected with OA1-myc and either treated with EGF for 30 min or exposed to UVC and incubated for 1 h. Ubiquitination-deficient EGFR-15KR (green) showed increased co-staining with OA1-myc (red) following EGF stimulation compared with EGFR-wt, to a similar level to that shown by UVC-internalized EGFR (-wt or -15KR). Data are mean±s.e.m. of three independent experiments, ***P<0.001 (Student's t-test). (f) Lysates from HeLa cells transfected with Hrs or Tsg101 siRNA were immunoblotted after 72 h for Hrs, Tsg101 and tubulin to assess knockdown efficiency (left). RNAi-treated cells were exposed to UVC, incubated for 1 h with anti-EGFR-gold (arrows) and processed for EM. Ultrathin sections (right) show enlarged MVBs containing reduced numbers of EGFR-positive ILVs in Hrs and Tsg101 siRNA-treated compared with control RNAi cells. Scale bars, 10 μm for confocal and 100 nm for EM images; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.
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f4: Ubiquitin-independent, ESCRT-dependent sorting of stress-internalized EGFR onto ILVs of MVBs.(a) Immunoprecipitation (IP) of EGFR from HeLa cell lysates and immunoblotting for ubiquitin and EGFR showed robust EGFR ubiquitination after EGF stimulation but not after exposure to UVC or cisplatin. (b) Treatment of HeLa cells with EGF 1 h post-UVC exposure induced strong EGFR ubiquitination, measured as in a, that was reduced compared with EGF alone, most likely because only 50% of EGFR was available for EGF stimulation after UVC exposure. (c) Stable PAE cell sublines expressing EGFR-wt or a ubiquitination-defective EGFR (EGFR-15KR) were exposed to UVC and incubated for 1 h with anti-EGFR-gold. Cells were fixed and processed for EM. Representative images of ultrathin sections with gold in ILVs (black arrows) and on the limiting membrane of MVBs (white arrows) from both sublines are shown. (d) Immunofluorescence shows perinuclear EGFR (green) accumulation 1 h after UVC exposure followed by recycling to the plasma membrane on subsequent p38 inhibition in both PAE EGFR-wt and -15KR cells, consistent with no role for ubiquitination in stress-induced EGFR traffic. (e) PAE EGFR-wt and -15KR cells were transfected with OA1-myc and either treated with EGF for 30 min or exposed to UVC and incubated for 1 h. Ubiquitination-deficient EGFR-15KR (green) showed increased co-staining with OA1-myc (red) following EGF stimulation compared with EGFR-wt, to a similar level to that shown by UVC-internalized EGFR (-wt or -15KR). Data are mean±s.e.m. of three independent experiments, ***P<0.001 (Student's t-test). (f) Lysates from HeLa cells transfected with Hrs or Tsg101 siRNA were immunoblotted after 72 h for Hrs, Tsg101 and tubulin to assess knockdown efficiency (left). RNAi-treated cells were exposed to UVC, incubated for 1 h with anti-EGFR-gold (arrows) and processed for EM. Ultrathin sections (right) show enlarged MVBs containing reduced numbers of EGFR-positive ILVs in Hrs and Tsg101 siRNA-treated compared with control RNAi cells. Scale bars, 10 μm for confocal and 100 nm for EM images; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.
Mentions: The best characterized MVB sorting machinery is the ESCRT complex system24, which recognizes ubiquitinated cargoes such as ligand-stimulated EGFR and sorts them onto intraluminal vesicles (ILVs) of MVBs that subsequently fuse with lysosomes for degradation. We found that, in contrast to EGF stimulation, UVC or cisplatin exposure did not trigger EGFR ubiquitination (Fig. 4a). Ultraviolet exposure may downregulate the cellular ubiquitination machinery25, but EGF-dependent EGFR ubiquitination was still present, although slightly reduced, in UVC-pre-treated cells, showing that the ubiquitination capacity was only marginally abridged (Fig. 4b). Consistent with the absence of a role for ubiquitination in stress-induced EGFR trafficking, ubiquitination-deficient mutant EGFR-15KR26 accumulated on both MVB-limiting membranes and ILVs following UVC exposure (Fig. 4c), and recycled to the plasma membrane following p38 inhibition (Fig. 4d), just as control EGFR-wt, when expressed in porcine aortic endothelial (PAE) cells. Moreover, EGFR-15KR co-segregated with transfected OA1 following both EGF and UVC exposure, whereas, as reported above in HeLa and flox/flox MEFs, EGFR-wt only co-localized with OA1 following UVC exposure (Fig. 4e), suggesting receptor ubiquitination as critical for its retention on the degradative pathway.

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