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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

ALIX is required for sorting into ILVs and retention in MVBs of stress-internalized, but not EGF-bound EGFR.(a, left) Co-staining of EGFR (green) with LBPA (red) showed little co-localization in serum-starved HeLa cells treated with EGF for 30 min, but considerable overlap in cells 1 h after UVC exposure. Note that, in serum-free conditions, LBPA does not accumulate in lysosomes, facilitating the detection of MVB-specific labelling. (a, right) Quantification of co-localization of EGFR with LBPA in EGF-treated versus UVC-exposed serum-starved HeLa cells. Data are mean±s.e.m., ***P<0.001 (Student's t-test). (b) Lysates from HeLa cells transfected with control or ALIX siRNA were immunoblotted after 72 h for ALIX and Rab11 (as a loading control) to assess knockdown efficiency. (c) ALIX siRNA-treated cells were stimulated with EGF for 30 min, or exposed to UVC and incubated for 1 h, in the presence of anti-EGFR-gold, before EM processing. Ultrathin sections show gold (arrows) on ILVs of densely packed MVBs after EGF stimulation, but mainly on the limiting membrane of enlarged MVBs containing few ILVs in UVC-exposed cells. (d) Control, Tsg101, ALIX or Tgs101+ALIX siRNA-treated HeLa cells were exposed to UVC, incubated for 1 h and fixed, or further treated with PITSTOPII for 1 h. Immunostaining for EGFR (green) shows that depletion of Tsg101 or ALIX inhibits perinuclear EGFR accumulation, whereas EGFR redistributes to the plasma membrane upon PITSTOPII treatment, indicating that Tsg101 and ALIX are required for intracellular retention of EGFR. EGFR is found in very large vacuoles in double Tsg101+ALIX knocked-down cells after UVC exposure, before redistribution to the plasma membrane upon PITSTOPII treatment. Scale bars, 10 μm for confocal and 100 nm for EM pictures; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.
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f5: ALIX is required for sorting into ILVs and retention in MVBs of stress-internalized, but not EGF-bound EGFR.(a, left) Co-staining of EGFR (green) with LBPA (red) showed little co-localization in serum-starved HeLa cells treated with EGF for 30 min, but considerable overlap in cells 1 h after UVC exposure. Note that, in serum-free conditions, LBPA does not accumulate in lysosomes, facilitating the detection of MVB-specific labelling. (a, right) Quantification of co-localization of EGFR with LBPA in EGF-treated versus UVC-exposed serum-starved HeLa cells. Data are mean±s.e.m., ***P<0.001 (Student's t-test). (b) Lysates from HeLa cells transfected with control or ALIX siRNA were immunoblotted after 72 h for ALIX and Rab11 (as a loading control) to assess knockdown efficiency. (c) ALIX siRNA-treated cells were stimulated with EGF for 30 min, or exposed to UVC and incubated for 1 h, in the presence of anti-EGFR-gold, before EM processing. Ultrathin sections show gold (arrows) on ILVs of densely packed MVBs after EGF stimulation, but mainly on the limiting membrane of enlarged MVBs containing few ILVs in UVC-exposed cells. (d) Control, Tsg101, ALIX or Tgs101+ALIX siRNA-treated HeLa cells were exposed to UVC, incubated for 1 h and fixed, or further treated with PITSTOPII for 1 h. Immunostaining for EGFR (green) shows that depletion of Tsg101 or ALIX inhibits perinuclear EGFR accumulation, whereas EGFR redistributes to the plasma membrane upon PITSTOPII treatment, indicating that Tsg101 and ALIX are required for intracellular retention of EGFR. EGFR is found in very large vacuoles in double Tsg101+ALIX knocked-down cells after UVC exposure, before redistribution to the plasma membrane upon PITSTOPII treatment. Scale bars, 10 μm for confocal and 100 nm for EM pictures; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.

Mentions: PMEL, which co-segregates with stress-activated EGFR, also undergoes ubiquitin-independent sorting onto ILVs, but independently of ESCRTs and requiring the tetraspanin CD63 (ref. 15). However, surprisingly, and despite lack of EGFR ubiquitination, knockdown of the ESCRT-0 component, Hrs, or the ESCRT-I component Tsg101 (ref. 24) resulted in inhibition of ILV formation and a reduced density of UVC-exposed EGFR on MVBs, where it predominantly localized to the limiting membranes (Fig. 4f, quantified in Supplementary Fig. 4a and validated with alternative siRNA sequences in Supplementary Fig. 4b). Presumably, for stress-internalized EGFR, the ESCRT-0 complex enables membrane recruitment of ESCRT-I rather than ubiquitinated cargo recognition24. Recent reports have shown that binding to any ESCRT can mediate ubiquitin-independent cargo sorting27, and the existence of a ESCRT-dependent, ubiquitin-independent pathway of ILV cargo sorting that depends on the ESCRT adaptor ALIX2829. However, no role for ALIX has been demonstrated in sorting of EGF-stimulated EGFR onto ILVs or its targeting to lysosomes for degradation30. ALIX is recruited to MVBs by binding to the rare lipid lyso-bisphosphatidic acid (LBPA)30, which we previously showed to be absent from EGF-stimulated EGFR-containing MVBs11, but present in MVBs containing OA1 (ref. 16). Here we found extensive co-localization of stress-internalized, but not EGF-stimulated EGFR with LBPA in serum-starved cells (Fig. 5a). Consistently, ALIX knockdown in UVC-exposed cells resulted in a similar phenotype to that of Hrs and Tsg101 knockdown, in that UVC-exposed EGFR localized predominantly to the limiting membrane of MVBs containing fewer ILVs and reduced overall EGFR density (Fig 5b,c, quantified in Supplementary Fig. 4a and validated with an alternative siRNA sequence in Supplementary Fig. 4c). In contrast, ALIX depletion had no clear effect on MVBs containing EGF-stimulated EGFR. This is the first demonstration that ALIX is required for sorting of stress-internalized EGFR onto ILVs, but, in agreement with previous studies, ALIX is dispensable for sorting of ubiquitinated EGF-bound EGFR onto ILVs30.


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)

ALIX is required for sorting into ILVs and retention in MVBs of stress-internalized, but not EGF-bound EGFR.(a, left) Co-staining of EGFR (green) with LBPA (red) showed little co-localization in serum-starved HeLa cells treated with EGF for 30 min, but considerable overlap in cells 1 h after UVC exposure. Note that, in serum-free conditions, LBPA does not accumulate in lysosomes, facilitating the detection of MVB-specific labelling. (a, right) Quantification of co-localization of EGFR with LBPA in EGF-treated versus UVC-exposed serum-starved HeLa cells. Data are mean±s.e.m., ***P<0.001 (Student's t-test). (b) Lysates from HeLa cells transfected with control or ALIX siRNA were immunoblotted after 72 h for ALIX and Rab11 (as a loading control) to assess knockdown efficiency. (c) ALIX siRNA-treated cells were stimulated with EGF for 30 min, or exposed to UVC and incubated for 1 h, in the presence of anti-EGFR-gold, before EM processing. Ultrathin sections show gold (arrows) on ILVs of densely packed MVBs after EGF stimulation, but mainly on the limiting membrane of enlarged MVBs containing few ILVs in UVC-exposed cells. (d) Control, Tsg101, ALIX or Tgs101+ALIX siRNA-treated HeLa cells were exposed to UVC, incubated for 1 h and fixed, or further treated with PITSTOPII for 1 h. Immunostaining for EGFR (green) shows that depletion of Tsg101 or ALIX inhibits perinuclear EGFR accumulation, whereas EGFR redistributes to the plasma membrane upon PITSTOPII treatment, indicating that Tsg101 and ALIX are required for intracellular retention of EGFR. EGFR is found in very large vacuoles in double Tsg101+ALIX knocked-down cells after UVC exposure, before redistribution to the plasma membrane upon PITSTOPII treatment. Scale bars, 10 μm for confocal and 100 nm for EM pictures; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.
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f5: ALIX is required for sorting into ILVs and retention in MVBs of stress-internalized, but not EGF-bound EGFR.(a, left) Co-staining of EGFR (green) with LBPA (red) showed little co-localization in serum-starved HeLa cells treated with EGF for 30 min, but considerable overlap in cells 1 h after UVC exposure. Note that, in serum-free conditions, LBPA does not accumulate in lysosomes, facilitating the detection of MVB-specific labelling. (a, right) Quantification of co-localization of EGFR with LBPA in EGF-treated versus UVC-exposed serum-starved HeLa cells. Data are mean±s.e.m., ***P<0.001 (Student's t-test). (b) Lysates from HeLa cells transfected with control or ALIX siRNA were immunoblotted after 72 h for ALIX and Rab11 (as a loading control) to assess knockdown efficiency. (c) ALIX siRNA-treated cells were stimulated with EGF for 30 min, or exposed to UVC and incubated for 1 h, in the presence of anti-EGFR-gold, before EM processing. Ultrathin sections show gold (arrows) on ILVs of densely packed MVBs after EGF stimulation, but mainly on the limiting membrane of enlarged MVBs containing few ILVs in UVC-exposed cells. (d) Control, Tsg101, ALIX or Tgs101+ALIX siRNA-treated HeLa cells were exposed to UVC, incubated for 1 h and fixed, or further treated with PITSTOPII for 1 h. Immunostaining for EGFR (green) shows that depletion of Tsg101 or ALIX inhibits perinuclear EGFR accumulation, whereas EGFR redistributes to the plasma membrane upon PITSTOPII treatment, indicating that Tsg101 and ALIX are required for intracellular retention of EGFR. EGFR is found in very large vacuoles in double Tsg101+ALIX knocked-down cells after UVC exposure, before redistribution to the plasma membrane upon PITSTOPII treatment. Scale bars, 10 μm for confocal and 100 nm for EM pictures; 4,6-diamidino-2-phenylindole (DAPI)-stained nuclei, blue.
Mentions: PMEL, which co-segregates with stress-activated EGFR, also undergoes ubiquitin-independent sorting onto ILVs, but independently of ESCRTs and requiring the tetraspanin CD63 (ref. 15). However, surprisingly, and despite lack of EGFR ubiquitination, knockdown of the ESCRT-0 component, Hrs, or the ESCRT-I component Tsg101 (ref. 24) resulted in inhibition of ILV formation and a reduced density of UVC-exposed EGFR on MVBs, where it predominantly localized to the limiting membranes (Fig. 4f, quantified in Supplementary Fig. 4a and validated with alternative siRNA sequences in Supplementary Fig. 4b). Presumably, for stress-internalized EGFR, the ESCRT-0 complex enables membrane recruitment of ESCRT-I rather than ubiquitinated cargo recognition24. Recent reports have shown that binding to any ESCRT can mediate ubiquitin-independent cargo sorting27, and the existence of a ESCRT-dependent, ubiquitin-independent pathway of ILV cargo sorting that depends on the ESCRT adaptor ALIX2829. However, no role for ALIX has been demonstrated in sorting of EGF-stimulated EGFR onto ILVs or its targeting to lysosomes for degradation30. ALIX is recruited to MVBs by binding to the rare lipid lyso-bisphosphatidic acid (LBPA)30, which we previously showed to be absent from EGF-stimulated EGFR-containing MVBs11, but present in MVBs containing OA1 (ref. 16). Here we found extensive co-localization of stress-internalized, but not EGF-stimulated EGFR with LBPA in serum-starved cells (Fig. 5a). Consistently, ALIX knockdown in UVC-exposed cells resulted in a similar phenotype to that of Hrs and Tsg101 knockdown, in that UVC-exposed EGFR localized predominantly to the limiting membrane of MVBs containing fewer ILVs and reduced overall EGFR density (Fig 5b,c, quantified in Supplementary Fig. 4a and validated with an alternative siRNA sequence in Supplementary Fig. 4c). In contrast, ALIX depletion had no clear effect on MVBs containing EGF-stimulated EGFR. This is the first demonstration that ALIX is required for sorting of stress-internalized EGFR onto ILVs, but, in agreement with previous studies, ALIX is dispensable for sorting of ubiquitinated EGF-bound EGFR onto ILVs30.

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