Limits...
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

EGFR internalization and intracellular retention in a specific subset of MVBs is required for EGFR TK activation and delays onset of stress-induced apoptosis.(a) Immunoblotting HeLa lysates showed transient, strong EGFR-T669 phosphorylation and gradually increased EGFR-Y1068 phosphorylation after UVC exposure, compared with weak T669 and rapid, strong Y1068 signal after EGF stimulation. (b) Immunoblotting HeLa lysates pre-incubated for 30 min with SB, exposed to UVC and incubated in the continuous presence of SB, showed that EGFR-Y1068 phosphorylation requires p38 activity. (c) Immunoblotting HeLa lysates showed that 30 min pre-incubation with dynasore prevented EGFR-Y1068 and ERK1/2 phosphorylation induced 1 h post UVC exposure, but not that induced by 30 min exposure to EGF. (d) Immunoblotting HeLa lysates showed that AP2α siRNA treatment inhibited EGFR-Y1068 phosphorylation up to 1 h post UVC exposure. (e, top) Immunoblotting PAE lysates showed that EGF-stimulated Y1068-phosphorylation is similar in PAE EGFR-ΔAP2 and -wt cells. However, although EGFR-wt showed increased Y1068-phosphorylation 15 min post UVC exposure, −ΔAP2 did not. Note that exposure time for p-EGFR Y1068 in EGF-stimulated samples has been reduced to avoid film saturation. (e, bottom) Quantification of EGFR-Y1068 phosphorylation from above. Data were normalized to control (untreated) EGFR-wt and are mean±s.e.m. of three independent experiments, *P<0.05 (Student's t-test). (f) Immunoblotting HeLa lysates showed that 30 min SB treatment after UVC-induced EGFR internalization reduced EGFR-Y1068 phosphorylation that was not rescued by Rab11 siRNA treatment. (g) Immunoblotting HeLa lysates showed that ALIX siRNA treatment reduced EGFR-Y1068 phosphorylation and prevented ERK1/2 phosphorylation 1 h post UVC exposure. (h, left) AP2α RNAi results in increased percentage of TUNEL-positive HeLa cells 2 h post UVC exposure compared with control RNAi. (h, right) PAE EGFR-ΔAP2 showed increased TUNEL-positive cells 8 h post UVC exposure compared with EGFR-wt cells. Data are mean±s.e.m. of three independent experiments, *P<0.05 (Student's t-test). (i) ALIX RNAi causes a similar increase compared with control RNAi to that caused by AP2α RNAi in the percentage of TUNEL-positive HeLa cells 2 h post UVC exposure. Data are mean±s.e.m. of three independent experiments, *P<0.05 (Student's t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4490399&req=5

f6: EGFR internalization and intracellular retention in a specific subset of MVBs is required for EGFR TK activation and delays onset of stress-induced apoptosis.(a) Immunoblotting HeLa lysates showed transient, strong EGFR-T669 phosphorylation and gradually increased EGFR-Y1068 phosphorylation after UVC exposure, compared with weak T669 and rapid, strong Y1068 signal after EGF stimulation. (b) Immunoblotting HeLa lysates pre-incubated for 30 min with SB, exposed to UVC and incubated in the continuous presence of SB, showed that EGFR-Y1068 phosphorylation requires p38 activity. (c) Immunoblotting HeLa lysates showed that 30 min pre-incubation with dynasore prevented EGFR-Y1068 and ERK1/2 phosphorylation induced 1 h post UVC exposure, but not that induced by 30 min exposure to EGF. (d) Immunoblotting HeLa lysates showed that AP2α siRNA treatment inhibited EGFR-Y1068 phosphorylation up to 1 h post UVC exposure. (e, top) Immunoblotting PAE lysates showed that EGF-stimulated Y1068-phosphorylation is similar in PAE EGFR-ΔAP2 and -wt cells. However, although EGFR-wt showed increased Y1068-phosphorylation 15 min post UVC exposure, −ΔAP2 did not. Note that exposure time for p-EGFR Y1068 in EGF-stimulated samples has been reduced to avoid film saturation. (e, bottom) Quantification of EGFR-Y1068 phosphorylation from above. Data were normalized to control (untreated) EGFR-wt and are mean±s.e.m. of three independent experiments, *P<0.05 (Student's t-test). (f) Immunoblotting HeLa lysates showed that 30 min SB treatment after UVC-induced EGFR internalization reduced EGFR-Y1068 phosphorylation that was not rescued by Rab11 siRNA treatment. (g) Immunoblotting HeLa lysates showed that ALIX siRNA treatment reduced EGFR-Y1068 phosphorylation and prevented ERK1/2 phosphorylation 1 h post UVC exposure. (h, left) AP2α RNAi results in increased percentage of TUNEL-positive HeLa cells 2 h post UVC exposure compared with control RNAi. (h, right) PAE EGFR-ΔAP2 showed increased TUNEL-positive cells 8 h post UVC exposure compared with EGFR-wt cells. Data are mean±s.e.m. of three independent experiments, *P<0.05 (Student's t-test). (i) ALIX RNAi causes a similar increase compared with control RNAi to that caused by AP2α RNAi in the percentage of TUNEL-positive HeLa cells 2 h post UVC exposure. Data are mean±s.e.m. of three independent experiments, *P<0.05 (Student's t-test).

Mentions: What is the functional consequence of the p38-, ESCRT- and ALIX-dependent sequestration of stress-exposed EGFR in this perinuclear subpopulation of MVBs? A previous study has suggested little EGFR activation and increased susceptibility to apoptosis following stress-induced EGFR internalization6, whereas others have reported opposing results31. We found that stress-induced EGFR internalization, which correlated with p38-specific EGFR-T669 phosphorylation previously shown to be a requisite for internalization31, triggered gradual EGFR TK activation, as shown by EGFR-Y1068 phosphorylation (Fig. 6a). EGFR TK activity was not required for its internalization, as cells pre-treated with the EGFR inhibitor gefitinib, which prevented UVC-induced EGFR-Y1068 phosphorylation (Supplementary Fig. 5a), still displayed receptor internalization following UVC, but not EGF, exposure (Supplementary Fig. 5b, quantified in Supplementary Fig. 5c). The gradual activation of stress-induced EGFR contrasted with the rapid, strong EGFR TK activity triggered by EGF binding, a situation that accounts for the above-mentioned difference in ubiquitination, known to require a threshold of receptor tyrosine phosphorylation32. Anisomycin-induced p38 activation was sufficient to trigger EGFR internalization and TK activity (Supplementary Fig. 5d). Conversely, p38 inhibition abrogated UVC-induced EGFR TK activity (Fig. 6b). To directly test the role of EGFR internalization in its activation after stress exposure, endocytosis was inhibited by dynamin inhibition (Fig. 6c and Supplementary Fig. 5f) or AP2α RNA interference (RNAi; Fig. 6d), as AP2α has previously been shown33, and confirmed in the present study (Supplementary Fig. 5g), to be required for UVC/cisplatin-dependent EGFR internalization. Both treatments abolished stress-induced EGFR activation. Conversely, dynamin inhibition did not affect EGF-stimulated EGFR activity or ERK1/2 phosphorylation, whereas the latter was abolished by dynasore in UVC-exposed cells (Fig. 6c). The specificity for EGFR internalization versus other endocytosed cargoes was confirmed in PAE cells expressing EGFR-wt versus EGFR-ΔAP2, an EGFR mutant for AP2 binding34 that failed to undergo UVC-induced internalization (Supplementary Fig. 5h). We detected increased EGFR activity in EGFR-wt but not -ΔAP2 cells upon UVC exposure, whereas EGF triggered similar EGFR activation in EGFR-ΔAP2 and -wt cells (Fig. 6e). Thus, we show for the first time that although EGF induces EGFR TK activation on the plasma membrane1, stress-induced EGFR TK activation/signalling require its internalization.


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)

EGFR internalization and intracellular retention in a specific subset of MVBs is required for EGFR TK activation and delays onset of stress-induced apoptosis.(a) Immunoblotting HeLa lysates showed transient, strong EGFR-T669 phosphorylation and gradually increased EGFR-Y1068 phosphorylation after UVC exposure, compared with weak T669 and rapid, strong Y1068 signal after EGF stimulation. (b) Immunoblotting HeLa lysates pre-incubated for 30 min with SB, exposed to UVC and incubated in the continuous presence of SB, showed that EGFR-Y1068 phosphorylation requires p38 activity. (c) Immunoblotting HeLa lysates showed that 30 min pre-incubation with dynasore prevented EGFR-Y1068 and ERK1/2 phosphorylation induced 1 h post UVC exposure, but not that induced by 30 min exposure to EGF. (d) Immunoblotting HeLa lysates showed that AP2α siRNA treatment inhibited EGFR-Y1068 phosphorylation up to 1 h post UVC exposure. (e, top) Immunoblotting PAE lysates showed that EGF-stimulated Y1068-phosphorylation is similar in PAE EGFR-ΔAP2 and -wt cells. However, although EGFR-wt showed increased Y1068-phosphorylation 15 min post UVC exposure, −ΔAP2 did not. Note that exposure time for p-EGFR Y1068 in EGF-stimulated samples has been reduced to avoid film saturation. (e, bottom) Quantification of EGFR-Y1068 phosphorylation from above. Data were normalized to control (untreated) EGFR-wt and are mean±s.e.m. of three independent experiments, *P<0.05 (Student's t-test). (f) Immunoblotting HeLa lysates showed that 30 min SB treatment after UVC-induced EGFR internalization reduced EGFR-Y1068 phosphorylation that was not rescued by Rab11 siRNA treatment. (g) Immunoblotting HeLa lysates showed that ALIX siRNA treatment reduced EGFR-Y1068 phosphorylation and prevented ERK1/2 phosphorylation 1 h post UVC exposure. (h, left) AP2α RNAi results in increased percentage of TUNEL-positive HeLa cells 2 h post UVC exposure compared with control RNAi. (h, right) PAE EGFR-ΔAP2 showed increased TUNEL-positive cells 8 h post UVC exposure compared with EGFR-wt cells. Data are mean±s.e.m. of three independent experiments, *P<0.05 (Student's t-test). (i) ALIX RNAi causes a similar increase compared with control RNAi to that caused by AP2α RNAi in the percentage of TUNEL-positive HeLa cells 2 h post UVC exposure. Data are mean±s.e.m. of three independent experiments, *P<0.05 (Student's t-test).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490399&req=5

f6: EGFR internalization and intracellular retention in a specific subset of MVBs is required for EGFR TK activation and delays onset of stress-induced apoptosis.(a) Immunoblotting HeLa lysates showed transient, strong EGFR-T669 phosphorylation and gradually increased EGFR-Y1068 phosphorylation after UVC exposure, compared with weak T669 and rapid, strong Y1068 signal after EGF stimulation. (b) Immunoblotting HeLa lysates pre-incubated for 30 min with SB, exposed to UVC and incubated in the continuous presence of SB, showed that EGFR-Y1068 phosphorylation requires p38 activity. (c) Immunoblotting HeLa lysates showed that 30 min pre-incubation with dynasore prevented EGFR-Y1068 and ERK1/2 phosphorylation induced 1 h post UVC exposure, but not that induced by 30 min exposure to EGF. (d) Immunoblotting HeLa lysates showed that AP2α siRNA treatment inhibited EGFR-Y1068 phosphorylation up to 1 h post UVC exposure. (e, top) Immunoblotting PAE lysates showed that EGF-stimulated Y1068-phosphorylation is similar in PAE EGFR-ΔAP2 and -wt cells. However, although EGFR-wt showed increased Y1068-phosphorylation 15 min post UVC exposure, −ΔAP2 did not. Note that exposure time for p-EGFR Y1068 in EGF-stimulated samples has been reduced to avoid film saturation. (e, bottom) Quantification of EGFR-Y1068 phosphorylation from above. Data were normalized to control (untreated) EGFR-wt and are mean±s.e.m. of three independent experiments, *P<0.05 (Student's t-test). (f) Immunoblotting HeLa lysates showed that 30 min SB treatment after UVC-induced EGFR internalization reduced EGFR-Y1068 phosphorylation that was not rescued by Rab11 siRNA treatment. (g) Immunoblotting HeLa lysates showed that ALIX siRNA treatment reduced EGFR-Y1068 phosphorylation and prevented ERK1/2 phosphorylation 1 h post UVC exposure. (h, left) AP2α RNAi results in increased percentage of TUNEL-positive HeLa cells 2 h post UVC exposure compared with control RNAi. (h, right) PAE EGFR-ΔAP2 showed increased TUNEL-positive cells 8 h post UVC exposure compared with EGFR-wt cells. Data are mean±s.e.m. of three independent experiments, *P<0.05 (Student's t-test). (i) ALIX RNAi causes a similar increase compared with control RNAi to that caused by AP2α RNAi in the percentage of TUNEL-positive HeLa cells 2 h post UVC exposure. Data are mean±s.e.m. of three independent experiments, *P<0.05 (Student's t-test).
Mentions: What is the functional consequence of the p38-, ESCRT- and ALIX-dependent sequestration of stress-exposed EGFR in this perinuclear subpopulation of MVBs? A previous study has suggested little EGFR activation and increased susceptibility to apoptosis following stress-induced EGFR internalization6, whereas others have reported opposing results31. We found that stress-induced EGFR internalization, which correlated with p38-specific EGFR-T669 phosphorylation previously shown to be a requisite for internalization31, triggered gradual EGFR TK activation, as shown by EGFR-Y1068 phosphorylation (Fig. 6a). EGFR TK activity was not required for its internalization, as cells pre-treated with the EGFR inhibitor gefitinib, which prevented UVC-induced EGFR-Y1068 phosphorylation (Supplementary Fig. 5a), still displayed receptor internalization following UVC, but not EGF, exposure (Supplementary Fig. 5b, quantified in Supplementary Fig. 5c). The gradual activation of stress-induced EGFR contrasted with the rapid, strong EGFR TK activity triggered by EGF binding, a situation that accounts for the above-mentioned difference in ubiquitination, known to require a threshold of receptor tyrosine phosphorylation32. Anisomycin-induced p38 activation was sufficient to trigger EGFR internalization and TK activity (Supplementary Fig. 5d). Conversely, p38 inhibition abrogated UVC-induced EGFR TK activity (Fig. 6b). To directly test the role of EGFR internalization in its activation after stress exposure, endocytosis was inhibited by dynamin inhibition (Fig. 6c and Supplementary Fig. 5f) or AP2α RNA interference (RNAi; Fig. 6d), as AP2α has previously been shown33, and confirmed in the present study (Supplementary Fig. 5g), to be required for UVC/cisplatin-dependent EGFR internalization. Both treatments abolished stress-induced EGFR activation. Conversely, dynamin inhibition did not affect EGF-stimulated EGFR activity or ERK1/2 phosphorylation, whereas the latter was abolished by dynasore in UVC-exposed cells (Fig. 6c). The specificity for EGFR internalization versus other endocytosed cargoes was confirmed in PAE cells expressing EGFR-wt versus EGFR-ΔAP2, an EGFR mutant for AP2 binding34 that failed to undergo UVC-induced internalization (Supplementary Fig. 5h). We detected increased EGFR activity in EGFR-wt but not -ΔAP2 cells upon UVC exposure, whereas EGF triggered similar EGFR activation in EGFR-ΔAP2 and -wt cells (Fig. 6e). Thus, we show for the first time that although EGF induces EGFR TK activation on the plasma membrane1, stress-induced EGFR TK activation/signalling require its internalization.

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