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UV induces tyrosine kinase-independent internalisation and endosome arrest of the EGF receptor

¹ Laboratory for Toxicopathology, Institute of Pathology, The National Hospital, University of Oslo, N-0027 Oslo, Norway
² Neurochemical Laboratory, Department Group of Basic Medical Sciences, University of Oslo, N-0317 Oslo, Norway
³ Center for Cellular Stress Responses, University of Oslo, N-0027 Oslo, Norway

View larger version (54K): [in a new window]   Fig. 1. UV-exposed cells did not show increased EGFR tyrosine phosphorylation. Tyrosine phosphorylation was analysed in whole cell lysates (A) and EGFR immunoprecipitates (B) subject to SDS-PAGE and western immunoblotting with rabbit anti-pY992, pY1068, pY1086, pY1148 and pY1173. Cells were either left untreated or exposed to the indicated UV-doses and chased for the indicated time intervals (minutes). The whole cell lysates (A) were also analysed for tyrosine phosphorylation with a general anti-phosphotyrosine antibody (4G10; pTyr). The total levels of EGFR, determined with anti-EGFR antibodies, are shown (bottom line). Cells stimulated with 5 nM EGF for 5 minutes were used as a positive control. C and E denote control and EGF-stimulated cells, respectively, and J denotes J/m2. The molecular weight markers presented at the left indicate 220 kDa.

View larger version (49K): [in a new window]   Fig. 2. UV-exposed cells did not reveal increased phosphate incorporation in the EGF receptor. In vivo labelling of cells were performed by preincubation of cells with phosphate-free medium (MEM) for 2 hours, followed by incubation with [32P] orthophosphate in DMEM for 2 hours. Cells were then left untreated or exposed to UV or EGF, and incubated for the indicated time intervals. Cell lysates were subject to immunoprecipitation with an anti-EGFR-protein G-sepharose conjugate. The EGFR immunoprecipitates were separated by SDS-PAGE, and identified by (A) autoradiography and (B) western immunoblotting with anti-EGFR. Cells stimulated with 5 nM EGF for 5 minutes were used as positive controls. C and E denote control and EGF-stimulated cells, respectively. The molecular weight markers presented at the left indicate 220 kDa.

View larger version (52K): [in a new window]   Fig. 3. UV-irradiation did not induce dimerisation of the EGFR. Cells were left untreated, or exposed to 50 J/m2 UV (lanes 1,2), 200 J/m2 UV (lanes 3,4), or EGF (10 nM on ice for 15 minutes). The UV-exposed cells were either irradiated at 37°C and incubated for 2 minutes at the same temperature (lanes 1,3), or irradiated on ice and further incubated on ice for 15 minutes (lanes 2,4). All samples were incubated with the crosslinker BS3 on ice for 20 minutes. Cell lysates were subject to SDS-PAGE and western immunoblotting with anti-EGFR. The band with molecular mass around 340 kDa representing dimeric EGFR is indicated (arrow). C and E denote control and EGF-stimulated cells, respectively. The molecular weight marker presented at the left indicates 220 kDa.

View larger version (35K): [in a new window]   Fig. 4. UV-exposure induces EGFR mobility shift. (A) Cells were either left untreated (lane 0) or exposed to 200 J/m2 UV (lanes 1,3) or 5 nM EGF (lanes 2,4), and incubated for 1 hour (lanes 1,2) or 2 hours (lanes 3,4). Cell lysates were subject to SDS-PAGE and western immunoblotting with anti-EGFR. Unstimulated cells were used as controls. (B) Cells were either left untreated, or exposed to 200 J/m2 UV or 5 nM EGF, and incubated for 10 minutes before immunoprecipitation with anti-EGFR. The immunoprecipitates were incubated in DEA buffer with or without alkaline phosphatase (AP). The immunoprecipitates were subject to SDS-PAGE and western immunoblotting with either anti-pY1173 or anti-EGFR. EGFR immunoprecipitates from unstimulated cells were incubated in DEA buffer and used as controls. C, control cells. The molecular weight markers presented at the left indicate 220 kDa.

View larger version (69K): [in a new window]   Fig. 5. UV-exposed cells showed no induced polyubiquitination of the EGF receptor. Cells were either left untreated or treated with 5 nM EGF or 200 J/m2 UV, and incubated for the indicated time intervals (minutes). (A) The cell lysates were subject to SDS-PAGE and western immunoblotting with anti-EGFR. Polyubiquitination, identified as smears, was observed only in EGF-stimulated cells incubated for 1-10 minutes (arrows). The molecular weight marker presented at the left indicates 220 kDa. (B) Cells were either left untreated or exposed to 200 J/m2 UV or 5 nM EGF, and incubated for 10 minutes before immunoprecipitation with anti-Cbl. The immunoprecipitates were subjected to SDS-PAGE and western immunoblotting with either anti-Cbl (left) or anti-pTyr (right). C, U and E denote control, UV-exposed and EGF-stimulated cells, respectively. The molecular weight markers presented at the left indicate 220 and 96 kDa.

View larger version (128K): [in a new window]   Fig. 6. UV-exposed EGFR was internalised to early endosomes. Cells were either left untreated (A) or exposed to 200 J/m2 UV, and incubated for 10 minutes. (B-D). Samples were subject to immunofluorescense confocal microscopy with anti-EGFR (green), and anti-EEA1 to identify early endosomes (red). (A) In unstimulated cells EGFR was located along the plasma membrane. (B-D) Exposure to UV resulted in a redistribution of EGFR to vesicles (B) containing EEA1 (C, and double stained in D). The yellow colour indicates co-localisation. Bar, 10 µm.

View larger version (114K): [in a new window]   Fig. 7. UV-exposed EGFR was arrested in endosomes. (A-C) Cells were exposed to 200 J/m2 UV, incubated for 20 minutes, and stained with anti-EGFR. Immunofluorescense confocal microscopy localised the receptor to vesicles (A; red) that were negative for the late endosome marker CD63 (B; green, and double stained in C). (D-F) Cells were exposed to 200 J/m2 UV and chased for 60 minutes, with FITC-labelled transferrin present in the last 20 minutes. Staining with anti-EGFR showed a redistribution of the receptor to vesicles (D; red) containing transferrin (E; green, and double stained in F). The yellow colour indicates co-localisation. Representative vesicle aggregates containing EGFR and transferrin are indicated (arrow). Bar, 10 µm.

View larger version (47K): [in a new window]   Fig. 8. UV-induced EGFR internalisation was independent of the receptor tyrosine kinase activity. (Upper panels) Cells were either left untreated, stimulated with 5 nM EGF for 10 minutes, with or without preincubation in 100 nM PD153035 for 2 hours. Cell lysates were subject to SDS-PAGE and western immunoblotting with anti-pY1173 (left) or anti-EGFR (right). The specific EGFR tyrosine kinase inhibitor PD153035 effectively inhibited the EGF-mediated receptor tyrosine phosphorylation (left). Whereas pretreatment with PD153035 did not inhibit the UV-induced EGFR gel mobility shift, the mobility shift induced by EGF was inhibited (right). C and E denote control and EGF-stimulated cells, respectively. (Lower panel) Cells were either left untreated (A), stimulated with 5 nM EGF for 10 minutes (B), with pretreatment with PD153035 (C), or pretreated with PD153035 followed by exposure to 200 J/m2 UV, and incubated for 10 minutes (D). Immunofluorescense confocal microscopy with anti-EGFR demonstrated an EGF-induced redistribution of EGFR from the plasma membrane (A) to vesicles (B). The EGF-mediated receptor internalisation was inhibited by pretreatment with 100 nM PD153035 for 2 hours (C). Inhibition of the UV-induced EGFR internalisation was not observed with pretreatment with PD153035 (D). Bar, 10 µM.

View larger version (117K): [in a new window]   Fig. 9. The adaptor proteins Shc and Grb2 were not redistributed to vesicles after UV-exposure. Cells were either left untreated (A and B), stimulated with 5 nM EGF for 10 minutes (C,D), or exposed to 200 J/m2 (E,F). Immunofluorescence confocal microscopy with anti-Shc (A,C,E) and anti-Grb2 (B,D,F) was used to study the distribution of the adaptor proteins. In unstimulated cells, Shc and Grb2 showed a diffuse cytosolic distribution (A and B, respectively). In cells stimulated with EGF for 10 minutes, Shc and Grb2 were redistributed to vesicles (C and D, respectively). In cells exposed to 200 J/m2 UV and incubated for 10 minutes, no redistribution of Shc and Grb2 was observed (E and F, respectively). Bar, 10 µM.

View larger version (76K): [in a new window]   Fig. 10. Effects of UV on signal proteins downstream of EGFR. Cells were either left untreated or treated with 10, 50 or 200 J/m2 UV, or 5 nM EGF, and incubated for the indicated time intervals (minutes). The cell lysates were subject to SDS-PAGE and western immunoblotting with antibodies to the indicated proteins. (A) The cells were pretreated with PD153035 or left untreated as indicated, before stimulation with UV or EGF. Anti-Raf1 displayed no mobility shift for Raf in UV-exposed cells, in contrast to that seen in EGF-stimulated cells. The EGF-induced mobility shift for Raf1 was inhibited by treatment with PD153035. Anti-pMEK showed a UV-induced MEK-activation, that was not inhibited by treatment with PD153035. By contrast, PD153035 inhibited the EGF-induced MEK-activation. (B) Cells were treated with increasing doses of UV, and incubated for the indicated time intervals (minutes), before immunblotting. MEK and ERK showed activation 20 minutes after irradiation (pMEK and pERK, respectively). Anti-EGFR was used to identify the protein levels in the different samples. C, control cells. The molecular weight markers presented at the left indicate 220 kDa.

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