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First published online 17 February 2004
doi: 10.1242/jcs.00951

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Epidermal growth factor regulates ubiquitination, internalization and proteasome-dependent degradation of connexin43

Institute for Cancer Research at The Norwegian Radium Hospital, N-0310 Oslo, Norway

View larger version (22K): [in a new window]   Fig. 1. Effects of EGF on GJIC and the Cx43 phosphorylation pattern in IAR20 cells. GJIC was measured by quantitative scrape loading. For Cx43 band-pattern examination, cell lysates were prepared as indicated and subjected to SDS-PAGE and western blotting with anti-Cx43 antibodies. (A) Cells were exposed to 50 ng ml–1 EGF for different lengths of time. (B) Cells were exposed to increasing concentrations of EGF for 30 minutes. (C) Cells were exposed to increasing concentrations of PD98059 for 30 minutes, then to EGF at a final concentration of 50 ng ml–1 and incubated for 30 minutes, in the sustained presence of PD98059.

View larger version (124K): [in a new window]   Fig. 2. Effect of EGF on Cx43 localization in IAR20 cells. IAR20 cells were either left untreated (A) or treated with 50 ng ml–1 EGF for 30 minutes (B) or 60 minutes (C). (D) IAR20 cells were preincubated with 50 µM PD98059 for 30 minutes, then incubated with EGF at a final concentration of 50 ng ml–1 and incubated for 30 minutes in the sustained presence of PD98059. The cells were fixed, immunostained with anti-Cx43 antibodies and visualized using fluorescence microscopy.

View larger version (125K): [in a new window]   Fig. 3. Effects of cycloheximide and MG132 on EGF-induced relocalization of Cx43. IAR20 cells were either left untreated (A) or treated with 50 ng ml–1 EGF for 30 minutes (B). (C) Cells were preincubated with 10 µg ml–1 cycloheximide for 2 hours, then exposed to EGF at a final concentration of 50 ng ml–1 and incubated for 30 minutes in the sustained presence of cycloheximide. (D) Cells were preincubated with 10 µM MG132 for 30 minutes, then exposed to EGF at a final concentration of 50 ng ml–1 and incubated for 30 minutes in the sustained presence of MG132. Cells were fixed, immunostained with anti-Cx43 antibodies and visualized using fluorescence microscopy.

View larger version (37K): [in a new window]   Fig. 4. EGF induces degradation of Cx43. Cell lysates were prepared as indicated and equal amounts of total cell protein were subjected to SDS-PAGE and western blotting with anti-Cx43 antibodies. As gel loading controls, the blots were stripped and reprobed with anti-actin antibodies. (A) IAR20 cells were treated with 10 µg ml–1 cycloheximide (chx) alone or in combination with 100 ng ml–1 EGF. (B) The Cx43 band intensities on gels shown in (A) were measured using Scion Image. The Cx43 band intensities were plotted relative to the intensity of Cx43 bands in untreated cells. (C) Cells were treated with 10 µg ml–1 cycloheximide and increasing concentrations of EGF for 4 hours. (D) Cells were preincubated with increasing concentrations of PD98059 (µm) for 30 minutes before treatment with 10 µg ml–1 cycloheximide and 100 ng ml–1 EGF for 4 hours in the sustained presence of PD98059.

View larger version (39K): [in a new window]   Fig. 5. Effect of MG132 and leupeptin on EGF-induced Cx43 degradation. (A) IAR20 cells were treated with 10 µg ml–1 cycloheximide (chx) in combination with either 100 ng ml–1 EGF, 100 ng ml–1 EGF and 10 µM MG132, or 100 ng ml–1 EGF and 100 µM leupeptin. Cell lysates were prepared at the indicated time points and equal amounts of total cell protein were subjected to SDS-PAGE. Cx43 was detected by western blotting with anti-Cx43 antibodies. (B) The Cx43 band intensities on gels shown in (A) were measured using Scion Image. The Cx43 band intensities were plotted relative to the intensity of Cx43 bands in untreated cells.

View larger version (47K): [in a new window]   Fig. 6. Effect of EGF on ubiquitination of Cx43. IAR20 cells were treated with 100 ng ml–1 EGF for different lengths of time or preincubated with 50 µM PD98059 for 15 minutes before addition of 100 ng ml–1 EGF. Cell lysates were subjected to immunoprecipitation with anti-Cx43 antibodies or preimmune serum (PI) as control. Equal amounts of total cell protein were subjected to SDS-PAGE and western blotting using affinity-purified anti-ubiquitin antibodies (top) or anti-Cx43 antibodies (bottom).

View larger version (130K): [in a new window]   Fig. 7. Effect of hypertonic sucrose medium on EGF-induced internalization of Cx43. IAR20 cells were incubated with 50 ng ml–1 EGF for 45 minutes at 37°C in Hepes-buffered DMEM without (A) or with (B) 0.45 M sucrose. The cells were fixed, immunostained with anti-Cx43 antibodies and visualized using fluorescence microscopy. Cells were incubated with 50 µg ml–1 Alexa-488-transferrin in the absence (C) or presence (D) of 0.45 M sucrose for 45 minutes at 37°C. The cells were fixed and transferrin was visualized using fluorescence microscopy.

View larger version (46K): [in a new window]   Fig. 8. Cx43 is hyperphosphorylated and ubiquitinated in the presence of hypertonic sucrose medium. (A) Cells were either left untreated or treated with 50 ng ml–1 EGF for 15 minutes in the presence or absence of 0.45 M sucrose as indicated. Cell lysates were prepared and equal amounts of total cell protein were subjected to SDS-PAGE. Cx43 was detected by western blotting with anti-Cx43 antibodies. (B) Cells were either left untreated or treated with 50 ng ml–1 EGF for 15 minutes in the presence or absence of 0.45 M sucrose as indicated. Cell lysates were subjected to immunoprecipitation with anti-Cx43 antibodies. Equal amounts of total cell protein were subjected to SDS-PAGE and ubiquitin and Cx43 were detected by western blotting using affinity-purified anti-ubiquitin antibodies (top) and anti-Cx43 antibodies (bottom).

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