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Downregulated AP-1 activity is associated with inhibition of Protein-Kinase-C-dependent CD44 and ezrin localisation and upregulation of PKC theta in A431 cells

¹ Cancer Research UK Beatson Laboratories, Garscube Estate, Switchback Road, Glasgow, G61 1BD Scotland
² The Netherlands Cancer Institute, Division of Cell Biology, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

View larger version (57K): [in a new window]   Fig. 1. CD44 and ezrin localisation in A431 cells. (A) A431 cells were serum starved for 2 days then processed for immunofluorescence using the E1/2 CD44 monoclonal antibody (a) and an anti-ezrin polyclonal antibody (b) (arrow indicates CD44-positive basal plaque; see text). (B) Serum-starved A431 cells were treated with 10 ng/ml EGF for 5 minutes before processing for immunofluorescence using the E1/2 CD44 antibody (a, b) or the ezrin antibody (c, d). Images (a, c and e) are confocal sections chosen to highlight membrane ruffling (open arrow), whereas images (b, d and f) are a higher confocal section showing microvilli (filled arrow). Panels e and f represent merged images (yellow) of a, c and b, d respectively. Bar, 10 µm.

View larger version (36K): [in a new window]   Fig. 2. CD44 localisation and expression in A431 cells expressing dominant-negative Jun. (A) NA and TA cells were serum starved for 2 days (a, c) or treated with EGF for 5 minutes (b, d), then processed for immunofluorescence using the E1/2 CD44 monoclonal antibody. Bar, 10 µm. (B) Whole cell extracts from serum-starved and EGF-treated NA and TA cells were prepared and analysed by western blotting using the E1/2 CD44 monoclonal antibody. CD44s is the standard 85 kDa isoform; CD44E indicates the 145 kDa, epithelial isoform.

View larger version (38K): [in a new window]   Fig. 3. Ezrin localisation and expression in A431 cells expressing TAM67. (A) NA and TA cells were serum starved for 2 days followed by treatment with 10 ng/ml EGF for 5 minutes. Cells were fixed then immunostained for ezrin distribution and counter-stained with phalloidin to visualise actin organization. Bar, 10 µm. (B) Whole cell extracts from serum-starved and EGF-treated NA and TA cells were prepared and analysed by western blotting using an ezrin polyclonal antibody.

View larger version (84K): [in a new window]   Fig. 4. Effect of Protein Kinase C inhibition on CD44 and ezrin localisation in A431 cells. NA cells were serum starved for 2 days then pretreated with or without 3 µM Ro-31-8220 for 30 minutes, followed by treatment with 10 ng/ml EGF for 5 minutes. Cells were fixed then immunostained for either CD44 (A) using the E1/2 monoclonal antibody and counterstaining with FITC-conjugated phalloidin or ezrin (B) using a polyclonal ezrin antibody and counterstaining with TRITC-conjugated phalloidin. Bar, 10 µm.

View larger version (63K): [in a new window]   Fig. 5. CD44 and ezrin localisation after phorbol ester treatment of NA and TA cells. NA cells (A, C) and TA cells (B, D) were serum starved for 2 days then treated with 100 ng/ml TPA for 5 minutes. After fixation, cells were immunostained for CD44 using the E1/2 monoclonal antibody (A, B, lower panels) or for ezrin using a polyclonal ezrin antibody (C, D). Cells were counterstained with phalloidin (A, B, upper panels) to visualise actin redistribution to cell-cell contacts (arrowheads). Bar, 10 µm.

View larger version (58K): [in a new window]   Fig. 6. Protein Kinase C isoform expression in NA and TA cells. (A) NA and TA cells were serum starved for 2 days then left untreated or treated with 10 ng/ml EGF for 5 minutes. Whole cell extracts were prepared and analysed by western blotting using antibodies for PKC, PKC, PKC and PKC. (B) Total RNA was prepared from NA and TA cells then 20 µg samples were electrophoresed, blotted then probed with a radiolabelled PCR fragment of human PKC. Equivalent loading of RNA samples was demonstrated using a 7S ribosomal cDNA probe.

View larger version (60K): [in a new window]   Fig. 7. Protein Kinase C activation in NA and TA cells. (A) NA and TA cells were serum starved for 2 days then either left untreated or treated with 10 ng/ml EGF or 100 ng/ml TPA for 5 minutes. Cells were scraped into lysis buffer and soluble (S) and insoluble (I) fractions were prepared as described in the Materials and Methods. Equal amounts of each fraction were electrophoresed on 10% SDS-PAGE gels and analysed by western blotting using antibodies recognising PKC, PKC, PKC and PKC. (B) PKC phosphorylation in NA and TA cells. Whole cell extracts were prepared from NA and TA cells that had been serum starved for 2 days then left either untreated or treated with 10 ng/ml EGF or 100 ng/ml TPA for 5 minutes. Western analysis followed using polyclonal antibodies recognising phosphoPKC (pan) antibody, which, in A431 cells, recognises phosphoPKC and phosphoPKC only. This was confirmed by probing adjacent lanes with PKC and PKC antibodies and showing co-migrating bands (data not shown).

View larger version (85K): [in a new window]   Fig. 8. PKC expression in A431 cells mimics aspects of TAM67 expression. (A) Protein lysates from A431, NA, TA and three A431- lines were analysed by western blotting to determine relative expression levels of PKC. The filter was reprobed with an Erk2 antibody to demonstrate equivalent protein levels in each lane. (B) Confluent monolayers of A431 and A431- cells (clones 24 and 36) were serum starved for 2 days, then wounded using a plastic tip. Cells were treated with 10 ng/ml EGF, then photographed after 36 hours.

View larger version (78K): [in a new window]   Fig. 9. PKC expression affects correct ezrin localisation in response to EGF. (A) A431 and A431- cells (clone 24) were plated onto coverslips, serum starved for 2 days, then left untreated or treated with 10 ng/ml EGF for 5 minutes. Cells were stained for actin (red) or ezrin (green) and analysed by confocal microscopy. (B) Confocal sections perpendicular to the substratum were taken to visualise ezrin localisation to microvilli (closed arrow) and membrane ruffles (open arrow).

View larger version (80K): [in a new window]   Fig. 10. EGF-induced CD44 localisation is disrupted in A431- cells. (A) A431 and A431- cells (clone 24) were plated onto coverslips, serum starved for 2 days, then left untreated or treated with 10 ng/ml EGF for 5 minutes. Cells were stained for actin (green) or CD44 (red) and analysed by confocal microscopy. (B) Confocal sections perpendicular to the substratum were taken to visualise CD44 distribution to apical structures.