Fig. 7. Involvement of PKC in the progastrin-induced cytoplasmic shift of ß-catenin. (A) Of an EGFP-tagged PKC isoform was transiently overexpressed in wild-type IMGE-5 cells (a). Treatment of EGFP-PKC transfected cells with 5nM progastrin_6-80 for 15 minutes (b) or 30 minutes (c), or with phorbol dibutyrate for 30 minutes (d), induced a delocalisation of EGFP-PKC to a perinuclear area and to the plasma membrane. Preincubation with the PI3-kinase inhibitor LY 294002 (f), for 30 minutes before, and during, progastrin stimulation prevented the delocalisation of EGFP-PKC to the membrane. Preincubation with the Src kinase inhibitor PP2 (e), or the PKC inhibitor calphostin C (g) had no effect on delocalisation. Control cells were transfected with an empty pEGFP vector similar to the vector used for PKC overexpression (h) and stimulated with 5 nM progastrin_6-80 for up to 45 minutes (i). Bars, 10 µm for a and c, 5 µm for all other panels. (B) In wild-type IMGE-5 cells, treatment with 5 nM progastrin_6-80 was found to increase transiently the association between ß-catenin (top line), the p85 subunit of PI3-kinase (filled bars) and PKC (open bars), as detected by densitometric scanning of western blots of anti-ß-catenin immunoprecipitates of cell lysates. (C) In IMGE-5/Src^-/- cells, preincubation with calphostin C reversed the progastrin_6-80-induced cytoplasmic shift of ß-catenin (ß-cat) shown in Fig. 5C. By contrast, the changes in ZO-1 and symplekin (SYM) localisation induced by progastrin_6-80 were barely affected by preincubation with calphostin C in wild-type (F.H., unpublished) and IMGE-5/PI3-k^-/- clones (Fig. 6B). Bar, 20 µm. (D) Preincubation with calphostin C (Calph) partially inhibited the increase in paracellular permeability for [³H]mannitol induced by 5 nM progastrin_6-80 (PG). The increase in permeability was almost completely blocked by the Src kinase inhibitor PP2, but was unaffected by preincubation with the PI3-kinase inhibitor LY294002. In B and D, densitometric analysis represents the average of at least three experiments, and statistical significance was assessed by Student's t test. *P<0.05, **P<0.01.

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