Fig. 6. PI3-kinase is involved in the progastrin-induced cytoplasmic shift of ß-catenin. (A) Basal Src kinase activity (black bars), assessed as described in the legend to Fig. 5, in IMGE-5 cells expressing a SH2 dominant-negative mutant of the p85 regulatory subunit of PI3-kinase (IMGE-5/PI3-k^-/-) (c) was similar to cells transfected with vector only (VO) (a). Furthermore, activation of Src kinase activity by 5 nM progastrin_6-80 (b,d, white bars) was not PI3-kinase dependent. (B) Treatment of IMGE-5/PI3-k^-/- cells with 5 nM progastrin_6-80 induced a partial or complete disappearance from the plasma membrane of ZO-1 and symplekin (SYM), respectively, compared with untreated (U) controls. The disappearance was prevented by preincubation with the Src kinase inhibitor PP2. Conversely, the cytoplasmic shift of ß-catenin (ß-cat) induced by progastrin_6-80 in wild-type or IMGE-5/Src^-/- cells was prevented in IMGE-5/PI3-k^-/- clones, as well as in IMGE-5/Src^-/- cells preincubated with the PI3-kinase inhibitor LY294002. In DLD-1/Src^-/- cells, ZO-1 was mostly located at the membrane, whereas DLD-1/PI3-k^-/- cells showed a largely cytoplasmic expression of E-cadherin (E-cad). Bar, 20 µm. (C) When compared with untreated cells (U, black bars), 5 nM progastrin_6-80 (PG, white bars) induced a partial dissociation of the occludin (Occl)/ZO-1 complex, assessed as described in the legend to Fig. 1, in IMGE-5/PI3-k^-/- cells (left panel), of similar amplitude to that observed in wild-type or IMGE-5/VO cells. However, dissociation of the E-cadherin (E-cad)/ß-catenin (ß-cat) complex after progastrin_6-80 treatment, assessed as described in the legend to Fig. 3, did not occur in IMGE-5/PI3-k^-/- clones (right panel). In A and C, densitometric analysis represents the average of at least three experiments, and statistical significance was assessed by Student's t test. *P<0.05.

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