Fig. 5. Involvement of Src kinase in the progastrin-induced delocalisation of junction proteins. (A) In DLD-1 colorectal carcinoma cells basal Src-kinase activity (black bars) in Src immunoprecipitates of cell lysates was higher in VO (a) than in ASG clones (c). Furthermore, 5 nM recombinant human progastrin_6-80 (5 minutes) stimulated Src-kinase activity (white bars) in ASG clones (d) but not in VO clones (b). The specificity of this effect was shown by the lack of stimulation of Src kinase activity by 5 nM progastrin_6-80 (f) in cells transfected with a dominant-negative mutant of Src (DLD-1/Src^-/-) (e). (B) In IMGE-5 cells, which do not produce progastrin-derived peptides, basal Src kinase activity (black bars) (a,c) was significantly increased by 5 nM progastrin_6-80 (5 minutes) (white bars) in VO cells (b) but not in cells transfected with dominant-negative Src (IMGE-5/Src^-/-) (d). Bar, 20 µm. (C) Activation of Src was essential for the effect of progastrin on the delocalisation of TJ proteins, as in IMGE-5/Src^-/- cells, progastrin_6-80 (PG) caused little, if any, delocalisation of ZO-1 and symplekin compared with untreated cells (U). By contrast, in IMGE-5/Src^-/- cells, 5 nM progastrin_6-80 treatment still caused delocalisation of the AJ protein ß-catenin (ß-cat). (D) The partial dissociation of the complex between ZO-1 (black bars) and occludin (Occl, white bars) induced in VO clones (a) by 5 nM progastrin_6-80 (b) was completely abolished in the IMGE-5/Src^-/- cells (c,d), as assessed by densitometric scanning of western blots of ZO-1 immunoprecipitates of cell lysates. (E) By contrast, expression of the Src dominant-negative mutant in IMGE-5 cells (c) did not prevent the dissociation of the complex between ß-catenin (ß-cat, black bars) and E-cadherin (E-cad, white bars) induced in VO cells (a) by 5 nM progastrin_6-80 (b,d), as assessed by densitometric scanning of western blots of ß-catenin immunoprecipitates of cell lysates. In A, B, D and E, 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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