THE SRC-FAMILY KINASE YES HAS A SELECTIVE ROLE IN THE REGULATION OF CELL-CELL ADHESIONS IN COLON CARCINOMA CELLS

INTRODUCTION. The activity of Src family kinases (SFKs) is progressively elevated in colon cancer from the colonic adenocarcinoma stage up to metastasis (1). This elevation is thought to be mainly due to deregulation of SFKs and not to mutations or over-expression. In HT29 colon carcinoma cells, SFK activity is high when compared to non-transformed cells and other colon cancer cell lines. Like Src, Yes is also deregulated in colon cancer, with higher Yes activity in adenomas with malignant potential (2). Using RNA interference we have examined the effect of a reduction in the levels of Yes or Src proteins on the morphology of HT29 cells. METHOD. HT29 cells (ATCC) were transiently transfected with siRNA duplexes targeting human Src or Yes (Upstate SmartPools). After 48h, cells were either photographed live or after fixing and labelling for immunofluorescence and confocal imaging. Parallel samples were used for immunoblotting and immunoprecipitation. RESULTS. Reduction of Yes protein levels is sufficient to induce changes in cell morphology although it only has a minor impact on the total SFK activity. Yes knock-down-induced changes are characterised by formation of multilayered cell aggregates (Figure 1) and an increase in cell-cell adhesion, similar to changes reported upon in vitro induction of HT29 enterocytic differentiation (3). This effect seems to be selective, and not due to a reduction in general SFK activity since Src knock-down only led to mild morphology changes, although it eliminates the majority of SFK activity. Multilayered aggregates are not caused by a reduction in the phosphorylation levels of either FAK or Paxillin, since only Src knock-down and not Yes knock-down had any impact on the phosphorylation of these SFK substrates. Conversely, Yes knock-