The Rho/Rho-kinase and the phosphatidylinositol 3-kinase pathways are essential for spontaneous locomotion of Walker 256 carcinosarcoma cells

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Abstract
Signal transduction pathways controlling spontaneous locomotion of Walker carcinosarcoma cells are not well understood. We have therefore investigated the role of signalling proteins in development of polarity and locomotion of these cells. Treatment of the cells with 100 ng/ml pertussis toxin had no significant effect on the percentage of polarized cells. In contrast, 2 different phosphatidylinositol 3-kinase inhibitors (wortmannin and LY-294002) markedly reduced the proportion of polarized cells. Spontaneous locomotion of the cells was also significantly inhibited by these two inhibitors. In agreement with these data, we observed localization of the p85α subunit of phosphatidylinositol 3-kinase predominantly in the membrane fraction of Walker carcinosarcoma cells, indicating constitutive activation of this enzyme. We also investigated a role of Rho family proteins. Spontaneous development of polarity was almost completely suppressed by electroporation of the cells in the presence of 4 μg/ml C3 exoenzyme, which specifically ADP-ribosylates and inactivates Rho. Two downstream targets of Rho, the Rho-activated kinases I and II, were also detected predominantly in the particulate membrane fraction, suggesting constitutive activation. A specific Rho-kinase inhibitor (Y-27632) blocked spontaneous polarization and migration in a concentration-dependent manner. Our results indicate that constitutive activation of the Rho/Rho-kinase pathway and of phosphatidylinositol 3-kinase plays an essential part in spontaneous development of polarity and cell locomotion of these cells.