ECHISTATIN INDUCES DECREASE OF PP125(FAK) PHOSPHORYLATION, DISASSEMBLY OF ACTIN CYTOSKELETON AND FOCAL ADHESIONS, AND DETACHMENT OF FIBRONECTIN-ADHERENT MELANOMA-CELLS

B16-BL6 mouse melanoma cells cultured on fibronectin-coated dishes wer e detached by treatment with echistatin, an RGD-containing disintegrin , Echistatin was active at micromolar concentrations and was not cytot oxic, Its effect was dose-dependent and reversible, Sequential morphol ogical changes leading to rounding up of the cells were detected by ph ase-contrast microscopy and by immunofluorescence analysis. A dramatic reduction in the number and size of focal adhesions and loss of cytop lasmic actin filaments were observed well before cell detachment occur red, Echistatin treatment down-regulated the phosphorylation of pp125( FAK) in fibronectin-adherent cells in a dose- and time-dependent fashi on, The reduction of pp125(FAK) phosphorylation preceded cell detachme nt and occurred even in the presence of orthovanadate, an inhibitor of protein tyrosine phosphatases. These results suggest that echistatin detaches cells from the fibronectin substratum by inducing a decrease of pp125(FAK) phosphorylation and that echistatin acts by inhibiting p rotein tyrosine kinase activity rather than activating protein tyrosin e phosphatases.