Echistatin inhibits pp125(FAK) autophosphorylation, paxillin phosphorylation and pp125(FAK)-paxillin interaction in fibronectin-adherent melanoma cells

Echistatin, a snake-venom RGD-containing protein, was previously shown to d isrupt cell-matrix adhesion by a mechanism that involves the reduction of p p125(FAK) tyrosine phosphorylation levels. The aim of this study was to est ablish the sequence of events downstream pp125(FAK) dephosphorylation that could be responsible for echistatin-induced disassembly of actin cytoskelet on and focal adhesions in fibronectin-adherent B16-BL6 melanoma cells. The results obtained show that echistatin induces a decrease of both autophosph orylation and kinase activity of pp125(FAK). One hour of cell exposure to e chistatin caused a 39% decrease of pp125(FAK) Tyr397 phosphorylation and a 31% reduction of pp125(FAK) autophosphorylation activity as measured by imm une-complex kinase assay. Furthermore, 1 h of cell treatment by echistatin produced a 63% decrease of paxillin phosphorylation, as well as a reduction in the amount of paxillin bound to pp125(FAK). Immunofluorescence analysis of echistatin treated cells showed the concomitant disappearance of both p axillin and pp125(FAK) from focal adhesions. The reduction of paxillin phos phorylation may represent a critical step in the pathway by which disintegr ins exert their biological activity, including the inhibition of experiment al metastasis in vivo.