GDP dissociation inhibitor D4-GDI (Rho-GDI 2), but not the homologous Rho-GDI 1, is cleaved by caspase-3 during drug-induced apoptosis

Different cytotoxic drugs induce cell death by activating the apoptotic pro gramme; a family of cysteinyl aspartate proteases named caspases has been s hown to be involved in the initiation as well as the execution of this kind of cell death. In the present study, cleavage of D4-GDI (Rho-GDI 2), an ab undant haemopoietic-cell GDP dissociation inhibitor for the Ras-related Rho family GTPases, was demonstrated after treatment of BJAB Burkitt-like lymp homa cells with taxol or epirubicin. The cleavage of D4-GDI occurred simult aneously with the activation of caspase-3 but preceded DNA fragmentation an d the morphological changes associated with apoptotic cell death. By using high-resolution two-dimensional gel electrophoresis it was shown that this cleavage is specific: whereas the level of the homologous protein Rho-GDI 1 was not significantly altered during drug-induced apoptosis and in cytochr ome c/dATP-activated cellular extracts, D4-GDI disappeared owing to proteol ytic cleavage. Inhibitor experiments with Z-DEVD-fmk (in which Z stands for benzyloxycarbonyl and fmk for fluoromethyl ketone) and microsequencing of the D4-GDI fragment revealed that this occurs at the caspase-3 cleavage sit e. Our results strongly suggest the differential regulation of the homologo us GDP dissociation inhibitors Rho-GDI 1 and D4-GDI during drug-induced apo ptosis by proteolysis mediated by caspase-3 but not by caspase-1. Owing to their crucial role as modulators of Rho GTPases, this might in turn have a significant impact on the mechanisms that induce the cytoskeletal and morph ological changes in apoptotic cells.