Filamin (280-kDa actin-binding protein) is a caspase substrate and is alsocleaved directly by the cytotoxic T lymphocyte protease granzyme B during apoptosis

We used yeast two-hybrid screening to identify the cytoskeletal protein fil amin as a ligand for the proapoptotic protease granzyme B, produced by cyto toxic T lymphocytes. Filamin was directly cleaved by granzyme B when target cells were exposed to granzyme B and the lytic protein perforin, but it wa s also cleaved in a caspase-dependent manner following the ligation of Fas receptors. A similar pattern of filamin cleavage to polypeptides of similar to 110 and 95 kDa was observed in Jurkat cells killed by either mechanism. However, filamin cleavage in response to granzyme B was not inhibited by t he caspase inhibitor z-Val-Ala-Asp-fluoromethylketone at concentrations tha t abolished DNA fragmentation. Filamin staining was redistributed from the cell membrane into the cytoplasm of Jurkat cells exposed to granzyme B and perforin and following Ligation of Fas receptors, coincident with the morph ological changes of apoptosis. Filamin-deficient human melanoma cells were significantly (although not completely) protected from granzyme B-mediated death compared with isogenic filamin-expressing cells, both in clonogenic s urvival and Cr-51 release assays, whereas death from multiple other stimuli was not affected by filamin deficiency. Thus, filamin is a functionally im portant substrate for granzyme B, as its cleavage may account at least part ly for caspase-independent cell death mediated by the granzyme.