Limited proteolysis of filamin is catalyzed by caspase-3 in U937 and Jurkat cells

Members of the caspase family have been implicated as key mediators of apop tosis in mammalian cells. However, few of their substrates are known to hav e physiological significance in the apoptotic process. We focused our scree ning for caspase substrates on cytoskeletal. proteins. We found that an act in binding protein, filamin, was cleaved from 280 kDa to 170, 150, and 120 kDa major N-terminal fragments, and 135,120, and 110 kDa major C-terminal f ragments when apoptosis was induced by etoposide in both the human monoblas tic leukemia cell line U937, and the human T lymphoblastic cell line Jurkat . The cleavage of filamin was blocked by a cell permeable inhibitor of casp ase-3-like protease, Ac-DEVD-cho, but not by an inhibitor of caspase-1-like protease, Ae-YVAD-cho. These results suggest that filamin is cleaved by a caspase-3-like protease. To examine whether caspase-3 cleaves filamin in vi tro, we prepared a recombinant active form of caspase-3 directly using a Pi chia pastoris overexpression system. When we applied recombinant active cas pase-3 to the cell lysate of U937 and Jurkat cells, filamin was cleaved int o the same fragments seen in apoptosis-induced cells in vivo. Platelet fila min was also cleaved directly from 280 kDa to 170, 150, and 120 kDa N-termi nal fragments, and the cleavage pattern was the same as observed in apoptot ic human cells in vivo. These results suggest that filamin is an in vivo su bstrate of caspase-3.