Simultaneous Degradation of αII- and βII-Spectrin by Caspase 3 (CPP32) in Apoptotic Cells

The degradation of αII- and βII-spectrin during apoptosis in cultured human neuroblastoma SH-SY5Y cells was investigated. Immunofluorescent staining showed that the collapse of the cortical spectrin cytoskeleton is an early event following staurosporine challenge. This collapse correlated with the generation of a series of prominent spectrin breakdown products (BDPs) derived from both αII- and βII-subunits. Major C-terminal αII-spectrin BDPs were detected at ≈150, 145, and 120 kDa (αII-BDP150, αII-BDP145, and αII-BDP120, respectively); major C-terminal βII-spectrin BDPs were at ≈110 and 85 kDa (βII-BDP110 and βII-BDP85, respectively). N-terminal sequencing of the major fragments produced in vitro by caspase 3 revealed that αII-BDP150 and αII-BDP120 were generated by cleavages at DETD1185*S1186 and DSLD1478*S1479, respectively. For βII-spectrin, a major caspase site was detected at DEVD1457*S1458, and both βII-BDP110 and βII-BDP85 shared a common N-terminal sequence starting with Ser1458. An additional cleavage site near the C terminus, at ETVD2146*S2147, was found to account for βII-BDP85. Studies using specific caspase or calpain inhibitors indicate that the pattern of spectrin breakdown during apoptosis differs from that during non-apoptotic cell death. We postulate that in concert with calpain, caspase rapidly targets critical sites in both αII- and βII-spectrin and thereby initiates a rapid dissolution of the spectrin-actin cortical cytoskeleton with apoptosis.