BCR-induced Apoptosis Involves Differential Regulation of C sub(16) and C sub(24)-Ceramide Formation and Sphingolipid-dependent Activation of the Proteasome

In this study, we describe an ordered formation of long- and very long-chain ceramide species in relation to the progression of B-cell receptor (BCR) triggering induced apoptosis. An early and caspase-independent increase in long-chain ceramide species, in which C sub(16)- ceramide predominated, was observed 6 h after BCR triggering. In contrast, very long-chain ceramide species were generated later, 12-24 h after BCR triggering. The formation of these very long-chain ceramide species, in which C sub(24)-ceramide predominated, required the activation of effector caspases. BCR-induced formation of long-chain ceramide species resulted in proteasomal activation and degradation of XIAP and subsequent activation of effector caspases, demonstrating an important cell-biological mechanism through which long-chain ceramides may be involved in the progression of BCR triggering induced apoptosis and subsequent formation of very long-chain ceramide species. BCR-induced activation of the proteasome was blocked with ISP-1/myriocin, a potent and selective inhibitor of serine palmitoyl transferase that catalyzes the first and rate-limiting step in the de novo formation of ceramide. Both ISP-1 and clasto-lactacystin beta -lactone, an irreversible inhibitor of the proteasome, prevented BCR cross-linking-induced XIAP degradation. Also, a mutant XIAP lacking the ubiquitin-ligating ring finger motif was completely resistant to proteasome-mediated degradation, and Ramos cells overexpressing XIAP became highly resistant to BCR cross-linking-induced activation of caspases. The formation of C sub(16)-ceramide in response to BCR cross-linking was found unaltered in XIAP overexpressing Ramos cells, whereas C sub(24)-ceramide formation was completely abolished. These results demonstrate how de novo generated long-chain ceramide species may be involved in the activation of downstream effector caspases and subsequent formation of very long-chain ceramide species. As such, these results provide novel and important insights into the significance of specific ceramide species in defined stages of apoptosis.