|Figure 1. Activation of caspase-8 involves dimerization and auto-proteolytic activation. Possible step-wise proteolytic cleavage during auto-activation.|
Active caspases proteolyze additional caspases generating a caspase cascade. Initiation of the caspase cascade leads to a form of controlled cell death termed apoptosis. Caspases are present in all cells as latent enzymes. Recruitment of caspases to oligomerized receptors (e.g., TNF receptors, FAS and TRAIL receptors) or intracellular adaptor proteins (e.g., APAF-1) leads to activation via dimerization or dimerization accompanied by autoproteolytic cleavage.
Caspase-8, for example, cleaves itself during or after dimerization.1-3 In staurosporine-treated Jurkat cells, the first proteolytic cleavage appears to occur at the junction of the small and large subunits (see Figure 1) to generate polypeptides of 12 and 41 kDa, detected respectively by an antibody against the small subunit and an antibody against the large subunit (see Figure 2). Subsequent cleavage between the pro-region and the large subunit (see Figure 1b-1c) would generate polypeptides of 17 and 24 kDa. The large 17 kDa subunit can be detected by the antibody generated against the large subunit. If the alternative step-wise autoproteolytic pathway (see Figure 1d-1f ) involves a primary cleavage at the junction of the pro-region and large subunit, then antibodies against the large and small subunits would detect polypeptides of 29 kDa. Polypeptides of 29 kDa are not detected (see Figure 2) nor are significant amounts of the 17 kDa polypeptide detected. Thus, the major proteolytic processing of caspase-8 is cleavage at the junction of the large and small subunits.
|Figure 2. Processing of caspase-8. Western blots using an antigen-purified antibody against the 12 kDa subunit and a mouse monoclonal antibody (Catalog # MAB704) against the 17 kDa subunit give results consistent with the first cleavage occurring at the junction between the large and small subunit in Jurkat cells induced to undergo apoptosis (STS, staurosporine-treated). The small subunits of caspase-8 (12 kDa) are detected by the antibody generated against the small subunit (Catalog # AF832) and polypeptides of 42/43 kDa and 17 kDa are detected by the antibody generated against the large subunit (Catalog # MAB704). Significant amounts of the 17 kDa polypeptide are not detected.||Figure 3. Processing of caspase-3. Jurkat cells were treated with staurosporine for various times and 5X extracts from 5 x 105 cells were blotted with a goat polyclonal anti-caspase-3 (Catalog # AF-605-NA).|
Based on western blot data, caspase subunits are generated and are often further degraded during apoptosis (see Figure 3). This has been observed in many cell types induced to undergo apoptosis by various methods. Therefore, when examining the possible cleavage of a caspase in a cell line, the criteria is both the disappearance of the precursor and detection of the cleavage products. If a decrease in the precursor is detected without an increase in the detection of subunits, earlier time points following induction of apoptosis should be examined.