A molecular approach for analysis of the effects of substrate cleavage

For polypeptides that are thought to be inactivated by caspase-mediated cleavage, expression of non-cleavable isoforms (e.g. in which the Pi aspartate has been mutated in vitro to an alanine) can provide some evidence for the role of cleavage. This is illustrated by the effect of non-cleavable lamins cited above.

For polypeptides that are thought to be activated by caspase-mediated cleavage (e.g. kinases or other enzymes), forced expression of the putative cleavage product can provide some insight into the biological role of the cleavage. For example, caspase-3 cleaves protein kinase C8 between its regulatory and catalytic domains to yield a C-terminal fragment with constitutive kinase activity (46). Overexpression of this C-terminal fragment induces apoptotic morphological changes in cells, whereas overexpression of the full-length polypeptide does not (47). By itself, this experiment simply demonstrates that the overexpressed C-terminal fragment is toxic to cells. However, when combined with antisense experiments demonstrating that the downregulation of protein kinase CS inhibits apoptosis (47), the net result is strong evidence supporting an active role for the protein kinase C8 fragment in apoptotic changes. A similar approach has been utilized to determine the biological effects of other caspase-generated enzyme fragments (1).

It should be noted, however, that the preceding experiments demonstrate a role for the caspase-generated enzyme fragment during apoptosis but do not elucidate that role. Although caspase activation has been shown to generate a variety of constitutively active kinase fragments (1), the substrates of these kinases remain to be identified; and the manner in which phosphorylation of these substrates contributes to the apoptotic process remains to be clarified. These are questions that are potentially difficult to address using a molecular approach.

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