Molecular mapping of the conserved physiological cell death pathway

Here I describe evidence of a conserved cell death mechanism. Genetic and quantitative biochemical analyses of T, B, and HeLa cell deaths reveal a conserved pattern of requisite components. I have dissected the role of caspases in cell death to reflect two obligate classes of cytoplasmic activities functioning in an amplifying cascade, with upstream ICE-like proteases activating downstream caspase 3-like caspases. Bcl-2 spares cells from death by punctuating this cascade, preventing activation of downstream caspases while leaving upstream activity undisturbed. This observation permits an operational definition of the stages of the cell death process. Upstream steps, which are necessary but not themselves lethal, are modulators of the death process. Downstream steps are effectors of, and not dissociable from, actual death; the irreversible commitment to cell death reflects the initiation of this downstream phase. The effector phase of death involves the activation in the nucleus of cell cycle kinases of the cyclin-dependent kinase (Cdk) family. Nuclear recruitment and activation of Cdk components is dependent on the caspase cascade, suggesting that catastrophic Cdk activity may be the actual effector of cell death. I demonstrate that cdk activity is necessary for the physiological cell death response in HeLa cells. The detection of active effector caspase in cells spared from death by overexpression of dominant negative cyclin dependent kinase mutants (DN-Cdks) is direct evidence that death associated cdk's are acting downstream of the requisite caspase cascade. in cells spared from death by inhibition of cdk activity, caspases are active and still functional as evidenced by the appearance of a subset of caspase dependent events. Despite the loss of mitochondrial membrane potential and destruction of the asymmetric distribution of phosphatidyl serine across the cytoplasmic membrane, the inhibition of cdk activity prevents death. This suggests that downstream effector caspase activity is necessary but not sufficient for the cell death response and that a requisite function of the proteolytic caspase cascade is the activation of effector cdk's. The ordered course of events that we have observed reflects essential thematic elements of a conserved sequence of modulatory and effector activities comprising a common pathway of physiological cell death.