Novel Mechanisms Controlling Caspase-9 Processing and Activation

University Of Minnesota-Twin Cities, Minneapolis MN

Investigators

Abstract

Apoptosis, an essential component of normal development in higher organisms, is a quick and neat form of cell suicide that causes minimal disruption to the surrounding tissue. Once activated, apoptosis is amplified by the controlled activation of a number of proteolytic enzymes, known as caspases. Two major pathways of caspase activation have been described - one involves the release of polypeptides, including cytochrome c, from mitochondria, and the other involves cell surface 'death' receptor activation by ligand binding. It has been widely accepted that the release of mitochondrial cytochrome c into the cytoplasm is essential for the activation of caspase-9, the 'initiator' caspase in the mitochondrial apoptotic pathway. Death receptor activated apoptosis, on the other hand, can follow either an extra-mitochondrial route, when large amounts of the initiator caspase-8 cleave and directly activate downstream 'effector' caspases in the cytosol, or a mitochondrial path, when low levels of active caspase-8 promote the release of mitochondrial cytochrome c by cleaving and activating a protein from the Bcl-2 family known as Bid. The pro-apoptotic effects of Bid are effectively neutralized in cells expressing high levels of survival proteins of the same family, such as the Bcl-xL protein, which can prevent cytochrome c release. However, some cell types that utilize the mitochondrial route in their primary response to ligand receptor-activated apoptosis die a rapid death despite the presence of high levels of the Bcl-xL protein. The research is directed towards understanding the mechanisms underlying this novel pathway to cell death. Early studies indicate that caspase-9 can be processed, by caspase-8, into an active form prior to cytochrome c release during death receptor-induced apoptosis. Preliminary observations also suggest that the processing site on caspase-9 is protected from mistimed or inappropriate cleavage by phosphorylation. The project is designed to test the hypothesis that the apoptosis initiator protein, caspase-9, can be processed and activated in a cytochrome c-independent manner by caspase-8 at a cleavage site that is regulated by phosphorylation. A variety of molecular, cellular and biochemical approaches will be utilized to dissect the two novel regulatory mechanisms i.e. the mechanism underlying cytochrome c-independent processing and activation of caspase-9 by active caspase-8, and the mechanism underlying the modulation of caspase-9 cleavage and activation by casein kinase 2, a ubiquitous cellular protein kinase. The research, in addition to providing a postdoctoral fellow and graduate student with crucial training, will expose undergraduate students of diverse backgrounds to state-of-the-art basic science techniques in an exciting research environment.