The proposed experiments will identify and characterize proteins that are regulated by oxidative stress, as this type of stress increases in human aging and often leads to apoptosis or cell death. We propose to characterize the activity of Bcl-XL after it is cleaved by caspases. Bcl-XL functions as an anti-apoptotic protein until it is proteolyzed by caspase activity. Once cleaved, Bcl-XL becomes a pro-apoptotic protein. The switch from an anti- to a pro-apoptotic protein is an irreversible commitment to cell death. In the absence of evidence to the contrary, it is generally believed that cleavage of Bcl-XL by caspases is a "loss of function" event. However, our preliminary data suggest that cleavage of Bcl-XL is a "gain of function" event, as we have discovered a physical interaction between delta61 Bcl-XL and MEKK3. Our hypothesis is that cleavage of Bcl-XL by caspases produces an intracellular ligand, delta61 Bcl-XL which interacts with the serine/threonine kinase, MEKK3. The specific aims of this proposal that will address our hypothesis are: I) Identify and characterize the signaling pathways that are downstream of delta61 Bcl-XL. We will express and purify a fusion protein from bacteria consisting of delta61 Bcl-XL and a cell permeable 16 amino acid sequence that is derived from the homeoprotein internalization domain of the Antennapedia protein. We will characterize the signaling pathways that are activated by delta61 Bcl-XL in the IL-2-dependent T lymphocyte cell line, CTLL-2; II) Characterize the activity of MEKK3 in the presence of its putative activator, delta61 Bcl-XL. We have identified a physical interaction between MEKK3 and delta61 Bcl-XL MEKK3 activates at least 3 different pathways, including the ERK, JNK, and NF-kappaB pathways. We will determine whether the interaction between delta61 Bcl-XL and MEKK3 affects the ability of MEKK3 to activate any of these pathways.
|Effective start/end date||4/1/00 → 3/31/01|
- National Institutes of Health: $75,750.00
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