Apoptosis is a program of cellular suicide wherein individual cells are removed from the midst of a living tissue without destroying overall tissue architecture. In response to apoptotic signals, cells activate a family of intracellular proteases known as caspases, which participate in the orderly dismantling and packaging of the dying cell. Under many circumstances (e.g. following treatment of cells with chemotherapeutic agents), caspase activation proceeds through a mitochondrial-dependent pathway, leading to translocation of the respiratory chain component, cytochrome c, from the mitochondria to the cytoplasm. Once cytoplasmic, cytochrome c interacts with an apoptotic regulator, Apaf-1 to promote ATP-dependent activation of a specific caspase, caspase 9. This complex of caspase 9/Apaf-1/cytochrome c is referred to as the apoptosome. It has been demonstrated that many signals promoting cell survival impinge upon the mitochondrial pathway, preventing the efflux of mitochondrial cytochrome c to the cytoplasm. This can effectively prevent caspase activation and forestall cell death. In this proposal, however, we focus on an alternative mode of cell survival, acting through post-cytochrome c inhibition of the apoptosome. Specifically, we have found that both activated Erk kinases and the oncoprotein Bcr-Abl can prevent activation of caspase 9, even in the presence of cytosolic cytochrome c. It is the goal of this proposal to elucidate the mechanism(s) underlying this inhibition. Towards this end we propose a careful characterization of the apoptosome (composition, post-translational modifications, binding partners) in the presence and absence of these activated kinases. Moreover, we propose to use a new methodology to identify and characterize direct substrates of these kinases in a cell-free apoptotic reconstitution system. Finally, we propose two screens to identify additional novel apoptosomal regulators. Collectively, these experiments should provide insight into cellular survival mechanisms acting after mitochondrial release of cytochrome c. ? ?

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
Project #
Application #
Study Section
Cancer Molecular Pathobiology Study Section (CAMP)
Program Officer
Spalholz, Barbara A
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Duke University
Schools of Medicine
United States
Zip Code
Matsuura, K; Huang, N-J; Cocce, K et al. (2017) Downregulation of the proapoptotic protein MOAP-1 by the UBR5 ubiquitin ligase and its role in ovarian cancer resistance to cisplatin. Oncogene 36:1698-1706
Yang, C-S; Sinenko, S A; Thomenius, M J et al. (2014) The deubiquitinating enzyme DUBAI stabilizes DIAP1 to suppress Drosophila apoptosis. Cell Death Differ 21:604-11
Kurokawa, Manabu; Ito, Takahiro; Yang, Chih-Sheng et al. (2013) Engineering a BCR-ABL-activated caspase for the selective elimination of leukemic cells. Proc Natl Acad Sci U S A 110:2300-5
Parrish, Amanda B; Freel, Christopher D; Kornbluth, Sally (2013) Cellular mechanisms controlling caspase activation and function. Cold Spring Harb Perspect Biol 5:
Kurokawa, Manabu; Kim, Jiyeon; Geradts, Joseph et al. (2013) A network of substrates of the E3 ubiquitin ligases MDM2 and HUWE1 control apoptosis independently of p53. Sci Signal 6:ra32
Chen, Chen; Zhang, Liguo; Huang, Nai-Jia et al. (2013) Suppression of DNA-damage checkpoint signaling by Rsk-mediated phosphorylation of Mre11. Proc Natl Acad Sci U S A 110:20605-10
Kim, Jiyeon; Parrish, Amanda B; Kurokawa, Manabu et al. (2012) Rsk-mediated phosphorylation and 14-3-3? binding of Apaf-1 suppresses cytochrome c-induced apoptosis. EMBO J 31:1279-92
Zhang, Liguo; Huang, Nai-Jia; Chen, Chen et al. (2012) Ubiquitylation of p53 by the APC/C inhibitor Trim39. Proc Natl Acad Sci U S A 109:20931-6
Huang, Nai-Jia; Zhang, Liguo; Tang, Wanli et al. (2012) The Trim39 ubiquitin ligase inhibits APC/CCdh1-mediated degradation of the Bax activator MOAP-1. J Cell Biol 197:361-7
Johnson, C E; Freel, C D; Kornbluth, S (2010) Features of programmed cell death in intact Xenopus oocytes and early embryos revealed by near-infrared fluorescence and real-time monitoring. Cell Death Differ 17:170-9

Showing the most recent 10 out of 15 publications