The APC tumor suppressor is a multifunctional protein involved in cell migration, proliferation, differentiation and apoptosis. It is a component of the Wnt signaling pathway and is best known for its ability to down-regulate β-catenin and consequent effects on transcriptional regulation. Previous work in our laboratory has demonstrated that APC accelerates apoptosis-associated caspase activity independently of transcription, suggesting novel tumor suppressor functions of APC.
In the present study, the transcription-independent mechanisms of APC function in apoptosis were investigated. We mapped the APC apoptosis-accelerating region to amino acids 1-760 by testing a series of non-overlapping APC segments. Interestingly, this segment corresponds to a stable group II caspase cleavage product of APC released during apoptosis that includes the amino-terminal amino acids 1-777. Mutation of the APC aspartic acid residue at position 777 in APC to an alanine completely abolished in vitro cleavage of APC by a recombinant group II caspase. This mutation also rendered the full length protein unable to accelerate apoptosis in vitro. A truncated APC protein associated with familial and sporadic colorectal cancer, and unable to accelerate apoptosis in vitro and in vivo, is resistant to group II caspase cleavage. These results demonstrate that cleavage of APC and the subsequent release of an amino-terminal segment are necessary for the transcription-independent mechanism of APC-mediated apoptosis.
Additionally, we have elucidated the mechanism of APC-mediated apoptosis by identifying and characterizing a downstream partner of APC in this apoptotic cascade. hTid-1, a homologue of the Drosophila tumor suppressor Tid 56, is an apoptosis modulator predominantly located in mitochondria. We have demonstrated that the amino-terminal segment of APC (APC 1-777) interacts with hTid-1 directly using co-immunoprecipitation, pull-down assays, and immunofluorescence. Immunofluorescence studies and subcellular fractionation show that the APC amino-terminus, exogenous and endogenous, which is released by caspase cleavage during apoptosis, is translocated to the mitochondria. Finally, over-expression of hTid1 partially rescues the HCT116 cells from apoptosis mediated by APC in the absence and presence of exogenous apoptosis stimuli. These data suggest that the amino-terminal segment of APC promotes cell sensitivity to apoptosis through physical and functional interactions with the apoptosis modulator hTid-1.
|School:||University of Cincinnati|
|Department:||Molecular Genetics, Biochemistry, and Microbiology|
|School Location:||United States -- Ohio|
|Source:||DAI-B 79/10(E), Dissertation Abstracts International|
|Keywords:||Apc, Apoptosis, Caspase, Htid-1, Transcription-independent|
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