Elucidating the Role of Phosphorylaction State and the Cytoplasmic Domanin in Mechanisms of TNF-alpha Converting Enzyme (TACE) Activation
Huang, Holly S.
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Alzheimer's disease is characterized by the progressive formation of insoluble neuritic/amyloid plaques in the brain. Cleavage of the amyloid-beta precursor protein (APP) by β-and y-secretases generates amyloid-beta (Aβ), the major component of neuritic plaques. Cleavage of APP by α-secretase prevents Aβ formation, resulting in non-amyloidogenic APP secretion products. Recent evidence has shown that phorbol 12- myristate, I3-acetate (PMA), affects protein kinase C (PKC) to upregulate α-secretase activity, suggesting that α-secretase activity and its phosphorylation state are in some way related. In order to substantiate the importance of the phosphorylation state, Chinese hamster ovary cells stably transfected with the 695 amino-acid isoform of APP (CH0695) were exposed to treatments of PMA in addition to varying concentrations of okadaic acid, a specific inhibitor of protein phosphatase (PP)-l and PP-2A. Through several experiments employing Western blot analysis of APP cleavage products secreted from CH0695 cells, we have been able to determine that phosphatase inhibition is another mechanism by which α-secretase activity is enhanced. Additional experiments showed that PP-I is the major phosphatase regulating α-secretases. Since the cytoplasmic domain of α-secretase is the likely site for interaction/regulation by PKC, and because TNF -alpha converting enzyme (TACE) is a known α-secretase, we designed cytoplasmic tail mutations of TACE and tested their ability to induce α-cleavage of APP following transfection into TACE knockout fibroblasts. Studies utilizing these cytoplasmic tail mutations are being conducted to give further insight into regulation of αl-secretase activity, which may represent a novel target for the discovery of new therapeutic agents to treat Alzheimer's disease.