TNF-α enhances serine/threonine phosphorylation of γ-secretase via a JNK-dependent MAPK pathway
| Autor: | Lan-Hsin Kuo, 郭嵐忻 |
|---|---|
| Rok vydání: | 2006 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 94 Amyloid-β (Aβ), which is generated through β- and γ-secretase-mediated proteolysis of amyloid precursor protein (APP), plays a critical role in the pathogenesis of Alzheimer’s disease (AD). γ-Secretase which cleaves Amyloid-β precursor protein (APP) in its transmembrane domain catalyzes the final proteolytic step in the generation of Aβ. Recent evidence has demonstrated that γ-secretase is a multiprotein complex composed of presenilin (PS), nicastrin (NCT), Aph-1 and Pen-2 and that all four proteins are essential and sufficient for the proteolytic activity of γ-secretase. Previous evidence has shown that several cytokines can stimulate γ-secretase activity, and tumor necrosis factor-α (TNF-α) is the most potent one. In this study, we initially show that TNF-α activates γ-secretase via a JNK-dependent MAPK pathway. We then seek to determine whether this cytokine-elicited regulation of γ-secretase is due to alteration in the JNK-dependent phosphorylation of this protease. To improve the efficiency on visualizing the alteration in phosphorylation of γ-secretase, we have generated a CHO-derived cell line (γNCT-36) that stably co-expresses a His-tagged NCT along with PS1, Aph-1, Pen-2 and APP. The complexes of γ-secretase can then be efficiently pulled down by HIS-SelectedTM Cobalt Affinity Gel through the affinity of His-tagged NCT. Here, we present evidence that TNF-α elicits JNK-dependent serine/threonine phosphorylation of PS1 and NCT in γNCT-36 cells, concomitant with the stimulation of γ-secretase activity. Blocking JNK activity with a potent JNK inhibitor (SP600125) can reduce TNF-α-triggered phosphorylation of PS1 and NCT. Consistently, the activated JNK can be co-purified with γ-secretase complexes, and promotes the phosphorylation of PS1 and NCT in an in vitro kinase assay. A putative JNK-induced phosphorylation site of PS1 has been identified, and cells harboring this PS1 mutant exhibit decreased γ-secretase activity. Our findings suggest that JNK is an intracellular mediator of TNF-α-elicited phosphorylation of γ-secretase and may directly interact with γ-secretase to modulate its phosphorylation levels and hence its activity. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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