Protein kinase C-dependent α-secretase competes with β-secretase for cleavage of amyloid-β precursor protein in the trans-Golgi network

Abstract

The release of amyloidogenic amyloid-β peptide (Aβ) from amyloid-β precursor protein (APP) requires cleavage by β- and γ-secretases. In contrast, α-secretase cleaves APP within the Aβ sequence and precludes amyloidogenesis. Regulated and unregulated α-secretase activities have been reported, and the fraction of cellular α-secretase activity regulated by protein kinase C (PKC) has been attributed to the ADAM (a disintegrin and metalloprotease) family members TACE and ADAM-10. Although unregulated α- secretase cleavage of APP has been shown to occur at the cell surface, we sought to identify the intracellular site of PKC-regulated α-secretase APP cleavage. To accomplish this, we measured levels of secreted ectodomains and C-terminal fragments of APP generated by α-secretase (sAPPα) (C83) versus β-secretase (sAPPβ) (C99) and secreted Aβ in cultured cells treated with PKC and inhibitors of TACE/ADAM-10. We found that PKC stimulation increased sAPPα but decreased sAPPβ levels by altering the competition between α- versus β-secretase for APP within the same organelle rather than by perturbing APP trafficking. Moreover, data implicating the trans-Golgi network (TGN) as a major site for β-secretase activity prompted us to hypothesize that PKC-regulated α-secretase(s) also reside in this organelle. To test this hypothesis, we performed studies demonstrating proteolytically mature TACE intracellularly, and we also showed that regulated α-secretase APP cleavage occurs in the TGN using an APP mutant construct targeted specifically to the TGN. By detecting regulated α-secretase APP cleavage in the TGN by TACE/ADAM-10, we reveal ADAM activity in a novel location. Finally, the competition between TACE/ADAM-10 and β-secretase for intracellular APP cleavage may represent a novel target for the discovery of new therapeutic agents to treat Alzheimer's disease.