Protein Kinase C-dependent α-Secretase Competes with β-Secretase for Cleavage of Amyloid-β Precursor Protein in the Trans-Golgi Network

doi:10.1074/jbc.275.4.2568

Protein Kinase C-dependent α-Secretase Competes with β-Secretase for Cleavage of Amyloid-β Precursor Protein in the Trans-Golgi Network*

From the Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

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.

Footnotes

↵* This work was supported by grants from the NIA, National Institutes of Health, and by the estate of Mona Schneidman.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵‡ These authors contributed equally to this work.
↵§ Recipient of a Medical Scientist Training Program Predoctoral Fellowship from the National Institutes of Health.
↵¶ Recipient of a Paul Beeson Faculty Scholar Award.
↵‖ To whom correspondence should be addressed: Center for Neurodegenerative Disease Research, Dept. of Pathology and Laboratory Medicine, Maloney 3, HUP, Philadelphia, PA 19104-4283. Tel.: 215-662-6427; Fax: 215-349-5909; E-mail: vmylee@mail.med.upenn.edu.
↵2 Chyung, A. C., Lee, V. M.-Y., and Doms, R. W., manuscript in preparation.
Abbreviations:

APP

amyloid-β precursor protein

Aβ

amyloid-β peptide

Aβ_1–40 and Aβ_1–42

40- and 42-amino acid-long forms of Aβ, respectively

AD

Alzheimer's disease

APP_WT

wild-type human APP₆₉₅ protein

APP_ΔKK

APP₆₉₅ modified to contain a dilysine ER retention motif

APP_ΔNL

APP₆₉₅ containing the KM/NL familial AD Swedish mutation

ADAM

a disintegrin and metalloprotease

TACE

tumor necrosis factor-α-converting enzyme

PKC

protein kinase C

PMA

phorbol 12-myristate 13-acetate

α_reg and α_unreg

regulated and uregulated α-secretase activities, respectively

TAPI

tumor necrosis factor-α protease inhibitor

C99

C89, and C83, the 99-, 89-, and 83-amino acid C-terminal fragments of APP, respectively

SFV

Semliki Forest virus

ELISA

enzyme-linked immunosorbent assay

CHO

Chinese hamster ovary

Tricine

N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine

TGN

trans-Golgi network

ER

endoplasmic reticulum

IC

intermediate compartment

mAb

monoclonal antibody

sAPPα

ectodomain of APP generated by α-secretase
- Received August 25, 1999.
- Revision received October 27, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.