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Membrane-associated epithelial cell adhesion molecule is slowly cleaved by -secretase prior to efficient proteasomal degradation of its intracellular domain.
J. Biol. Chem. 294, 3051-3064 (2019)
Verlagsversion Preprint Forschungsdaten DOI
Regulated intramembrane proteolysis (RIP) is a key mechanism for activating transmembrane proteins such as epithelial cell adhesion molecule (EpCAM) for cellular signaling and degradation. EpCAM is highly expressed in carcinomas and progenitor and embryonic stem cells and is involved in the regulation of cell adhesion, proliferation, and differentiation. Strictly sequential cleavage of EpCAM through RIP involves initial shedding of the extracellular domain by -secretase (ADAM) and -secretase (BACE) sheddases, generating a membrane-tethered C-terminal fragment EpCTF. Subsequently, the rate-limiting -secretase complex catalyzes intramembrane cleavage of EpCTF, generating an extracellular EpCAM-A-like fragment and an intracellular EpICD fragment involved in nuclear signaling. Here, we have combined biochemical approaches with live-cell imaging of fluorescent protein tags to investigate the kinetics of -secretase-mediated intramembrane cleavage of EpCTF. We demonstrate that -secretase-mediated proteolysis of exogenously and endogenously expressed EpCTF is a slow process with a 50% protein turnover in cells ranging from 45 min to 5.5 h. The slow cleavage was dictated by -secretase activity and not by EpCTF species, as indicated by cross-species swapping experiments. Furthermore, both human and murine EpICDs generated from EpCTF by -secretase were degraded efficiently (94-99%) by the proteasome. Hence, proteolytic cleavage of EpCTF is a comparably slow process, and EpICD generation does not appear to be suited for rapidly transducing extracellular cues into nuclear signaling, but appears to provide steady signals that can be further controlled through efficient proteasomal degradation. Our approach provides an unbiased bioassay to investigate proteolytic processing of EpCTF in single living cells.
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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Schlagwörter Epithelial Cell Adhesion Molecule (epcam) ; Gamma-secretase ; Proteasome Degradation ; Regulated Intramembrane Proteolysis ; Protein Processing ; Live-cell Imaging ; Sheddase ; Cell Adhesion ; Proteolytic Processing ; Transmembrane Protein ; Cell Signaling; Regulated Intramembrane Proteolysis; Amyloid Precursor Protein; Gamma-secretase; Epcam Expression; Ep-cam; Antigen; Tumor; Cancer; Contributes; Maintenance
ISSN (print) / ISBN 0021-9258
Zeitschrift Journal of Biological Chemistry, The
Quellenangaben Band: 294, Heft: 9, Seiten: 3051-3064
Verlag American Society for Biochemistry and Molecular Biology
Verlagsort 9650 Rockville Pike, Bethesda, Md 20814-3996 Usa
Begutachtungsstatus Peer reviewed
Institut(e) CCG Personalized Radiotherapy in Head and Neck Cancer (KKG-KRT)