Membrane-associated epithelial cell adhesion molecule is slowly cleaved by γ-secretase prior to efficient proteasomal degradation of its intracellular domain.
J Biol Chem
; 294(9): 3051-3064, 2019 03 01.
Article
em En
| MEDLINE
| ID: mdl-30598504
ABSTRACT
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 α-secretase (ADAM) and ß-secretase (BACE) sheddases, generating a membrane-tethered C-terminal fragment EpCTF. Subsequently, the rate-limiting γ-secretase complex catalyzes intramembrane cleavage of EpCTF, generating an extracellular EpCAM-Aß-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 γ-secretase-mediated intramembrane cleavage of EpCTF. We demonstrate that γ-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 γ-secretase activity and not by EpCTF species, as indicated by cross-species swapping experiments. Furthermore, both human and murine EpICDs generated from EpCTF by γ-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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Membrana Celular
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Espaço Intracelular
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Complexo de Endopeptidases do Proteassoma
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Secretases da Proteína Precursora do Amiloide
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Proteólise
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Molécula de Adesão da Célula Epitelial
Tipo de estudo:
Risk_factors_studies
Limite:
Animals
/
Humans
Idioma:
En
Revista:
J Biol Chem
Ano de publicação:
2019
Tipo de documento:
Article