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Regulated intramembrane proteolysis and degradation of murine epithelial cell adhesion molecule mEpCAM.

Hachmeister M, Bobowski KD, Hogl S, Dislich B, Fukumori A, Eggert C, Mack B, Kremling H, Sarrach S, Coscia F, Zimmermann W, Steiner H, Lichtenthaler SF, Gires O - PLoS ONE (2013)

Bottom Line: Additional EpCAM orthologs have been unequivocally identified in silico in 52 species.Sequence comparisons across species disclosed highest homology of BACE1 cleavage sites and in presenilin-dependent γ-cleavage sites, whereas strongest heterogeneity was observed in metalloprotease cleavage sites.In summary, EpCAM is a highly conserved protein present in fishes, amphibians, reptiles, birds, marsupials, and placental mammals, and is subject to shedding, γ-secretase-dependent regulated intramembrane proteolysis, and proteasome-mediated degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, Head and Neck Surgery, Ludwig-Maximilians-University, Munich, Germany.

ABSTRACT
Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is highly and frequently expressed in carcinomas and (cancer-)stem cells, and which plays an important role in the regulation of stem cell pluripotency. We show here that murine EpCAM (mEpCAM) is subject to regulated intramembrane proteolysis in various cells including embryonic stem cells and teratocarcinomas. As shown with ectopically expressed EpCAM variants, cleavages occur at α-, β-, γ-, and ε-sites to generate soluble ectodomains, soluble Aβ-like-, and intracellular fragments termed mEpEX, mEp-β, and mEpICD, respectively. Proteolytic sites in the extracellular part of mEpCAM were mapped using mass spectrometry and represent cleavages at the α- and β-sites by metalloproteases and the b-secretase BACE1, respectively. Resulting C-terminal fragments (CTF) are further processed to soluble Aβ-like fragments mEp-β and cytoplasmic mEpICD variants by the g-secretase complex. Noteworthy, cytoplasmic mEpICD fragments were subject to efficient degradation in a proteasome-dependent manner. In addition the γ-secretase complex dependent cleavage of EpCAM CTF liberates different EpICDs with different stabilities towards proteasomal degradation. Generation of CTF and EpICD fragments and the degradation of hEpICD via the proteasome were similarly demonstrated for the human EpCAM ortholog. Additional EpCAM orthologs have been unequivocally identified in silico in 52 species. Sequence comparisons across species disclosed highest homology of BACE1 cleavage sites and in presenilin-dependent γ-cleavage sites, whereas strongest heterogeneity was observed in metalloprotease cleavage sites. In summary, EpCAM is a highly conserved protein present in fishes, amphibians, reptiles, birds, marsupials, and placental mammals, and is subject to shedding, γ-secretase-dependent regulated intramembrane proteolysis, and proteasome-mediated degradation.

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Cleavage and proteasomal degradation of human EpCAM.HEK293 cells were stably transfected with hEpCAM-YFP and used to determine cleavage products of hEpCAM in membrane assays. Membranes of stable transfectants were isolated and either kept at 0°C (0 h) or incubated at 37°C in reaction buffer for the indicated time points. Thereafter, pellets and supernatant were collected upon differential centrifugation. Pellets (A) and supernatants (B) of membrane assays were separated in a 10% SDS-PAGE and probed with hEpICD- and YFP-specific antibodies. (C) HEK293 hEpCAM-YFP transfectants were treated with DMSO (control) or the γ-secretase inhibitor DAPT before being subjected to a membrane assay. The total fraction of the membrane assay was separated in a 10% SDS-PAGE, and probed with a YFP-specific antibody. Treatment with DAPT resulted in the accumulation of CTF-YFP and in the inhibition of hEpICD-YFP formation. (D) HEK293 human Myc-CTF-TF-YFP transfectants were treated with DMSO (control), the γ-secretase inhibitor DAPT, the proteasome inhibitor β-lacto-lactocystin (β-Lac), or combination of both. Thereafter, whole cell lysates were separated in a 10% SDS-PAGE, and probed with a YFP-specific antibody. Treatment with β-lacto-lactocystin resulted in an accumulation of hEpICD-YFP. Similar loading of protein lysates was visualised upon staining of tubulin on the same blots. Protein bands corresponding to human Myc-CTF-TF-YFP and mEpICD-YFP are indicated in each immunoblot. Shown are the representative results of three independent experiments. (E) YFP fluorescence was analysed in dependency of the treatment of HEK293 human Myc-CTF-TF-YFP transfectants. DMSO treatment served as a reference and values were normalised to one. Shown are the mean values with standard deviations from three independent experiments. (F) HEK293 cells stably expressing hEpCAM-YFP were transiently transfected with expression plasmids for luciferase (Luc) as a control or BACE1 (BACE1). After 24 hours, supernatants were removed and cells treated with the indicated inhibitors of BACE1 (C3), γ-secretase (DAPT) or combinations thereof. After additional 24 hours, supernatants were collected and hEpEX was immunoprecipitated and visualised upon immunoblotting with specific antibodies. Shown are two representative results with exposure times (1 s and 10 s). Over-expression of BACE1 and equal protein loading were verified upon immunobloting (lower left and right panel, respectively).
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pone-0071836-g006: Cleavage and proteasomal degradation of human EpCAM.HEK293 cells were stably transfected with hEpCAM-YFP and used to determine cleavage products of hEpCAM in membrane assays. Membranes of stable transfectants were isolated and either kept at 0°C (0 h) or incubated at 37°C in reaction buffer for the indicated time points. Thereafter, pellets and supernatant were collected upon differential centrifugation. Pellets (A) and supernatants (B) of membrane assays were separated in a 10% SDS-PAGE and probed with hEpICD- and YFP-specific antibodies. (C) HEK293 hEpCAM-YFP transfectants were treated with DMSO (control) or the γ-secretase inhibitor DAPT before being subjected to a membrane assay. The total fraction of the membrane assay was separated in a 10% SDS-PAGE, and probed with a YFP-specific antibody. Treatment with DAPT resulted in the accumulation of CTF-YFP and in the inhibition of hEpICD-YFP formation. (D) HEK293 human Myc-CTF-TF-YFP transfectants were treated with DMSO (control), the γ-secretase inhibitor DAPT, the proteasome inhibitor β-lacto-lactocystin (β-Lac), or combination of both. Thereafter, whole cell lysates were separated in a 10% SDS-PAGE, and probed with a YFP-specific antibody. Treatment with β-lacto-lactocystin resulted in an accumulation of hEpICD-YFP. Similar loading of protein lysates was visualised upon staining of tubulin on the same blots. Protein bands corresponding to human Myc-CTF-TF-YFP and mEpICD-YFP are indicated in each immunoblot. Shown are the representative results of three independent experiments. (E) YFP fluorescence was analysed in dependency of the treatment of HEK293 human Myc-CTF-TF-YFP transfectants. DMSO treatment served as a reference and values were normalised to one. Shown are the mean values with standard deviations from three independent experiments. (F) HEK293 cells stably expressing hEpCAM-YFP were transiently transfected with expression plasmids for luciferase (Luc) as a control or BACE1 (BACE1). After 24 hours, supernatants were removed and cells treated with the indicated inhibitors of BACE1 (C3), γ-secretase (DAPT) or combinations thereof. After additional 24 hours, supernatants were collected and hEpEX was immunoprecipitated and visualised upon immunoblotting with specific antibodies. Shown are two representative results with exposure times (1 s and 10 s). Over-expression of BACE1 and equal protein loading were verified upon immunobloting (lower left and right panel, respectively).

Mentions: In order to specify the cleavage of human EpCAM, membrane assays were performed with HEK293 cells stably expressing hEpCAM-YFP. Over time, an accumulation of two C-terminal fragments of human EpCAM, which preceded the generation of hEpICD-YFP, was observed (Figure 6A and B). Similarly to mEpCAM-YFP, the amount of CTF-YFP and hEpICD-YFP in pellets and soluble fractions of membrane assays were reciprocal, with hEpICD amounts being highest in the soluble fraction (Figure 6B). Treatment of stable transfectants of HEK293 cells with the γ-secretase inhibitor DAPT resulted in a loss of hEpICD, confirming the involvement of γ-secretase in the cleavage of CTF-YFP to hEpICD-YFP (Figure 6C).


Regulated intramembrane proteolysis and degradation of murine epithelial cell adhesion molecule mEpCAM.

Hachmeister M, Bobowski KD, Hogl S, Dislich B, Fukumori A, Eggert C, Mack B, Kremling H, Sarrach S, Coscia F, Zimmermann W, Steiner H, Lichtenthaler SF, Gires O - PLoS ONE (2013)

Cleavage and proteasomal degradation of human EpCAM.HEK293 cells were stably transfected with hEpCAM-YFP and used to determine cleavage products of hEpCAM in membrane assays. Membranes of stable transfectants were isolated and either kept at 0°C (0 h) or incubated at 37°C in reaction buffer for the indicated time points. Thereafter, pellets and supernatant were collected upon differential centrifugation. Pellets (A) and supernatants (B) of membrane assays were separated in a 10% SDS-PAGE and probed with hEpICD- and YFP-specific antibodies. (C) HEK293 hEpCAM-YFP transfectants were treated with DMSO (control) or the γ-secretase inhibitor DAPT before being subjected to a membrane assay. The total fraction of the membrane assay was separated in a 10% SDS-PAGE, and probed with a YFP-specific antibody. Treatment with DAPT resulted in the accumulation of CTF-YFP and in the inhibition of hEpICD-YFP formation. (D) HEK293 human Myc-CTF-TF-YFP transfectants were treated with DMSO (control), the γ-secretase inhibitor DAPT, the proteasome inhibitor β-lacto-lactocystin (β-Lac), or combination of both. Thereafter, whole cell lysates were separated in a 10% SDS-PAGE, and probed with a YFP-specific antibody. Treatment with β-lacto-lactocystin resulted in an accumulation of hEpICD-YFP. Similar loading of protein lysates was visualised upon staining of tubulin on the same blots. Protein bands corresponding to human Myc-CTF-TF-YFP and mEpICD-YFP are indicated in each immunoblot. Shown are the representative results of three independent experiments. (E) YFP fluorescence was analysed in dependency of the treatment of HEK293 human Myc-CTF-TF-YFP transfectants. DMSO treatment served as a reference and values were normalised to one. Shown are the mean values with standard deviations from three independent experiments. (F) HEK293 cells stably expressing hEpCAM-YFP were transiently transfected with expression plasmids for luciferase (Luc) as a control or BACE1 (BACE1). After 24 hours, supernatants were removed and cells treated with the indicated inhibitors of BACE1 (C3), γ-secretase (DAPT) or combinations thereof. After additional 24 hours, supernatants were collected and hEpEX was immunoprecipitated and visualised upon immunoblotting with specific antibodies. Shown are two representative results with exposure times (1 s and 10 s). Over-expression of BACE1 and equal protein loading were verified upon immunobloting (lower left and right panel, respectively).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3756971&req=5

pone-0071836-g006: Cleavage and proteasomal degradation of human EpCAM.HEK293 cells were stably transfected with hEpCAM-YFP and used to determine cleavage products of hEpCAM in membrane assays. Membranes of stable transfectants were isolated and either kept at 0°C (0 h) or incubated at 37°C in reaction buffer for the indicated time points. Thereafter, pellets and supernatant were collected upon differential centrifugation. Pellets (A) and supernatants (B) of membrane assays were separated in a 10% SDS-PAGE and probed with hEpICD- and YFP-specific antibodies. (C) HEK293 hEpCAM-YFP transfectants were treated with DMSO (control) or the γ-secretase inhibitor DAPT before being subjected to a membrane assay. The total fraction of the membrane assay was separated in a 10% SDS-PAGE, and probed with a YFP-specific antibody. Treatment with DAPT resulted in the accumulation of CTF-YFP and in the inhibition of hEpICD-YFP formation. (D) HEK293 human Myc-CTF-TF-YFP transfectants were treated with DMSO (control), the γ-secretase inhibitor DAPT, the proteasome inhibitor β-lacto-lactocystin (β-Lac), or combination of both. Thereafter, whole cell lysates were separated in a 10% SDS-PAGE, and probed with a YFP-specific antibody. Treatment with β-lacto-lactocystin resulted in an accumulation of hEpICD-YFP. Similar loading of protein lysates was visualised upon staining of tubulin on the same blots. Protein bands corresponding to human Myc-CTF-TF-YFP and mEpICD-YFP are indicated in each immunoblot. Shown are the representative results of three independent experiments. (E) YFP fluorescence was analysed in dependency of the treatment of HEK293 human Myc-CTF-TF-YFP transfectants. DMSO treatment served as a reference and values were normalised to one. Shown are the mean values with standard deviations from three independent experiments. (F) HEK293 cells stably expressing hEpCAM-YFP were transiently transfected with expression plasmids for luciferase (Luc) as a control or BACE1 (BACE1). After 24 hours, supernatants were removed and cells treated with the indicated inhibitors of BACE1 (C3), γ-secretase (DAPT) or combinations thereof. After additional 24 hours, supernatants were collected and hEpEX was immunoprecipitated and visualised upon immunoblotting with specific antibodies. Shown are two representative results with exposure times (1 s and 10 s). Over-expression of BACE1 and equal protein loading were verified upon immunobloting (lower left and right panel, respectively).
Mentions: In order to specify the cleavage of human EpCAM, membrane assays were performed with HEK293 cells stably expressing hEpCAM-YFP. Over time, an accumulation of two C-terminal fragments of human EpCAM, which preceded the generation of hEpICD-YFP, was observed (Figure 6A and B). Similarly to mEpCAM-YFP, the amount of CTF-YFP and hEpICD-YFP in pellets and soluble fractions of membrane assays were reciprocal, with hEpICD amounts being highest in the soluble fraction (Figure 6B). Treatment of stable transfectants of HEK293 cells with the γ-secretase inhibitor DAPT resulted in a loss of hEpICD, confirming the involvement of γ-secretase in the cleavage of CTF-YFP to hEpICD-YFP (Figure 6C).

Bottom Line: Additional EpCAM orthologs have been unequivocally identified in silico in 52 species.Sequence comparisons across species disclosed highest homology of BACE1 cleavage sites and in presenilin-dependent γ-cleavage sites, whereas strongest heterogeneity was observed in metalloprotease cleavage sites.In summary, EpCAM is a highly conserved protein present in fishes, amphibians, reptiles, birds, marsupials, and placental mammals, and is subject to shedding, γ-secretase-dependent regulated intramembrane proteolysis, and proteasome-mediated degradation.

View Article: PubMed Central - PubMed

Affiliation: Department of Otorhinolaryngology, Head and Neck Surgery, Ludwig-Maximilians-University, Munich, Germany.

ABSTRACT
Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein, which is highly and frequently expressed in carcinomas and (cancer-)stem cells, and which plays an important role in the regulation of stem cell pluripotency. We show here that murine EpCAM (mEpCAM) is subject to regulated intramembrane proteolysis in various cells including embryonic stem cells and teratocarcinomas. As shown with ectopically expressed EpCAM variants, cleavages occur at α-, β-, γ-, and ε-sites to generate soluble ectodomains, soluble Aβ-like-, and intracellular fragments termed mEpEX, mEp-β, and mEpICD, respectively. Proteolytic sites in the extracellular part of mEpCAM were mapped using mass spectrometry and represent cleavages at the α- and β-sites by metalloproteases and the b-secretase BACE1, respectively. Resulting C-terminal fragments (CTF) are further processed to soluble Aβ-like fragments mEp-β and cytoplasmic mEpICD variants by the g-secretase complex. Noteworthy, cytoplasmic mEpICD fragments were subject to efficient degradation in a proteasome-dependent manner. In addition the γ-secretase complex dependent cleavage of EpCAM CTF liberates different EpICDs with different stabilities towards proteasomal degradation. Generation of CTF and EpICD fragments and the degradation of hEpICD via the proteasome were similarly demonstrated for the human EpCAM ortholog. Additional EpCAM orthologs have been unequivocally identified in silico in 52 species. Sequence comparisons across species disclosed highest homology of BACE1 cleavage sites and in presenilin-dependent γ-cleavage sites, whereas strongest heterogeneity was observed in metalloprotease cleavage sites. In summary, EpCAM is a highly conserved protein present in fishes, amphibians, reptiles, birds, marsupials, and placental mammals, and is subject to shedding, γ-secretase-dependent regulated intramembrane proteolysis, and proteasome-mediated degradation.

Show MeSH
Related in: MedlinePlus