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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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Stability of mEpICD species.(A) HEK293 and mF9 cells stably expressing murine Myc-CTF-FT-YFP were treated with DMSO, DAPT, lactacystin-β-lactone, or DAPT and lactacystin-β-lactone. Protein lysates were separated in a 10% SDS-PAGE and probed with a YFP-specific antibody. As a control, lysates from vector control HEK293 cells were used (WT). CTF-YFP and EpICD-YFP are annotated. As a loading control, the same immunoblot membranes were probed with GAPDH-specific antibodies. (B) mF9 cells stably expressing murine Myc-CTF-FT-YFP were treated with DMSO, DAPT, lactacystin-β-lactone, or DAPT and lactacystin-β-lactone. YFP fluorescence intensities were assessed upon flow cytometry and normalised to values of DMSO-treated controls. Shown are mean values with standard deviations from three independent experiments. (C) mF9 cells stably expressing murine Myc-CTF-FT-YFP were subjected to mass spectrometric analysis. To do so, lysates of cells treated with DMSO or the proteasome inhibitor lactacystin-β-lactone and lysates from membrane assays were used. Representative mass spectrometry spectrum is depicted and five major peaks are annotated. Membrane assay probes and treatment with proteasome inhibitor led to the increase of peak ε1 and to the appearance of two peaks ε3 and ε5. (D) HEK293 cells stably expressing murine Myc-CTF-FT-YFP were treated with DMSO or the proteasome inhibitor lactacystin-b-lactone. Representative mass spectrometry spectrum is depicted and five major peaks are annotated. (E) Tabular overview of γ-secretase cleavage sites within mEpCAM as determined upon mass spectrometric analysis and alignment to potential molecular weights. Calculated and determined masses are given in Dalton including error of the peptide. (F) Sequence alignment of murine and human EpCAM (top), and murine EpCAM and murine Trop-2 (bottom). γ-Secretase cleavages at ε-position are indicated. Solid triangle marks the cleavage site of the stable EpICD variant, grey triangles cleavage sites of labile EpICD variants, and open triangle of N-terminally trimmed EpICD. (G) HEK293 cells stably expressing murine MVI-EpICD-YFP and MVLVI-EpICD-YFP mutants were subjected to flow cytometry assessment of YFP fluorescence. Shown are representative graphs of both stable transfectants after treatment with DMSO or lactacystin-β-lactone. (H) YFP fluorescence intensities of HEK293 transfectants stably expressing murine MVI-EpICD-YFP and MVLVI-EpICD-YFP mutants are given as mean fluorescence intensity ratios. Cells treated with lactacystin-β-lactone served as reference and values were set to one for comparison.
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pone-0071836-g005: Stability of mEpICD species.(A) HEK293 and mF9 cells stably expressing murine Myc-CTF-FT-YFP were treated with DMSO, DAPT, lactacystin-β-lactone, or DAPT and lactacystin-β-lactone. Protein lysates were separated in a 10% SDS-PAGE and probed with a YFP-specific antibody. As a control, lysates from vector control HEK293 cells were used (WT). CTF-YFP and EpICD-YFP are annotated. As a loading control, the same immunoblot membranes were probed with GAPDH-specific antibodies. (B) mF9 cells stably expressing murine Myc-CTF-FT-YFP were treated with DMSO, DAPT, lactacystin-β-lactone, or DAPT and lactacystin-β-lactone. YFP fluorescence intensities were assessed upon flow cytometry and normalised to values of DMSO-treated controls. Shown are mean values with standard deviations from three independent experiments. (C) mF9 cells stably expressing murine Myc-CTF-FT-YFP were subjected to mass spectrometric analysis. To do so, lysates of cells treated with DMSO or the proteasome inhibitor lactacystin-β-lactone and lysates from membrane assays were used. Representative mass spectrometry spectrum is depicted and five major peaks are annotated. Membrane assay probes and treatment with proteasome inhibitor led to the increase of peak ε1 and to the appearance of two peaks ε3 and ε5. (D) HEK293 cells stably expressing murine Myc-CTF-FT-YFP were treated with DMSO or the proteasome inhibitor lactacystin-b-lactone. Representative mass spectrometry spectrum is depicted and five major peaks are annotated. (E) Tabular overview of γ-secretase cleavage sites within mEpCAM as determined upon mass spectrometric analysis and alignment to potential molecular weights. Calculated and determined masses are given in Dalton including error of the peptide. (F) Sequence alignment of murine and human EpCAM (top), and murine EpCAM and murine Trop-2 (bottom). γ-Secretase cleavages at ε-position are indicated. Solid triangle marks the cleavage site of the stable EpICD variant, grey triangles cleavage sites of labile EpICD variants, and open triangle of N-terminally trimmed EpICD. (G) HEK293 cells stably expressing murine MVI-EpICD-YFP and MVLVI-EpICD-YFP mutants were subjected to flow cytometry assessment of YFP fluorescence. Shown are representative graphs of both stable transfectants after treatment with DMSO or lactacystin-β-lactone. (H) YFP fluorescence intensities of HEK293 transfectants stably expressing murine MVI-EpICD-YFP and MVLVI-EpICD-YFP mutants are given as mean fluorescence intensity ratios. Cells treated with lactacystin-β-lactone served as reference and values were set to one for comparison.

Mentions: Therefore, we addressed the cleavage and stability of mEpICD using the mCTF-FT-YFP construct, which is readily processed by γ-secretase, in stable transfectants of HEK293 cells. In line with an anticipated poor stability of mEpICD, mCTF-YFP was very weakly and mEpICD-YFP was not detectable in immunoblot experiments with whole cell lysates of HEK293 and mF9 cells stably expressing Myc-CTF-FT-YFP in the absence of any treatment (Figure 5A, DMSO lane). Treatment of cells with the proteasome inhibitor lactacystin-β-lacton or MG132 strongly stabilised mEpICD and allowed for the detection of substantial amounts of cleaved mEpICD (Figure 5A, lanes 4 and data not shown). Further experiments were conducted with lactacystin-β-lacton because MG132 was reported to be a pleiotropic drug, which affects the enzymatic activity of β- and β-secretase to substantial degree, too [39], [40], [41]. Interestingly, treatment of cells with the β-secretase inhibitor DAPT resulted in strong stabilisation and accumulation of Myc-CTF-FT-YFP, suggesting that primarily mEpICD and not mCTF is prone to proteasomal degradation (Figure 5A, lanes 2 and 3). Accordingly, treatment of cells with lactacystin-β-lacton induced only a minor stabilisation of Myc-CTF-FT-YFP (Figure 5A, lanes 4). The specificity of all protein bands was confirmed using lysates from HEK293 cells transfected with the empty vector only (Figure 5A).


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)

Stability of mEpICD species.(A) HEK293 and mF9 cells stably expressing murine Myc-CTF-FT-YFP were treated with DMSO, DAPT, lactacystin-β-lactone, or DAPT and lactacystin-β-lactone. Protein lysates were separated in a 10% SDS-PAGE and probed with a YFP-specific antibody. As a control, lysates from vector control HEK293 cells were used (WT). CTF-YFP and EpICD-YFP are annotated. As a loading control, the same immunoblot membranes were probed with GAPDH-specific antibodies. (B) mF9 cells stably expressing murine Myc-CTF-FT-YFP were treated with DMSO, DAPT, lactacystin-β-lactone, or DAPT and lactacystin-β-lactone. YFP fluorescence intensities were assessed upon flow cytometry and normalised to values of DMSO-treated controls. Shown are mean values with standard deviations from three independent experiments. (C) mF9 cells stably expressing murine Myc-CTF-FT-YFP were subjected to mass spectrometric analysis. To do so, lysates of cells treated with DMSO or the proteasome inhibitor lactacystin-β-lactone and lysates from membrane assays were used. Representative mass spectrometry spectrum is depicted and five major peaks are annotated. Membrane assay probes and treatment with proteasome inhibitor led to the increase of peak ε1 and to the appearance of two peaks ε3 and ε5. (D) HEK293 cells stably expressing murine Myc-CTF-FT-YFP were treated with DMSO or the proteasome inhibitor lactacystin-b-lactone. Representative mass spectrometry spectrum is depicted and five major peaks are annotated. (E) Tabular overview of γ-secretase cleavage sites within mEpCAM as determined upon mass spectrometric analysis and alignment to potential molecular weights. Calculated and determined masses are given in Dalton including error of the peptide. (F) Sequence alignment of murine and human EpCAM (top), and murine EpCAM and murine Trop-2 (bottom). γ-Secretase cleavages at ε-position are indicated. Solid triangle marks the cleavage site of the stable EpICD variant, grey triangles cleavage sites of labile EpICD variants, and open triangle of N-terminally trimmed EpICD. (G) HEK293 cells stably expressing murine MVI-EpICD-YFP and MVLVI-EpICD-YFP mutants were subjected to flow cytometry assessment of YFP fluorescence. Shown are representative graphs of both stable transfectants after treatment with DMSO or lactacystin-β-lactone. (H) YFP fluorescence intensities of HEK293 transfectants stably expressing murine MVI-EpICD-YFP and MVLVI-EpICD-YFP mutants are given as mean fluorescence intensity ratios. Cells treated with lactacystin-β-lactone served as reference and values were set to one for comparison.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0071836-g005: Stability of mEpICD species.(A) HEK293 and mF9 cells stably expressing murine Myc-CTF-FT-YFP were treated with DMSO, DAPT, lactacystin-β-lactone, or DAPT and lactacystin-β-lactone. Protein lysates were separated in a 10% SDS-PAGE and probed with a YFP-specific antibody. As a control, lysates from vector control HEK293 cells were used (WT). CTF-YFP and EpICD-YFP are annotated. As a loading control, the same immunoblot membranes were probed with GAPDH-specific antibodies. (B) mF9 cells stably expressing murine Myc-CTF-FT-YFP were treated with DMSO, DAPT, lactacystin-β-lactone, or DAPT and lactacystin-β-lactone. YFP fluorescence intensities were assessed upon flow cytometry and normalised to values of DMSO-treated controls. Shown are mean values with standard deviations from three independent experiments. (C) mF9 cells stably expressing murine Myc-CTF-FT-YFP were subjected to mass spectrometric analysis. To do so, lysates of cells treated with DMSO or the proteasome inhibitor lactacystin-β-lactone and lysates from membrane assays were used. Representative mass spectrometry spectrum is depicted and five major peaks are annotated. Membrane assay probes and treatment with proteasome inhibitor led to the increase of peak ε1 and to the appearance of two peaks ε3 and ε5. (D) HEK293 cells stably expressing murine Myc-CTF-FT-YFP were treated with DMSO or the proteasome inhibitor lactacystin-b-lactone. Representative mass spectrometry spectrum is depicted and five major peaks are annotated. (E) Tabular overview of γ-secretase cleavage sites within mEpCAM as determined upon mass spectrometric analysis and alignment to potential molecular weights. Calculated and determined masses are given in Dalton including error of the peptide. (F) Sequence alignment of murine and human EpCAM (top), and murine EpCAM and murine Trop-2 (bottom). γ-Secretase cleavages at ε-position are indicated. Solid triangle marks the cleavage site of the stable EpICD variant, grey triangles cleavage sites of labile EpICD variants, and open triangle of N-terminally trimmed EpICD. (G) HEK293 cells stably expressing murine MVI-EpICD-YFP and MVLVI-EpICD-YFP mutants were subjected to flow cytometry assessment of YFP fluorescence. Shown are representative graphs of both stable transfectants after treatment with DMSO or lactacystin-β-lactone. (H) YFP fluorescence intensities of HEK293 transfectants stably expressing murine MVI-EpICD-YFP and MVLVI-EpICD-YFP mutants are given as mean fluorescence intensity ratios. Cells treated with lactacystin-β-lactone served as reference and values were set to one for comparison.
Mentions: Therefore, we addressed the cleavage and stability of mEpICD using the mCTF-FT-YFP construct, which is readily processed by γ-secretase, in stable transfectants of HEK293 cells. In line with an anticipated poor stability of mEpICD, mCTF-YFP was very weakly and mEpICD-YFP was not detectable in immunoblot experiments with whole cell lysates of HEK293 and mF9 cells stably expressing Myc-CTF-FT-YFP in the absence of any treatment (Figure 5A, DMSO lane). Treatment of cells with the proteasome inhibitor lactacystin-β-lacton or MG132 strongly stabilised mEpICD and allowed for the detection of substantial amounts of cleaved mEpICD (Figure 5A, lanes 4 and data not shown). Further experiments were conducted with lactacystin-β-lacton because MG132 was reported to be a pleiotropic drug, which affects the enzymatic activity of β- and β-secretase to substantial degree, too [39], [40], [41]. Interestingly, treatment of cells with the β-secretase inhibitor DAPT resulted in strong stabilisation and accumulation of Myc-CTF-FT-YFP, suggesting that primarily mEpICD and not mCTF is prone to proteasomal degradation (Figure 5A, lanes 2 and 3). Accordingly, treatment of cells with lactacystin-β-lacton induced only a minor stabilisation of Myc-CTF-FT-YFP (Figure 5A, lanes 4). The specificity of all protein bands was confirmed using lysates from HEK293 cells transfected with the empty vector only (Figure 5A).

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