Role of Protein Kinase C in Endothelin Converting Enzyme-1 trafficking and shedding from endothelial cells

Sanjaya Kuruppu, Nathalie Tochon-Danguy, Alexander Ian Smith

Research output: Contribution to journalArticleResearchpeer-review

Abstract

This study aimed to determine the consequences of Protein Kinase C (PKC) mediated Endothelin Converting Enzyme-1 (ECE-1) phosphorylation and its relationship to ECE-1 expression and shedding. The proteins on the surface of EA.hy926 cells were labelled with EZ-Link NHS-SS-Biotin both prior to (control) and following stimulation by 2 muM phorbol 12-myristate 13-acetate (PMA) which activates PKC. The biotinylated proteins were isolated using neutravidin beads, resolved by gel electrophoresis and analysed by western blotting using anti-ECE-1 antibodies. Significant increase in ECE-1 expression at the cell surface was observed following stimulation by PMA, compared to unstimulated control cells (170 +/- 32.3 of control, n=5). The ECE-1 activity (expressed as muM substrate cleaved/min) was determined by monitoring the cleavage of a quenched fluorescent substrate. The specificity of cleavage was confirmed using the ECE-1 inhibitor CGS35066. The stimulation of cells by PMA (1 muM, 6 hr) significantly increased the ECE-1 activity (0.28 +/- 0.02; n=3) compared to the control (0.07 +/- 0.02; n=3). This increase was prevented by prior incubation with the PKC inhibitor bisindolymaleimide (BIM; 2 muM for 1 hr; 0.10 +/- 0.01; n=3). Treatment with PMA also increased the activity of ECE-1 in the media (0.18 +/- 0.01; n=3) compared to control (0.08 +/- 0.01; n=3). In addition, this study confirmed by western immunoblotting that only the extracellular region of ECE-1 is released from the cell surface. These data indicate for the first time that PKC activation induces the trafficking and shedding of ECE to and from the cell surface respectively.
Original languageEnglish
Pages (from-to)173 - 177
Number of pages5
JournalBiochemical and Biophysical Research Communications
Volume398
Issue number2
DOIs
Publication statusPublished - 2010

Cite this

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title = "Role of Protein Kinase C in Endothelin Converting Enzyme-1 trafficking and shedding from endothelial cells",
abstract = "This study aimed to determine the consequences of Protein Kinase C (PKC) mediated Endothelin Converting Enzyme-1 (ECE-1) phosphorylation and its relationship to ECE-1 expression and shedding. The proteins on the surface of EA.hy926 cells were labelled with EZ-Link NHS-SS-Biotin both prior to (control) and following stimulation by 2 muM phorbol 12-myristate 13-acetate (PMA) which activates PKC. The biotinylated proteins were isolated using neutravidin beads, resolved by gel electrophoresis and analysed by western blotting using anti-ECE-1 antibodies. Significant increase in ECE-1 expression at the cell surface was observed following stimulation by PMA, compared to unstimulated control cells (170 +/- 32.3 of control, n=5). The ECE-1 activity (expressed as muM substrate cleaved/min) was determined by monitoring the cleavage of a quenched fluorescent substrate. The specificity of cleavage was confirmed using the ECE-1 inhibitor CGS35066. The stimulation of cells by PMA (1 muM, 6 hr) significantly increased the ECE-1 activity (0.28 +/- 0.02; n=3) compared to the control (0.07 +/- 0.02; n=3). This increase was prevented by prior incubation with the PKC inhibitor bisindolymaleimide (BIM; 2 muM for 1 hr; 0.10 +/- 0.01; n=3). Treatment with PMA also increased the activity of ECE-1 in the media (0.18 +/- 0.01; n=3) compared to control (0.08 +/- 0.01; n=3). In addition, this study confirmed by western immunoblotting that only the extracellular region of ECE-1 is released from the cell surface. These data indicate for the first time that PKC activation induces the trafficking and shedding of ECE to and from the cell surface respectively.",
author = "Sanjaya Kuruppu and Nathalie Tochon-Danguy and Smith, {Alexander Ian}",
year = "2010",
doi = "10.1016/j.bbrc.2010.06.045",
language = "English",
volume = "398",
pages = "173 -- 177",
journal = "Biochemical and Biophysical Research Communications",
issn = "0006-291X",
publisher = "Elsevier",
number = "2",

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Role of Protein Kinase C in Endothelin Converting Enzyme-1 trafficking and shedding from endothelial cells. / Kuruppu, Sanjaya; Tochon-Danguy, Nathalie; Smith, Alexander Ian.

In: Biochemical and Biophysical Research Communications, Vol. 398, No. 2, 2010, p. 173 - 177.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Role of Protein Kinase C in Endothelin Converting Enzyme-1 trafficking and shedding from endothelial cells

AU - Kuruppu, Sanjaya

AU - Tochon-Danguy, Nathalie

AU - Smith, Alexander Ian

PY - 2010

Y1 - 2010

N2 - This study aimed to determine the consequences of Protein Kinase C (PKC) mediated Endothelin Converting Enzyme-1 (ECE-1) phosphorylation and its relationship to ECE-1 expression and shedding. The proteins on the surface of EA.hy926 cells were labelled with EZ-Link NHS-SS-Biotin both prior to (control) and following stimulation by 2 muM phorbol 12-myristate 13-acetate (PMA) which activates PKC. The biotinylated proteins were isolated using neutravidin beads, resolved by gel electrophoresis and analysed by western blotting using anti-ECE-1 antibodies. Significant increase in ECE-1 expression at the cell surface was observed following stimulation by PMA, compared to unstimulated control cells (170 +/- 32.3 of control, n=5). The ECE-1 activity (expressed as muM substrate cleaved/min) was determined by monitoring the cleavage of a quenched fluorescent substrate. The specificity of cleavage was confirmed using the ECE-1 inhibitor CGS35066. The stimulation of cells by PMA (1 muM, 6 hr) significantly increased the ECE-1 activity (0.28 +/- 0.02; n=3) compared to the control (0.07 +/- 0.02; n=3). This increase was prevented by prior incubation with the PKC inhibitor bisindolymaleimide (BIM; 2 muM for 1 hr; 0.10 +/- 0.01; n=3). Treatment with PMA also increased the activity of ECE-1 in the media (0.18 +/- 0.01; n=3) compared to control (0.08 +/- 0.01; n=3). In addition, this study confirmed by western immunoblotting that only the extracellular region of ECE-1 is released from the cell surface. These data indicate for the first time that PKC activation induces the trafficking and shedding of ECE to and from the cell surface respectively.

AB - This study aimed to determine the consequences of Protein Kinase C (PKC) mediated Endothelin Converting Enzyme-1 (ECE-1) phosphorylation and its relationship to ECE-1 expression and shedding. The proteins on the surface of EA.hy926 cells were labelled with EZ-Link NHS-SS-Biotin both prior to (control) and following stimulation by 2 muM phorbol 12-myristate 13-acetate (PMA) which activates PKC. The biotinylated proteins were isolated using neutravidin beads, resolved by gel electrophoresis and analysed by western blotting using anti-ECE-1 antibodies. Significant increase in ECE-1 expression at the cell surface was observed following stimulation by PMA, compared to unstimulated control cells (170 +/- 32.3 of control, n=5). The ECE-1 activity (expressed as muM substrate cleaved/min) was determined by monitoring the cleavage of a quenched fluorescent substrate. The specificity of cleavage was confirmed using the ECE-1 inhibitor CGS35066. The stimulation of cells by PMA (1 muM, 6 hr) significantly increased the ECE-1 activity (0.28 +/- 0.02; n=3) compared to the control (0.07 +/- 0.02; n=3). This increase was prevented by prior incubation with the PKC inhibitor bisindolymaleimide (BIM; 2 muM for 1 hr; 0.10 +/- 0.01; n=3). Treatment with PMA also increased the activity of ECE-1 in the media (0.18 +/- 0.01; n=3) compared to control (0.08 +/- 0.01; n=3). In addition, this study confirmed by western immunoblotting that only the extracellular region of ECE-1 is released from the cell surface. These data indicate for the first time that PKC activation induces the trafficking and shedding of ECE to and from the cell surface respectively.

UR - http://www.ncbi.nlm.nih.gov/pubmed/20558134

U2 - 10.1016/j.bbrc.2010.06.045

DO - 10.1016/j.bbrc.2010.06.045

M3 - Article

VL - 398

SP - 173

EP - 177

JO - Biochemical and Biophysical Research Communications

JF - Biochemical and Biophysical Research Communications

SN - 0006-291X

IS - 2

ER -