Applicability of green fluorescence protein in the study of endothelin converting enzyme-1c trafficking

DM Sanjaya Kuruppu, Nathalie Tochon-Danguy, Alexander Ian Smith

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Endothelin-1 (ET-1) is one of the most potent peptide vasoconstrictors known. It is produced upon the cleavage of its precursor Big Endothelin-1 by Endothelin Converting Enzyme-1 (ECE-1).Production of ET-1 is thought to be dependent upon the expression of ECE-1 at the cell surface. Therefore mechanisms inducing the trafficking of ECE-1 to the cell surface havebeen the focus of recent research.This research has identified phosphorylation of the cytoplasmic region of ECE-1as a main cellular signal inducing its trafficking to the cell surface. Previous studies have used green fluorescent protein (GFP) tagged ECE-1 to monitor phosphorylation induced trafficking of ECE-1 to the cell surface. However, it has been speculated that the addition of the GFP tag can itself alter enzyme activity and phosphorylation of ECE-1, and hence the suitability of GFP or any other protein tag in studying ECE-1 distribution and trafficking. ECE-1c is the most widely expressed isoform in endothelial cells. We thereforeexpressedECE-1c with a GFP tag either at the N or C-terminus of ECE-1c.Catalytic activity and effect on Protein Kinase C (PKC) induced phosphorylation was compared between the two chimerasand wild type ECE-1c. Our results indicate that positioning of the GFP tag on the C-terminus abrogates activity without effecting PKC induced phosphorylation. However, GFP tag on the N-terminus has the opposite effect. Results of this study shed light on the applicability of GFP or perhaps other protein tags in studying ECE-1c distribution and trafficking.
Original languageEnglish
Pages (from-to)306 - 313
Number of pages8
JournalProtein Science
Volume22
Issue number3
DOIs
Publication statusPublished - 2013

Cite this

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title = "Applicability of green fluorescence protein in the study of endothelin converting enzyme-1c trafficking",
abstract = "Endothelin-1 (ET-1) is one of the most potent peptide vasoconstrictors known. It is produced upon the cleavage of its precursor Big Endothelin-1 by Endothelin Converting Enzyme-1 (ECE-1).Production of ET-1 is thought to be dependent upon the expression of ECE-1 at the cell surface. Therefore mechanisms inducing the trafficking of ECE-1 to the cell surface havebeen the focus of recent research.This research has identified phosphorylation of the cytoplasmic region of ECE-1as a main cellular signal inducing its trafficking to the cell surface. Previous studies have used green fluorescent protein (GFP) tagged ECE-1 to monitor phosphorylation induced trafficking of ECE-1 to the cell surface. However, it has been speculated that the addition of the GFP tag can itself alter enzyme activity and phosphorylation of ECE-1, and hence the suitability of GFP or any other protein tag in studying ECE-1 distribution and trafficking. ECE-1c is the most widely expressed isoform in endothelial cells. We thereforeexpressedECE-1c with a GFP tag either at the N or C-terminus of ECE-1c.Catalytic activity and effect on Protein Kinase C (PKC) induced phosphorylation was compared between the two chimerasand wild type ECE-1c. Our results indicate that positioning of the GFP tag on the C-terminus abrogates activity without effecting PKC induced phosphorylation. However, GFP tag on the N-terminus has the opposite effect. Results of this study shed light on the applicability of GFP or perhaps other protein tags in studying ECE-1c distribution and trafficking.",
author = "Kuruppu, {DM Sanjaya} and Nathalie Tochon-Danguy and Smith, {Alexander Ian}",
year = "2013",
doi = "10.1002/pro.2212",
language = "English",
volume = "22",
pages = "306 -- 313",
journal = "Protein Science",
issn = "0961-8368",
publisher = "Wiley-Blackwell",
number = "3",

}

Applicability of green fluorescence protein in the study of endothelin converting enzyme-1c trafficking. / Kuruppu, DM Sanjaya; Tochon-Danguy, Nathalie; Smith, Alexander Ian.

In: Protein Science, Vol. 22, No. 3, 2013, p. 306 - 313.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - Applicability of green fluorescence protein in the study of endothelin converting enzyme-1c trafficking

AU - Kuruppu, DM Sanjaya

AU - Tochon-Danguy, Nathalie

AU - Smith, Alexander Ian

PY - 2013

Y1 - 2013

N2 - Endothelin-1 (ET-1) is one of the most potent peptide vasoconstrictors known. It is produced upon the cleavage of its precursor Big Endothelin-1 by Endothelin Converting Enzyme-1 (ECE-1).Production of ET-1 is thought to be dependent upon the expression of ECE-1 at the cell surface. Therefore mechanisms inducing the trafficking of ECE-1 to the cell surface havebeen the focus of recent research.This research has identified phosphorylation of the cytoplasmic region of ECE-1as a main cellular signal inducing its trafficking to the cell surface. Previous studies have used green fluorescent protein (GFP) tagged ECE-1 to monitor phosphorylation induced trafficking of ECE-1 to the cell surface. However, it has been speculated that the addition of the GFP tag can itself alter enzyme activity and phosphorylation of ECE-1, and hence the suitability of GFP or any other protein tag in studying ECE-1 distribution and trafficking. ECE-1c is the most widely expressed isoform in endothelial cells. We thereforeexpressedECE-1c with a GFP tag either at the N or C-terminus of ECE-1c.Catalytic activity and effect on Protein Kinase C (PKC) induced phosphorylation was compared between the two chimerasand wild type ECE-1c. Our results indicate that positioning of the GFP tag on the C-terminus abrogates activity without effecting PKC induced phosphorylation. However, GFP tag on the N-terminus has the opposite effect. Results of this study shed light on the applicability of GFP or perhaps other protein tags in studying ECE-1c distribution and trafficking.

AB - Endothelin-1 (ET-1) is one of the most potent peptide vasoconstrictors known. It is produced upon the cleavage of its precursor Big Endothelin-1 by Endothelin Converting Enzyme-1 (ECE-1).Production of ET-1 is thought to be dependent upon the expression of ECE-1 at the cell surface. Therefore mechanisms inducing the trafficking of ECE-1 to the cell surface havebeen the focus of recent research.This research has identified phosphorylation of the cytoplasmic region of ECE-1as a main cellular signal inducing its trafficking to the cell surface. Previous studies have used green fluorescent protein (GFP) tagged ECE-1 to monitor phosphorylation induced trafficking of ECE-1 to the cell surface. However, it has been speculated that the addition of the GFP tag can itself alter enzyme activity and phosphorylation of ECE-1, and hence the suitability of GFP or any other protein tag in studying ECE-1 distribution and trafficking. ECE-1c is the most widely expressed isoform in endothelial cells. We thereforeexpressedECE-1c with a GFP tag either at the N or C-terminus of ECE-1c.Catalytic activity and effect on Protein Kinase C (PKC) induced phosphorylation was compared between the two chimerasand wild type ECE-1c. Our results indicate that positioning of the GFP tag on the C-terminus abrogates activity without effecting PKC induced phosphorylation. However, GFP tag on the N-terminus has the opposite effect. Results of this study shed light on the applicability of GFP or perhaps other protein tags in studying ECE-1c distribution and trafficking.

UR - http://www.ncbi.nlm.nih.gov/pubmed/23281075

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JF - Protein Science

SN - 0961-8368

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ER -