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 have been the focus of recent research. This research has identified phosphorylation of the cytoplasmic region of ECE-1 as 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 therefore expressed ECE-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 chimeras and 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.
authors.affiliation: Department of Biochemistry & Molecular Biology, Monash University, Wellington Rd, Clayton, Victoria 3800, Australia. Sanjaya.Kuruppu@monash.edu
authors.forename: Sanjaya
authors.initials: S
authors.lastname: Kuruppu
authors.forename: Nathalie
authors.initials: N
authors.lastname: Tochon-Danguy
authors.forename: A Ian
authors.initials: AI
authors.lastname: Smith
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000187:drug effects / Q000378:metabolism*; D020536:Enzyme Activators / Q000494:pharmacology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008666:Metalloendopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010766:Phosphorylation / Q000187:drug effects; D054730:Protein Interaction Domains and Motifs; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000031:analogs & derivatives / Q000494:pharmacology
chemical_list: D020536:Enzyme Activators; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; C016658:phorbolol myristate acetate; D011493:Protein Kinase C; C492359:ECEL1 protein, human; D008666:Metalloendopeptidases; D013755:Tetradecanoylphorbol Acetate