Protein Kinase C recognition sites in the cytoplasmic domain of Endothelin Converting Enzyme-1c

Kuruppu, Sanjaya; Tochon-Danguy, Nathalie; Smith, A. Ian

doi:10.1016/j.bbrc.2012.09.105

Cited by 8 publications

(11 citation statements)

References 28 publications

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“…BigET 18 -34 is a truncated version of the physiological substrate BigET-1 and is known to undergo 60% cleavage by ECE-1 (8). Previously we have used this substrate to monitor ECE-1 activity in cell membrane preparations and culture media (9). The N-terminal fragment resulting from the cleavage of both BigET-1 and BigET 18 -34 could not be detected by MALDI ToF mass spectrometry.…”

Section: Resultsmentioning

confidence: 99%

Soluble and Catalytically Active Endothelin Converting Enzyme-1 is Present in Cerebrospinal Fluid of Subarachnoid Hemorrhage Patients

Kuruppu

Chou

Feske

et al. 2014

Molecular & Cellular Proteomics

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Endothelin converting Enzyme-1 (ECE-1) is essential for the production of Endothelin-1 (ET-1), which is associated with vasospasm following subarachnoid hemorrhage (SAH). We have previously demonstrated the presence of a catalytically active soluble form of ECE-1 in the media of endothelial cells. We aimed to determine if this form of ECE-1 exists in vivo, in cerebrospinal fluid (CSF) of SAH patients. We examined CSF taken from SAH subjects for the presence of soluble ECE-1 using a bradykinin based quenched fluorescent substrate assay. We obtained further confirmation by characterizing the CSF mediated cleavage products of BigET-1 and BigET 18 -34 (6 g/ml) using mass spectrometry. The specificity of cleavage was confirmed using the ECE-1 inhibitor CGS35066 5nmol/L. SAH CSF samples had mean ECE-1 activity of 0.127 ؎ 0.037 mols of substrate cleaved/ l of CSF/24 h. The C-terminal peptides generated upon the cleavage of BigET-1 and BigET 18 -34 were detected 48 h after incubation of these substrates with CSF. Cleavage of these substrates was inhibited by CGS35066. Results of Western blots also produced strong evidence for the presence of truncated soluble ECE-1 in CSF. These results strongly suggest the presence of a truncated but catalytically active form of ECE-1 in the CSF of SAH subjects. Further studies are necessary to determine the biological significance of soluble ECE-1 in CSF of SAH subjects, including an association with vasospasm after SAH. Endothelin-1 (ET-1) 1 is the most potent vasoconstrictor in the central nervous system. Elevated levels of ET-1 in cerebrospinal fluid (CSF) have been implicated in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH) (1). However, it is not known whether the production of ET-1 within the CSF space contributes to the pathogenesis of vasospasm. ET-1 is produced upon the cleavage of its precursor BigET-1 by the highly specific metalloprotease Endothelin Converting Enzyme-1 (ECE-1). The rate-limiting step of ET-1 production is the expression and localization of ECE-1, whose catalytic activity is confined to the extracellular C-terminal domain. Previously, we have demonstrated that the catalytically active C terminus can be shed from the cell surface (2). This results in the release of a truncated but catalytically active form of ECE-1 into the extracellular milieu.Although the presence of a soluble form of ECE-1 has been demonstrated in vitro, it is yet to be shown in any human biological fluid. In this study, we have used a combination of mass spectrometry, Western blotting as well as quenched fluorescent substrate (QFS) based enzyme assays to demonstrate for the first time the presence of catalytically active, soluble form of ECE-1 in CSF of SAH subjects. EXPERIMENTAL PROCEDURESCSF Sample Collection-CSF samples were obtained from a prospective biorepository of SAH and non-SAH hydrocephalus human subjects in accordance with local institutional review board approved protocol. Detailed descriptions of biorepository methods have been previously pu...

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Section: Resultsmentioning

confidence: 99%

Soluble and Catalytically Active Endothelin Converting Enzyme-1 is Present in Cerebrospinal Fluid of Subarachnoid Hemorrhage Patients

Kuruppu

Chou

Feske

et al. 2014

Molecular & Cellular Proteomics

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“…Recent research has shown that phosphorylation is a key factor stimulating the trafficking of ECE-1 to the cell surface 8 where it catalyzes the production of ET-1. Furthermore, ECE-1 can be shed from the cell surface to produce a soluble counterpart of the membrane bound form which retains catalytic activity.…”

Section: Discussionmentioning

confidence: 99%

“…Mutation of either residue significantly inhibited PKC induced ECE-1c phosphorylation. 8 It is likely the addition of the Nterminal GFP tag mainly inhibits phosphorylation of the Tyr4 residue reflecting its close proximity to the GFP tag. The effects of GFP addition on ECE-1c localization was also analyzed.…”

Section: Discussionmentioning

confidence: 99%

“…7 The activation of protein kinase C (PKC) by phorbol esters such as phorbol 12-myristate 13-acetate (PMA), results in the phosphorylation of Tyr4 and Ser35 residues within the N-terminus of ECE-1. 8 This has been widely accepted as a mechanism which induces the trafficking of ECE-1 to the cell surface. 2,[8][9][10] Disease stimuli such as high glucose have been proven to increase the trafficking of ECE-1c to the cell surface via this mechanism.…”

Section: Introductionmentioning

confidence: 99%

“…8 This has been widely accepted as a mechanism which induces the trafficking of ECE-1 to the cell surface. 2,[8][9][10] Disease stimuli such as high glucose have been proven to increase the trafficking of ECE-1c to the cell surface via this mechanism. 9 Increased cell surface expression of ECE-1 has been implicated in the pathogenesis of cardiovascular diseases [11][12][13] and in female malignancies.…”

Section: Introductionmentioning

confidence: 99%

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

Kuruppu

Tochon-Danguy²,

Smith

2013

Protein Science

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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.

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TIMAP, the versatile protein phosphatase 1 regulator in endothelial cells

Boratkó

Csortos

2017

IUBMB Life

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Transforming growth factor (TGF)-β inhibited membrane associated protein, TIMAP, is the member of the myosin phosphatase targeting protein (MYPT) family of protein phosphatase 1 (PP1) regulatory subunits. The N-terminal part of TIMAP has a typical MYPT family structure with a sequence element called MyPhone (myosin phosphatase N-terminal element), a putative bipartite nuclear localization signal, a PP1 catalytic subunit binding motif, and five ankyrin repeats. The C-terminal half of TIMAP is intrinsically disordered, but ends with a functional CAAX box for lipid modification which allows localization of TIMAP at the plasma membrane. TIMAP is prenylated by farnesyl transferase with the contribution of the anchoring protein, RACK1 in the cytoplasm. The controlling effect of TIMAP on PP1 is moderated by PKA/GSK3β and PKC mediated phosphorylation of TIMAP, the sites are located in the disordered region of the protein. TIMAP is abundant in endothelial cells. A growing body of evidence attained through characterization of newly identified protein partners calls attention to its critical role in normal and pathological activities of the endothelium via regulation of PP1. TIMAP binds the non-integrin laminin receptor 1 and the endothelin converting enzyme 1, which may connect TIMAP to angiogenesis, tumor invasion and metastasis. Barrier protecting role of TIMAP was shown for pulmonary artery endothelial cells. ERM (ezrin-radixin-moesin) proteins, as potential in vivo PP1-TIMAP substrates, are critical targets in the barrier maintenance. TIMAP affects phosphorylation level and subcellular localization of merlin and eukaryotic elongation factor-1A1. Merlin is a key component of signaling pathways regulating cell proliferation, membrane domain formation and cell-cell junction organization. Noncanonical functions of the elongation factor include a role in organization of cytoskeleton dynamics and in apoptosis. The interacting/binding partners identified so far demonstrate a rather complex role of TIMAP in key functions of the endothelium offering TIMAP as a plausible target in pathological issues. © 2017 IUBMB Life, 69(12):918-928, 2017.

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Protein Kinase C recognition sites in the cytoplasmic domain of Endothelin Converting Enzyme-1c

Cited by 8 publications

References 28 publications

Soluble and Catalytically Active Endothelin Converting Enzyme-1 is Present in Cerebrospinal Fluid of Subarachnoid Hemorrhage Patients

Soluble and Catalytically Active Endothelin Converting Enzyme-1 is Present in Cerebrospinal Fluid of Subarachnoid Hemorrhage Patients

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

TIMAP, the versatile protein phosphatase 1 regulator in endothelial cells

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