Constitutive Phosphorylation of Human Endothelin-converting Enzyme-1 Isoforms

Abstract

To investigate the phosphorylation of human endothelin-converting enzyme-1 (hECE-1) and identify potential residues involved, both in vivo and in vitro phosphorylation labeling assays of hECE-1 isoforms were performed in combination with site-directed mutagenesis and mass spectrometric analyses. Initial studies found that endogenous hECE-1 was constitutively phosphorylated in a primary endothelial cell line. The four known isoforms of hECE-1 expressed in this cell line (1a, 1b, 1c, and 1d) were then cloned by reverse transcription-PCR to determine which isoform(s) may be phosphorylated. The isoforms differ only in the first portion of their short aminoterminal cytoplasmic domains whereas their transmembrane domains and ectodomains of the proteins are identical. Isoforms 1b, 1c, and 1d but not 1a, were constitutively phosphorylated in vivo when expressed in Chinese hamster ovary cells and casein kinase I readily phosphorylated the immunopurified isoforms in vitro. Site-directed mutagenesis established that two conserved serine residues, Ser 18 and Ser 20, (numbering based on isoform 1c) form at least one phosphorylation site in these three isoforms. Mutant forms of 1b, 1c, and 1d were constructed in which a single alanine was introduced at either serine residue and a double mutant for each isoform was constructed as well in which both serines were replaced with alanine. Phosphorylation of the single mutants was greatly reduced and was nearly abolished in the double mutants in both in vivo and in vitro labeling assays. Analysis by MALDI-MS of 32 P-labeled proteolytic peptides derived from wild type 1c and the 1c mutants supported both Ser 18 and Ser 20 as phosphorylated residues. These data demonstrate the first finding that hECE-1 is constitutively phosphorylated within its cytoplasmic domain in an isoform-specific manner.

Endothelin-converting enzyme inhibitors and their potential for cardiovascular and renal therapeutics

“…Thus, the isoforms of ECE-1 differ only in the first portion of their short amino-terminal cytoplasmic domains, whereas their transmembranal domain and ectodomains are identical [69]. The isoforms of 1b, 1c and 1d, but not 1a, are constitutively phosphorylated [69].…”

“…Thus, the isoforms of ECE-1 differ only in the first portion of their short amino-terminal cytoplasmic domains, whereas their transmembranal domain and ectodomains are identical [69]. The isoforms of 1b, 1c and 1d, but not 1a, are constitutively phosphorylated [69]. An understanding of the differences in these isoforms may allow the development of selective isoform inhibitors, which may allow a better targeting of individual functions/disease.…”

Biochemical applications of mass spectrometry in pharmaceutical drug discovery

“…Kinases can function in cascades that amplify signals governing cell function, and high-level members (i.e., early signalers) of the system are low in abundance and challenging subjects for MS. Mutagenesis of potential phosphorylation sites is the established method to identify them, and can be supplemented by MALDI-TOF MS performed on digests of small quantities of protein, such as those obtained in immunoprecipitates from cell lysates (MacLeod et al, 2002).…”

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

“…6 The cytoplasmic N-terminus of ECE-1 is constitutively phosphorylated at Ser18 and Ser20. 7 ECE-1a isoform lacks these residues and hence is not constitutively phosphorylated. 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.…”

“…7 ECE-1a isoform lacks these residues and hence is not constitutively phosphorylated. 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.…”