Constitutive phosphorylation of human endothelin-converting enzyme-1 isoforms.
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 amino-terminal 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.
MacLeod
Kathryn J
KJ
Department of CNS Molecular Sciences, Pfizer Global Research and Development, Ann Arbor Laboratories, Ann Arbor, Michigan 48105, USA. Kathryn.MacLeod@Pfizer.com
Husain
Rhonda D
RD
Gage
Douglas A
DA
Ahn
Kyunghye
K
D000595:Amino Acid Sequence; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D016466:CHO Cells; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D006224:Cricetinae; D000072396:Endothelin-Converting Enzymes; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008666:Metalloendopeptidases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D007527:Isoenzymes; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; C000606923:ECE1 protein, human; D000072396:Endothelin-Converting Enzymes