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

Endothelin Converting Enzyme-1 (ECE-1) is essential for the production of the potent vasoconstrictor Endothelin-1 (ET-1). The activation of Protein Kinase C (PKC) by phorbol 12-myristate 13-acetate (PMA) can increase ECE-1 phosphorylation, which in turn promotes ECE-1c trafficking to the cell surface where ET-1 production occurs. This study has identified the specific residues in the N-terminal cytoplasmic tail of ECE-1c isoform that are phosphorylated upon the activation of PKC. Levels of phosphorylation are expressed as a % phosphorylation in untreated CHO-K1 cells. We transfected CHO-K1 cells with wild type and mutant forms of ECE-1c (Ala4-ECE-1c, Ala35ECE-1c and Ala4/35ECE-1c) to confirm the involvement of Thr4 and Ser35 residues in PMA induced phosphorylation of ECE-1c. Phosphorylation of wild type ECE-1c increased in response to PMA treatment (150 ± 13%, unpaired t-test, P < 0.05, significantly different compared to untreated control). The two single mutants and one combined mutant significantly reduced the PMA induced phosphorylation (103–117 ± 6–13%; unpaired t-test; n = 8; P < 0.05 significantly different compared to untreated control). The mutations had no effect on the basal ECE-1c phosphorylation. In addition, they had no effect on the catalytic activity as evidenced by the similar rate of substrate cleavage compared to wild type. This study is the first to confirm the residues phosphorylated upon the activation of PKC by PMA. The results complete a gap in our understanding of the mechanism(s) behind PKC induced trafficking of ECE-1.

► Tyr4 and Ser35 in ECE-1c form putative recognition sites for PKC.
► Mutation of Tyr4/Ser35 has no effect on activity or expression of ECE-1c.
► Mutation of Tyr4/Ser35 reduces PKC induced phosphorylation of ECE-1c.
► The activation of PKC results in the phosphorylation of Tyr4 and Ser35 in ECE-1c.