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(Circulation Research. 2002;91:749.)
© 2002 American Heart Association, Inc.

Integrative Physiology

CK2 Phosphorylates the Angiotensin-Converting Enzyme and Regulates Its Retention in the Endothelial Cell Plasma Membrane

Karin Kohlstedt, Firouzeh Shoghi, Werner Müller-Esterl, Rudi Busse, Ingrid Fleming

From the Institut für Kardiovaskuläre Physiologie (K.K., R.B., I.F.), and Institut für Biochemie II (F.S., W.M.-E.), Klinikum der J.W.G.-Universität, Frankfurt am Main, Germany.

Correspondence to Ingrid Fleming, PhD, Institut für Kardiovaskuläre Physiologie, Klinikum der J.W.G.-Universität, Theodor-Stern-Kai 7, D-60590 Frankfurt am Main, Germany. E-mail fleming{at}em.uni-frankfurt.de

Soluble angiotensin-converting enzyme (ACE) is derived from the membrane-bound form by proteolytic cleavage of its C-terminal domain. Because intracellular events might be involved in the regulation of the cleavage process, we determined whether the cytoplasmic tail of ACE is phosphorylated and whether this process regulates secretion. Immunoprecipitation of ACE (180 kDa) from ³²P-labeled endothelial cells revealed that ACE is phosphorylated. Phosphorylation was not observed in endothelial cells overexpressing a mutant form of ACE (ACES, all five cytoplasmic serine residues replaced by alanine). CK2 coprecipitated with ACE from endothelial cells, and CK2 phosphorylated both ACE and a peptide corresponding to the cytoplasmic tail. Mutation of serine¹²⁷⁰ within the CK2 consensus sequence almost abolished ACE phosphorylation. In ACE-overexpressing endothelial cells, ACE was mostly localized to the plasma membrane. However, no ACE was detected in the plasma membrane of ACES-overexpressing cells, although a precursor ACE (170 kDa) was prominent in the endoplasmic reticulum and the cell supernatant contained substantial amounts of the soluble protein (175 kDa). A correlation between ACE-phosphorylation and secretion was confirmed in endothelial cells treated with the CK2-inhibitor, 5,6-dichloro-1-ß-D-ribofuranosylbenzimidazole, which time-dependently decreased the phosphorylation of ACE and increased its shedding. These results indicate that the CK2-mediated phosphorylation of ACE regulates its retention in the plasma membrane and may determine plasma ACE levels.

Key Words: angiotensin-converting enzyme • casein kinase 2 • phosphorylation • protein cleavage • ACE secretase

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