Calmodulin binds to the cytoplasmic domain of angiotensin-converting enzyme and regulates its phosphorylation and cleavage secretion.
The rate of cleavage secretion of the enzymatically active ectodomain of angiotensin-converting enzyme (ACE) is regulated by tyrosine phosphorylation of the protein and by the phorbol ester, phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. Here, we report that both calmodulin inhibitor (CaMI) and calmodulin kinase inhibitor could also enhance cleavage secretion of ACE. This effect was accompanied by the dissociation of calmodulin from a specific region within the cytoplasmic domain of ACE to which it had been bound. The same domain of ACE was phosphorylated, and both CaMI and PMA caused dephosphorylation of ACE as well. Mass spectrometric and mutational analyses identified Ser730 as the only phosphorylated residue in the cytoplasmic domain of ACE. The Ser730 --> Ala mutant of ACE was not phosphorylated, but it still bound calmodulin, and its cleavage secretion was enhanced by both CaMI and PMA. Similarly, when Ser730 was replaced by the phosphoserine mimetic, Asp, cleavage secretion of the resultant mutant remained susceptible to the enhancing effect of CaMI and PMA. These results demonstrate that, although CaMI and PMA can enhance both cleavage secretion of ACE and its dephosphorylation, the two effects are not mutually interdependent.
Chattopadhyay
Saurabh
S
Department of Molecular Cardiology, Lerner Research Institute, The Cleveland Clinic Foundation, Cleveland, Ohio 44195, USA.
Santhamma
Kizhakkekara R
KR
Sengupta
Saubhik
S
McCue
Brian
B
Kinter
Michael
M
Sen
Ganes C
GC
Sen
Indira
I
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002147:Calmodulin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002460:Cell Line; D003593:Cytoplasm; D004252:DNA Mutational Analysis; D004847:Epithelial Cells; D013058:Mass Spectrometry; D008565:Membrane Proteins; D051379:Mice; D007703:Peptidyl-Dipeptidase A / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D012694:Serine / Q000737:chemistry; D013755:Tetradecanoylphorbol Acetate
D002147:Calmodulin; D008565:Membrane Proteins; D012694:Serine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D007703:Peptidyl-Dipeptidase A; D013755:Tetradecanoylphorbol Acetate