Abstract
Both germinal (gACE) and somatic (sACE) isozymes of angiotensin-converting enzyme (ACE) are type I ectoproteins whose enzymatically active ectodomains are cleaved and shed by a membrane-bound protease. Here, we report a role of protein tyrosine phosphorylation in regulating this process. Strong enhancements of ACE cleavage secretion was observed upon enhancing protein Tyr phosphorylation by treating gACE- or sACE-expressing cells with pervanadate, an inhibitor of protein Tyr phosphatases. Secreted gACE, cell-bound mature gACE and its precursors were all Tyr-phosphorylated, as was the endoplasmic reticulum protein, immunoglobulin heavy chain-binding protein, that co-immunoprecipitated with ACE. The enhancement of cleavage secretion by pervanadate did not require the presence of the cytoplasmic domain of ACE, and it was not accomplished by enhancing the rate of intracellular processing of the protein. The observed enhancement of cleavage secretion of ACE in pervanadate-treated cells was specifically blocked by an inhibitor of the p38 mitogen-activated protein (MAP) kinase but not by inhibitors of many other Ser/Thr and Tyr protein kinases, including a specific inhibitor of protein kinase C that, however, could block the enhancement of cleavage secretion elicited by phorbol ester. These results indicate that ACE Tyr phosphorylation, probably in the endoplasmic reticulum, enhances the rate of its cleavage secretion at the plasma membrane using a regulatory pathway that may involve p38 MAP kinase.
Highlights
Renin-angiotensin system is considered a major regulator of fluid homeostasis, electrolyte balance and blood pressure
The observed enhancement of cleavage secretion of angiotensin-converting enzyme (ACE) in pervanadate-treated cells was blocked by an inhibitor of the p38 mitogen-activated protein (MAP) kinase but not by inhibitors of many other Ser/Thr and Tyr protein kinases, including a specific inhibitor of protein kinase C that, could block the enhancement of cleavage secretion elicited by phorbol ester
The binding of binding protein (BiP) with ACE inhibits the rate of ACE shedding by retaining ACE precursors in the endoplasmic reticulum (ER) [18]. (ii) Specific protein kinase C (PKC) isozymes associate with ACE in unstimulated cells
Summary
Materials—Compound 3 ([N-[DL-[2-(hydroxyaminocarbonyl)methyl]4-methylpentanoyl]-L-3-(tert-butyl)alanyl-L-alanine, 2-aminoethyl amide) was generously supplied by Dr Roy Black (Immunex Corp., Seattle, WA). ACE was immunoprecipitated from aliquots of cell extract and culture medium by polyclonal ACE antiserum, analyzed by SDS-PAGE, and transferred to nitrocellulose membranes. In a complementary assay, biotinylated ACE from similar aliquots of cell extract and culture medium were precipitated by immobilized streptavidin gel (Pierce), analyzed by SDS-PAGE, Western blotted with anti-ACE antibody, and quantified as mentioned above. At the end of the incubation, the culture medium were discarded, and cell lysates were prepared in radioimmune precipitation assay buffer (1% Triton X-100 in 50 mM Tris-HCl buffer, pH 7.4, 0.15 M NaCl, 1 mM EDTA and 0.1% SDS [8]), containing 1 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and a protease inhibitor mixture. Immunoprecipitation with anti-tyrosine phosphate antibody (5 l) and SDS-PAGE analysis was conducted, as mentioned above, except that radioimmune precipitation assay buffer described above was used for immunoprecipitation.
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