Role of Tyrosine Phosphorylation in the Regulation of Cleavage Secretion of Angiotensin-converting Enzyme

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 sACEexpressing 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.Renin-angiotensin system is considered a major regulator of fluid homeostasis, electrolyte balance and blood pressure. Angiotensin-converting enzyme (ACE), 1 a key component of this system, converts the inactive precursor angiotensin I to the active pressor hormone angiotensin II (1). It also inactivates bradykinin, a vasodilator. The role of ACE within the reninangiotensin system is well established, and inhibition of this enzyme is routinely used for therapeutic management of hypertension. ACE-like proteins have been identified in lower organisms, such as Drosophila (2, 3), indicating that it is an evolutionarily conserved protein that also shows an overall sequence homology of 80 -90% across mammalian species. This evolutionary importance of ACE, coupled with its widespread distribution in many organs within a species, suggests that over and above its role in the renin-angiotensin system, ACE probably plays a much broader role in general physiology. This notion is supported by the severely abnormal phenotype of ACE knockout mouse (4 -6).There are two different isozymic forms of ACE. These are derived from the same gene by tissue specific choice of alternative transcription initiation sites. The larger somatic ACE (sACE), a component of the renin-angiotensin system, is expressed in vascular endothelial cells, renal proximal tubule epithelial (RTE) cells, intestinal brush border cells, macrophages, and monocytes. The smaller germinal ACE (gACE) is expressed exclusively in the maturing sperm and is involved in male reproductio...

Pulsatile Mechanical Pressure Promotes Angiotensin-Converting Enzyme Expression in Aortic Smooth Muscle Cells

“…The concentration of AII in the culture media was not increased under the pressurized condition in our experiments, which may due to the absence of angiotensin I (data not shown). Although other mechanisms may be involved [29,30], our findings in this study suggest that the expression and activity of ACE in HASMCs were partly controlled by local AII produced by the ACE in HASMCs themselves, indicating the existence of a local negative feedback effect in addition to the systemic feedback produced by circulating AII.…”

Calmodulin Binds to the Cytoplasmic Domain of Angiotensin-converting Enzyme and Regulates Its Phosphorylation and Cleavage Secretion

“…Recently we also showed that tyrosine phosphorylation of the ectodomain of ACE regulates ACE shedding (28). Here we show that ACE is also phosphorylated on a specific serine residue of the cytoplasmic domain.…”

“…Therefore, the regulation of ACE cleavage secretion might be necessary for maintaining this balance. Our previous studies have demonstrated that ACE shedding is a regulated process, which could be stimulated by PMA, an activator of protein kinase C (24), or pervanadate, an inhibitor of tyrosine phosphatases (28), and inhibited by inhibitors of specific class of metalloproteases (22). Specific cellular proteins are associated with ACE molecules and regulate the ACE shedding process (27).…”

Signaling via the Angiotensin-Converting Enzyme Results in the Phosphorylation of the Nonmuscle Myosin Heavy Chain IIA

“…However, there are some differences in the cytoplasmic domains of the two proteins; for example, the cleavage/secretion of rabbit testicular ACE has been reported to be regulated by the tyrosine phosphorylation of its cytoplasmic tail (Santhamma et al, 2004), whereas the cytoplasmic domain of human somatic ACE does not contain a tyrosine residue. We were also unable to detect a direct effect of PKC on the phosphorylation of ACE-associated MYH9.…”