Phosphorylation of endothelin converting enzyme-1 isoforms: relevance to subcellular localization.

Endothelin-converting enzyme (ECE)-1 is a metalloenzyme with four subisoforms, which differ only in their amino-terminal domain. ECE-1a and c are the most common isoforms and are found at the plasma membrane and in the Golgi complex, whereas ECE-1b displays lysosomal localization. We have recently shown that ECE-1a but not ECE-1b also colocalizes with nuclear membrane markers, and that maintenance of cells in high glucose (25 mM) promotes relocalization of ECE-1a from the membrane to the intracellular compartment. To investigate the mechanisms involved in this process, we conducted a search for potential phosphorylation sites, which yielded a different number of putative sites for protein kinase (PK)-C and PKA in the amino-terminal region. Stimulation of Chinese hamster ovary (CHO) cells expressing a green fluorescent protein (GFP)-tagged human ECE-1a or ECE-1b with 100 nM phorbol myristate acetate (PMA) resulted in phosphorylation of ECE-1a, as determined by immunoprecipitation with an antibody to GFP followed by immunoblotting with an antibody to phosphoserine. Stimulation of cells with PMA also promoted intracellular relocalization, as seen in cells grown under high-glucose conditions. Incubation of cells grown in 25 mM glucose with the PKC inhibitor, calphostin C (100 nM), partially prevented the relocalization of ECE-1a from the plasma membrane to intracellular compartments. Stimulation of cells with 100 nM forskolin caused phosphorylation of ECE-1b and not ECE-1a, which is consistent with the lack of a putative PKA site in the ECE-1a amino-terminal sequence. Although phosphorylation is not required for ECE-1 enzymatic activity, these results suggest that ECE-1 isoforms are phosphorylated and that phosphorylation might play an important role in the regulation of intracellular trafficking of ECE-1 subisoforms.

Constitutive ERK1/2 activation by a chimeric neurokinin 1 receptor-beta-arrestin1 fusion protein. Probing the composition and function of the G protein-coupled receptor "signalsome".

The beta-arrestins, a small family of G protein-coupled receptor (GPCR)-binding proteins involved in receptor desensitization, have been shown to bind extracellular signal-regulated kinases 1 and 2 (ERK1/2) and function as scaffolds for GPCR-stimulated ERK1/2 activation. To better understand the mechanism of beta-arrestin-mediated ERK1/2 activation, we compared ERK1/2 activation by the wild-type neurokinin 1 (NK1) receptor with a chimeric NK1 receptor having beta-arrestin1 fused to the receptor C terminus (NK1-betaArr1). The NK1 receptor couples to both G(s) and G(q/11), resides on the plasma membrane, and mediates rapid ERK1/2 activation and nuclear translocation in response to neurokinin A. In contrast, NK1-betaArr1 is a G protein-uncoupled "constitutively desensitized" receptor that resides almost entirely in an intracellular endosomal compartment. Despite its inability to respond to neurokinin A, we found that NK1-betaArr1 expression caused robust constitutive activation of cytosolic ERK1/2 and that endogenous Raf, MEK1/2, and ERK1/2 coprecipitated in a complex with NK1-betaArr1. While agonist-dependent ERK1/2 activation by the NK1 receptor was independent of protein kinase A (PKA) or PKC activity, NK1-betaArr1-mediated ERK1/2 activation was completely inhibited when basal PKA and PKC activity were blocked. In addition, the rate of ERK1/2 dephosphorylation was slowed in NK1-betaArr1-expressing cells, suggesting that beta-arrestin-bound ERK1/2 is protected from mitogen-activated protein kinase phosphatase activity. These data suggest that beta-arrestin binding to GPCRs nucleates the formation of a stable "signalsome" that functions as a passive scaffold for the ERK1/2 cascade while confining ERK1/2 activity to an extranuclear compartment.

Acute effects of PGF2alpha on MMP-2 secretion from human ciliary muscle cells: a PKC- and ERK-dependent process.

PURPOSE: Studies were designed to evaluate the cellular mechanisms associated with prostaglandin (PG)F(2alpha)-induced matrix metalloproteinase (MMP)-2 secretion from human ciliary muscle (HCM) cells. METHODS: The secretion and activity of MMP-2 was determined by Western blot analysis and zymography, using conditioned medium and HCM cells. ERK1/2 activity was measured by in-gel kinase assay and Western blot analysis with anti-phospho-ERK1/2 antibodies. RESULTS: PGF(2alpha) increased the secretion of MMP-2 in a dose-dependent manner with an EC(50) of 2.7 x 10(-8) M. The addition of 1 muM PGF(2alpha) also increased MMP-2 secretion in a time-dependent manner with maximum secretion occurring at 4 hours after administration. At 4 hours, the maximum increase in MMP-2 secretion and activity were 112% +/- 32% and 88% +/- 18%, respectively. The secretory action of PGF(2alpha) was inhibited by pretreatment with a protein kinase C (PKC) inhibitor, chelerythrine chloride; the FP receptor antagonist, AL-8810; and the MEK inhibitor, PD-98059. The addition of PGF(2alpha) and latanoprost acid increased ERK1/2 activity by 117% +/- 12% and 75% +/- 9%, respectively. The PGF(2alpha)- and latanoprost-acid-induced ERK1/2 activation was blocked by the presence of PKC inhibitors and downregulation of PKC by prolonged incubation with a phorbol ester. CONCLUSIONS: These data provide evidence that FP receptor activation leads to an increase in the secretion and activation of MMP-2 through PKC- and ERK1/2-dependent pathways. FP-agonist-induced activation of ERK1/2 was blocked by PKC inhibitors, indicating that PKC activation is required for ERK1/2 activation and MMP-2 secretion from HCM cells. In the ciliary muscle, the functional responses to ERK1/2 activation include secretion of MMP-2, supporting the hypothesis that increases in uveoscleral outflow facility induced by PG administration involves the secretion and activation of MMP-2.

Nuclear localization of endothelin-converting enzyme-1: subisoform specificity.

OBJECTIVE: The biosynthesis of endothelin-1 (ET-1), the most potent vasoconstrictor with mitogenic properties, involves the processing of intermediate protein big ET-1 by a unique metalloprotease, endothelin-converting enzyme-1 (ECE-1). ECE-1 has 4 subisoforms that possess the same catalytic properties but different localization patterns on the plasma membrane and cytosol. We investigated the trafficking of ECE-1 subisoforms using green fluorescent protein-tagged recombinant enzymes in target and nontarget cells. METHODS AND RESULTS: ECE-1 localization was studied using confocal microscopy, which provides evidence for the first time that both ET-1 and ECE-1a are also found in the nuclear compartment in transiently transfected cells as well as in native endothelial cells that endogenously possess the ET system. In cells maintained in high-glucose medium, ECE-1a-specific staining shifted from plasma membrane to intracellular compartments. ECE-1b subisoform, however, is mainly in the cytosolic compartment, indicating a subisoform specificity for nuclear localization. CONCLUSIONS: Our findings define a novel localization pattern for the ET system, which may be differentially regulated under pathophysiological conditions.

Vascular endothelin converting enzyme-1 expression and activity is upregulated in clinical diabetes.

Circulating and vascular endothelin-1 (ET-1) levels are elevated in diabetes, but the molecular components of the enzymatic activation of ET-1 in the vasculature remains unknown. Furthermore, the distribution of ET receptors favors a contractile phenotype in African Americans with diabetes. Whether there is any difference in local ET-1 activation in this population is unknown. This study examined the expression and activity of ET converting enzyme-1 subisoforms (ECE-1) in the internal mammary artery specimens obtained from patients undergoing coronary artery bypass grafting. The study groups included African-American (AA) and Caucasian (CA), nondiabetic (ND) and diabetic (D) patients: AAND N = 10, CAND N = 9, AAD N = 9, and CAD N = 11. The expression of ECE-1 a, ECE-1 b and ECE-1c subisoforms was studied by RT-PCR. ECE-1 a was upregulated 2- and 4-fold in the CAD and MD groups, respectively (P < .05). In African-American patient groups, ECE-1 activity (fmol/ mg protein.h) was augmented from 2,804 +/- 185 in nondiabetic tissue samples to 6,857 +/- 393 in the diabetic tissue (P < .05). There was a similar increase in the CAD group, which did not significantly differ from AA diabetics. ECE-1 inhibitors, phosphoramidon and FR-901533, inhibited vascular ECE-1 activity by more than 80%. While neutral endopeptidase (NEP) and matrix metalloproteinase-2 (MMP-2) are able to process big ET-1, inhibitors of NEP (thiorphan) and MMP (batimistat) did not affect ECE-1 activity. In conclusion, the enzymatic pathway essential for generating vascular ET-1 is activated in the vasculature of both AA and CA diabetic patients and this activation is highly specific for ECE-1.

Prostaglandin F(2alpha) stimulates tyrosine phosphorylation of phospholipase C-gamma1.

In this study, we investigated the ability of prostaglandin F(2alpha) (PGF(2alpha)) to induce tyrosine phosphorylation of phospholipase C-gamma1 (PLC-gamma1) in cat iris sphincter smooth muscle (CISM) cells. PGF(2alpha)(1 microM) stimulated PLC-gamma1 tyrosine phosphorylation in a time- and dose-dependent manner with a maximum increase of 3-fold at 0.5min. The protein tyrosine kinase inhibitors, genistein, and tyrphostin A-25, blocked the stimulatory effects of PGF(2alpha), suggesting involvement of protein tyrosine kinase activity in the physiological actions of the PGF(2alpha). Furthermore, PGF(2alpha)-induced p42/p44 MAP kinase activation was also completely blocked by protein tyrosine kinase inhibitors. In summary, these findings show that PGF(2alpha) stimulates tyrosine phosphorylation of PLC-gamma1 in CISM cells and indicate that PGF(2alpha)-stimulated tyrosine phosphorylation is responsible for an early signal transduction event.