Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The ubiquitous plasma membrane Na+/H+ exchanger (termed NHE1) is activated by diverse hormonal signals, with the notable exception of hormones acting through cAMP as second messenger. Therefore, the Na+/H+ exchanger found in the nucleated trout red cell is of particular interest since it is activated by catecholamines, forskolin, and cAMP analogues. We report here that a cloned cDNA encoding the red cell exchanger restores functional Na+/H+ activity when transfected into Na+/H+ antiporter-deficient fibroblasts (i.e., it regulates intracellular pH in a Na-dependent and amiloride-sensitive manner). This red cell exchanger represents an additional form of Na+/H+ exchanger (termed beta NHE), which is characterized by a specific cytoplasmic domain involved in activation by the cAMP-dependent signaling pathway. After transfection in the same cellular context, beta NHE, but not NHE1, is activated by cAMP or by hormones that increase cAMP levels. Comparison of the amino acid sequences of exchangers shows that beta NHE, but not NHE1, contains two clustered consensus motifs for phosphorylation by a cAMP-dependent protein kinase (protein kinase A; PKA). A deletion mutant devoid of the C-terminal region of the cytoplasmic loop containing the two PKA sites restores Na+/H+ activity but is no longer activated by cAMP analogues or catecholamines. In red blood cells, the Na+/H+ exchanger is also activated by another pathway involving protein kinase C (PKC). Expression of beta NHE in fibroblasts shows that these two independent signaling pathways impinge on two distinct domains of the exchanger. The cytoplasmic segment containing PKA consensus sites, which is crucial for cAMP activation, is unnecessary for stimulation by PKC activators.
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PMID:Cloning and expression of a cAMP-activated Na+/H+ exchanger: evidence that the cytoplasmic domain mediates hormonal regulation. 137 18

All cloned members of the mammalian Na+/H+ exchanger gene family encode proteins that consist of two functionally distinct domains: a membrane-bound N terminus and a cytoplasmic C terminus, which are required for ion transport and regulation of transport, respectively. Despite their similarity in structure, three members of this family, designated NHE1, NHE2, and NHE3, exhibit different kinetic mechanisms in response to growth factors and protein kinases. For instance, growth factors stimulate NHE1 by a change in the affinity constant for intracellular H+, K'(Hi+), and regulate NHE2 and NHE3 by a change in Vmax. We have constructed chimeric Na+/H+ exchangers by exchanging the N and C termini among three cloned rabbit Na+/H+ exchangers (NHE1 to NHE3) to determine which domain is responsible for the above Vmax-vs.-K'(H(i)+) effect of the Na+/H+ isoforms. All of the chimeras had functional exchange activity and basal kinetic properties similar to those of wild-type exchangers. Studies with serum showed that the N terminus is responsible for the Vmax-vs.-K'(H(i)+) stimulation of the Na+/H+ exchanger isoforms. Moreover, phorbol 12-myristate 13-acetate and fibroblast growth factor altered Na+/H+ exchange only in chimeras that had an epithelial N-terminal domain matched with an epithelial C-terminal domain. Therefore, the protein kinase-induced regulation of Na+/H+ exchangers is mediated through a specific interaction between the N- and C-termini, whcih is restricted so that epithelial N- and epithelial N-and C-terminal portions of the exchangers are required for regulation.
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PMID:Chimeric Na+/H+ exchangers: an epithelial membrane-bound N-terminal domain requires an epithelial cytoplasmic C-terminal domain for regulation by protein kinases. 747 72

Na+/H+ exchanger (NHE) activity is regulated by several types of receptors directly coupled to distinct classes (i.e. Gs, Gi, Gq, and G12) of heterotrimeric (alpha beta gamma) GTP-binding proteins (G proteins), which, upon activation, modulate production of various second messengers (e.g. cAMP, cGMP, diacylglycerol, inositol trisphosphate, and Ca2+). Recently, four isoforms of the rat Na+/H+ exchanger were identified by molecular cloning. To examine their intrinsic responsiveness to G protein and second messenger stimulation, three of these isoforms, NHE-1, -2, and -3, were stably expressed in mutant Chinese hamster ovary cells devoid of endogenous NHE activity (AP-1 cells). Incubation of cells with either AIF4-, a general agonist of G proteins, or cholera toxin, a selective activator of G alpha s that stimulates adenylate cyclase, accelerated the rates of amiloride-inhibitable 22Na+ influx mediated by NHE-1 and -2, whereas they inhibited that by NHE-3. Similarly, short term treatment with phorbol 12-myristate 13-acetate, which mimics diacylglycerol activation of protein kinase C (PKC), or with agents (i.e. forskolin, 8-(4-chlorophenylthio)-cAMP, and isobutylmethylxanthine) that lead to activation of cAMP-dependent protein kinase (PKA) also stimulated transport by NHE-1 and NHE-2 but depressed that by NHE-3. The effects of phorbol 12-myristate 13-acetate were blocked by depleting cells of PKC or by inhibiting PKC using chelerythrine chloride, confirming a role for PKC in modulating NHE isoform activities. Likewise, the PKA antagonist, H-89, attenuated the effects of elevated cAMPi on NHE-1, -2, and -3, further demonstrating the regulation by PKA. Unlike cAMPi, elevation of cGMPi by treatment with dibutyryl-cGMP or 8-bromo-cGMP had no influence on NHE isoform activities, thereby excluding the possibility of a role for cGMP-dependent protein kinase in these cells. These data support the concept that the NHE isoforms are differentially responsive to agonists of the PKA and PKC pathways.
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PMID:Plasma membrane Na+/H+ exchanger isoforms (NHE-1, -2, and -3) are differentially responsive to second messenger agonists of the protein kinase A and C pathways. 749 49

Parathyroid hormone (PTH) and parathyroid hormone-related peptide (PTHRP) regulate Na+/H+ exchanger activity in osteoblastic cells, although the signaling components involved are not precisely defined. Since these peptide hormones can stimulate production of diverse second messengers (i.e. cAMP and diacylglycerol) that activate protein kinase A (PKA) and protein kinase C (PKC) in target cells, it is conceivable that either one or both of these pathways can participate in modulating exchanger activity. To discriminate among these possibilities, a series of synthetic PTH and PTHRP fragments were used that stimulate adenylate cyclase and/or PKC. In the osteoblastic cell line UMR-106, human PTH(1-34) and PTHRP(1-34) augmented adenylate cyclase activity, whereas PTH(3-34), PTH(28-42), and PTH(28-48) had no effect. Nevertheless, all these peptide fragments were found to enhance PKC translocation from the cytosol to the membrane in a dose-dependent (10(-11) to 10(-7) M) manner. PTHRP(1-16), a biologically inert fragment, was incapable of influencing either the PKA or PKC pathway. PTH(1-34) and PTHRP(1-34), but not PTH(3-34), PTH(28-42), PTH(28-48), or PTHRP(1-16), elevated Na+/H+ exchanger activity, implicating cAMP as the transducing signal. In accordance with this observation, forskolin (10 microM), which directly stimulates adenylate cyclase, also activated Na+/H+ exchanger activity. The involvement of PKA was verified when the highly specific PKA inhibitor, H-89, completely abolished the stimulatory effect of PTH(1-34) and forskolin on Na+/H+ exchange. In addition, Northern blot analysis revealed the presence of only the NHE-1 isoform of the Na+/H+ exchanger in UMR-106 cells. In summary, these results indicated that PTH and PTHRP activate the Na+/H+ exchanger NHE-1 isoform in osteoblastic UMR-106 cells exclusively via a cAMP-dependent pathway.
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PMID:Parathyroid hormone and parathyroid hormone-related peptide activate the Na+/H+ exchanger NHE-1 isoform in osteoblastic cells (UMR-106) via a cAMP-dependent pathway. 755 63

Na+/H+ exchanger isoform and the effect of high osmolality on its function was studied in cultured renal epithelial cells (LLC-PK1 and OK). Using NHE-3-specific antibody, immunoblots of luminal membranes from LLC-PK1 and OK cells specifically labeled proteins with molecular masses 90 and 95 kDa, indicating that NHE-3 is the isoform expressed on the luminal membranes of these epithelia. Proximal tubular suspensions from rabbit kidney cortex were incubated in control (310 mosm/liter) or high osmolality (510 mosm/liter) medium for 45 min and utilized for brush border membrane vesicle preparation. Influx of amiloride-sensitive 22Na+ at 10 s (pHo 7.5, pHi 6.0) into brush border membrane vesicles was 37% lower in the high osmolality group (p < 0.03). LLC-PK1 or OK cells were grown to confluence and examined for Na+/H+ exchange activity. An increase in medium osmolality to 510 mosm following acid loading decreased the 5-min uptake of the amiloride-sensitive 22Na+ in LLC-PK1 and OK cells (p < 0.04 and < 0.03 for LLC-PK1 cell OK cells, respectively). An increase in medium osmolality to 510 mosm in vascular smooth muscle cells, which express NHE-1, produced 45 and 64% stimulation of the amiloride-sensitive 22Na+ influx at base-line pHi and acid-loaded condition, respectively (p < 0.03 and < 0.01). Down-regulation of protein kinase C by preincubation with phorbol 12-myristate 13-acetate or inhibition of Ca(2+)-calmodulin-dependent protein kinase (calmodulin-kinase II) by N-6-aminohexyl-5-chloro-1-naphthalenesulfonamide (W-7) in LLC-PK1 cells did not block the inhibitory effect of high osmolality on Na+/H+ exchange activity. We conclude that renal proximal tubule epithelial cells express Na+/H+ exchange isoform NHE-3 on their luminal membranes and that hyperosmolality decreases transporter activity during cell acidification. This inhibitory effect might be unique to the NHE-3 isoform, since vascular smooth muscle cells which express NHE-1 exhibit an increase in Na+/H+ exchange activity in response to high osmolality.
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PMID:Effect of high osmolality on Na+/H+ exchange in renal proximal tubule cells. 819 9

Studies of the effect of cAMP on the cloned Na+/H+ exchangers (NHEs) are difficult to interpret as variable results have been reported for the different isoforms when expressed in various cell types. We took advantage of the fact that the human NHE1 and the trout erythrocyte beta NHE, when expressed in the same cell line, PS120, respond differently to cAMP (NHE1 is insensitive, beta NHE is activated) to analyze the molecular mechanisms of cAMP activation. We constructed both a chimera between NHE1 and beta NHE and a set of beta NHE mutants to delineate the critical parts of the molecule involved in the activation process. NHE1 becomes cAMP stimulated when its cytoplasmic domain is replaced by the cytoplasmic domain of beta NHE; thus, the cytoplasmic C terminus of beta NHE, which contains two cAMP-dependent consensus sequences, is essential to confer cAMP dependence. Serine to glycine substitution of only one of the two protein kinase A (PKA) consensus sites decreased by 60% the ability of cAMP to activate Na+/H+ exchange. Simultaneous Ser to Gly substitution of the two PKA consensus sites decreased the cAMP-mediated activation by 72%. The residual activation required a cytoplasmic fragment (aa 559-661) that contains four sequences considered likely as putative PKA consensus sites. The results obtained with the chimeric NHE also demonstrated that if the cytoplasmic C terminus is crucially involved in the hormonal activation, the rate of Na+/H+ exchange so induced can be modulated by the nature of the interaction between the N- and C-terminal domains.
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PMID:The cytoplasmic domain of the Na+/H+ exchangers (NHEs) dictates the nature of the hormonal response: behavior of a chimeric human NHE1/trout beta NHE antiporter. 820 3

Renal epithelial cells may express apical and basolateral Na/H exchangers which are different in their physiological regulation and different in their sensitivities to the inhibitor amiloride. In the present study RKPC-2 cells [a Simian virus 40 (SV-40) transformed cell line of rabbit S2 proximal tubular origin] were examined for localization (apical vs basolateral) and regulation of Na/H-exchange activity(ies) by parathyroid hormone (PTH). In addition, using specific cDNA probes we determined the expression of multiple isoforms of Na/H exchangers in RKPC-2 cells. By the use of BCECF [2',7',bis(2-carboxyethyl)-5,6-carboxyfluorescein intracellular pH (pHi) indicator] and single cell fluorescence microscopy, Na/H-exchange activities (defined as initial rate of Na-dependent pHi recovery) were found on the apical and basolateral membrane of RKPC-2 cells; apical and basolateral transport activities differed in sensitivity to dimethylamiloride, the basolateral being more sensitive. Northern blot analysis demonstrated the presence of a 5.2-kb transcript, related to Na/H-exchanger activity NHE-1, and a 3.2-kb transcript, related to Na/H-exchanger activity NHE-2. PTH (10(-8) M) inhibited apically and basolaterally located Na/H-exchanger activities. The inhibitory effect of PTH was mimicked by 8-bromo-adenosine 3'5'-cyclic monophosphate (cAMP); it was blunted in the presence of H-89 (inhibitor of protein kinase A) and was unaffected by calphostin C (inhibitor of protein kinase C). In contrast to 8-bromo-cAMP (and PTH), exposure of RKPC-2 cells to phorbol 12-myristate 13-acetate (TPA) caused a significant stimulation of both Na/H-exchange activities.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Identification of PTH-responsive Na/H-exchanger isoforms in a rabbit proximal tubule cell line (RKPC-2). 825 19

The human fibroblast, "amiloride-sensitive" Na/H exchanger (NHE1) was transfected into opossum kidney cells (OK cells) (OK/NHE1 cells). Northern blot analysis confirmed that the NHE1 message is expressed in OK/NHE1 cells. In immunoblot analysis, an anti-human NHE1 antibody labelled a membrane protein only present in OK/NHE1 cells. In contrast to the parental cell line containing only an apically located, "amiloride-resistant" Na/H exchange activity, OK/NHE1 cells contain apically and basolaterally located Na/H exchange activities, the apical activity being "amiloride resistant" and the basolateral being "amiloride sensitive". Parathyroid hormone (PTH) inhibited apical transport activity (OK and OK/NHE1 cells) but had no effect on basolateral transport activity (OK/NHE1 cells). Pharmacological activation of protein kinase A (forskolin) decreased both apical and basolateral Na/H exchange activity. Incubation with phorbol ester (exogenous activation of protein kinase C) reduced apical Na/H exchange activity (OK and OK/NHE1 cells) but had only a moderate, inhibitory effect on basolateral Na/H exchange activity (OK/NHE1 cells). These results indicate that transfection of OK cells with human fibroblast NHE1 cDNA encoding an "amiloride-sensitive" form of the Na/H exchanger results in expression of basolaterally located "NHE1-related" transport activity. Regulatory control of intracellular Na/H exchange activities (apically versus basolaterally located) and intercellular Na/H exchange activities (NHE1-related) differs. This may relate to cell-specific properties as well as to exchanger-specific properties.
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PMID:Na/H exchange activities in NHE1-transfected OK-cells: cell polarity and regulation. 827 82

This review focuses on studies from our laboratory investigating the mechanisms of chronic regulation of the Na/H antiporter in renal and nonrenal cells. Tissue culture provides an ideal tool for investigating this problem because it avoids many complicating effects that would occur in an intact animal during a chronic study. Chronic decreases in extracellular fluid pH cause an increase in Na/H antiporter activity that is dependent on protein synthesis and associated with an increase in NHE-1 (isoform of the sodium-hydrogen antiporter) mRNA abundance. This effect is associated with acid-induced increases in a number of immediate early genes, including c-fos, c-jun, junB, and egr-1. In primary cultures of rabbit proximal tubule cells, activation of protein kinase C for 2 hours causes an increase in Na/H antiporter activity that persists 24 hours later, is dependent on transcription and translation, and is associated with an increase in NHE-1 mRNA abundance. Chronic activation of protein kinase A in opossum kidney (OKP) cells causes an increase in Na/H antiporter activity that persists 16 to 20 hours later and is dependent on protein synthesis. This latter effect is of particular interest because it is opposite in direction to the acute inhibitory effect of protein kinase A on the Na/H antiporter in these cells.
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PMID:Chronic regulation of the Na/H antiporter. 839 72

The Na+/H+ exchangers belong to a family of transport proteins involved in intracellular pH regulation and vectorial sodium transport across various epithelial tissues. We have cloned a unique isoform (NHE-2) by screening a rat intestinal cDNA library utilizing a human NHE-1 fragment. In this report we describe molecular cloning of the human Na+/H+ exchanger (NHE-2, HGMW-approved symbol SLC9A2). The cDNA encodes a protein of 698 amino acid residues. Human NHE-2 is widely distributed in tissues of the gastrointestinal tract, kidney, heart, testes, uterus, and adrenal glands. Hydropathy plot indicates that the translated protein is predicted to have 10 transmembrane domains with three potential N-glycosylation sites and four potential cAMP- and cGMP-dependent protein kinase phosphorylation sites. This cDNA maps the location of the gene encoding NHE-2 to human chromosome 2q11.2.
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PMID:Molecular cloning, sequencing, chromosomal localization, and tissue distribution of the human Na+/H+ exchanger (SLC9A2). 859 99


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