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)

A H(+)-coupled amino acid transporter has been characterised functionally at the brush border membrane of the human intestinal cell line Caco-2. This carrier, hPAT1 (human Proton-coupled Amino acid Transporter 1) or SLC36A1, has been identified recently at the molecular level and hPAT1 protein is localised to the brush border membrane of human small intestine. hPAT1 transports both amino acids (e.g., beta-alanine) and therapeutic agents (e.g., D-cycloserine). In human Caco-2 cells, hPAT1 function (H(+)/amino acid symport) is associated with a decrease in intracellular pH (pH(i)), which selectively activates the Na(+)/H(+) exchanger NHE3, and thus maintains pH(i) and the driving force for hPAT1 function (the H(+) electrochemical gradient). This study provides the first evidence for regulation of hPAT1 function. Activation of the cAMP/protein kinase A pathway in Caco-2 cell monolayers either using pharmacological tools (forskolin, 8-br-cAMP, [(11,22,28)Ala]VIP) or physiological activators (the neuropeptides VIP and PACAP) inhibited hPAT1 function (beta-alanine uptake) at the apical membrane. Under conditions where NHE3 is inactive (the absence of Na(+), apical pH 5.5, the presence of the NHE3 inhibitor S1611) no regulation of beta-alanine uptake is observed. Forskolin and VIP inhibit pH(i) recovery (NHE3 function) from beta-alanine-induced intracellular acidification. Immunocytochemistry localises NHERF1 (NHE3 regulatory factor 1) to the apical portion of Caco-2 cells where it will interact with NHE3 and allow PKA-mediated phosphorylation of NHE3. In conclusion, we have shown that amino acid uptake via hPAT1 is inhibited by activators of the cAMP pathway indirectly through inhibition of NHE3 activity.
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PMID:Indirect regulation of the intestinal H+-coupled amino acid transporter hPAT1 (SLC36A1). 1575 24

The Na(+)/H(+) exchanger 3 (NHE3) is expressed in the brush border membrane (BBM) of proximal tubules (PT). Its activity is down-regulated on increases in intracellular cAMP levels. The aim of this study was to investigate the contribution of the protein kinase A (PKA) and the exchange protein directly activated by cAMP (EPAC) dependent pathways in the regulation of NHE3 by adenosine 3',5'-cyclic monophosphate (cAMP). Opossum kidney cells and murine kidney slices were treated with cAMP analogs, which selectively activate either PKA or EPAC. Activation of either pathway resulted in an inhibition of NHE3 activity. The EPAC-induced effect was independent of PKA as indicated by the lack of activation of the kinase and the insensitivity to the PKA inhibitor H89. Both PKA and EPAC inhibited NHE3 activity without inducing changes in the expression of the transporter in BBM. Activation of PKA, but not of EPAC, led to an increase of NHE3 phosphorylation. In contrast, activation of PKA, but not of EPAC, inhibited renal type IIa Na(+)-coupled inorganic phosphate cotransporter (NaPi-IIa), another Na-dependent transporter expressed in proximal BBM. PKA, but not EPAC, induced the retrieval of NaPi-IIa from BBM. Our results suggest that EPAC activation may represent a previously unrecognized mechanism involved in the cAMP regulation of NHE3, whereas regulation of NaPi-IIa is mediated by PKA but not by EPAC.
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PMID:Regulation of sodium-proton exchanger isoform 3 (NHE3) by PKA and exchange protein directly activated by cAMP (EPAC). 1640 44

Although aldosterone influences a variety of cellular processes through nongenomic mechanisms, the significance of nongenomic pathways for aldosterone-induced regulation of epithelial function is not understood. Recently, we demonstrated that aldosterone inhibits transepithelial HCO(3)(-) absorption in the medullary thick ascending limb (MTAL) through a nongenomic pathway. This inhibition is mediated through a direct cellular action of aldosterone to inhibit the apical membrane NHE3 Na(+)/H(+) exchanger. The present study was designed to identify the intracellular signaling pathway(s) responsible for this aldosterone-induced transport regulation. In rat MTALs perfused in vitro, addition of 1 nM aldosterone to the bath decreased HCO(3)(-) absorption by 30%. This inhibition was not mediated by cAMP/PKA and was not prevented by inhibitors of PKC or PI3-K, pertussis toxin, or rapamycin. The inhibition of HCO(3)(-) absorption by aldosterone was largely eliminated by the MEK/ERK inhibitors U-0126 and PD-98059. Aldosterone increased ERK activity 1.8-fold in microdissected MTALs. This ERK activation is rapid (</=5 min) and is blocked by U-0126 or PD-98059 but is unaffected by spironolactone or actinomycin D. Pretreatment with U-0126 to block ERK activation prevented the effect of aldosterone to inhibit apical NHE3. These data demonstrate that aldosterone inhibits NHE3 and HCO(3)(-) absorption in the MTAL through rapid activation of the ERK signaling pathway. The results identify NHE3 as a target for nongenomic regulation by aldosterone and establish a role for ERK in the acute regulation of NHE3 and its epithelial absorptive functions.
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PMID:Aldosterone inhibits apical NHE3 and HCO3- absorption via a nongenomic ERK-dependent pathway in medullary thick ascending limb. 1675 29

Diarrhea associated with inflammatory bowel disease has been attributed to stimulated secretion of proinflammatory cytokines like IFN-gamma and TNF-alpha, which have been shown to downregulate the expression of the sodium-hydrogen exchanger-3 (NHE3) gene. In this study, we have investigated the mechanism of NHE3 gene regulation by IFN-gamma and TNF-alpha in C2BBe1 cells. In response to both IFN-gamma (30 ng/ml) and TNF-alpha (20 ng/ml), the construct containing the bp -95 to +5 region of the human NHE3 promoter, which harbors a number of cis-elements including four potential Sp1 binding sites, showed a maximum repression of 60%. Knockdown of Sp1 and Sp3 expression using small interfering RNA resulted in a significant inhibition of the NHE3 promoter activity and resistance to cytokines effects. These cytokines showed no effects on the expression of Sp1 and Sp3 mRNA and protein levels as assessed by RT-PCR and Western blot analyses, respectively. After treatment with cytokines, the binding of Sp1 and Sp3 proteins to NHE3 promoter decreased significantly, as seen by gel mobility shift assays and chromatin immunoprecipitation assays. The inhibitory effects of both cytokines on the NHE3 promoter were completely blocked by the broad-range kinase inhibitor staurosporine and the selective protein kinase A (PKA) inhibitor 8-bromoadenosine-3',5'-cyclic monophosphorothioate, Rp-isomer. The binding affinity of Sp1 and Sp3 proteins for NHE3 Sp1 probe was significantly decreased after in vitro phosphorylation of nuclear proteins by the alpha-catalytic subunit of PKA. Our data indicate that IFN-gamma and TNF-alpha may repress the NHE3 promoter activity in C2BBe1 cells by PKA-mediated phosphorylation of Sp1 and Sp3 transcription factors.
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PMID:IFN-gamma and TNF-alpha regulate human NHE3 gene expression by modulating the Sp family transcription factors in human intestinal epithelial cell line C2BBe1. 1676 Feb 59

It has been shown that when CFTR and NHE3 are co-expressed on the apical membrane of the A6-NHE3 cell monolayers, the two transporters interact via a shared regulatory complex composed of NHERF2, ezrin, and PKA. We observe here that co-expression of NHE3 reduced both PKA-dependent apical CFTR expression and its activation once in place by approximately 50%. To analyze the role of NHERF2 in this process, we transfected NHE3 expressing and non-expressing A6 monolayers with NHERF2 cDNA in which its binding domains had been deleted. When only CFTR is expressed on the apical membrane, deletion of any of the NHERF2 binding domains inhibited both PKA-dependent apical CFTR expression and its activation, while when NHE3 was co-expressed with CFTR PDZ2 deletion was without effect on CFTR sorting and activity. This suggests that when the PDZ2 domain is "sequestered" by interacting with NHE3 it can no longer participate in CFTR functional expression.
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PMID:NHE3 inhibits PKA-dependent functional expression of CFTR by NHERF2 PDZ interactions. 1682 84

The serum- and glucocorticoid-inducible kinase-1 (SGK1) is ubiquitously expressed and under genomic control by cell stress (including cell shrinkage) and hormones (including gluco- and mineralocorticoids). Similar to its isoforms SGK2 and SGK3, SGK1 is activated by insulin and growth factors via phosphatidylinositol 3-kinase and the 3-phosphoinositide-dependent kinase PDK1. SGKs activate ion channels (e.g., ENaC, TRPV5, ROMK, Kv1.3, KCNE1/KCNQ1, GluR1, GluR6), carriers (e.g., NHE3, GLUT1, SGLT1, EAAT1-5), and the Na+-K+-ATPase. They regulate the activity of enzymes (e.g., glycogen synthase kinase-3, ubiquitin ligase Nedd4-2, phosphomannose mutase-2) and transcription factors (e.g., forkhead transcription factor FKHRL1, beta-catenin, nuclear factor kappaB). SGKs participate in the regulation of transport, hormone release, neuroexcitability, cell proliferation, and apoptosis. SGK1 contributes to Na+ retention and K+ elimination of the kidney, mineralocorticoid stimulation of salt appetite, glucocorticoid stimulation of intestinal Na+/H+ exchanger and nutrient transport, insulin-dependent salt sensitivity of blood pressure and salt sensitivity of peripheral glucose uptake, memory consolidation, and cardiac repolarization. A common ( approximately 5% prevalence) SGK1 gene variant is associated with increased blood pressure and body weight. SGK1 may thus contribute to metabolic syndrome. SGK1 may further participate in tumor growth, neurodegeneration, fibrosing disease, and the sequelae of ischemia. SGK3 is required for adequate hair growth and maintenance of intestinal nutrient transport and influences locomotive behavior. In conclusion, the SGKs cover a wide variety of physiological functions and may play an active role in a multitude of pathophysiological conditions. There is little doubt that further targets will be identified that are modulated by the SGK isoforms and that further SGK-dependent in vivo physiological functions and pathophysiological conditions will be defined.
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PMID:(Patho)physiological significance of the serum- and glucocorticoid-inducible kinase isoforms. 1701 87

Direct phosphorylation of sodium hydrogen exchanger type 3 (NHE3) is a well-established physiological phenomenon; however, the exact role of NHE3 phosphorylation in its regulation remains unclear. The objective of this study was to evaluate whether NHE3 phosphorylation at serines 552 and 605 is physiologically regulated in vivo and, if so, whether changes in phosphorylation at these sites are tightly coupled to changes in transport activity. To this end, we directly compared PKA-induced NHE3 inhibition with site-specific changes in NHE3 phosphorylation in vivo and in vitro. In vivo, PKA was activated using an intravenous infusion of parathyroid hormone in Sprague-Dawley rats. In vitro, PKA was activated directly in opossum kidney (OKP) cells using forskolin and IBMX. NHE3 activity was assayed in microvillar membrane vesicles in the rat model and by (22)Na uptake in the OKP cell model. In both cases, NHE3 phosphorylation at serines 552 and 605 was determined using previously characterized monoclonal phosphospecific antibodies directed to these sites. In vivo, we found dramatic changes in NHE3 phosphorylation at serines 552 and 605 with PKA activation but no corresponding alteration in NHE3 activity. This dissociation between NHE3 phosphorylation and activity was further verified in OKP cells in which phosphorylation clearly preceded transport inhibition. We conclude that although phosphorylation of NHE3 at serines 552 and 605 is regulated by PKA both in vivo and in vitro, phosphorylation of these sites does not directly alter Na(+)/H(+) exchange activity.
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PMID:NHE3 phosphorylation at serines 552 and 605 does not directly affect NHE3 activity. 1740 82

The multi-PDZ domain containing protein Na(+)/H(+) Exchanger Regulatory Factor 1 (NHERF1) binds to Na(+)/H(+) exchanger 3 (NHE3) and is associated with the brush border (BB) membrane of murine kidney and small intestine. Although studies in BB isolated from kidney cortex of wild type and NHERF1(-/-) mice have shown that NHERF1 is necessary for cAMP inhibition of NHE3 activity, a role of NHERF1 in NHE3 regulation in small intestine and in intact kidney has not been established. Here a method using multi-photon microscopy with the pH-sensitive dye SNARF-4F (carboxyseminaphthorhodafluors-4F) to measure BB NHE3 activity in intact murine tissue and use it to examine the role of NHERF1 in regulation of NHE3 activity. NHE3 activity in wild type and NHERF1(-/-) ileum and wild type kidney cortex were inhibited by cAMP, whereas the cAMP effect was abolished in kidney cortex of NHERF1(-/-) mice. cAMP inhibition of NHE3 activity in these two tissues is mediated by different mechanisms. In ileum, a protein kinase A (PKA)-dependent mechanism accounts for all cAMP inhibition of NHE3 activity since the PKA antagonist H-89 abolished the inhibitory effect of cAMP. In kidney, both PKA-dependent and non-PKA-dependent mechanisms were involved, with the latter reproduced by the effect on an EPAC (exchange protein directly activated by cAMP) agonist (8-(4-chlorophenylthio)-2'O-Me-cAMP). In contrast, the EPAC agonist had no effect in proximal tubules in NHERF1(-/-) mice. These data suggest that in proximal tubule, NHERF1 is required for all cAMP inhibition of NHE3, which occurs through both EPAC-dependent and PKA-dependent mechanisms; in contrast, cAMP inhibits ileal NHE3 only by a PKA-dependent pathway, which is independent of NHERF1 and EPAC.
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PMID:Tissue-specific regulation of sodium/proton exchanger isoform 3 activity in Na(+)/H(+) exchanger regulatory factor 1 (NHERF1) null mice. cAMP inhibition is differentially dependent on NHERF1 and exchange protein directly activated by cAMP in ileum versus proximal tubule. 1758 Mar 7

Secretin stimulates ductal secretion by activation of cAMP --> PKA --> CFTR --> Cl(-)/HCO(3)(-) exchanger in cholangiocytes. We evaluated the expression of alpha(2A)-, alpha(2B)-, and alpha(2C)-adrenergic receptors in cholangiocytes and the effects of the selective alpha(2)-adrenergic agonist UK 14,304, on basal and secretin-stimulated ductal secretion. In normal rats, we evaluated the effect of UK 14,304 on bile and bicarbonate secretion. In bile duct-ligated (BDL) rats, we evaluated the effect of UK 14,304 on basal and secretin-stimulated 1) bile and bicarbonate secretion; 2) duct secretion in intrahepatic bile duct units (IBDU) in the absence or presence of 5-(N-ethyl-N-isopropyl)amiloride (EIPA), an inhibitor of the Na(+)/H(+) exchanger isoform NHE3; and 3) cAMP levels, PKA activity, Cl(-) efflux, and Cl(-)/HCO(3)(-) exchanger activity in purified cholangiocytes. alpha(2)-Adrenergic receptors were expressed by all cholangiocytes in normal and BDL liver sections. UK 14,304 did not change bile and bicarbonate secretion of normal rats. In BDL rats, UK 14,304 inhibited secretin-stimulated 1) bile and bicarbonate secretion, 2) expansion of IBDU luminal spaces, and 3) cAMP levels, PKA activity, Cl(-) efflux, and Cl(-)/HCO(3)(-) exchanger activity in cholangiocytes. There was decreased lumen size after removal of secretin in IBDU pretreated with UK 14,304. In IBDU pretreated with EIPA, there was no significant decrease in luminal space after removal of secretin in either the absence or presence of UK 14,304. The inhibitory effect of UK 14,304 on ductal secretion is not mediated by the apical cholangiocyte NHE3. alpha(2)-Adrenergic receptors play a role in counterregulating enhanced ductal secretion associated with cholangiocyte proliferation in chronic cholestatic liver diseases.
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PMID:The alpha2-adrenergic receptor agonist UK 14,304 inhibits secretin-stimulated ductal secretion by downregulation of the cAMP system in bile duct-ligated rats. 1763 18

The activity of the Na(+)/H(+) exchanger NHE3 is regulated by a number of factors including parathyroid hormone (PTH). In the current study, we used a renal epithelial cell line, the opossum kidney (OKP) cell, to elucidate the mechanisms underlying the long-term effects of PTH on NHE3 transport activity and expression. We observed that NHE3 activity was reduced 6 h after addition of PTH, and this reduction persisted almost unaltered after 24 h. The decrease in activity was associated with diminished NHE3 cell surface expression at 6, 16, and 24 h after PTH addition, total cellular NHE3 protein at 16 and 24 h, and NHE3 mRNA abundance at 24 h. The lower levels of NHE3 mRNA were associated to a small, but significant, decrease in mRNA stability. Additionally, by analyzing the rat NHE3 gene promoter activity in OKP cells, we verified that the regulatory region spanning the segment -152 to +55 was mildly reduced under the influence of PTH. This effect was completely abolished by the presence of the PKA inhibitor KT 5720. In conclusion, long-term exposure to PTH results in reduction of NHE3 mRNA levels due to a PKA-dependent inhibitory effect on the NHE3 promoter and a small reduction of mRNA half-life, and decrease in the total amount of protein which is preceded by endocytosis of the apical surface NHE3. The decreased NHE3 expression is likely to be responsible for the reduction of sodium, bicarbonate, and fluid reabsorption in the proximal tubule consistently perceived in experimental models of PTH disorders.
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PMID:Mechanisms underlying the long-term regulation of NHE3 by parathyroid hormone. 1832 24


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