Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.1 (protein kinase)
 81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The epithelial brush border Na+/H+ exchanger isoform 3 (NHE3) is regulated by growth factors and protein kinases. When stably expressed in PS120 fibroblasts, NHE3 is stimulated by serum and fibroblast growth factor (FGF) and inhibited by phorbol esters. To examine the role of phosphorylation of NHE3 in growth factor/protein kinase regulation, NHE3 was C-terminally tagged with an 11-amino acid epitope of the vesicular stomatitis virus glycoprotein (VSVG) and stably expressed in Na+/H+ exchanger null PS120 fibroblasts (PS120/NHE3V). NHE3V was regulated by serum, FGF, and phorbol ester in a manner identical to wild type non-VSVG-tagged NHE3. Phosphorylation of NHE3V was evaluated via immunoprecipitation with anti-VSVG antibody after in vivo labeling of PS120/NHE3V cells with [32P]orthophosphate. NHE3V was phosphorylated under basal conditions. However, FGF and PMA, under conditions in which these agonists regulate NHE3V, altered neither the amount of phosphorylation of NHE3V as analyzed by one-dimensional SDS-polyacrylamide gel electrophoresis and autoradiography nor two-dimensional phosphopeptide maps of tryptic digests of NHE3V. In contrast, while changes in NHE3V phosphorylation were not observed with serum exposure by one-dimensional SDS-polyacrylamide gel electrophoresis, two-dimensional studies showed increases in two phosphopeptides. Under all these conditions, phosphoamino acid analysis showed that NHE3V was phosphorylated only on serine residues. By cell surface protein biotinylation studies under basal conditions, at least 27% of the NHE3V was expressed on the cell surface. To further analyze the phosphorylation status of the surface and intracellular forms of NHE3V under basal conditions and determine whether the amount of phosphorylation of the surface form changes upon serum, FGF, and PMA regulation, the surface form of NHE3V was separated from intracellular form by biotinylation/avidin-agarose precipitation. Under basal conditions, both intracellular and surface forms of NHE3V were phosphorylated. However, the amount of phosphorylation of the surface form of NHE3V did not change upon stimulation by serum and FGF and inhibition by PMA based on one-dimensional SDS-polyacrylamide gel electrophoresis and autoradiography. Thus, we conclude that when expressed in PS120 cells, while NHE3 is a phosphoprotein under basal conditions, its regulation by FGF and PMA is not by changes in the phosphorylation of NHE3, while regulation by serum may involve changes in its phosphorylation. Regulation of NHE3 probably involves intermediate associated regulatory proteins. The function of basal phosphorylation of NHE3 is not known.
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PMID:Regulation of the epithelial brush border Na+/H+ exchanger isoform 3 stably expressed in fibroblasts by fibroblast growth factor and phorbol esters is not through changes in phosphorylation of the exchanger. 921 92

As potential autocrine or paracrine factors, extracellular nucleotides are known to be important regulators of renal ion transporters by activating cell surface receptors and intracellular signaling pathways. We investigated the influence of extracellular adenine nucleotides on Na+/H+ exchanger isoform 3 (NHE3) activity in A6-NHE3 cells. This is a polarized cell line obtained by stable transfection of A6 cells with the cDNA encoding the rat isoform of NHE3, which is expressed on the apical membrane. Basolateral addition of the P2Y(1) agonist, 2-MeSADP, induced an inhibition of NHE3 activity, which was prevented by preincubation with selective P2Y(1) antagonists, MRS 2179 (N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate) and MRS 2286 (2-[2-(2-chloro-6-methylamino-purin-9-yl)-ethyl]-propane-1,3-bisoxy(diammoniumphosphate)). NHE3 activity was also significantly inhibited by ATP and ATP-gamma-S but not by UTP. 2-MeSADP induced a P2Y(1) antagonist-sensitive increase in both [Ca2+]i and cAMP production. Pre-incubation with a PKC inhibitor, Calphostin C, or the calcium chelator BAPTA-AM, had no effect on the 2-MeSADP-dependent inhibition of NHE3 activity, whereas this inhibition was reversed by either incubation with the PKA inhibitor H89 or by mutation of two PKA target serines (S552 and S605) on NHE3. Pre-incubation of the A6-NHE3 cells with the synthetic peptide, Ht31, which prevents the binding between AKAPs and the regulatory PKA subunits RII, also prevented the 2-MeSADP-induced inhibition of NHE3. We conclude that only the cAMP/PKA pathway is involved in the inhibition of NHE3 activity.
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PMID:Extracellular adenine nucleotides regulate Na+/H+ exchanger NHE3 activity in A6-NHE3 transfectants by a cAMP/PKA-dependent mechanism. 1218 15

The serum and glucocorticoid-induced protein kinase gene (sgk-1) encodes a multifunctional kinase that can be phosphorylated and activated through a phosphatidylinositol 3-kinase-dependent signaling pathway. In many cell types, endogenous SGK-1 steady-state protein levels are very low but can be acutely up-regulated after glucocorticoid receptor-mediated transcriptional activation; in breast epithelial and cancer cell lines, this up-regulation is associated with promotion of cell survival. We and others have noted that ectopically introduced full-length SGK-1 is poorly expressed, although SGK-1 lacking the first 60 amino acids (delta60SGK-1) is expressed at much higher-fold protein levels than wild-type SGK-1 in both human embryonic kidney 293T and MCF10A mammary epithelial cells. In this report, we demonstrate for the first time that the low steady-state expression level of SGK-1 is due to polyubiquitination and subsequent degradation by the 26S proteasome. Deletion of the amino-terminal 60 amino acids of SGK-1 results in a mutant SGK-1 protein that is neither efficiently polyubiquitinated nor degraded by the 26S proteasome, accounting for the higher steady-state levels of the truncated protein. We also demonstrate that a subset of SGK-1 localizes to the plasma membrane and that the polyubiquitin-modified SGK-1 localizes to a membrane-associated fraction of the cell. Taken together, these data suggest that a significant fraction of SGK-1 is membrane-associated and ubiquitinated. These findings are consistent with the recently described role of SGK-1 in phosphorylating the membrane-associated protein Nedd4-2 and the integral membrane Na+/H+ exchanger isoform 3 (NHE3) and suggest a novel mechanism of regulation of SGK-1.
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PMID:Ubiquitin modification of serum and glucocorticoid-induced protein kinase-1 (SGK-1). 1221 62

Dopamine D1-like receptors are linked via G proteins to multiple cellular signaling pathways, namely adenylyl cyclase (AC) and phospholipase C (PLC). We have previously shown that the D1-mediated inhibition of Na+-K+-ATPase activity in OK cells involves the sequential activation of the AC-protein kinase A (AC-PKA) and the PLC-protein kinase C (PLC-PKC) pathways. The present study evaluated signaling cascades involved in dopamine-mediated inhibition of Na+/H+ exchanger isoform 3 (NHE3) in rat and opossum renal cells. Na+/H+ exchanger activity was assayed as the initial rate of intracellular pH (pHi) recovery after an acid load. Vmax values (in pH units/s) for Na+-dependent pHi recovery in rat cells (0.0097+/-0.0007) were greater (P<0.05) those in opossum cells (0.0063+/-0.0007), with similar Km values (in mM) for Na+ (rat, 35+/-9; opossum, 24+/-9). The IC50 values for EIPA and amiloride induced decrease in NHE activity in rat and opossum kidney cells are in agreement with the observation that rat renal proximal tubules and opossum kidney cells express mainly the NHE3 isoform. The D1-like receptor agonist SKF 38393 inhibited NHE3 activity in a concentration-dependent manner in both rat and opossum cells. The D1-mediated inhibition of NHE3 was prevented either by the D1-like receptor antagonist SKF 83566 (1 microM), overnight treatment with cholera toxin (500 ng/ml) and the PKA antagonist H-89 (10 microM) in rat and opossum kidney cells. The effect of SKF 38393 was abolished by the PKC antagonist chelerythrine (1 microM), or the PLC inhibitor U-73,122 (3 microM) in opossum cells, but not in rat cells. In addition, dibutyril cAMP (dB-cAMP; 500 microM) was found to increase PLC activity in OK cells but not in rat cells. The effect of D1-like dopamine agonist was accompanied by increases in cyclic AMP production in rat and opossum cells. The inhibitory effect of SKF 38393 (1 microM) on NHE3 activity was abolished in rat and opossum cells pre-treated with the anti-GSalpha antibody, but not in cells treated with the anti-Gq/11 alpha antibody. It is concluded that D1 agonists decrease NHE3 activity by classical stimulation of AC and PKA via GSalpha proteins in rat kidney cells. By contrast, the D1-mediated inhibition of NHE3 in renal opossum cells involves a peculiar mechanism with AC-PKA and PLC-PKC pathways.
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PMID:Distinct signalling cascades downstream to Gsalpha coupled dopamine D1-like NHE3 inhibition in rat and opossum renal epithelial cells. 1497 10