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)

In the renal medulla during antidiuresis, the extracellular fluid becomes hyperosmotic. Madin-Darby canine kidney (MDCK) epithelial cells adapt in hyperosmotic conditions and serve as a useful tissue culture model for cellular responses to hyperosmolality. We demonstrate that hyperosmolality stimulates phospholipase C, Raf-1 kinase mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase activities and that it increases phosphorylation of Raf-1 kinase, and p42 MAP kinase in MDCK cells. Stimulation of these kinases is osmolality-dependent (from 300 to 600 mosm/kg H2O). The time course of activation is sequential; the peak stimulation for Raf-1 kinase is at 5 min, at 10 min for MAP kinase kinase and MAP kinase, and at 20 min for S6 kinase. The activation of Raf-1 kinase and MAP kinase is inhibited by phorbol 12-myristate 13-acetate pretreatment in the presence of calphostin C or H-7. Tyrosine kinase inhibitors (genistein, herbimycin) do not significantly suppress hyperosmolality-induced MAP kinase activity. The increase of Ins-1,4,5-P3 levels by hyperosmolality suggests that activation of these kinases is mediated at least partially via activation of phospholipase C. Thus, hyperosmolality stimulates the serine/threonine kinases, Raf-1 kinase, MAP kinase kinase, MAP kinase, and S6 kinase, via predominantly protein kinase C-dependent, tyrosine kinase-independent pathways in MDCK cells.
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PMID:Sequential activation of Raf-1 kinase, mitogen-activated protein (MAP) kinase kinase, MAP kinase, and S6 kinase by hyperosmolality in renal cells. 752 42

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

The rate of uptake of 2-aminoisobutyrate (AIB), a non-metabolizable analogue of alanine, by Leishmania donovani was investigated as a function of culture age and osmolality in the presence of protein kinase inhibitors and of glucose, glutamate or proline. Hyperosmolality inhibited AIB uptake by cells of both growth stages, but to a greater extent for log than for stationary cells. Staurosporine, a protein kinase C inhibitor, had no effect on AIB uptake by cells of either culture age, but it reduced the rate of AIB release in response to hypo-osmolality, and more so in stationary cells than in log cells. Genistein, a protein-tyrosine kinase inhibitor, caused a small increase in AIB uptake by log cells under both iso- and hyperosmotic conditions, but had no effect on AIB uptake by stationary cells. Genistein also caused a small increase in the rate of release of AIB in response to a decrease in osmolality. Brief preincubation with glucose, glutamate or proline of cells that had been depleted of their osmolytes by prior exposure to hypo-osmolality caused an increase in AIB release by log cells, but a decrease by stationary cells. Similarly, whereas glucose and glutamate increased the rate of AIB uptake by log cells, and proline inhibited it, they had no effect on AIB uptake by stationary cells. Thus AIB uptake and release are sensitive to changes in osmolality, to protein kinase inhibitors, and to certain nutrients in a manner that changes markedly with culture age.
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PMID:Uptake and release of 2-aminoisobutyrate by Leishmania donovani: culture-age dependent effects of osmolality and of protein kinase inhibitors. 900 89

Inner medullary collecting duct (IMCD) cells adapt to a hypertonic environment by synthesizing transporters that allow for accumulation of organic osmolytes. To examine for activation of additional mitogen-activated protein (MAP) kinases, extracts of IMCD-3 cells subjected to a hypertonic medium (600 mosmol/kgH2O) for 15 min were fractionated by Mono Q fast-performance liquid chromatography and assayed with the epidermal growth factor receptor [EGFR-(662-681)] peptide as substrate. Three peaks of activity were identified. Western blotting revealed that these peaks coincided with Jun NH2-terminal kinase (JNK), extracellular signal-regulated protein kinases, ERK1 and ERK2, and p38 MAP kinase. To assess the functional significance of ERK2 activation in IMCD-3 cells, the effect of PD-098059, an inhibitor of the upstream regulatory protein kinase MAP/ERK kinase (MEK) was assessed. PD-098059 inhibited ERK activation by hypertonicity. Yet, the stimulation of inositol uptake, a marker of adaptation, after 16 h was unaltered. Direct measurements of JNK activity [phosphorylation of GST-cJun-(1-79)] revealed a marked (20- to 40-fold) increase in activity as medium osmolality was increased from 300 to 900 mosmol/kgH2O with either NaCl or mannitol. Urea induced a more modest increase in activity. The response is prompt and detected as early as 2 min after exposure, reaching a maximum activation at 10-15 min. Downregulation of cellular protein kinase C (PKC) by chronic exposure to phorbol esters only minimally attenuated the JNK response to hyperosmolality, indicating a lack of involvement of PKC. We conclude that, in IMCD-3 cells, inhibition of ERK activation by hyperosmolality does not prevent osmoregulatory increase in inositol transport. This is not consistent with a role for ERKs in the response. The roles for JNK and p38 have not been ruled out, and these pathways may represent the initiating event in the subsequent transcription of organic osmolyte transporter genes and adaptation to extracellular hypertonicity.
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PMID:Multiple mitogen-activated protein kinases are regulated by hyperosmolality in mouse IMCD cells. 908 72

Na(+)/H(+) exchanger regulatory factor (NHERF), an essential protein cofactor in cAMP-mediated inhibition of Na(+)/H(+) exchange transporter 3 (NHE3), facilitates the formation of a signal complex of proteins that includes NHE3, NHERF, and ezrin. This model for NHE3 regulation was developed in fibroblasts and its applicability to epithelial cells remains to be established. Opossum kidney (OK) cells were transfected with either empty vector (control), full-length mouse (m) NHERF(1-355), or a truncated mNHERF(1-325) that lacked ezrin binding and had been demonstrated in fibroblasts to bind NHE3 but not mediate its cAMP-associated inhibition. 8-Bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) at 10(-4) M inhibited Na(+)/H(+) exchange activity in control and OK cells expressing wild-type mNHERF(1-355) by >60% but by <10% in cells expressing mNHERF(1-325). NHE3 coimmunoprecipitated with mNHERF(1-325), but cAMP phosphorylation of NHE3 was impaired in cells expressing mNHERF(1-325). The inhibitory effect of hyperosmolality on NHE3 activity and the uptake of 3-O-methyl-D-glucose was the same in all three cell lines. Cell surface expression of NHE3 was not changed by cAMP in any of the cells lines. These data indicate that disruption of the NHERF-ezrin signal complex attenuates the inhibitory effect of cAMP on NHE3 activity in OK cells and provides evidence supporting the proposed model of protein kinase A regulation of NHE3 in epithelial cells.
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PMID:Ezrin binding domain-deficient NHERF attenuates cAMP-mediated inhibition of Na(+)/H(+) exchange in OK cells. 1145 30

Hyperosmolality in the renal medullary interstitium is generated by the renal countercurrent multiplication system, in which the medullary thick ascending limb (MAL) and the outer medullary collecting duct (OMCD) primarily participate. Since arginine vasopressin (AVP) regulates Na-K-ATPase activity directly via protein kinase A and indirectly via hyperosmolality, we investigated the acute and chronic effects of hyperosmolality on Na-K-ATPase and AVP-dependent cAMP generation in the MAL and OMCD. Microdissected MAL and OMCD from control and dehydrated rats were used for the measurement of Na-K-ATPase activity, mRNA expression of alpha-1, beta-1, and beta-2 subunits of Na-K-ATPase, and AVP-dependent cAMP generation. Na-K-ATPase activity in the MAL from dehydrated rats, as measured in isotonic medium, was higher than that of control rats. Moreover, incubation of samples in hypertonic medium (490 mOsm/kg H2O) further increased Na-K-ATPase activity. Dehydration increased alpha-1, beta-1, and beta-2 mRNA expression in the MAL without changing that in the OMCD. Western blot analysis revealed that in the outer medulla, the expression of beta-1, but not that of alpha-1 or beta-2, was stimulated by dehydration. Incubation of MAL or OMCD in hypertonic medium increased AVP-dependent cAMP generation. Higher levels of AVP-dependent cAMP were generated in the MAL from dehydrated rats than that of controls, although incubation in hypertonic medium did not lead to additional increases in AVP-dependent cAMP accumulation. In contrast, AVP-dependent cAMP generation in the OMCD was stimulated by dehydration, and was further stimulated by incubation in hypertonic medium. These findings demonstrate that Na-K-ATPase is upregulated short- and long-term hyperosmolality in the MAL, but not in OMCD.
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PMID:Differential effects of hyperosmolality on Na-K-ATPase and vasopressin-dependent cAMP generation in the medullary thick ascending limb and outer medullary collecting duct. 1639 72

Arginine vasopressin (AVP) is expressed in paraventricular, supraoptic, and suprachiasmatic nuclei of the hypothalamus, where transcription of the AVP gene is activated by various forms of stress, such as hyperosmolality, inflammation, and photic stimulation. In vasopressinergic neurons, the expression of the Fos/Jun family proteins is known to be rapidly induced after these stimuli as well. However, it is still unknown whether these proteins actually mediate AVP gene expression. In this study we examined in vitro the role of Fos/Jun protein in transcriptional regulation of the AVP gene using the BE(2)M17 neuroblastoma cell line. We found that 5'-promoter activity of the rat AVP gene (-803/+26) markedly increased when all combinations of the Fos/Jun family proteins were overexpressed. Coexpression of the cAMP-responsive element-binding protein-binding protein and steroid receptor coactivator-1a further enhanced the Fos/Jun-mediated transcription. Using site-directed mutagenesis and EMSA techniques, we identified an activation protein 1 (AP1)-like element (-134/-128; TGAATCA) in the AVP gene 5'-promoter region, which is the sole responsible site for the Fos/Jun-mediated transcription. We also found that 12-O-tetradecarbonyl phorbol 13-acetate stimulates AVP gene transcription partly via the AP1 site through the activation of ERK signaling. Together, these results suggest that a variety of Fos/Jun family member proteins stimulate transcription of the AVP gene through the AP1 site we identified. Furthermore, this effect may be activated by both protein kinase A and protein kinase C signaling pathways.
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PMID:Identification of a functional AP1 element in the rat vasopressin gene promoter. 1654 67

Cyooxygenase-2 (COX-2)-derived PGE2 is critical for the integrity and function of renal medullary cells during antidiuresis. The present study extended our previous finding that tonicity-induced COX-2 expression is further stimulated by the major COX-2 product PGE2 and investigated the underlying signaling pathways and the functional relevance of this phenomenon. Hyperosmolality stimulated COX-2 expression and activity in Madin-Darby canine kidney (MDCK) cells, a response that was further increased by PGE2-cAMP signaling, suggesting the existence of a positive feedback loop. This effect was diminished by AH-6809, an EP2 antagonist, and by the PKA inhibitor H-89, but not by AH-23848, an EP4 antagonist. The effect of PGE2 was mimicked by forskolin and dibutyryl-cAMP, suggesting that the stimulatory effect of PGE2 on COX-2 is mediated by a cAMP-PKA-dependent mechanism. Accordingly, cAMP-responsive element (CRE)-driven reporter activity paralleled the effects of PGE2, AH-6809, AH-23848, H-89, forskolin, and dibutyryl-cAMP on COX-2 expression. In addition, the stimulatory effect of PGE2 on tonicity-induced COX-2 expression was blunted in cells transfected with dominant-negative CRE binding (CREB) protein, as was the case in a COX-2 promoter reporter construct in which a putative CRE was deleted. Furthermore, PGE2 resulted in PKA-dependent phosphorylation of the pro-apoptotic protein Bad at Ser155, a mechanism that is known to inactivate Bad, which coincided with reduced caspase-3 activity during osmotic stress. Conversely, pharmacological interruption of the PGE2-EP2-cAMP-PKA pathway abolished Ser155 phosphorylation of Bad and blunted the protective effect of PGE2 on cell survival during osmotic stress. These observations indicate the existence of a positive feedback loop of PGE2 on COX-2 expression during osmotic stress, an effect that apparently is mediated by EP2-cAMP-PKA signaling, and that contributes to cell survival under hypertonic conditions.
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PMID:PGE2 potentiates tonicity-induced COX-2 expression in renal medullary cells in a positive feedback loop involving EP2-cAMP-PKA signaling. 1900 64

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