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)

To confirm the intracellular signal transduction in regulation of alkaline phosphatase (ALP) activity by calcitonin in kidney tubular cells, effects of several inhibitors of cyclic nucleotide phosphodiesterase (PDE) isoenzymes and cyclic AMP-dependent protein kinase (PKA) on the action of salmon calcitonin in porcine kidney tubular epithelial cells LLC-PK1 were examined. A confluent culture of LLC-PK1 cells was treated with calcitonin and inhibitors in Dulbecco's modified Eagle's medium supplemented with 0.1% bovine serum albumin, and intracellular cyclic AMP content and ALP activity were measured after incubation for 30 min and 48 hr, respectively. Calcitonin and PDE 4 inhibitors increased cyclic AMP level and ALP activity in the cells, and PDE 4 inhibitors synergistically potentiated the effects of calcitonin. Calcitonin induced ALP activation by treatment for the first 1 hr, as well as continuous treatment for 48 hr, while it never increased the enzyme activity just after 1-hr exposure. Rolipram, an inhibitor of PDE 4 isoenzyme, induced ALP activation by itself and in combination with calcitonin by only a long term treatment (48 hr). The activation of ALP by calcitonin and rolipram each alone and in combination was completely abolished by a PKA inhibitor, H-89. These results confirm that calcitonin induces ALP activation through the cyclic AMP-PKA pathway and that PDE 4 isoenzyme is closely associated with the calcitonin-receptor system and plays a major role in hydrolysis of cyclic AMP produced in the kidney tubular cells.
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PMID:Role of phosphodiesterase 4 isoenzyme in alkaline phosphatase activation by calcitonin in porcine kidney LLC-PK1 cells. 954 Dec 82

The calcitonin receptor expressed by the porcine LLC-PK1 renal tubule cells is a seven-transmembrane domain, G protein-coupled receptor activating adenylyl-cyclase and phospholipase C. Salmon calcitonin stimulated dose- and time-dependent release of the phospholipase D-dependent phosphatidylcholine product [3H] choline with an EC50 = 2.5 +/-0.3 x 10(-8) M, similar to that determined for phosphoinositide metabolism (EC50 = 4.5 +/-1.0 x 10(-8)M). The hormone failed to induce release of [3H]phosphocholine and [3H]glycerophosphocholine, ruling out activation of phosphatydilcholine-specific phospholipase C and phospholipase A. Calcitonin stimulated phosphatidic acid, a product of phospholipase D-dependent phosphatydilcholine hydrolysis. Activation of phospholipase D was confirmed by release of [3H]phosphatydilethanol, a specific and stable product in the presence of a primary alcohol. Activation of calcitonin receptor induced diacylglycerol formation, with a rapid peak followed by a prolonged increase, due to activation of phospholipase C and of phospholipase D. Consequently, the protein kinase-C alpha, but not the delta isoenzyme, was cytosol-to-membrane translocated by approximately 50% after 20 min exposure to calcitonin, whereas protein kinase-C zeta, which was approximately 40% membrane-linked in unstimulated cells, translocated by approximately 19%. The human calcitonin receptor expressed by BIN-67 ovary tumor cells, although displaying higher affinity for calcitonin, failed to activate phospholipase D and protein kinase-C in response to the hormone. This receptor lacks the G protein binding consensus site due to the presence of a 48-bp cassette encoding for a 16-amino acid insert in the predicted first intracellular loop. This modification is likely to prevent the calcitonin receptor from associating to phospholipase-coupled signaling.
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PMID:Phospholipase D- and protein kinase C isoenzyme-dependent signal transduction pathways activated by the calcitonin receptor. 964 99

The regulation of transport of the fluorescent organic cation 4-(4-dimethylaminostyryl)-N-methylpyridinium (ASP+) by renal proximal tubular organic cation transport was studied in IHKE-1 and LLC-PK1 cells with a recently established fluorometric technique (Stachon et al., 1996, 1997). Stimulation of Ca++/diacylglycerol-dependent protein kinase by 1,2-dioctanoyl glycerol (DOG; 0.01-1 mumol/l, n = 7), ATP (0.1 mmol/l, n = 9), oxytocin (0.1 mumol/l, n = 6) and bradykinin (1 mumol/l, n = 7) resulted in an increase of ASP+ accumulation in IHKE-1 cells by 35 +/- 9% (DOG), 65 +/- 30% (ATP), 66 +/- 14% (bradykinin) and 70 +/- 20% (oxytocin) as compared with basal conditions, whereas ASP+ accumulation was slightly reduced in LLC-PK1 cells after stimulation with DOG (1 mumol/l, -20 +/- 7%, n = 10) and angiotensin II (0.1 nmol/l, -20 +/- 5%, n = 6). ASP+ accumulation in IHKE-1 cells also was increased by 0.5 mumol/l (20 +/- 8%, n = 8) and 1 mumol/l forskolin (35 +/- 13%, n = 19), and by 8-bromo-cAMP (1 mumol/l, 125 +/- 25%, n = 9), both activators of the cAMP-dependent protein kinase (PKA). Activation of the cGMP-dependent protein kinase (PKG) by human atrial natriuretic peptide (10 nmol/l, n = 10) or 8-bromo-cGMP (0.1 mmol/l, n = 12) resulted in an increase of 35 +/- 5% and 28 +/- 6%, respectively. Activation of PKA and PKG had no influence on ASP+ transport in LLC-PK1 cells. Regulation of ASP+ uptake by these two cell lines may be caused by direct phosphorylation of the organic cation transporters involved or by regulation of trafficking of the transporters to the membrane. Differences in the organic cation transporter isoforms or alternatively, in the trafficking may contribute to the distinct regulation of ASP+ transport in IHKE-1 and LLC-PK1 cells.
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PMID:Regulation of organic cation transport in IHKE-1 and LLC-PK1 cells. Fluorometric studies with 4-(4-dimethylaminostyryl)-N-methylpyridinium. 965 73

Glycogen is the principal storage form of glucose in animal cells. It accumulates in electron-dense cytoplasmic granules and is synthesized by glycogen synthase (GS), the rate-limiting enzyme of glycogen deposition. Glycogen synthase kinase-3 (GSK-3) is a protein kinase that phosphorylates GS. Two nearly identical forms of GSK-3 exist: GSK-3 alpha and GSK-3 beta. Both are constitutively active in resting cells and their activity can be modulated by hormones and growth factors. GSK-3 is implicated in the regulation of many physiological responses in mammalian cells by phosphorylating substrates including neuronal cell adhesion molecule, neurofilaments, synapsin I, and tau. Recent observations point to functions for glycogen and glycogen metabolism in the nucleus. GSK-3 phosphorylates several transcription factors, and we have recently shown that it modifies the major nuclear pore protein p62. It also regulates PK1, a protein kinase required for maintaining the interphase state and for DNA replication in cycling Xenopus egg extracts. Recently, glycogen was shown to be required for nuclear reformation in vitro using ovulated Xenopus laevis egg lysates. Because neither glycogen nor GSK-3 has been localized to the nuclear envelope or intranuclear sites, glycogen and GSK-3 activites were measured in rat liver nuclei and nuclear reformation extracts. Significant quantities of glycogen-like material co-purified with the rat-liver nuclear envelope. GSK-3 is also highly enriched in the glycogen pellet of egg extracts of Xenopus that is required for nuclear assembly in vitro. Based on the finding that enzymes of glycogen metabolism copurify with glycogen, we propose that glycogen may serve a structural role as a scaffold for nuclear assembly and sequestration of critical kinases and phosphatases in the nucleus.
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PMID:Nuclear glycogen and glycogen synthase kinase 3. 971 12

Several recent studies have provided clear evidence that angiotensin-converting enzyme (ACE)-inhibitors slow the progression of renal disease. These effects are mainly independent from a comitant reduction in systemic blood pressure. Thus, angiotensin II (Ang II) exerts other effects on the kidney which are involved in the loss of renal function. Ang II induces proliferation of cultured mesangial and glomerular endothelial cells. Our group was the first to demonstrate that Ang II stimulates hypertrophy of cultured proximal tubular cells. Ang II stimulates bioactivation and expression of transforming growth factor-beta (TGF-beta) in tubular MCT cells. This Ang II-mediated expression of TGF-beta is due to an increase in transcriptional activity. A neutralizing anti-TGF-beta antibody attenuates the Ang II-induced increase in protein synthesis in MCT cells suggesting that the hypertrophy is mediated by synthesis and activation of endogenous TGF-beta. Proximal tubular cells undergoing Ang II-mediated hypertrophy are arrested in the G1-phase of the cell cycle and express typical G1-phase-associated genes. Induction of such G1-phase-associated early growth response genes have been also described in vivo after infusion of Ang II into the renal artery. This G1-phase arrest depends on the induction of the cyclin-dependent kinase (CdK) inhibitor p27Kip1. p27Kip1 expression is stimulated after incubation of LLC-PK1 cells with Ang II or TGF-beta and binds to cyclin D1-CdK4 complexes, inhibits their kinase activity, and hampers G1-phase exit. Ang II stimulates transcription of collagen type IV in MCT cells. In addition to the classical a1 (IV) chain, a3 (IV) collagen, which has normally a restricted localization in the kidney, is also induced. This stimulation is mediated by endogenous synthesis and autocrine action of TGF-beta because a neutralizing anti-TGF-beta antibody as well as TGF-beta antisense oligonucleotides attenuate Ang II-induced collagen type IV transcription and synthesis. In addition, Ang II exerts immunomodulatory effects on the kidney through the induction of chemokines such as MCP-1 and RANTES. In conclusion, Ang II has emerged as a multifunctional acting as a growth factor and a profibrogenic cytokine, and even having inflammatory properties.
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PMID:Angiotensin II is involved in the progression of renal disease: importance of non-hemodynamic mechanisms. 985 83

The 25-hydroxyvitamin D3 1alpha-hydroxylase, also referred to as CYP27B1, is a mitochondrial cytochrome P450 enzyme that catalyzes the biosynthesis of 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) from 25-hydroxyvitamin D3 in renal proximal tubular cells. Recently, human, mouse, and rat CYP27B1 cDNA have been cloned, however the gene regulation has not been fully elucidated. In the present study, porcine CYP27B cDNA was cloned, and the effects of cAMP and vitamin D3 on the regulation of CYP27B1 mRNA expression in LLC-PK1 cells were examined. PCR cloning revealed that porcine CYP27B1 cDNA consisted of 2316 bp, encoding a protein of 504 amino acids. The deduced amino acid sequence showed over 80% identity to the human, mouse, and rat enzyme. LLC-PK1 cells were incubated with humoral factors, and expression of CYP27B1 mRNA was measured by a quantitative reverse transcription-PCR. At the completion of 3-, 6-, 12-, and 24-h incubations, 500 micromol/L 8-bromo-cAMP had significantly increased CYP27B1 mRNA expression (260 to 340%). The adenylate cyclase activator forskolin at 50 micromol/L also had a stimulatory effect at 6 h (190%). Moreover, the protein kinase A inhibitor H-89 reduced the cAMP effect. On the other hand, 1alpha,25(OH)2D3 had no effect on CYP27B1 mRNA expression at 10 and 100 nmol/L, whereas expression of 25-hydroxyvitamin D3 24-hydroxylase (CYP24) mRNA was markedly increased by 1alpha,25(OH)2D3. These findings suggest that LLC-PK1 cells express CYP27B1 mRNA, and that cAMP is an upregulating factor of the CYP27B1 gene in vitro.
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PMID:Cloning of porcine 25-hydroxyvitamin D3 1alpha-hydroxylase and its regulation by cAMP in LLC-PK1 cells. 1023 81

ERM (Ezrin-Radixin-Moesin) proteins function as plasma membrane-actin cytoskeleton linkers and participate in the formation of specialized domains of the plasma membrane. We have investigated ezrin function in tubulogenesis of a kidney-derived epithelial cell line, LLC-PK1. Here we show that cells overproducing a mutant form of ezrin in which Tyr-353 was changed to a phenylalanine (Y353F) undergo apoptosis when assayed for tubulogenesis. While investigating the mechanism responsible for this apoptosis, we found that ezrin interacts with p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase). Two distinct sites of ezrin are involved in this interaction, the amino-terminal domain containing the first 309 aa and the phosphorylated Tyr-353 residue, which binds to the carboxyl-terminal SH2 domain of p85. Cells producing Y353F ezrin are defective in activation of the protein kinase Akt, a downstream target of PI 3-kinase that protects cells against apoptosis. Furthermore, the apoptotic phenotype of these cells is rescued by production of a constitutively activated form of PI 3-kinase. Taken together, these results establish a novel function for ezrin in determining survival of epithelial cells by activating the PI 3-kinase/Akt pathway.
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PMID:Ezrin, a plasma membrane-microfilament linker, signals cell survival through the phosphatidylinositol 3-kinase/Akt pathway. 1037 9

The biosynthesis of 1alpha, 25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1) in renal proximal tubules. It was recently demonstrated that LLC-PK1 cells express CYP27B1 mRNA, which is regulated by intracellular cAMP but not vitamin D3. To clarify the effect of calcitonin on vitamin D3 metabolism in vitro, LLC-PK1 cells were incubated with hormonal factors, and expression of CYP27B1 mRNA was measured by quantitative reverse transcription-PCR. Calcitonin at 100 nmol/L significantly increased CYP27B1 mRNA expression by 24 h (271 +/- 21% of control). Incubation with calcitonin over a range of 1 micromol/L to 1 pmol/L resulted in a concentration-dependent increase in CYP27B1 mRNA levels. It is known that the calcitonin receptor has dual intracellular signaling pathways, via protein kinases A and C. Both 500 micromol/L 8-bromo-cAMP, a protein kinase A activator, and 100 nmol/L phorbol 12-myristate 13-acetate, a protein kinase C activator, increased CYP27B1 mRNA levels at 24 h (207 +/- 54 and 246 +/- 58% of control, respectively). However, calcitonin-induced CYP27B1 mRNA expression was only inhibited by the protein kinase C inhibitors staurosporine and calphostin C. The protein kinase A inhibitors Rp-cAMPS at 10 and 100 micromol/L and H-89 at 10 micromol/L had no effect on the action of calcitonin, in spite of cAMP-activation by calcitonin. The present data suggest that calcitonin upregulates CYP27B1 mRNA expression via the protein kinase C pathway in LLC-PK1 cells.
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PMID:Calcitonin induces 25-hydroxyvitamin D3 1alpha-hydroxylase mRNA expression via protein kinase C pathway in LLC-PK1 cells. 1058 84

Nitric oxide (NO) reduces the molecular activity of Na+-K+-ATPase in opossum kidney (OK) cells, a proximal tubule cell line. In the present study, we investigated the cellular mechanisms for the inhibitory effect of NO on Na+-K+-ATPase. Sodium nitroprusside (SNP), a NO donor, inhibited Na+-K+-ATPase in OK cells, but not in LLC-PK1 cells, another proximal tubule cell line. Similarly, phorbol 12-myristate 13-acetate, a protein kinase C (PKC) activator, inhibited Na+-K+-ATPase in OK, but not in LLC-PK1, cells. PKC inhibitors staurosporine or calphostin C, but not the protein kinase G inhibitor KT-5823, abolished the inhibitory effect of NO on Na+-K+-ATPase in OK cells. Immunoblotting demonstrated that treatment with NO donors caused significant translocation of PKCalpha from cytosolic to particulate fractions in OK, but not in LLC-PK1, cells. Furthermore, the translocation of PKCalpha in OK cells was attenuated by either the phospholipase C inhibitor U-73122 or the soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one. U-73122 also blunted the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. The phospholipase A2 inhibitor AACOCF3 did not blunt the inhibitory effect of SNP on Na+-K+-ATPase in OK cells. AACOCF3 alone, however, also decreased Na+-K+-ATPase activity in OK cells. In conclusion, our results demonstrate that NO activates PKCalpha in OK, but not in LLC-PK1, cells. The activation of PKCalpha in OK cells by NO is associated with inhibition of Na+-K+-ATPase.
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PMID:Nitric oxide activates PKCalpha and inhibits Na+-K+-ATPase in opossum kidney cells. 1060 Sep 32

To study the membrane mobility of aquaporin water channels, clones of stably transfected LLC-PK1 cells were isolated with plasma membrane expression of GFP-AQP1 and GFP-AQP2, in which the green fluorescent protein (GFP) was fused upstream and in-frame to each aquaporin (AQP). The GFP fusion did not affect AQP tetrameric association or water transport function. GFP-AQP lateral mobility was measured by irreversibly bleaching a spot (diameter 0.8 microm) on the membrane with an Argon laser beam (488 nm) and following the fluorescence recovery into the bleached area resulting from GFP translational diffusion. In cells expressing GFP-AQP1, fluorescence recovered to >96% of its initial level with t(1/2) of 38 +/- 2 s (23 degrees C) and 21 +/- 1 s (37 degrees C), giving diffusion coefficients (D) of 5.3 and 9.3 x 10(-11) cm(2)/s. GFP-AQP1 diffusion was abolished by paraformaldehyde fixation, slowed >50-fold by the cholesterol-binding agent filipin, but not affected by cAMP agonists. In cells expressing GFP-AQP2, fluorescence recovered to >98% with D of 5.7 and 9.0 x 10(-11) cm(2)/s at 23 degrees C and 37 degrees C. In contrast to results for GFP-AQP1, the cAMP agonist forskolin slowed GFP-AQP2 mobility by up to tenfold. The cAMP slowing was blocked by actin filament disruption with cytochalasin D, by K(+)-depletion in combination with hypotonic shock, and by mutation of the protein kinase A phosphorylation consensus site (S256A) at the AQP2 C-terminus. These results indicate unregulated diffusion of AQP1 in membranes, but regulated AQP2 diffusion that was dependent on phosphorylation at serine 256, and an intact actin cytoskeleton and clathrin coated pit. The cAMP-induced immobilization of phosphorylated AQP2 provides evidence for AQP2-protein interactions that may be important for retention of AQP2 in specialized membrane domains for efficient membrane recycling.
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PMID:cAMP regulated membrane diffusion of a green fluorescent protein-aquaporin 2 chimera. 1065 16


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