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)

Calu-3, a cell line derived from a lung adenocarcinoma, forms tight junctions, expresses cystic fibrosis transmembrane conductance regulator (CFTR), and secretes Cl- in response to adenosine 3',5'-cyclic monophosphate (cAMP)-elevating agents. Anion conductance of Calu-3 cells was assessed with isotopic flux and patch-clamp methods at 22 degrees C. Iodide efflux was increased by cAMP-elevating agents and brief trypsin treatment. A 7.1 +/- 0.4-pS voltage-independent Cl- channel with linear current-voltage relation was the most common channel observed in cell-attached recordings and was identified as CFTR on the basis of shared features with recombinant CFTR. In unstimulated cells, the mean minimum number of active CFTR channels per patch was 1 +/- 1 (n = 12), increasing to 6 +/- 8 (n = 40) after stimulation with cAMP-elevating agents or after brief trypsin treatment. Channel closure after excision was biexponential with tau 1 approximately 4 s and tau 2 approximately 79 s; typically channels were open continuously until closing permanently. In 11 of 12 excised patches, channels were reactivated by exposure to cAMP-dependent protein kinase (PKA) plus ATP. Efficacy of reactivation was inversely related to the duration from excision to addition of PKA. Channels were blocked by 20-40 microM 5-nitro-2-(3-phenylpropylamino)benzoate on cytosolic but not external side. Active CFTR channels were recorded in 83% of total patches. Other types of Cl- channels were observed in 5 of 52 (10%) cell-attached patches and in 17 of 34 (50%) excised patches, including an outwardly rectifying channel in 2 patches. CFTR channels are the predominant pathway for cAMP-stimulated Cl- conductance in Calu-3 cells; the long open times in the absence of ATP are not explained by present models of CFTR activation.
 ...
PMID:CFTR in Calu-3 human airway cells: channel properties and role in cAMP-activated Cl- conductance. 751 79

Chloride channel activity of cystic fibrosis transmembrane conductance regulator (CFTR) requires activation of protein kinase A (PKA) by 3'-5'-cyclic adenosine monophosphate (cAMP). The level of cAMP is controlled by the balance between cAMP synthesis and hydrolysis by adenylate cyclase and phosphodiesterases (PDEs), respectively. CFTR channel activity appears to be most sensitive to the activity of type III cyclic nucleotide PDEs in Calu-3 and 16HBE cells, both derived from airway epithelium and expressing wild-type CFTR. Type III PDEs can be identified by their sensitivity to specific inhibitors such as milrinone and amrinone. In Calu-3 cells, specific inhibition of type III PDEs increased chloride efflux up to 13.7-fold, whereas neither rolipram nor Ro20-1724 (type IV PDE inhibitors) nor 3-isobutyl-1-methylxanthine (IBMX, a nonspecific PDE inhibitor) elicited significant increases. None of these compounds had an appreciable effect on total cellular cAMP levels, yet the effects of milrinone and amrinone on chloride efflux were blocked by treatment of cells with Rp-cAMPS, a cAMP analog that inhibits PKA at the site of cAMP binding. Similarly, H-8, an inhibitor of PKA, reduced milrinone-stimulated chloride efflux, indicating that efflux is mediated through the cAMP/PKA pathway. Whole-cell patch clamp analysis revealed that milrinone generated chloride conductances with properties consistent with those of CFTR. Milrinone elicited chloride currents in a dose-dependent manner and induced CFTR activity in the absence of adenylate cyclase agonists. These data suggest that type III PDEs are specifically involved in CFTR activation in airway epithelial cells and that PDE regulation of CFTR may involve subcellular compartments of cAMP.
 ...
PMID:CFTR-mediated chloride permeability is regulated by type III phosphodiesterases in airway epithelial cells. 757 3

p16INK4A, a specific inhibitor of cyclin-dependent kinase (cdk)4 and cdk6, is a candidate tumor suppressor in malignancies with wild-type retinoblastoma (Rb). Loss of p16INK4A frees these cdks from inhibition, permitting constitutive phosphorylation of Rb and inactivation of its growth suppressive properties. Consistent with this model, Rb-positive non-small cell lung cancers (NSCLCs) have little or no detectable p16INK4A protein, whereas Rb-negative lung cancers have abundant p16INK4A. However, only some NSCLCs have homozygous deletions or nonsense mutations in a remaining p16INK4A allele, suggesting that other mechanisms must account for absent or low levels of p16INK4A protein. Here, we analyzed 9 Rb-positive NSCLC cell lines for the controls governing p16INK4A activity. Four lines had homozygous deletions of p16INK4A (SK-LU-1, SK-MES-1, A-427, and SW900), and three had a point mutation in a single allele. First, in H520 cells, the previously reported deletion at codon 45 results in a frameshift that produces no detectable protein. Second, in Calu-3 cells, a His to Tyr substitution at codon 83 produced a variant with a shortened half-life that was unable to form complexes with cdk4 or cdk6. Third, in H661 cells, the previously reported point mutation in the second intron splice donor site resulted in a smaller p16INK4A protein. Although this variant formed complexes with cdk4 and cdk6, it had a profoundly reduced half-life, producing low steady-state levels of p16INK4A and abundant levels of free cdks. Finally, Calu-1 and Calu-6 cells transcribed no detectable mRNA encoding authentic p16INK4A. These cell lines displayed methylation of the CpG island surrounding the first exon of p16INK4A and expressed abundant levels of a nontranslated mRNA containing an alternative first exon (E1 beta), as did all other cell lines in which the p16INK4A locus was not deleted. These data indicate that Rb-positive NSCLC cells have evolved a variety of pathways to suppress p16INK4A expression. Reintroduction of p16INK4A into these cell lines by retroviral transfer resulted in a reduced growth rate, increased abundance of hypophosphorylated Rb, accumulation of cells in G1, and a less transformed morphology in Rb-positive, but not Rb-negative cells, suggesting that loss of p16INK4A is essential for maintenance of the transformed phenotype.
 ...
PMID:Multiple mechanisms of p16INK4A inactivation in non-small cell lung cancer cell lines. 852 14

Cell cycle progression requires activation of different cyclin-dependent kinases (CDKs) which are positively regulated by cyclins and negatively regulated by CDK inhibitors. Growth inhibition of the Calu-1 lung carcinoma cells induced with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent activator of protein kinase C, is associated with G2/M arrest and induction of expression of a novel, faster-migrating form of p21(WAF1/CIP1/SDI1) (p21) protein, an inhibitor of cyclin-dependent kinases. This faster-migrating p21 protein was also expressed in TPA-treated A549 lung carcinoma cells which also exhibited G2/M arrest but not in TPA-treated U937 leukemia cells, which only expressed a slower-migrating form of p21 protein. However, reverse transcriptase-polymerase chain reaction and Southern analysis demonstrated no evidence of novel splice in TPA-treated Calu-1 cells. On the other hand, immunoblotting analysis demonstrated that the faster-migrating p21 protein could be detected only by peptide antibody directed against the N terminus but not the C terminus, suggestive of truncation of the latter or protein modification that results in the loss of the C-terminal epitope. Correlation of G2/M arrest with expression of the faster-migrating p21 protein suggests that this novel form of p21 protein may be a mediator of G2/M arrest and growth inhibition.
 ...
PMID:Novel form of p21(WAF1/CIP1/SDI1) protein in phorbol ester-induced G2/M arrest. 893 83

We examined the DNA sequence and transcription factor requirements for cAMP-induced transactivation of the human renin promoter using Calu-6 cells that express human renin mRNA endogenously. A series of constructs containing 896 base pairs of human renin 5'-flanking DNA fused to the luciferase gene and containing either the native, a consensus, or a nonfunctional cAMP response element (CRE) were used to assess DNA sequence requirements mediating the cAMP response. Expression vectors encoding the CREB-1 transcription factor, a dominant negative mutant form of CREB-1, and the catalytic subunit of protein kinase A (PKA) were used to assess transcription factor requirements mediating the cAMP response. Forskolin treatment alone only caused a 2-3-fold activation of the HREN promoter in Calu-6 cells, but nearly a 10-fold activation in JEG-3 cells, which do not express renin but are highly responsive to cAMP. Gel shift assays revealed the binding of five specific DNA-protein complexes consisting of the ATF-1 and CREB-1 transcription factors, one of which was an ATF-1.CREB-1 heterodimer suggesting the potential for regulation of CREB-1 activity by ATF-1. However, over-expression of CREB-1 did not significantly enhance forskolin-induced human renin transcriptional activity. Transfection of both Calu-6 and JEG-3 cells with a PKA expression vector resulted in a 10-fold induction of human renin transcriptional activity in constructs containing the native or consensus CRE and 5-fold activation in a construct containing a nonfunctional CRE. We confirmed that the PKA response has both a CREB-dependent and CREB-independent component by demonstrating that the PKA response was abolished by co-transfection of a dominant negative mutant form of CREB-1 into cells containing the native or consensus CRE construct but not in cells containing the nonfunctional CRE construct. We therefore conclude that the human renin promoter can be transcriptionally activated in a renin expressing cell line through the cAMP-PKA pathway and is mediated by both a CREB-dependent and CREB-independent mechanism.
 ...
PMID:Transactivation of the human renin promoter by the cyclic AMP/protein kinase A pathway is mediated by both cAMP-responsive element binding protein-1 (CREB)-dependent and CREB-independent mechanisms in Calu-6 cells. 899 53

C-type natriuretic peptide (CNP), a hormone which stimulates particulate guanylate cyclase activity, was studied for its ability to stimulate chloride permeability through the cystic fibrosis transmembrane conductance regulator (CFTR) in airway epithelial cells. Two cell lines, Calu-3 and CF-T43, were used as models of normal and cystic fibrosis (CF) airway epithelial cells, respectively. Calu-3 cells, derived from a lung carcinoma, express relatively high levels of wild-type CFTR. CF-T43 is a transformed line derived from a nasal polyp and expresses the mutant CFTR, deltaF508. Calu-3 cells exposed to the nucleotide guanosine-3',5'-monophosphate (cGMP) analogue 8-Br-cGMP exhibit increased 36Cl- efflux, demonstrating that cGMP can mediate changes in chloride permeability. CNP induces a bumetanide-sensitive short circuit current across Calu-3 monolayers. Whole-cell currents stimulated by CNP display linear current-voltage relationships and have inhibitor pharmacology and ion selectivity consistent with CFTR channel activity. Sodium nitroprusside (SNP), an activator of soluble guanylate cyclase, and CNP both increase cGMP levels and short circuit current in Calu-3 cells. In contrast, exposure of CF-T43 cells to CNP resulted in an increased 36Cl- efflux rate only when combined with the adenylate cyclase agonist isoproterenol and the response was sensitive to kinase inhibitors. CF-T43 cells exposed to isoproterenol and SNP showed no increase in chloride efflux. Together, these data indicate that CNP can activate wild-type and mutant CFTR through a cAMP-dependent protein kinase pathway and that the sensitivity of Calu-3 cells for this stimulation is greater than that of the CF-T43 cells.
 ...
PMID:C-type natriuretic peptide increases chloride permeability in normal and cystic fibrosis airway cells. 911 58

The activity of the CFTR Cl- channel is dependent on its phosphorylation status set by kinases and phosphatases. We report here that protein phosphatase 2B (PP2B) and protein kinase C (PKC) are potential regulators of the cystic fibrosis conductance regulator (CFTR). Treating CFTR-expressing 3T3 cells with either of the two specific PP2B blockers cyclosporin A (CsA, 1 microM) or deltamethrin (DM, 30 nM) caused rapid activation of CFTR in cell-attached patches. As determined by noise analysis of multi channel patches, DM- or CsA-activated CFTR displayed gating kinetics comparable to those of forskolin-activated CFTR. After activation of CFTR by blocking PP2B, CFTR still inactivated. CFTR-mediated currents were, on average, 6.1 times larger when cells were stimulated by forskolin during PP2B block compared to stimulation by forskolin alone. This suggests that, in CFTR-expressing 3T3 cells, a phosphorylation site of CFTR is regulated by cellular PKA, PP2B and another phosphatase. However, in the epithelial cell lines Calu-3 and HT-29/B6, CsA and DM had no effect on CFTR activity in both cell-attached patch-clamp and transepithelial experiments. In contrast, when exogenous PP2B was added to patches excised from 3T3 or Calu-3 cells, PKA-activated CFTR currents were quickly inactivated. This indicates that free exogenous PP2B can inactivate CFTR in patches from both cell types. We propose that in order to regulate CFTR in an intact cell, PP2B may require a selective subcellular localization to become active. When excised patches were PKC-phosphorylated, the gating kinetics of CFTR were significantly different from those of PKA-phosphorylated CFTR. Addition of PP2B also inactivated PKC-activated CFTR showing the indiscriminate dephosphorylation of different phosphorylation sites by PP2B.
 ...
PMID:Regulation of CFTR by protein phosphatase 2B and protein kinase C. 959 16

Protein kinase C (PKC) regulates cystic fibrosis transmembrane conductance regulator (CFTR) channel activity but the PKC signaling mechanism is not yet known. The goal of these studies was to identify PKC isotype(s) required for control of CFTR function. CFTR activity was measured as 36Cl efflux in a Chinese hamster ovary cell line stably expressing wild-type CFTR (CHO-wtCFTR) and in a Calu-3 cell line. Chelerythrine, a PKC inhibitor, delayed increased CFTR activity induced with phorbol 12-myristate 13-acetate or with the cAMP-generating agents (-)-epinephrine or forskolin plus 8-(4-chlorophenylthio)adenosine 3',5'- cyclic monophosphate. Immunoblot analysis of Calu-3 cells revealed that PKC-alpha, -betaII, -delta, -epsilon, and -zeta were expressed in confluent cell cultures. Pretreatment of cell monolayers with Lipofectin plus antisense oligonucleotide to PKC-epsilon for 48 h prevented stimulation of CFTR with (-)-epinephrine, reduced PKC-epsilon activity in unstimulated cells by 52.1%, and decreased PKC-epsilon mass by 76.1% but did not affect hormone-activated protein kinase A activity. Sense oligonucleotide to PKC-epsilon and antisense oligonucleotide to PKC-delta and -zeta did not alter (-)-epinephrine-stimulated CFTR activity. These results demonstrate the selective regulation of CFTR function by constitutively active PKC-epsilon.
 ...
PMID:Antisense oligonucleotide to PKC-epsilon alters cAMP-dependent stimulation of CFTR in Calu-3 cells. 981 85

The geranylgeranyltransferase I inhibitor GGTI-298 has recently been shown to arrest human tumor cells in the G1 phase of the cell cycle, induce apoptosis, and inhibit tumor growth in nude mice. In the present manuscript, we provide a possible mechanism by which GGTI-298 mediates its tumor growth arrest. Treatment of the human lung carcinoma cell line Calu-1 with GGTI-298 results in inhibition of the phosphorylation of retinoblastoma protein, a critical step for G1/S transition. The kinase activities of two G1/S cyclin-dependent kinases, CDK2 and CDK4, are inhibited in Calu-1 cells treated with GGTI-298. Furthermore, GGTI-298 has little effect on the expression levels of CDK2, CDK4, CDK6, cyclins D1 and E, but decreases the levels of cyclin A. GGTI-298 increases the levels of the cyclin-dependent kinase inhibitors p21 and p15 and had little effect on those of p27 and p16. Most interesting is the ability of GGTI-298 to induce partner switching for several CDK inhibitors. GGTI-298 promotes binding of p21 and p27 to CDK2 while decreasing their binding to CDK6. Reversal of partner switching and G1 block was observed after removal of GGTI-298. Furthermore, GGTI-298 treatment results in an increased binding of p15 to CDK4, which is paralleled with decreased binding to p27. The results demonstrate that the GGTI-298-mediated G1 block in Calu-1 cells involves increased expression and partner switching of CDK inhibitors resulting in inhibition of CDK2 and CDK4, and retinoblastoma protein phosphorylation.
 ...
PMID:The geranylgeranyltransferase I inhibitor GGTI-298 induces hypophosphorylation of retinoblastoma and partner switching of cyclin-dependent kinase inhibitors. A potential mechanism for GGTI-298 antitumor activity. 1006 46

Delivery of IgA to the mucosal surface occurs via transcytosis of polymeric IgA (pIgA) across the epithelium, a process mediated by the pIgR. Several factors increase pIgR expression in human epithelial cells, including IL-4 and IFN-gamma. Using an RNase protection assay, we found that IL-4 and IFN-gamma increase steady state levels of pIgR mRNA in both human intestinal (HT29) and airway (Calu-3) epithelial cells. Time course studies in HT29 clone 19A cells showed that with each cytokine alone and with both together: 1) there was a significant lag before mRNA levels increased; 2) maximal levels were not reached until 48-72 h after the addition of cytokines; 3) mRNA levels remained elevated in the continued presence of cytokines; and 4) addition of actinomycin D or removal of cytokines led to decreases in mRNA levels with a half-life of approximately 20-28 h. Cytokine-dependent increases in steady state levels of pIgR mRNA were inhibited by cycloheximide and by protein tyrosine kinase inhibitors but not by inhibitors of protein kinase C or cAMP-dependent protein kinase A. Both IFN-gamma and IL-4 increased expression of the inducible transcription factor IFN regulatory factor-1 (IRF-1), but levels of IRF-1 only weakly correlated with levels of pIgR mRNA, suggesting that additional transcription factors are required. These studies provide additional insights into the mechanisms by which cytokines regulate expression of the pIgR, a central player in mucosal immunity.
 ...
PMID:IL-4 and IFN-gamma increase steady state levels of polymeric Ig receptor mRNA in human airway and intestinal epithelial cells. 1022 81


1 2 3 4 5 Next >>