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)

The cystic fibrosis transmembrane conductance regulator (CFTR) functions as a Cl- channel that becomes activated after phosphorylation by cAMP-dependent protein kinase (PKA). We demonstrate that PKA also plays a crucial role in maintaining basal expression of the CFTR gene in the human colon carcinoma cell line T84. Inhibition of PKA activity by expression of a dominant-negative regulatory subunit or treatment with the PKA-selective inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) caused a complete suppression of CFTR gene expression without affecting other constitutively active genes. Basal expression of a 2.2-kb region of the CFTR promoter linked to a luciferase reporter gene (CFTR-luc) exhibited the same dependence on PKA. The ability of cAMP to induce CFTR over basal levels is cell-type specific. In T84 cells, both the endogenous CFTR gene and CFTR-luc exhibited only a modest inducibility (approximately 2-fold), whereas in the human choriocarcinoma cell line JEG-3, CFTR-luc could be induced at least 4-fold. A variant cAMP-response element is present at position -48 to -41 in the CFTR promoter, and mutation of this sequence blocks basal expression. We conclude that cAMP, acting through PKA, is an essential regulator of basal CFTR gene expression and may mediate an induction of CFTR in responsive cell types.
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PMID:Basal expression of the cystic fibrosis transmembrane conductance regulator gene is dependent on protein kinase A activity. 754 84

The human colon carcinoma cell lines Caco-2 and HT-29 take up taurine actively. Treatment of Caco-2 cells with Escherichia coli heat-stable enterotoxin (STa) or with guanylin inhibited taurine uptake by approximately 40%. In contrast, neither STa nor guanylin changed the uptake of taurine in HT-29 cells. The inhibition in Caco-2 cells was associated with a decrease in the maximal velocity as well as in the affinity of the transporter. STa caused a 21-fold increase in guanosine 3',5'-cyclic monophosphate (cGMP) levels in Caco-2 cells with no change in cAMP levels. Neither cGMP nor cAMP levels were affected by STa treatment in HT-29 cells. Experiments with protein kinase inhibitors suggested that protein kinase A may mediate the observed effects of STa on taurine uptake. In accordance with this suggestion, treatment of Caco-2 cells with cholera toxin, which elevated intracellular cAMP levels, was found to inhibit taurine uptake. The steady state levels of the taurine transporter mRNA transcripts were not altered as a result of STa treatment. Studies with Caco-2 cells grown on permeable filters revealed that STa acts from the apical side. The taurine uptake from the apical side was inhibited by STa, but the taurine uptake from the basolateral side remained unaffected. It is suggested that the activity of the intestinal taurine transporter may be regulated by protein kinase A at a posttranslational level and that the intestinal absorption of taurine may be impaired during infection with enterotoxigenic strains of E. coli.
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PMID:Regulation of taurine transport by Escherichia coli heat-stable enterotoxin and guanylin in human intestinal cell lines. 761 6

Trypanosoma cruzi invades most nucleated mammalian cells by as yet unknown mechanisms. We report here that while T. cruzi attaches to epithelial cells lacking signaling transforming growth factor beta (TGF beta) receptor I or II, the adherent parasites cannot penetrate and replicate inside the mutant cells, as they do in parental cells. Invasion of the mutants is restored by transfection with the TGF beta receptor genes, as are biological responses to TGF beta. Similar rescue of both TGF beta antiproliferative response and T. cruzi invasion was demonstrated in a hybrid of TGF beta-resistant bladder and colon carcinoma cells. In addition, T. cruzi did not efficiently invade epithelial cells with dysfunction of the intracellular signaling cascade caused by the constitutive expression of the cyclin-dependent kinase cdk4 or of the oncogene H-ras. Treatment with TGF beta, but not with other antiproliferative agents of non-phagocytic cells, greatly enhances T. cruzi invasion. Moreover, infective, but not noninfective, trypanosomes strongly induce a TGF beta-responsive reporter gene in TGF beta-sensitive, but not in TGF beta-insensitive, cell lines. Thus, T. cruzi itself may directly trigger activation of the TGF beta signaling pathway required for parasite entry into the mammalian cells.
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PMID:Trypanosome invasion of mammalian cells requires activation of the TGF beta signaling pathway. 762 17

We have investigated the expression of protein kinase C (PKC) and protein kinase A (PKA) during the phases of growth and differentiation of the human colon carcinoma Caco-2 cells. We studied whether differentiation correlated with the responsiveness to cAMP and with an increased transport of the catalytic subunit of PKA into the nucleus. Also, we evaluated whether this phenomenon was affected by PKC activity. High levels of activated PKC were found in the plasma membranes of replicating cells. When the cells began to differentiate, plasma membrane-activated PKC decreased, while the cytosolic fraction increased. On the contrary, PKA holoenzyme increased during differentiation, along with the transport of its catalytic subunit into the nucleus. Both types I and II kinase A holoenzymes increased during differentiation, with maximal type II activity found when cells were fully differentiated. In replicating preconfluent cells, the inhibition of PKC by high dose phorbol 12-myristate 13-acetate or sphingosine increased the amount of both PKA catalytic subunit in the nucleus and sucrase activity. During differentiation, 8-Bromo-cAMP increased PKA catalytic subunit in the nucleus and apoliprotein A1 mRNA levels. These effects were inhibited by low-dose phorbol 12-myristate 13-acetate, which activates PKC in the plasma membranes. Our data suggest that PKC is activated in proliferating Caco-2 cells. The inhibition of PKC induces the transport of PKA catalytic subunit into the nucleus and the expression of the differentiation markers. Differentiated Caco-2 cells show a lower activation of PKC and an increased transport of the catalytic subunit of PKA into the nucleus.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:The enterocyte-like differentiation of the Caco-2 tumor cell line strongly correlates with responsiveness to cAMP and activation of kinase A pathway. 781 34

The differentiated clone 19A of the HT-29 human colon carcinoma cell line was used as a model to study the intracellular electrophysiological effects of interaction of the cAMP, the protein kinase C (PKC) and the Ca2+ pathways. (a) A synergistic effect between ionomycin and forskolin was observed. From intracellular responses it was concluded that the synergistic effect is caused by activation of an apical Cl- conductance by protein kinase A and a basolateral K+ conductance by Ca2+. (b) A transient synergistic effect of ionomycin and the phorbol ester phorbol dibutyrate (PDB) was found. The decrease of the response appeared to be due to PKC-dependent inactivation of the basolateral K+ conductance. The synergism is caused by PKC-dependent increase of the apical Cl- conductance and Ca(2+)-dependent increase of the basolateral K+ conductance. (c) The effects of carbachol and PDB were not fully additive presumably because of their convergence on PKC activation. (d) Forskolin and PDB, when added in this order, had a less than additive effect. Results of cell-attached patch-clamp studies, presented in the accompanying paper, showed a synergistic effect of forskolin and PDB on non-rectifying small-conductance Cl- channels. Assuming that these channels are involved in the transepithelial responses it is suggested that forskolin and PDB induce a modulatory, synergistic increase of the apical Cl- conductance when both pathways are activated simultaneously. (e) The HT-29cl.19A cells differ from T84 cells in that the latter did n ot respond with an increase of the short-circuit current to addition of phorbol ester. this may be due to a very low expression of PKA alpha.
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PMID:Activation of ion transport by combined effects of ionomycin, forskolin and phorbol ester on cultured HT-29cl.19A human colonocytes. 827 89

Chloride (Cl-) channels are important in the regulation of salt and water transport in secretory epithelial cells. A disturbed Cl- secretion is the most consistent characteristic in the genetic disease cystic fibrosis. An outwardly rectifying Cl- channel (OR) with a conductance of 25-50 pS had been proposed to play a major role in Cl- secretion. Activation by Ca2+ and the protein kinases (PK) A and C (at less than 10 nM Ca2+) as well as inhibition by PKC (at 1 microM Ca2+) has been reported. In the present study, we have identified and characterized the OR in HT29.cl19A human colon carcinoma cells. The OR displayed a conductance of 31 +/- 4 pS (n = 25). Its open probability in 10 nM Ca2+ was voltage-dependent in 50% of the patches, starting from 0.2 at -70 mV to 0.8 at 70 mV. The spontaneous activation in excised inside-out patches at -60 mV was Ca(2+)-dependent and decreased from 29% in 1 mM Ca2+ to 2% in 10 nM Ca2+. Active OR were found in (a) 25% of patches exposed to 10 nM Ca2+, ATP and cAMP only, (b) 42% of the patches exposed to 10 nM Ca2+, ATP and the catalytic subunit of PKA (CAK) and (c) 67% of the patches exposed to 1 mM Ca2+, ATP plus CAK. Inhibition of voltage-activated channels by addition of PKC in 1 microM or 1 mM Ca2+ was not observed. Attempts to activate the OR in cell-attached patches by increasing cAMP levels under different experimental conditions were unsuccessful.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of chloride channels in the human colon carcinoma cell line HT29.cl19A. 838 68

UCN-01 (7-hydroxy-staurosporine) is a potent and selective inhibitor of protein kinase C (PKC), one of several protein kinases examined. UCN-01 itself was shown to exhibit antitumor activity in vitro and in vivo in oncogene-activated human and murine tumor cell lines. Since the mechanism(s) of action of UCN-01 is thought to be different from those of alkylating agents, including mitomycin C (MMC), we tested the combined effect of UCN-01 with MMC on human epidermoid carcinoma A431 cells. UCN-01 potentiated the antiproliferative activity of MMC and yet it did not affect the growth of the cells in vitro. However, other nonselective protein kinase inhibitors, such as staurosporine, K-252a, KT6124 (a derivative of K-252a) and H7, did not enhance the activity of MMC. Isobologram analysis revealed that the interaction of UCN-01 with MMC was synergistic in its antiproliferative activity. A DNA histogram of A431 cells treated with both UCN-01 and MMC showed a block in the cell cycle at the G1/S phase. However, a histogram of cells treated with UCN-01 or MMC alone showed a G1 or a G2M block, respectively. The combined effect of UCN-01 with MMC was further examined in vivo in xenografted A431 cells in nude mice. The combination of both drugs in a single i.v. injection exhibited greater antitumor activity than MMC and UCN-01 alone (P < 0.01). This synergistic antitumor effect was also confirmed in two other solid tumor cell lines, i.e. human xenografted colon carcinoma Co-3 and murine sarcoma 180. The same was observed in the i.v.-inoculated P388 leukemia model, in which we saw an increased lifespan of mice when UCN-01 was combined with MMC. These results suggests the feasibility of using UCN-01 in clinical oncology, especially in combination with alkylating agents such as MMC. In addition, this combination therapy might be a novel chemotherapeutic approach to MMC-insensitive tumors in clinical trials.
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PMID:Enhancement of antitumor activity of mitomycin C in vitro and in vivo by UCN-01, a selective inhibitor of protein kinase C. 850 Feb 22

LS-174T human colon carcinoma cells that contain approximately equal amounts of cAMP-dependent protein kinase (PKA) isozymes, PKA-I and PKA-II, were infected with retroviral vectors coding for regulatory (R) and catalytic (C) subunits of human PKA. In cells overexpressing RII alpha, RII beta and RII beta-P (a RII beta mutant at the autophosphorylation site), PKA-II levels increased while PK-A levels decreased. PKA-I was almost completely eliminated in cells overexpressing RII beta or RII beta-P. In contrast, overexpression of either RI alpha or C alpha had little or no effect on PKA isozyme levels. Although all infectants expressed high levels of PKA subunit mRNAs in accordance with gene introduction, the R subunit protein expression was reflected in PKA isozyme levels rather than in subunit mRNA levels. Only RII beta infectants demonstrated marked growth inhibition in monolayer culture, reduced thymidine incorporation into DNA, and inability to grow in semisolid medium or in serum-free medium. Conversely, all other infectants displayed growth properties similar to uninfected parental cells. The growth-retardation properties of RII beta infectants were reflected in their altered phenotypic appearances. Our findings that the mutant RII beta-P could not mimic the growth-inhibitory effect of RII beta suggest the functional importance of the authophosphorylation site in RII beta. Our results suggest a role for RII beta in the suppression of neoplastic cell growth, and thus abnormal expression of R subunit isoforms of PKA may be involved in neoplastic transformation.
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PMID:Overexpression of RII beta regulatory subunit of protein kinase A in human colon carcinoma cell induces growth arrest and phenotypic changes that are abolished by site-directed mutation of RII beta. 865 92

Human colon carcinoma cell line HCT116 harbors an oncogenic Ki-ras gene. Introduction of an exogenous full-length NF1 gene or its GTPase-activating protein (GAP)-related domain suppressed the tumor-forming ability of this cell line in nude mice. A GAP-related domain peptide carrying a K1423E mutation, which shows greatly diminished GAP activity but a normal binding affinity for p2lras-GTP, was also tested. This construct was able to suppress tumor formation by the HCT116 cell line, thus ruling out the possibility that the observed tumor suppression is due to the GAP activity of NF1. Reduced Raf-1 kinase activity in cells which expressed these NF1 constructs suggested that neurofibromin may interfere with the interaction between Ras and Raf. Introduction of a mutationally activated Raf-1 kinase domain reversed tumor suppression by neurofibromin, implicating Raf-1 as the primary downstream transducer of the oncogenic Ras signal. An increase in apoptotic cell death, which could be delayed by activated Raf-1 kinase, was also seen in cells carrying the exogenous NF1 gene.
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PMID:Suppression of a human colon cancer cell line by introduction of an exogenous NF1 gene. 866 28

The secreted proteoglycan decorin has been implicated in the negative control of cell proliferation primarily by virtue of its ability to block transforming growth factor-beta. Moreover, decorin expression is markedly up-regulated during quiescence but suppressed upon viral transformation, whereas de novo decorin expression in colon carcinoma cells abrogates the malignant phenotype by arresting the cells in the G1 phase of the cell cycle. Here we show that this decorin-induced growth arrest is associated with up-regulation of p21 mRNA and protein in a transforming growth factor-beta- and p53-independent pathway. The augmented p21 protein is present as a multimeric complex with various cyclins and cyclin-dependent kinases in the nuclei of decorin-expressing cells, thereby leading to suppression of cyclin-dependent kinase activity and block of cell division. Through the usage of decorin-specific antisense oligodeoxynucleotide treatment, we demonstrate that the expression of decorin is closely linked to that of p21 and that abrogation of decorin leads to suppression of p21 and restoration of cell division. Collectively, our results provide a plausible mechanism by which decorin may contribute to retard and suppress the growth of tumor cells in vivo.
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PMID:Decorin-induced growth suppression is associated with up-regulation of p21, an inhibitor of cyclin-dependent kinases. 870 60


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