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)

A novel "cAMP-resistant" variant of LLC-PK1 renal epithelial cells which is impaired in in vivo down-regulation of response following hormonal stimulation of adenylate cyclase (AC) is described. Compared to parental cells, the BIB27 mutant exhibited markedly higher in vivo activation of cAMP-dependent protein kinase (cAMP-PK) in response to the hormones salmon calcitonin (SCT) or [Arg8]-vasopressin (AVP) or the AC activator forskolin. The activation of cAMP-PK subsequent to agonist stimulation also persisted much longer in the mutant than in LLC-PK1 cells, although the cAMP-PK of BIB27 cells was normal in terms of both absolute levels and regulation by cAMP in vitro. Intracellular cAMP accumulation was also much higher in BIB27 than in LLC-PK1 cells following agonist stimulation. Production of cAMP could be detected in BIB27 cells even 12 h after treatment with AVP or SCT, whereas cAMP production in LLC-PK1 had returned to basal within 1 and 8 h, respectively. High levels of free cAMP-PK catalytic (C) subunit in BIB27 persisted even 12 h after hormone addition, meaning that the higher cAMP production in BIB27 did not result in the normal down-regulation of cAMP-PK C subunit levels. In vitro AC activity in BIB27 cell homogenates could be stimulated by hormones or receptor-independent agonists, but to a lesser extent than in LLC-PK1 cell homogenates. The SCT and AVP concentrations promoting half-maximal AC activation in BIB27 cells were about 10- and 3-fold higher than parental, respectively. BIB27 accordingly appeared to possess a mutation in AC responsible for the impairment of both in vitro response to agonists and the normal in vivo down-regulation processes following hormonal stimulation.
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PMID:A novel LLC-PK1 renal epithelial cell mutant impaired in in vivo down-regulation of cAMP-mediated hormonal response. 171 64

Expression of the urokinase-type plasminogen activator (uPA) gene in LLC-PK1 cells can be induced by signals mediated by both cAMP-dependent protein kinase (PKA) and Ca(2+)- and phospholipid-dependent protein kinase (PKC). We have utilized the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) to down-regulate PKC, in order to test for an effect on the PKA-mediated induction of the uPA gene expression. Incubation of cells for 24 h with 100 ng/ml TPA caused a marked decrease of PKC protein, both in cytosolic and particulate fractions, and an 85% reduction of total PKC activity. After down-regulation of PKC, uPA mRNA accumulation induced by 8-Br-cAMP was 5-10-fold higher than in control cells. Both uPA mRNA stability and uPA gene transcription rates induced by 8-Br-cAMP were increased by PKC down-regulation (6- and 1.8-fold, respectively). Although total PKA activity was reduced by 20% in extracts from PKC-depleted cells, activation of PKA by 8-Br-cAMP was 2.5-fold higher than in control cells. This enhanced activation of PKA in PKC-depleted cells also occurred in response to other cAMP derivatives and to cAMP induced endogenously by the activation of adenylate cyclase with forskolin, but was not due to down-regulation-associated changes in the rate of cAMP synthesis. Our results demonstrate that in LLC-PK1 cells, down-regulation of PKC results in an enhanced induction of uPA gene expression by cAMP-mediated signals without alterations in adenylate cyclase activity, suggesting a mechanism distal to adenylate cyclase.
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PMID:Protein kinase C down-regulation enhances cAMP-mediated induction of urokinase-type plasminogen activator mRNA in LLC-PK1 cells. 171 70

Urokinase-type plasminogen activator (uPA) gene expression in LLC-PK1 cells is induced by activation of cAMP-dependent protein kinase (cAMP-PK) or protein kinase C (PK-C). To determine whether protein phosphatases can also modulate uPA gene expression, we tested okadaic acid, a potent specific inhibitor of protein phosphatases 1 and 2A, in the presence and absence of cAMP-PK and PK-C activators. Okadaic acid by itself induced uPA mRNA accumulation. This induction was strongly attenuated by the inhibition of protein synthesis. In contrast, the inhibition of protein synthesis enhanced induction by 8-bromo-cAMP and only delayed induction by 12-O-tetradecanoylphorbol-13-acetate (TPA). In addition, down-regulation of PK-C by chronic treatment with TPA did not abrogate the okadaic acid-dependent induction. These results provide evidence for a novel signal transduction pathway leading to gene regulation that involves protein phosphorylation but is independent of both cAMP-PK and PK-C.
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PMID:Okadaic acid induction of the urokinase-type plasminogen activator gene occurs independently of cAMP-dependent protein kinase and protein kinase C and is sensitive to protein synthesis inhibition. 184 95

Two Dictyostelium discoideum protein kinase(PK)-encoding cDNAs (Dd PK1 and Dd PK2) have been isolated by hybridization with an oligodeoxyribonucleotide derived from a highly conserved region of eukaryotic PKs. The two nucleotide (nt) sequences encode new putative serine/threonine-specific PKs. Dd PK1 is a partial cDNA covering the entire catalytic domain. The derived amino acid (aa) sequence is about 30% identical to both cAMP-dependent protein kinase (cAPK) and protein kinase C. The Dd PK2 sequence was extended through the isolation of a genomic fragment encoding a complete putative protein. A single intron is present, as deduced from sequence comparison with the cDNA. The catalytic domain appears more closely related to the catalytic subunit of cAPK (54% sequence identity). However, our nt sequence potentially codes for a much larger protein (648 vs. about 350 aa for most cAPKs) with a N-terminal half containing long homopolymers of threonines, glutamines and asparagines. Similar repeats occur at the C terminus of Dd PK1, Dd PK1 is expressed in vegetatively growing cells and during development. Dd PK1 RNA decreases after 6 h of starvation to re-accumulate once the cells have aggregated. Dd PK2 transcripts, present at a low amount in growing cells, rise upon starvation. A switch to a shorter form of transcripts occurs between 3 and 6 h into development.
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PMID:Isolation of two genes encoding putative protein kinases regulated during Dictyostelium discoideum development. 186 10

The precise mechanistic role of the cAMP-dependent protein kinase (cAMP-PK) in cAMP-mediated gene induction remains unclear. Renal epithelial cell mutants were compared to the LLC-PK1 parental cell line for induction of the cAMP-responsive urokinase-type plasminogen activator (uPA) gene, as quantitated by the technique of mRNA solution hybridization. The FIB4 and FIB6 mutants, which possess less than 10% parental cAMP-PK catalytic (C) subunit activity, showed markedly diminished uPA mRNA induction in response to agents elevating intracellular cAMP such as the cAMP analogue 8-bromo-cAMP and the adenylate cyclase-stimulating hormones vasopressin and calcitonin. In contrast, the mutant cells responded to a similar or greater extent than the parental cells in terms of uPA mRNA induction following treatment with the Ca2+/phospholipid-dependent protein kinase activator phorbol 12-myristate 13-acetate (PMA). Elevation of intracellular cAMP was found to induce a translocation of the cAMP-PK C subunit from the perinuclear Golgi region to the nucleus in both parental and mutant cell lines, as shown by immunocytochemical techniques. Results argue for the role of the cAMP-PK C subunit activity and possibly nuclear translocation of the C subunit in cAMP-mediated uPA induction, which is mechanistically distinct from the PMA-stimulated response.
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PMID:Mechanisms of cAMP-mediated gene induction: examination of renal epithelial cell mutants affected in the catalytic subunit of the cAMP-dependent protein kinase. 189 92

Biotinyl analogues of [Arg8]vasopressin were synthesized with the biotinyl moiety at position 4. This involved the substitution of 2, 4-diaminobutyric acid (Dab) for Gln4 in [1-deamino-Arg8]vasopressin to give the parent peptide des-[Dab4,Arg8]vasopressin. Two biotinyl analogues with different spacers between the side chain of Dab4 and the biotinyl residue were then prepared and characterized in detail. The analogues retained high binding affinities for the V2-receptor in both bovine kidney membranes and LLC-PK1 renal epithelial cells and for the V1-receptor in rat liver membranes. Both analogues were as potent as [Arg8] vasopressin in stimulating the cAMP-dependent protein kinase and the production of urokinase-type plasminogen activator in LLC-PK1 cells, with concentration dependence consistent with receptor binding affinities. Avidin or streptavidin did not appear to reduce receptor binding or biological activity of the biotinyl analogues. The use of the biotinylated vasopressin analogue des-[Dab-(biotinylamido)hexanoyl4, Arg8]vasopressin together with fluorescein-labeled streptavidin as a fluorescent probe for the V2-receptor in LLC-PK1 cells demonstrated the following: 1) Specific binding of the biotinyl analogue shown by quantitative single-cell fluorescence measurements using the technique of fluorescence microphotolysis; 2) the V2-receptor visualized by fluorescence microscopy; and 3) the expression of the V2-receptor detected by flow cytometry.
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PMID:Biotinyl analogues of vasopressin as biologically active probes for vasopressin receptor expression in cultured cells. 214 64

We have studied the regulation of urokinase-type plasminogen activator gene expression by cAMP in LLC-PK1 cells. We found a cAMP responsive region 3.4 kb upstream of the transcription initiation site, which comprised three protein-binding domains designated A, B, and C. Domains A and B both contain a sequence, TGACG, homologous to a consensus cAMP response element (CRE; TGACGTCA). Effective cAMP-mediated induction was achieved when these two domains were linked with domain C, which by itself did not confer cAMP responsiveness to a heterologous promoter nor contained CRE-like sequence, suggesting a functional cooperation among these domains. Results of competition studies using gel retardation and DNase I footprinting assays suggest that there is a protein-protein interaction between a CRE binding protein and a domain C binding protein. In gel retardation assays, binding of a nuclear protein to domains A and B was strongly augmented by addition of the catalytic subunit of cAMP-dependent protein kinase, whereas the protein binding to domain C was slightly inhibited, suggesting that protein phosphorylation is involved in the regulation of protein-DNA interaction.
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PMID:Macromolecular interaction on a cAMP responsive region in the urokinase-type plasminogen activator gene: a role of protein phosphorylation. 215 33

The cAMP-dependent protein kinase from LLC-PK1 cells can be activated in vivo by calcitonin and vasopressin, or forskolin. Continuous treatment of cells with these agents results in a decrease of total cAMP-PK activity. The loss of kinase activity was enhanced when either of these three agents was incubated in the presence of isobutylmethylxanthine. Results obtained using affinity purified antibodies to the catalytic subunit show that the loss of kinase was due to specific proteolysis of this subunit.
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PMID:cAMP mediated proteolysis of the catalytic subunit of cAMP-dependent protein kinase. 241 85

We report here the isolation and sequence of a cDNA for the type II regulatory subunit of the cAMP-dependent protein kinase (cAMP-PK) from a lambda gt-11 cDNA library derived from a porcine epithelial cell line (LLC-PK1). The cDNA was detected by immunological screening using an affinity purified polyclonal antibody for bovine RII. DNA sequence analysis of the 467 bp EcoRI insert confirmed the identity of the clone, because the deduced amino acid sequence corresponded to the published sequence for the bovine RII protein. Northern analysis of total RNA from the LLC-PK1 cells indicated a single mRNA species of about 6.0 kb, probably derived from a single copy gene.
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PMID:Expression cloning of a cDNA encoding the type II regulatory subunit of the cAMP-dependent protein kinase. 243 26

The catalytic (C) subunit activity of the cAMP-dependent protein kinase (cAMP-PK) from the mutant cell lines, FIB4 and FIB6, is only 10% compared with the parent cell line, LLC-PK1 [Jans and Hemmings (1986) FEBS Lett. 205, 127-131]. In order to understand the nature of the mutant phenotypes the cAMP-PK from parent and mutant cell lines was studied in more detail. Analysis of mutant cAMP-PK activity by ion-exchange chromatography revealed that kinase activity associated with type I holoenzyme of both FIB4 and FIB6 was only 5% parental, and the activity of the type II holoenzyme was about 20% parental. The type I regulatory (RI) subunits associated with the type I were also found to be reduced by 70-80% in both mutants, whereas the type II R subunit levels were similar to that of the parent. The residual kinase activity associated with the type I holoenzyme from FIB4 and FIB6 could not be activated by cAMP whereas the type II holoenzyme was activated by cAMP (Ka of 5.5 X 10(-8) M), and showed normal affinities for Kemptamide and ATP. A polyclonal antibody to the catalytic subunit was used to quantify the level of this protein in wild-type and mutant cells. This analysis showed that FIB4 and FIB6 had nearly normal levels of C subunit, suggesting that the C subunit synthesized by the mutants was mostly inactive. As both type I and type II cAMP-PK holoenzymes were abnormal, the most likely explanation of the mutant phenotype is a defect either in the structural gene for the C subunit or in an enzyme involved in its posttranslational processing. However, a second lesion affecting the RI subunit cannot be ruled out at this moment.
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PMID:Characterization of two mutants of the LLC-PK1 porcine kidney cell line affected in the catalytic subunit of the cAMP-dependent protein kinase. 243 50


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