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)

LLC-PK1/PKE20 cells (a continuous epithelial cell line) has two different Na/H exchange activities: Na/H-1 located in the basolateral membrane and Na/H-2 located in the apical membrane [Casavola et al. (1989) Biochem Biophys Res Commun 165:833-837; Haggerty et al. (1988) Proc Natl Acad Sci USA 86:6797-6801]. In the present report we have studied hormone regulation of these exchange activities by measuring Na-dependent recovery of pHi from an acid load (by using microspectrofluorometry and 2,7-bis(carboxyethyl)-5,6-carboxyfluorescein) in response to activation of regulatory cascades by either pharmacological agents or by vasopressin or calcitonin. Agents leading to activation of protein kinase A (cAMP-dependent), such as forskolin (10 microM), 8-Br-cAMP (0.25 mM), and isobutylmethylxanthine (0.5 mM), inhibited Na/H-2 and Na/H-1 by an average of 49%. Stimulation of protein kinase C by a phorbol ester (phorbol 12-myristate 13-acetate, TPA, 100 nM) inhibited Na/H-2 (by an average of 48%) and stimulated Na/H-1 (by an average of 38%); these effects of TPA were also observed in the presence of forskolin (100 microM). Addition of either vasopressin (2 microM) or calcitonin (0.3 microM) onto both sides of the monolayer decreased the activity of Na/H-2 by an average of 26.3% and 27.7% respectively, and stimulated the activity of Na/H-1 by an average of 17.4% and 38.7% respectively; exposure of cells to either hormone stimulated production of cAMP and inositol trisphosphate, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Polarized expression of Na+/H+ exchange activity in LLC-PK1/PKE20 cells: II. Hormonal regulation. 131 51

Calcitonin (CT) activates both the cAMP and the protein kinase C (PKC) pathways in the kidney cell line LLC-PK1. Although CT also activates cAMP in osteoclasts, its effects on PKC in this cell type are unknown. In order to determine whether the response of osteoclasts to CT also involves the PKC pathway, the effects of activators and inhibitors of PKC on bone resorption and cell surface area were analyzed in isolated rat osteoclasts. As expected, CT inhibited in a dose-dependent manner bone resorption by rat osteoclasts cultured for 24 h on devitalized bovine bone slices and this effect could be mimicked by cAMP. The inhibitory effect of CT could however also be mimicked by phorbol-12,13-dibutyrate (PDBu) and blocked by the PKC inhibitor sphingosine, as well as by the less specific inhibitors H7 and H8, none of which had detectable effects in the absence of CT. No changes in the number of attached osteoclasts were observed under any of these conditions. These results indicate that CT activates PKC in osteoclasts and that this activation, like the activation of cAMP-dependent protein kinase, leads to an inhibition of bone resorption. Quantitative time-lapse videomicroscopy showed that the CT-induced retraction of osteoclasts also involved activation of the PKC pathway and could therefore be induced by phorbol esters. In contrast, (Bu)2 cAMP (1-200 microM) failed to induce rapid cell retraction. It is concluded that, in osteoclasts, CT receptors are coupled to both the cAMP-dependent protein kinase and the PKC pathways. Although these two second messengers can have additive inhibitory effects on bone resorption, only activation of the PKC pathway induces rapid cell retraction. These two effects of calcitonin on osteoclasts are therefore independent and may be functionally unrelated.
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PMID:Differential effects of the 3',5'-cyclic adenosine monophosphate and protein kinase C pathways on the response of isolated rat osteoclasts to calcitonin. 132 63

Phosphorylation of glial fibrillary acidic protein (GFAP) induces disassembly of the filaments. An amino-terminal fragment of bovine GFAP (G-Hf) was produced by lysylendopeptidase digestion. G-Hf formed ribbon-like filaments in the presence of GFAP even in low ionic strength, whereas the fragment itself did not form any structures. Only one (PK3) of the five V8 protease fragments of G-Hf accelerated GFAP assembly to the same degree as G-Hf did, whereas the other fragments did not. When PK3 was cleaved into two fragments, it lost the assembly-accelerating property. The sequence of PK3 was determined as RRRVTSATRRSYVSSSE, which corresponded to residues 3-19 of porcine GFAP. It was concluded that PK3 contains a sequence indispensable for GFAP assembly and that neither PK1 (RRRVTS) nor PK2 (ATRRSYVSSSE) included all of the sequence. A single phosphorylation of PK3 by cyclic AMP-dependent protein kinase diminished its assembly-accelerating property. The phosphorylation site was determined as Ser-12 of porcine GFAP. It was shown that single phosphorylation of the amino-terminal head domain, which contains an indispensable sequence for GFAP assembly, might be sufficient for GFAP disassembly.
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PMID:Assembly regulatory domain of glial fibrillary acidic protein. A single phosphorylation diminishes its assembly-accelerating property. 142 73

The present studies examined effects of ATP depletion and calmodulin antagonism on stimulation of Na(+)-H+ exchange by cytosolic acidification in renal epithelial cells (LLC-PK1). ATP depletion significantly inhibited both amiloride-sensitive 22Na+ uptake (P less than 0.001; n = 12) and Na(+)-dependent intracellular pH (pHi) recovery in 2',7'-bis (carboxyethyl)-5(6)-carboxyfluorescein acetoxymethylester (BCECF/AM)-loaded cells. Calmodulin antagonists, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) and calmidazolium, both caused a concentration-dependent inhibition of Na(+)-H+ exchange activity. The W-7-induced inhibition of Na(+)-H+ exchange occurred in cells incubated for 24 h with phorbol 12-myristate 13-acetate, indicating that the effect of W-7 was not mediated by protein kinase C inhibition. Both W-7 and ATP depletion shifted the pHi dependence of the antiporter, and ATP depletion also reduced the maximal activity. In LLC-PK1/CL4 cells grown on permeable filters, W-7 inhibited the cytosolic acidification-stimulated basolateral exchanger by 54 +/- 5% (P less than 0.005; n = 7) and, in contrast, stimulated the apical exchanger by 28 +/- 13% (P less than 0.05; n = 6). ATP depletion significantly inhibited apical Na(+)-H+ exchange. These results suggest that an ATP-Ca(2+)-calmodulin-dependent process is involved in regulation of Na(+)-H+ exchange in LLC-PK1 cells. A Ca(2+)-calmodulin-dependent process activated the amiloride-sensitive basolateral Na(+)-H+ exchanger and inhibited the amiloride-resistant apical antiporter. Phosphorylation of these two Na(+)-H+ exchangers or regulatory proteins by a Ca(2+)-calmodulin-dependent protein kinase may mediate this differential regulation.
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PMID:Regulation of Na(+)-H+ exchange by ATP depletion and calmodulin antagonism in renal epithelial cells. 165 80

Exposure of intact LLC-PK1 cells to the phorbol ester 4-phorbol 12-myristate 13-acetate (PMA) increases basal, arginine vasopressin-stimulated, and forskolin-stimulated adenylate cyclase activity in LLC-PK1 membranes. This observation suggests that protein kinase C can increase adenosine 3',5'-cyclic monophosphate (cAMP) in LLC-PK1 cells. To determine whether cAMP regulates protein kinase C activity in LLC-PK1 cells, intact cells were exposed to either forskolin or to soluble cAMP analogues. Acute (5 and 30 min) exposure to either forskolin or cAMP analogues increases protein kinase C activity as observed by two different methods for measuring protein kinase C. Acute exposure to PMA translocates protein kinase C from a soluble to a particulate cell fraction, whereas acute exposure to cAMP increases both soluble and particulate forms of protein kinase C. Longer exposure (18 h) to PMA results in a loss of protein kinase C activity, whereas 18-h exposure to cAMP results in a further increase in protein kinase C activity. The effect of cAMP but not of PMA to stimulate protein kinase C activity can be attenuated by the pro-R diastereoisomer of adenosine 3',5'-cyclic phosphorothioate, suggesting a protein kinase A-mediated effect. These results suggest the presence of a monodirectional mode of signal transduction system interaction in LLC-PK1 cells in which protein kinase C and protein kinase A can potentiate each other.
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PMID:cAMP stimulates protein kinase C activity in cultured renal LLC-PK1 cells. 166 Oct 84

Urokinase-type plasminogen activator (uPA) is expressed at higher levels in many transformed cells as compared with their non-transformed counterparts. The transformed phenotype is associated with changes in the cytoskeleton. Therefore, we have investigated whether alterations in the cytoskeleton can trigger changes in the expression of the uPA gene. To this end we analyzed the expression of the uPA gene following exposure of porcine kidney cells, LLC-PK1, to agents that modify the organization of specific components of the cytoskeleton. These cells exhibited increased uPA mRNA and protein after disruption of microtubules by colchicine or nocodazole treatment or after disruption of microfilaments by cytochalasin B treatment. Colchicine, nocodazole, and cytochalasin B did not cause alterations in the level of cAMP-dependent protein kinase in LLC-PK1 cells. In contrast, down-regulation of protein kinase C by phorbol myristate acetate, reduced, but did not fully prevent the induction of uPA mRNA when LLC-PK1 cells were subsequently exposed to colchicine, nocodazole, or cytochalasin B. Apparently, a signal transduction pathway in part involving protein kinase C but not cAMP-protein kinase mediates the regulatory changes at the transcriptional level of the uPA gene. Inhibition of protein synthesis by cycloheximide prior to the exposure of LLC-PK1 cells to colchicine, nocodazole, or cytochalasin B, largely prevented the induction of uPA mRNA.
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PMID:Disruption of cytoskeletal structures results in the induction of the urokinase-type plasminogen activator gene expression. 169 7

The role of cyclic adenosine monophosphate (cAMP) dependent protein kinase (PKA) in modulating functions of differentiated renal cells is well established. Its importance in controlling their growth and differentiation is less clear. We have used somatic cell genetic techniques to probe the role of PKA in controlling morphology and behavior of a renal epithelial cell line, LLC-PK1, which acquires many properties characteristic of the renal proximal tubular cell. Mutants of this line altered in PKA activity have been isolated and their behavior compared to that of the parent line. The results indicate that PKA is involved, either directly or indirectly, in maintenance of cell morphology, cell-cell and cell-substratum interactions, density-dependent growth regulation, and expression of one function characteristic of the renal proximal tubular cell, Na-hexose symport. The relevance of these results to the role of PKA in controlling growth and differentiation of renal epithelial cells in vivo is discussed.
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PMID:Cyclic adenosine monophosphate modulates cell morphology and behavior of a cultured renal epithelial. 169 40

In the porcine renal epithelial cell line, LLC-PK1, activation of the cAMP-dependent signal transduction pathway induces the urokinase-type plasminogen activator (uPA) gene. We show here that the cAMP response is enhanced when the intracellular calcium concentration is increased. When LLC-PK1 cells were treated with the calcium ionophore ionomycin alone, there was no uPA mRNA accumulation. However, in the presence of ionomycin the dose-response of 8-bromo-cAMP (Br-cAMP) with respect to uPA mRNA accumulation was shifted toward the lower concentrations of Br-cAMP. A Northern blot analysis after the inhibition of RNA synthesis and nuclear run-on assays showed that the synergistic effect of Ca2+ could be attributed to increases in uPA gene transcription and mRNA stability. In the presence of cycloheximide, a protein synthesis inhibitor, uPA mRNA was stabilized, but the effect of ionomycin on Br-cAMP-induced mRNA accumulation was still maintained. The result suggests that the Ca2+, at least on transcription, does not require new protein synthesis. Ionomycin treatment did not modify the activity of the cAMP-dependent protein kinase, suggesting that Ca2+ either affects a step in the pathway between the kinase and the uPA gene, or acts independently of the cAMP-dependent protein kinase pathway. The effect of ionomycin was not suppressed by protein kinase C down-regulation nor by inhibitors of calmodulin. Synergism was also observed when Br-cAMP was replaced with calcitonin, a peptide hormone which is coupled to adenylate cyclase, and when ionomycin was replaced with another ionophore A23187, suggesting that the synergism is due to an interaction between cAMP-dependent and Ca2(+)-dependent signal transduction pathways.
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PMID:Ca2+ potentiates cAMP-dependent expression of urokinase-type plasminogen activator gene through a calmodulin- and protein kinase C-independent mechanism. 170 Nov 76

The relationship between activation of the cAMP-dependent protein kinase (cAMP-PK) and ligand binding and internalization by the vasopressin renal (V2-type) receptor of LLC-PK1 renal epithelial cells was examined. Upon cAMP-PK activation through 1 h treatment with the cAMP analogue 8-bromo-cAMP (BrcA), a marked reduction in V2-receptor steady state number and internalization in LLC-PK1 cells was effected. In cells treated for 17 h with BrcA and hence down-regulated for cAMP-PK, the V2-receptor number was normal but internalization was markedly reduced. Cells of the LLC-PK1 mutant FIB4, which possesses about 10% parental cAMP-PK catalytic subunit activity, exhibited lower V2-receptor steady state number and internalization in comparison to untreated LLC-PK1 cells. A negative correlation was thus evident between cAMP-PK activation and V2-receptor number, and internalization. Phosphorylation by cAMP-PK may effect ligand-independent removal of receptor from the plasma membrane.
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PMID:cAMP-dependent protein kinase activation affects vasopressin V2-receptor number and internalization in LLC-PK1 renal epithelial cells. 170 31

Previous studies have implicated adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase (PKA) in regulation of both growth and expression of differentiated function in the pig renal epithelial cell, LLC-PK1. To investigate this possible regulatory mechanism, we compared growth behavior, morphology, and appearance of two differentiated functions, Na-hexose symport (SYMP) and gamma-glutamyl transpeptidase (gamma-GT), in the LLC-PK1 line and two PKA-deficient mutants (FIB4 and FIB6). Compared with the wild-type cell line, the mutant lines continued to proliferate at higher population densities and exhibited altered cell morphology, poorer formation of the brush-border structure, and decreased or lack of expression of SYMP and gamma-GT activities. Wild-type and mutant cells exhibit an identical logarithmic growth rate. Both lines form cell-cell junctions and exhibit identical kinetic properties of expressed SYMP activity. These results strongly support the hypothesis that PKA modulates a defined subset of cellular processes, including aspects of growth control and expression of the differentiated phenotype, in this renal epithelial cell line.
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PMID:cAMP-dependent protein kinase regulates renal epithelial cell properties. 171 77


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