Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Ca2+-independent delta-isoform of protein kinase C (PKC-delta) was overexpressed in LLC-PK1 epithelia and placed under control of a tetracycline-responsive expression system. In the absence of tetracycline, the exogenous PKC-delta is expressed. Western immunoblots show that the overexpressed PKC-delta is found in the cytosolic, membrane-associated, and Triton-insoluble fractions. Overexpression of PKC-delta produced subconfluent and confluent epithelial morphologies similar to that observed on exposure of wild-type cells to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. Transepithelial electrical resistance (RT) in cell sheets overexpressing PKC-delta was only 20% of that in cell sheets incubated in the presence of tetracycline, in which the amount of PKC-delta and RT were similar to those in LLC-PK1 parental cell sheets. Overexpression of PKC-delta also elicited a significant increase in transepithelial flux of D-[14C]mannitol and a radiolabeled 2 x 10(6)-molecular-weight dextran, suggesting with the RT decrease that overexpression increased paracellular, tight junctional permeability. Electron microscopy showed that PKC-delta overexpression results in a multilayered cell sheet, the tight junctions of which are almost uniformly permeable to ruthenium red. Freeze-fracture electron microscopy indicates that overexpression of PKC-delta results in a more disorganized arrangement of tight junctional strands. As with LLC-PK1 cell sheets treated with 12-O-tetradecanoylphorbol-13-acetate, the reduced RT, increased D-mannitol flux, and tight junctional leakiness to ruthenium red that are seen with PKC-delta overexpression suggest the involvement of PKC-delta in regulation of tight junctional permeability.
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PMID:Overexpression of protein kinase C-delta increases tight junction permeability in LLC-PK1 epithelia. 968 9

The addition of phorbol 12-myristate 13-acetate (PMA) to renal LLC-PK1-F+ cells caused a rapid decrease in the level of phosphoenolpyruvate carboxykinase (PCK) mRNA and reversed the stimulatory effects of exposure to acidic medium (pH 6.9, 10 mM HCO-3) or cAMP. In contrast, prolonged treatment with PMA increased the levels of PCK mRNA. The two effects correlated with the membrane translocation and downregulation of the alpha-isozyme of protein kinase C and were blocked by pretreatment with specific inhibitors of protein kinase C. The rapid decrease in PCK mRNA caused by PMA occurred with a half-life (t1/2 = 1 h) that is significantly faster than that measured during recovery from acid medium or following inhibition of transcription (t1/2 = 4 h). The effect of PMA was reversed by staurosporine, which apparently acts by inhibiting a signaling pathway other than protein kinase C. Staurosporine had no effect on the half-life of the PCK mRNA, but it stimulated the activity of a chloramphenicol acetyltransferase gene that was driven by the initial 490 base pairs of the PCK promoter and transiently transfected into LLC-PK1-F+ cells. This effect was additive to that of cAMP, and neither stimulation was reversed by PMA. The stimulatory effect of staurosporine was mapped to the cAMP response element (CRE-1) and P3(II) element of the PCK promoter. The data indicate that, in LLC-PK1-F+ cells, activation of protein kinase C decreases the stability of the PCK mRNA, whereas transcription of the PCK gene may be suppressed by a kinase that is inhibited by staurosporine.
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PMID:PMA and staurosporine affect expression of the PCK gene in LLC-PK1-F+ cells. 972 8

The carboxyl(C)-truncated human (h) PTH (hPTH) analog hPTH(1-31), which activates adenylyl cyclase (AC), but not protein kinase C, in rat osteosarcoma cells, exerts an anabolic effect on rat bone in vivo similar to that of hPTH(1-34). It has been proposed, therefore, that this action of PTH(1-34) is mediated exclusively by stimulation of AC via the rat type-1 PTH/PTH-related peptide (PTHrP) receptor (PTH1R). To determine whether this selective signaling pattern also might be a property of the hPTH1R, we studied signal transduction via heterologously expressed hPTH1Rs in response to activation by hPTH(1-34), hPTH(1-31), and a C-truncated analog that does not increase rat bone mass in vivo, hPTH(1-30). In porcine LLC-PK1 cells that stably expressed recombinant hPTH1Rs, these three peptides activated AC identically (EC50 = 1-2 nM). In cells with comparable expression of rat PTH1Rs, AC activation by hPTH(1-34) and hPTH(1-31) again was identical, whereas full activation by hPTH(1-30) required higher concentrations (EC50 = 10 nM vs. 1 nM). Surprisingly, hPTH(1-31) fully stimulated phospholipase C (PLC), via both species of PTH1Rs, with potency that was similar (hPTH1Rs) or slightly reduced (rat PTH1Rs), relative to that of hPTH(1-34). hPTH(1-30), however, was 5-fold less potent than hPTH(1-34) in activating PLC via hPTH1Rs and showed weak and only partial activity via the rat PTH1R. Comparable results were obtained when human and rat PTH1Rs were transiently expressed heterologously in COS-7 cells or homologously in HEK 293 and UMR 106-01 cells, respectively. Binding affinities of these C-truncated peptides to human and rat PTH1Rs were concordant with their relative potencies in activating PLC. We conclude that hPTH(1-31) and, to a lesser extent, hPTH(1-30) can activate PLC, as well as AC, via both rat and human PTH1Rs. Accordingly, a role for PLC activation in the anabolic action of PTH in vivo cannot be excluded.
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PMID:Type-1 parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptors activate phospholipase C in response to carboxyl-truncated analogs of PTH(1-34). 975 12

Norepinephrine (NE) transporters (NETs) found in the neuronal plasma membrane mediate the removal of NE from the extracellular space, limiting the activation of adrenoceptors at noradrenergic synapses. Our previous studies with the noradrenergic neuroblastoma SK-N-SH have revealed a muscarinic receptor-triggered regulation of NET surface density and transport capacity, mediated in part by protein kinase C activation. Low abundance of NET proteins in this native cell model, however, preclude direct confirmation of altered trafficking of NET proteins. In our study, we monitored the activity and surface distribution of human NET proteins in transient and stably-transfected cell lines after application of kinase activators and inhibitors. Using hNET stably transfected HEK-293 and LLC-PK1 cells, as well as transiently transfected COS-7 cells, we demonstrate that PKC-activating phorbol esters, beta-PMA or beta-PDBu selectively diminish l-NE transport capacity (Vmax) with little change in NE Km. Effects of phorbol esters are rapid, stereospecific and blocked by protein kinase C inhibitors, staurosporine and bisindolylmaleimide I. As in SK-N-SH cells, beta-PMA induces a reduction in intact cell [3H]nisoxetine binding sites with no change in nisoxetine Kd or total membrane NET density. Cell-surface biotinylation and confocal immunofluorescence techniques confirm that protein kinase C-dependent reductions in NE transport capacity and whole-cell antagonist binding density are accompanied by reductions in cell-surface human NET protein expression. Together these findings argue for kinase-modulated protein trafficking as a potential route for acute regulation of antidepressant-sensitive NE clearance.
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PMID:Acute regulation of norepinephrine transport: II. PKC-modulated surface expression of human norepinephrine transporter proteins. 980 5

Parathyroid hormone (PTH) activates PTH/PTH-related peptide-related receptors (PTHRs) to stimulate both adenylyl cyclase (AC) and phospholipase C (PLC). How these parallel signals mediate specific cellular and tissue responses to PTH, such as the complex anabolic versus catabolic actions of PTH on bone, remains unsettled. Previous studies of PTHR signaling and function employed mainly rodent or other cell lines that express endogenous PTHRs and, possibly, alternate species of PTH receptors. To preclude confounding effects of such receptors, we stably expressed recombinant human PTHRs (hPTHRs) at different levels of surface density in LLC-PK1 porcine renal epithelial cells that lack endogenous PTH responsiveness. hPTH(1-34) induced concentration-dependent activation of both AC and PLC via transfected hPTHRs. Maximal intensity of each signal increased with receptor density, but more hPTHRs were required for PLC than for AC activation. Coupling to AC was saturated at receptor densities too low to detect sustained PLC activation. hPTH(3-34), found by others to be a PLC/protein kinase C (PKC)-selective peptide in rat cells, did not activate PLC via human (or rat) PTHRs under conditions (1 microM peptide, 106 hPTHRs/cell) where hPTH(1-34) stimulated PLC severalfold. Other cellular responses that require PKC activation in these cells, such as sodium-dependent phosphate transport and cAMP-independent secretion of plasminogen activator, were induced by PTH(1-34) but not by hPTH(3-34) or hPTH(7-34). We conclude that amino-truncated PTH analogs reported to activate PKC cannot directly activate phosphatidylinositol-specific PLC via the human or rat PTHR and therefore that PTH receptors may access alternate, PLC-independent pathways of PKC activation in some target cells. The relative intensity of AC and PLC signaling via the hPTHR may be strongly regulated by changes in its surface expression.
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PMID:Dual signaling and ligand selectivity of the human PTH/PTHrP receptor. 989 61

The alpha6beta1 integrin heterodimer has been implicated in the mediation of renal epithelial cell binding to laminin, and it has been suggested that this binding is important for renal morphogenesis and development. Studies of nonrenal cells have suggested that the functional activity of alpha6beta1 integrin is regulated by protein kinase C (PKC) activity. In this study, the binding of a renal epithelial cell line, LLC-PK1, to laminin was characterized and the role of PKC activity in the modulation of binding was investigated. LLC-PK1 cells bound to laminin-coated surfaces in a time- and laminin concentration-dependent manner. Binding was strongly inhibited by anti-beta1 integrin antibodies and by anti-alpha6 integrin antibodies. Antibodies against alpha2 integrin and a3 integrin had little inhibitory effect. Cells bound to both whole laminin and laminin fragment E8, i.e., the fragment to which the alpha6beta1 integrin heterodimer binds. Exposure of cells to PKC activators for as little as 2 h enhanced cell binding to laminin approximately twofold, in a protein synthesis-dependent manner. PKC inhibitors antagonized this effect. PKC-stimulated binding was also inhibited by anti-beta1 integrin and anti-alpha6 integrin antibodies. PKC activation did not alter expression of beta1 integrin subunits at the cell surface after short time periods (2 to 4 h), but expression was increased after longer time periods (24 h). These results indicate that the renal epithelial cell line LLC-PK1 binds to laminin via the alpha6betal integrin heterodimer and binding is enhanced by PKC activation. The PKC-mediated enhancement of binding requires protein synthesis and is mediated in part by activation of surface alpha6beta1 integrin.
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PMID:Binding of the renal epithelial cell line LLC-PK1 to laminin is regulated by protein kinase C. 1036 59

The role of protein kinase C (PKC) in the regulation of the cytoskeleton of epithelial cells with tightly sealed contacts, poor contacts, and without contacts were investigated by incubating them with a protein kinase C activator phorbol myristoyl acetate (PMA). The morphology and organization of the membrane skeleton and stress fibers as well as the localization of an actin-bundling PKC substrate MARCKS in confluent MDCK cells originating from the distal tubulus of dog kidney, LLC-PK1 cells originating from the proximal tubulus of pig kidney, src-transformed MDCK cells, epidermoid carcinoma A431 cells, and MDCK cells grown in low calcium medium (LC medium) in low density were visualized with phase contrast and immunofluorescence microscopy. Four different responses to the PMA-treatment in actin-based structures of cultured epithelial cells were observed: 1) disintegration of the membrane skeleton in confluent MDCK cells; 2) depolymerization of the stress fibers in confluent MDCK and LLC-PK1 cells; 3) formation of the membrane skeleton in A431 cells, and 4) formation of the stress fibers and membrane skeleton in LC-MDCK cells. Thus, it seems that in fully confluent tightly sealed epithelium, activation of PKC has a deleterious effect on actin-based structures, whereas in cells without contacts or loose contacts, activation of PKC by PMA results in improvement of actin-based cytoskeletal structures. The main difference between the two kidney cell lines used is their selectivity to ion transport: the monolayer of LLC-PK1 cells is anion selective and MDCK cells cation selective. We propose a model where alterations in the ionic milieu within the MDCK cells by means of cation channels affect the disintegration of the membrane skeleton. The distribution of MARCKS followed the distribution of fodrin in both cell lines upon PMA-treatment, suggesting that phosphorylation of MARCKS by PKC may contribute in the regulation of the integrity of the membrane skeleton.
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PMID:Translocation of MARCKS and reorganization of the cytoskeleton by PMA correlates with the ion selectivity, the confluence, and transformation state of kidney epithelial cell lines. 1045 56

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) activate the PTH/PTHrP receptor to trigger parallel increases in adenylyl cyclase (AC) and phospholipase C (PLC). The amino (N)-terminal region of PTH-(1-34) is essential for AC activation. Ligand domains required for activation of PLC, PKC, and other effectors have been less well-defined, although some studies in rodent systems have identified a core region [hPTH-(29-32)] involved in PKC activation. To determine the critical ligand domain(s) for PLC activation, a series of truncated hPTH-(1-34) analogues were assessed using LLC-PK1 cells that stably express abundant transfected human or rat PTH/PTHrP receptors. Phospholipase C signaling and ligand-binding affinity were reduced by carboxyl (C)-terminal truncation of hPTH-(1-34) but were coordinately restored when a binding-enhancing substitution (Glu(19) --> Arg(19)) was placed within hPTH-(1-28), the shortest hPTH peptide that could fully activate both AC and PLC. Phospholipase C, but not AC, activity was reduced by substituting Gly(1) for Ser(1) in hPTH-(1-34) and was eliminated entirely by removing either residue 1 or the alpha-amino group alone. These changes did not alter binding affinity. These findings led to design of an analogue, [Gly(1),Arg(19)]hPTH-(1-28), that was markedly signal-selective, with full AC but no PLC activity. Thus, the extreme N-terminus of hPTH constitutes a critical activation domain for coupling to PLC. The C-terminal region, especially hPTH-(28-31), contributes to PLC activation through effects upon receptor binding but is not required for full PLC activation. The N-terminal determinants of AC and PLC activation in hPTH-(1-34) overlap but are not identical, as subtle modifications in this region may dissociate activation of these two effectors. The [Gly(1),Arg(19)]hPTH-(1-28) analogue, in particular, should prove useful in dissociating AC- from PLC-dependent actions of PTH.
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PMID:Amino-terminal modifications of human parathyroid hormone (PTH) selectively alter phospholipase C signaling via the type 1 PTH receptor: implications for design of signal-specific PTH ligands. 1052 Dec 52

We evaluated the role of epidermal growth factor (EGF) in the regulation of L-alanine transport in LLC-PK1 renal epithelia. After 2 h of incubation, EGF had no significant effect on L-alanine uptake by LLC-PK1 cells. However, prolonged (16 h) incubation with 2 and 20 ng/ml of EGF resulted in significant increases in sodium-dependent L-alanine uptake as compared with controls. Treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA; 20 ng/ ml) caused a marked increase in sodium-dependent L-alanine uptake after both 2 and 16 h of incubation, and the treatment with TPA (20 ng/ml) EGF (20 ng/ml) for 16 h resulted in significant acceleration of the TPA-stimulated increase in L-alanine uptake by LLC-PK1 cells. Coincubation with H-7 (20 microM) inhibited both EGF- and TPA-stimulated increases in L-alanine uptake, and genistein (20 microg/ml) blocked the stimulatory effect of EGF in L-alanine transport to the control level. Furthermore, coincubation with cycloheximide (20 microg/ml) for 16 h inhibited both EGF- and TPA-stimulated increases in L-alanine transport to a great extent. The sodium- independent L-alanine uptake was not affected by treatment with either EGF or TPA. These results suggest that the activation of protein kinase C through tyrosine kinase activation plays a role in the EGF effect of stimulating L-alanine transport in LLC-PK1 cells and that the effect is mainly due to increased protein de novo synthesis which occurs after protein kinase C activation.
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PMID:Effect of epidermal growth factor on sodium-dependent L-alanine transport in LLC-PK1 cells. 1056 48

Cell adhesion to individual macromolecules of the extracellular matrix has dramatic effects on the subcellular localization of the actin-bundling protein fascin and on the ability of cells to form stable fascin microspikes. The actin-binding activity of fascin is down-regulated by phosphorylation, and we used two differentiated cell types, C2C12 skeletal myoblasts and LLC-PK1 kidney epithelial cells, to examine the hypothesis that cell adhesion to the matrix components fibronectin, laminin-1, and thrombospondin-1 differentially regulates fascin phosphorylation. In both cell types, treatment with the PKC activator 12-tetradecanoyl phorbol 13-acetate (TPA) or adhesion to fibronectin led to a diffuse distribution of fascin after 1 h. C2C12 cells contain the PKC family members alpha, gamma, and lambda, and PKCalpha localization was altered upon cell adhesion to fibronectin. Two-dimensional isoelectric focusing/SDS-polyacrylamide gels were used to determine that fascin became phosphorylated in cells adherent to fibronectin and was inhibited by the PKC inhibitors calphostin C and chelerythrine chloride. Phosphorylation of fascin was not detected in cells adherent to thrombospondin-1 or to laminin-1. LLC-PK1 cells expressing green fluorescent protein (GFP)-fascin also displayed similar regulation of fascin phosphorylation. LLC-PK1 cells expressing GFP-fascin S39A, a nonphosphorylatable mutant, did not undergo spreading and focal contact organization on fibronectin, whereas cells expressing a GFP-fascin S39D mutant with constitutive negative charge spread more extensively than wild-type cells. In contrast, C2C12 cells coexpressing S39A fascin with endogenous fascin remained competent to form microspikes on thrombospondin-1, and cells that expressed fascin S39D attached to thrombospondin-1 but did not form microspikes. Blockade of PKCalpha activity by TPA-induced down-regulation led to actin association of wild-type fascin in fibronectin-adherent C2C12 and LLC-PK1 cells but did not alter the distribution of S39A or S39D fascins. The association of fascin with actin in fibronectin-adherent cells was also evident in the presence of an inhibitory antibody to integrin alpha5 subunit. These novel results establish matrix-initiated PKC-dependent regulation of fascin phosphorylation at serine 39 as a mechanism whereby matrix adhesion is coupled to the organization of cytoskeletal structure.
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PMID:Cell-matrix adhesions differentially regulate fascin phosphorylation. 1058 51


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