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)

A new member of the connexin gene family has been identified and designated rat connexin-31 (Cx31) based on its predicted molecular mass of 30,960 daltons. Cx31 is 270 amino acids long and is coded for by a single copy gene. It is expressed as a 1.7-kilobase mRNA that is detected in placenta, Harderian gland, skin, and eye. Cx31 is highly conserved and can be detected in species as distantly related to rat as Xenopus laevis. It exhibits extensive sequence similarity to the previously identified connexins, 58, 50, and 40% amino acid identity to Cx26, Cx32, and Cx43, respectively. When conservation of predicted phosphorylation sites is used to adjust the alignment of Cx31 to other connexins, a unique alignment of three predicted protein kinase C phosphorylation sites near the carboxyl terminus of Cx31 with three sites at the carboxyl terminus of Cx43 is revealed.
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PMID:Molecular cloning and characterization of a new member of the gap junction gene family, connexin-31. 170 19

A reduction of gap-junctional intercellular communication (GJIC) often accompanies neoplastic transformation. The present work demonstrates that transformation by the oncogenic human DNA virus, human papilloma virus 16(HPV16), also reduces GJIC between L6 rat myoblasts. HPVs are associated with anogenital cancers, the incidence of which is increasing in HIV positive patients of both sexes. Using videofluorescence imaging of Fura-2 loaded cells a lack of GJIC between transformed HPV16-L6 cells was first indicated by uncoordinated brief [Ca2+]i spikes in clusters of DMSO-treated HPV16-L6 cells instead of the synchronous, sustained [Ca2+]i surges in clusters of DMSO-treated L6 cells. Reduced GJIC between HPV16-L6 cells was demonstrated directly by a much reduced transfer of lucifer yellow dye from HPV16-L6 cells, which had been loaded with the dye through electroporation with an EPIZAP II in situ electroporator, to neighbouring nonelectroporated HPV16-L6 cells. One reason for this reduced GJIC between HPV16-L6 cells could have been their dramatically enhanced activity of membrane-associated PKC which is known to phosphorylate connexins and down-regulate gap junctions. However, the main reason was the viral-induced inhibition of the expression of a major gap junction component, Cx43 (Connexin 43), in the transformed myoblasts.
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PMID:Alterations in cell-cell communication in human papillomavirus type 16 (HPV16) transformed rat myoblasts. 754 85

We have examined the effect of protein kinase (PKC) depletion in SV40-transformed Djungarian hamster fibroblasts (DM15 cells) on the level of gap junction permeability. Cx43 electrophoretic mobility, and cell sensitivity to different uncoupling stimuli. After 24 hr exposure to 12-O-tetradecanoyl-phorbol-13-acetate (TPA), the total PKC activity in DM15 cells was reduced to 20-25% in comparison with intact cells. In PKC-depleted cells the level of dye coupling was 30-40% higher than in the same untreated cultures. Western blot analysis revealed multiple forms of the gap junction protein connexin 43, which correspond to known phosphorylated and dephosphorylated forms of this protein. No decrease in the level of connexin 43 phosphorylation after PKC depletion was observed. TPA (10(-7) g/ml), mezerein (10(-7) g/ml), teleocidin (10(-8) g/ml), Ca-ionophore A23187 (10(-6) g/ml), insecticide 1,1,1-trichloro-2,2-bis-(p-chlorphenyl)-ethane (DDT) (10(-4) g/ml), and nigericin (10(-5) M in hydrolysate lactalbumin solution, pH 6.3) induced a four-to six-fold decrease in the number of recipient cells in the dye-coupling assay. PKC-depleted cells became almost completely resistant to the uncoupling effect of mezerein, teleocidin, and A23187, as well as to new exposure to TPA, and became partially resistant to the effect of DDT. Nigericin inhibited intercellular communication between PKC-depleted cells to the same extent as between control cells. Thus, in the cell system studied, PKC plays a certain role in maintaining the basal level of gap junction permeability and has an important significance as a mediator of the uncoupling effects of such substances as TPA, mezerein, teleocidin, and Ca2+.
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PMID:Role of protein kinase C in the regulation of gap junctional communication. 770 63

The effect of 12-O-tetradeconylphorbol-13-acetate (TPA) on gap junction assembly between Novikoff hepatoma cells was examined. Cells were dissociated with EDTA to single cells and then reaggregated to form new junctions. When TPA (25 nM) was added to the cells at the onset of the 60-min reaggregation, dye transfer was detected at only 0.6% of the cell-cell interfaces compared to 72% for the untreated control and 74% for 4-alpha TPA, an inactive isomer of TPA. Freeze-fracture electron microscopy of reaggregated control cells showed interfaces containing an average of more than 600 aggregated intramembranous gap junction particles, while TPA-treated cells had no gap junctions. However, Lucifer yellow dye transfer between nondissociated cells via gap junctions was unaffected by 60 min of TPA treatment. Therefore, TPA dramatically inhibited gap junction assembly but did not alter channel gating nor enhance disassembly of preexisting gap junction structures. Short term TPA treatment (< 30 min) increased phosphorylation of the gap junction protein molecular weight of 43,000 (Cx43), but did not change the cellular level of Cx43. Cell surface biotinylation experiments suggested that TPA did not substantially reduce the plasma membrane concentration of Cx43. Therefore, the simple presence of Cx43 in the plasma membrane is not sufficient for gap junction assembly, and protein kinase C probably exerts an effect on assembly of gap junctions at the plasma membrane level.
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PMID:Analyzing phorbol ester effects on gap junctional communication: a dramatic inhibition of assembly. 780 68

The aim of this study was to evaluate the rapid regulation of cell-cell communication by using the microinjection of purified cAMP-dependent protein kinase (protein kinase A), the Ca(2+)-phospholipid-dependent protein kinase (protein kinase C), or the inhibitor proteins (PKI and CKI) that are, respectively, specific for each of these enzymes. Gap junction phenotypes of myometrial tissue and cells were studied by means of immunocytochemistry with antibody to connexin 43 (alpha 1; Cx43). Cells were enzymatically disaggregated from myometrium of nonpregnant, mid-pregnant (Day 14), and late-pregnant (Day 29) rabbit uteri (n = 8 per group) and seeded at high density such that after 4 days, cultures had the appearance of a cross-sectioned myometrium. Purified proteins and their subunits were microinjected, and intercellular communication was evaluated by monitoring Lucifer Yellow dye transfer. Cultures were treated with 0.5 mM 8Br-cAMP (8-bromo adenosine 3',5' cyclic monophosphate) or 10 microM OAG (1-oleoyl-2-acetyl-sn-glycerol), which, respectively, activate protein kinase A and protein kinase C. Immunoreactive Cx43 and cell-cell communication were examined 5 min to 2 h later. Cx43 was detected in myometrial cryosections and cultured cells by indirect immunofluorescence, and its expression increased with gestation. Exposure to 8Br-cAMP increased the amount of immunoreactive Cx43. Basal dye transfer was minimal in nonpregnant cells, increased in cells of mid-pregnant uteri, and was maximal in late-pregnant cells. Treatment with 8Br-cAMP enhanced transfer in mid- and late-pregnant cells but had no obvious effect on cells from nonpregnant animals. OAG treatment inhibited dye transfer in greater than 95% of the cells tested irrespective of pregnancy status. PKI inhibited cell-cell communication within 2 min and up to 40 min. Injection of free catalytic subunit of protein kinase A following PKI inhibition restored communication within 2-3 min, with maximal transfer in 4-5 min. Protein kinase C inhibited communication, which resumed in < 3 min after injection of CKI. We conclude that rabbit myometrial cells engage in Cx43-mediated cell-cell communication and that this process increases during pregnancy. Further, activators of protein kinase A or injected free catalytic subunit rapidly enhances cell-cell communication, whereas activators of protein kinase C or the enzyme itself diminishes this process.
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PMID:Regulation of cell-cell communication mediated by connexin 43 in rabbit myometrial cells. 814 55

Studies on physiological modulation of intercellular communication mediated by protein kinases are often complicated by the fact that cells express multiple gap junction proteins (connexins; Cx). Changes in cell coupling can be masked by simultaneous opposite regulation of the gap junction channel types expressed. We have examined the effects of activators and inhibitors of protein kinase A (PKA), PKC, and PKG on permeability and single channel conductance of gap junction channels composed of Cx45, Cx43, or Cx26 subunits. To allow direct comparison between these Cx, SKHep1 cells, which endogenously express Cx45, were stably transfected with cDNAs coding for Cx43 or Cx26. Under control conditions, the distinct types of gap junction channels could be distinguished on the basis of their permeability and single channel properties. Under various phosphorylating conditions, these channels behaved differently. Whereas agonists/antagonist of PKA did not affect permeability and conductance of all gap junction channels, variable changes were observed under PKC stimulation. Cx45 channels exhibited an additional conductance state, the detection of the smaller conductance states of Cx43 channels was favored, and Cx26 channels were less often observed. In contrast to the other kinases, agonists/antagonist of PKG affected permeability and conductance of Cx43 gap junction channels only. Taken together, these results show that distinct types of gap junction channels are differentially regulated by similar phosphorylating conditions. This differential regulation may be of physiological importance during modulation of cell-to-cell communication of more complex cell systems.
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PMID:Differential regulation of distinct types of gap junction channels by similar phosphorylating conditions. 859 Aug

Short term (15 min) effects of activators of protein kinase A (PKA), PKC and PKG on cardiac macroscopic (g(j)) and single channel (gamma j) gap junctional conductances were studied in pairs of neonatal rat cardiomyocytes. Under dual whole-cell voltage-clamp, PKC activation by 100 nM TPA increased g(j) by 16 +/- 2% (mean +/- S.E.M, n = 9), 1.5 mM of the PKG activator 8-bromo-cGMP (8Br-cGMP) decreased g(j) by 26 +/- 2% (n = 4), whereas 1.5 mM of the PKA activator 8Br-cAMP did not affect g(j) (1 +/- 5%, n = 11). Single cardiac gap junction channel events, resolved in the presence of heptanol, indicated two gamma j sizes of 20 pS and 40-45 pS. Under control conditions, the larger events were most frequently observed. Whereas 8Br-cAMP did not change this distribution, TPA or 8Br-cGMP shifted the gamma j distribution to the lower sizes. Diffusion of 6-carboxyfluorescein (6-CF), a gap junction permeant tracer, from the injected cell to neighboring cells was studied on small clusters of neonatal rat cardiomyocytes. Under control conditions, 6-CF labeled 8.4 +/- 0.4 cells (mean +/- S.E.M, n = 31). Whereas 8Br-cAMP did not change the extent of dye transfer (8.1 +/- 0.5 cells, n = 10), TPA restricted the diffusion of 6-CF to 2.2 +/- 0.2 cells (n = 30) and 8Br-cGMP to 3.5 +/- 0.3 cells (n = 10). This suggests that permeability and single channel conductance of Cx43 gap junction channels are parallel related. Altogether, these results point to the differential modulation of electrical and metabolic coupling of cardiac cells by various phosphorylating conditions.
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PMID:Regulation of cardiac gap junction channel permeability and conductance by several phosphorylating conditions. 873 33

In this study, we investigated whether the tumor promoters, 12-O-tetradecanoylphorbol-13-acetate (TPA), phenobarbital (PB), and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (DDT), inhibited gap junctional intercellular communication (GJIC) in a cell-specific or connexin-specific manner and whether protein kinase C was involved. To do this, we used highly communicating WB-F344 rat liver epithelial cells, which express connexin43 as their predominant gap junction protein, WB-aB1 cells, which are a GJIC-incompetent mutant line of WB-F344 cells and that express connexin43, WB-a/32-10 cells, which are a highly communicating derivative of WB-aB1 cells generated by stable transduction with a connexin32 retroviral expression vector, and primary cultured rat hepatocytes, which express conexin32 predominantly. Treatment of WB-F344 and WB-a/32-10 cells, but not hepatocytes, with TPA inhibited GJIC (assayed by Lucifer Yellow dye microinjection). This inhibition involved protein kinase C because (i) inhibition was prevented by co-treatment of the cells with a specific protein kinase C inhibitor, bis-indolylmaleimide, and (ii) treatment with TPA for 24 h had no effect on dye-coupling in agreement with the downregulation of protein kinase C. TPA also caused the internalization of Cx43-containing gap junctions and the formation of a hyperphosphorylated form of Cx43, Cx43-P3, in WB-F344 cells only, but TPA had no effect on Cx32-containing gap junctions or protein mobility. In contrast, PB inhibited GJIC only in hepatocytes and DDT inhibited GJIC in all three types of cells; bis-indolylmaleimide did not block the effects of either agent. These results indicate that the inhibitory actions of TPA and PB on GJIC are cell-specific rather than connexin-specific and that TPA inhibits connexin43 and connexin32-mediated GJIC through a protein kinase C-dependent mechanism.
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PMID:Inhibition of gap junctional intercellular communication by tumor promoters in connexin43 and connexin32-expressing liver cells: cell specificity and role of protein kinase C. 947 9

Although there is general agreement that gap junction channels formed by the connexin43 (Cx43; alpha 1) protein most likely have important roles during heart development, evidence to support this view has been equivocal. Lacking this information, it is difficult to understand the basis of heart malformations found in the Cx43 knockout mice and in children with a severe form of visceroatrial heterotaxia that coincides with missense mutations of the Cx43 gene. To address this issue we used a combination of western blots to follow the emergence of Cx43 in heart, and in vitro and in vivo phosphorylation to assess the effect of mutation on Cx43 phosphorylation. We evaluated the activity ratios of cAMP-dependent protein kinase and protein kinase C in hearts of 8.5-day-old mouse embryos through to birth. The results demonstrate that Cx43 is present in the native phosphorylated species in day 8.5 hearts and thereafter. Further, the activities of cAMP-dependent protein kinase and protein kinase C are mirror images of each other during the 8.5-10.5 days of early heart development. From these results we conclude that Cx43 gap junction channels are present and capable of being regulated by day 8.5 of embryonic heart development.
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PMID:Misregulation of connexin43 gap junction channels and congenital heart defects. 1020 6

Calcium is one of the most universal signal-transduction elements in a large variety of cells ranging from bacteria to specialized neurons. Ca2+ acts as a second messenger controlling such processes as secretion, cell differentiation or signal transmission. In order to be able to execute their specific functions and to react in a coordinated way to stimuli, multicellular organs need a precise orchestration of cellular functions. For this purpose cells have developed different forms of intercellular communication (IC). In this study we investigated a number of mechanisms of intracellular propagation and IC using experiments with fluorescent Ca(2+)-indicators, confocal microscopy and digital imaging techniques. In ROS 17/2.8 osteoblasts, retinal pigment epithelial cells (RPE) and CPAE endothelial cells, a small mechanical deformation of the plasma membrane results in a transient increase of free cytoplasmic Ca2+ concentration ([Ca2+]i). This Ca(2+)-rise starts at the site of stimulation and propagates concentrically to neighboring cell layers. The intracellular Ca(2+)-wave in RPE and ROS cells is caused by Ca(2+)-influx followed by Ca(2+)-release from the intracellular stores and by intercellular propagation of the Ca(2+)-wave. The [Ca2+]i-transient upon mechanical stimulation of LLC-PK1 epithelial cells, C6 glioma cells and MLO-Y4 osteocytes was limited and/or variable. In CPAE cells only the intracellular release is important for evoking the Ca(2+)-transient, and is followed by IC. IC can occur via gap junctions (GJ) consisting of membrane-spanning proteins, connexins (Cx). It was demonstrated that IC and GJ in RPE and ROS cells can be reversibly blocked by gap-junction inhibitors such as heptanol or halothane. We demonstrated important differences in modulation of gap junctional communication between these cell types. While in RPE cells stimulation of PKC activity was able to inhibit IC, this was not the case in ROS cells. We screened LE-RPE cDNA via PCR using specific primers for different connexins and found no effect of high glucose solutions, which cause decreased intercellular communication, on the Cx-isoforms expressed. Cx43 is the only Cx-isoform present at the protein level for which Western blot analysis revealed the presence of different forms corresponding to different phosphorylated states. Increased phosphorylation of Cx43 was only seen after direct PKC activation by PMA, but not by indirect PKC activation by high glucose levels. The decreased communication by high glucose concentrations was however associated by a decreased expression of cellular Cx43 to about 3/4 of the level in control conditions. High glucose concentrations therefore decrease Cx43 at the protein level via a PKC effect that appears to be independent of the direct activation of PKC by phorbolesters. Mechanical stimulation did not evoke intercellular Ca(2+)-waves in LLC-PK1 epithelial cells, C6 glioma cells and MLO-Y4 osteocytes. In CPAE-endothelial cells, the contribution of gap junctions to IC following mechanical stimulation is negligible, and modulation of gap junctions via phosphorylation or high glucose solutions is absent. Perfusion experiments and pharmacological studies demonstrated that IC following mechanical stimulation of these cells occurs via release of an extracellular mediator. Our experiments provide strong evidence in favor of purinergic agonists as mediators, such as ATP but mainly ADP. In conclusion we can say that cells contain a wide spectrum of mechanisms for intra- and intercellular communication, and that widely different mechanisms can evoke the same phenomenon of intra- and intercellular Ca(2+)-waves.
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PMID:[Intra- and intercellular Ca(2+)-signal transduction]. 1119 79


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