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)

Alcoholics frequently suffer from moderate to severe bone loss that results in bone fractures. Both decreased bone production and increased bone resorption have been postulated to contribute to ethanol (ETOH)-mediated bone loss. Bone resorption is induced by several proinflammatory cytokines such as interleukin-1 and -6. The expression of these cytokines is induced by the transcription factor NFkappaB, which, in turn, is activated by several kinases. It follows that protein kinase and NFkappaB activation may contribute to ETOH-induced bone loss. Accordingly, we sought to determine if ETOH activates protein tyrosine kinases (PTK) and NFkappaB DNA binding in a human osteoblast-like cell line (HOBIT). Ethanol at 50 and 100 mmol/L (reflective of blood ethanol levels reached in chronic alcoholics) for 24 h did not alter HOBIT cell viability. In contrast, 200 mmol/L ethanol decreased cell viability by 40%. Treatment of HOBIT cells with 100 mmol/L ETOH induced nuclear NFkappaB:DNA complex formation and NFkappaB activity. Incubation of HOBIT cells with ETOH at 50 and 100 mmol/L for 30 min induced a 2.5- and 4.2-fold increase in PTK activity, respectively. Preincubation of HOBIT cells with damnacanthal (DAM), which inhibits p56lck, blocked ETOH-mediated PTK activity; whereas, preincubation with herbimycin A, which inhibits pp60src, did not. DAM inhibited both ethanol-induced NFkappaB activation in HOBIT cells and interleukin-6 expression in primary human osteoblasts. Finally, preincubation with the protein kinase C inhibitor, bisindolylmaleimide I HCl (BIS), diminished ETOH-mediated PTK activity; whereas, preincubation with the protein kinase A inhibitor, H89, did not. These data demonstrate that ETOH induces NFkappaB nuclear translocation through p56lck in HOBIT cells. BIS' inhibition of PTK activation suggests that ETOH activates PTK through a protein kinase C-dependent pathway. These data suggest that ETOH may contribute to bone loss through activation of signal transduction that results in production of an osteoclastogenic cytokine (i.e., interleukin-6) in osteoblasts.
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PMID:Ethanol activates NFkappaB DNA binding and p56lck protein tyrosine kinase in human osteoblast-like cells. 1118 74

Cell membranes contain sphingolipids and cholesterol, which cluster together in distinct domains called rafts. The outer-membrane leaflet of these peculiar membrane domains contains glycosylphosphatidylinositol-anchored proteins, while the inner leaflet contains proteins implicated in signalling, such as the acylated protein kinase p56(lck) and the palmitoylated adaptator LAT (linker for activation of T-cells). We present here an approach to study the lipid composition of rafts and its change upon T-cell activation. Our method is based on metabolic labelling of Jurkat T-cells with different precursors of glycerophospholipid synthesis, including glycerol and fatty acids with different lengths and degrees of saturation as well as phospholipid polar head groups. The results obtained indicate that lipid rafts isolated by the use of sucrose density-gradient centrifugation after Triton X-100 extraction in the cold, besides sphingolipids and cholesterol, contain unambiguously all classes of glycerophospholipids: phosphatidylserine, phosphatidylinositol, phosphatidylethanolamine and phosphatidylcholine. Fatty acid labelling shows that lipid rafts are labelled preferentially with saturated fatty acids while the rest of the plasma membrane incorporates mostly long-chained polyunsaturated fatty acids. To see whether the raft composition as measured by metabolic labelling of phospholipids is involved in T-cell activation, we investigated the production of sn-1,2-diacylglycerol (DAG) in CD3-activated cells. DAG production occurs within rafts, confirming previous demonstration of protein kinase C translocation into membrane microdomains. Our data demonstrate that raft disorganization by methyl-beta-cyclodextrin impairs both CD3-induced DAG production and changes in cytosolic Ca(2+) concentration. These lines of evidence support the conclusion that the major events in T-cell activation occur within or due to lipid rafts.
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PMID:Metabolic labelling of membrane microdomains/rafts in Jurkat cells indicates the presence of glycerophospholipids implicated in signal transduction by the CD3 T-cell receptor. 1196 65

Transformation of cells by src -like kinases leads to altered cell morphology associated with the disassembly of focal contacts and concomitant increase in tyrosine phosphorylation of pp125(FAK) x p56(lck) is a lymphocyte-specific member of the src family of protein tyrosine kinases that associates with cell surface glycoproteins such as CD4 and CD8. It phosphorylates and activates pp125(FAK) and increases its autokinase activity, thus pretreatment of pp125(FAK) with protein kinase C (PKC) markedly attenuates its phosphorylation and activation, suggesting a potential regulatory pathway of pp125(FAK) activation in focal contacts. p56(lck) further phosphorylates and activates actin binding protein (ABP-280; filamin) and controls its association with cell surface receptors such as beta-2 integrins, actin filament cross-linking, and possibly lipid membrane insertion.
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PMID:p56(lck) Controls phosphorylation of filamin (ABP-280) and regulates focal adhesion kinase (pp125(FAK)). 1217 Oct 35

The human major histocompatibility complex (HLA) encodes two sets of HLA class I molecules, which have been termed class Ia (or classical) and class Ib (or nonclassical) molecules. The class Ia molecules include the gene products of HLA-A, HLA-B, and HLA-C loci and are characterized by broad tissue expression and by a high degree of polymorphism. The class Ib molecules include the gene products of HLA-E, HLA-F, and HLA-G loci and are characterized by a restricted tissue distribution and by limited polymorphism. Besides being expressed on nucleated cells, classical and nonclassical HLA class I molecules are present in serum in soluble form (sHLA-I). The serum level of sHLA-I molecules is significantly increased in a variety of physiological and pathological conditions such as pregnancy, acute rejection episodes following organ allografts, acute graft-versus-host-disease (GVHD) following bone marrow transplantation, autoimmune diseases, viral infections, and malignant melanoma. Because of the statistically significant association with clinical parameters, the level of sHLA-I antigens has been suggested to represent a useful marker to predict the evolution of viral infections and to monitor the clinical course of allografts. Moreover, elevated levels of functional sHLA-I and soluble Fas-ligand molecules have been detected by our group in blood components and might play a role in the immunomodulatory effect of autologous and allogeneic transfusions. Several lines of evidence suggest that sHLA-I molecules are immunologically functional and may play an immunoregulatory role. In fact, they have been shown to elicit antibodies in both allogeneic and xenogeneic combinations, to inhibit the activity of alloreactive cytotoxic T lymphocytes (CTL), and to induce apoptosis in alloreactive and virus-specific CTL, in activated autologous and allogeneic CD8+ T cells, and in CD8+ NK cells. There is general agreement about the mechanism underlying the inhibition of CTL activity by sHLA antigens. This inhibition appears to be mediated by interactions of sHLA-I antigens a1 and a2 domains with T cell receptor (TCR). By contrast, there is conflicting information about the mechanism underlying induction of apoptosis of activated T cells by sHLA-I antigens. Several authors reported that sHLA-I molecules induced apoptosis of alloreactive CD8+ cytotoxic T lymphocytes through interaction with their TCR. However, our own data and those other groups indicate that classical and nonclassical sHLA-I molecules trigger Fas/Fas-ligand mediated apoptosis of phytohemoagglutinin (PHA)-activated and virus-specific CD8+ T lymphocytes as well as of CD8+ NK cells by interacting with CD8 coreceptor. Recently, we performed a series of experiments in our laboratory to clarify the intracellular mechanism(s) leading to Fas-ligand upregulation and secretion. These unpublished data indicate that sHLA-I/CD8 ligation elicits the phosphorylation of p56lck protein thyrosin kinase (PTK) associated with CD8 cytoplasmic domain in the absence of any other TCR-derived signal, the activation of syk-like ZAP-70 PTK and protein kinase C, and extracellular calcium influx. Then, activation and nuclear translocation of NF-kB and NF-AT occurs, leading to Fas-ligand mRNA transcription and soluble Fas-ligand secretion, which delivers the death signal. Interestingly, soluble Fas-ligand secretion and CD8+ cell apoptosis, but not CD8+ cell cytolitic activity, are completely inhibited by Cyclosporin A, which specifically blocks the activation of the calcineurin/calmodulin pathway. Taken together, these data suggest that sHLA-I molecules are involved in a signal-transduction pathway leading to Fas-ligand expression, soluble Fas-ligand secretion, and CD8+ cells apoptosis.
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PMID:Soluble HLA class I molecules/CD8 ligation trigger apoptosis of CD8+ cells by Fas/Fas-ligand interaction. 1280 26

We have previously shown that bisperoxovanadium (bpV) phosphotyrosyl phosphatase inhibitors can potently activate NF-kappaB. We have already determined that p56(lck), ZAP-70, SLP-76, capacitative entry of calcium, and calcium-regulated effectors are important in bpV-induced NF-kappaB activation. In this study, we evaluated whether other signal transducers previously reported in NF-kappaB induction by T cell activating stimuli are also activated by bpV compounds. Nuclear translocation of NF-kappaB was evaluated in cell lines deficient for either CD45 or p36(LAT) to assess the role of these signal transducers in bpV-mediated NF-kappaB activation. A deficiency of either protein greatly reduced the extent of NF-kappaB nuclear translocation following bpV treatment. Isoform-specific PKC inhibitors were then used to show that bpV compounds activate NF-kappaB through both calcium-sensitive and -insensitive PKC isoforms. The implication of the IkappaB-kinase complex was then investigated through the use of an IkappaBalpha-specific kinase assay and plasmids expressing catalytically inactive forms of IKKalpha and IKKbeta. Upstream kinases involved in IKK complex activation such as TPL-2/COT, NIK, and IKKepsilon were also shown to play an important role in bpV-mediated NF-kappaB activation. Finally, reporter gene transcriptional assays and gel shift assays were performed to compare the kinetics of activation of NF-kappaB by bpV with those of antigenic and TNFalpha stimulation. We demonstrate, both in Jurkat cells and in primary T cells, that bpV-mediated NF-kappaB activation kinetics are comparable to those of an antigenic stimulation but occur much slower than the kinetics seen upon TNFalpha treatment.
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PMID:NF-kappaB induction by bisperoxovanadium compounds requires CD45, p36(LAT), PKC, and IKK activity and exhibits kinetics of activation comparable to those of TCR/CD28 coengagement. 1284 75

Gangliosides, sialic acid containing glycosphigolipids, are ubiquitous constituents of cell plasma membranes. Each cell type shows a peculiar ganglioside expression pattern. In human T lymphocytes monosialoganglioside GM3 represents the main ganglioside constituent of cell plasma membrane where it is concentrated in glycosphingolipid-enriched microdomains (GEM). The presence of tyrosine kinase receptors, mono- (Ras, Rap) and heterotrimeric G proteins, Src-like tyrosine kinases (lck, lyn, fyn), PKC isozymes, glycosylphosphatidylinositol (GPI)-anchored proteins and, after T cell activation, the Syk-family kinase Zap-70, prompts these portions of the plasma membrane to be considered as "glycosignaling domains." In particular, during T cell activation and/or other dynamic functions of the cell, such as apoptosis, key signaling molecules are recruited to these microdomains, where they strictly interact with GM3. The association of transducer proteins with GM3 in microdomains suggests that this ganglioside is the main marker of GEM in human lymphocytes and is a component of a cell plasma membrane multimolecular signaling complex involved in cell-cell interaction, signal transduction, and cell activation.
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PMID:Role of GM3-enriched microdomains in signal transduction regulation in T lymphocytes. 1499 37

To elucidate the molecular mechanisms underlying stimulation of rat organic cation transporter type 1 (rOCT1) by protein kinase C (PKC) activation, functional properties and regulation of rOCT1 stably expressed in HEK293 cells after site-directed mutagenesis of putative PKC phosphorylation-sites were compared with wild-type (WT) rOCT1 using microfluorometric measurements with the fluorescence organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)). Either substitutions of single (S286A, S292A, T296A, S328A, and T550A) or of all five PKC-sites (5x-PKC) with alanine suppressed PKC-induced stimulation of ASP(+) uptake, whereas regulation by p56(lck) tyrosine kinase was conserved in all mutants. Remarkably, the apparent affinities for TEA(+), TPA(+), and quinine were changed differently in each mutant (EC(50) in WT, S286A, S292A, T296A, S328A, T550A, and 5x-PKC in mumol: TEA(+): 105, 153, 56, 1135, 484, 498, 518; TPA(+): 0.1, 2.1, 0.3, 1.0, 43, 0.3, 2.2; quinine: 1.5, 3.0, 2.5, 4.8, 81, 7.6, 8.9, respectively). After mutations, no effects of PKC activation on apparent affinity of rOCT1 for these substrates could be detected, in contrast to what was observed in WT. PKC activation had no significant effect on rOCT1 trafficking from intracellular pools to the cell membrane. Substitution of all PKC sites suppressed PKC-induced phosphorylation of rOCT1. In conclusion, it was found that the presence of all five potential PKC phosphorylation sites is necessary for the PKC-induced stimulation of rOCT1. The different effects on the EC(50) values by the different mutations suggest that the large intracellular loop participates in building the substrate binding pocket of rOCT1 or specifically modulates its structure.
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PMID:Individual PKC-phosphorylation sites in organic cation transporter 1 determine substrate selectivity and transport regulation. 1585 19

Glucocorticoids (GCs) are effective immunosuppressive agents and mediate well-defined transcriptional effects via GC receptors. There is increasing evidence that GCs also initiate rapid nongenomic signaling events. Using activated human CD4(+) lymphocytes and a peptide array containing 1176 different kinase consensus substrates, we generated a comprehensive profile of GC-induced rapid effects on signal transduction. The results show marked early differences in phosphorylation between GC-pretreated cells and control cells, including impaired phosphorylation of p56lck/p59fyn (Lck/Fyn) consensus substrates. Immunoprecipitation and in vitro kinase assays reveal rapid GC-induced down-modulation of Lck and Fyn kinases using SAM68 (Src [pp60c-src]-associated in mitosis 68 kDa) as a substrate. Additionally, immunoprecipitation experiments revealed reduced Lck-CD4 and Fyn-CD3 associations, suggesting GC inhibited recruitment of these kinases to the T-cell receptor complex. Western blot analysis revealed reduced phosphorylation of a series of downstream signaling intermediates following GC treatment, including protein kinase B (PKB), protein kinase C (PKC), and mitogen-activated protein kinases (MAPKs). Experiments with GC receptor-negative Jurkat cells and a pharmacologic GC receptor ligand (RU486) indicated that rapid inhibition of Lck and Fyn kinases is GC receptor dependent. Parallel experiments conducted following the application of GCs in healthy individuals confirmed suppression of Lck/Fyn in T cells within 1 hour in vivo. These results identify the inhibition of Lck and Fyn kinases as rapid targets of GCs, mediated via a GC receptor-dependent pathway.
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PMID:Rapid immunosuppressive effects of glucocorticoids mediated through Lck and Fyn. 1589 16

The binding of soluble HLA class I (sHLA-I) molecules to CD8 on EBV-specific CTL induced up-regulation of Fas ligand (FasL) mRNA and consequent sFasL protein secretion. This, in turn, triggered CTL apoptosis by FasL/Fas interaction. Molecular analysis of the biochemical pathways responsible for FasL up-regulation showed that sHLA-I/CD8 interaction firstly induced the recruitment of src-like p56(lck) and syk-like Zap-70 protein tyrosine kinases (PTK). Interestingly, p59(fyn) was activated upon the engagement of CD3/TCR complex but not upon the interaction of sHLA-I with CD8. In addition, sHLA-I/CD8 interaction, which is different from signaling through the CD3/TCR complex, did not induce nuclear translocation of AP-1 protein complex. These findings suggest that CD8- and CD3/TCR-mediated activating stimuli can recruit different PTK and transcription factors. Indeed, the engagement of CD8 by sHLA-I led to the activation of Ca2+ calmodulin kinase II pathway, which eventually was responsible for the NF-AT nuclear translocation. In addition, we found that the ligation of sHLA-I to CD8 recruited protein kinase C, leading to NF-kappaB activation. Both NF-AT and NF-kappaB were responsible for the induction of FasL mRNA and consequent CTL apoptosis. Moreover, FasL up-regulation and CTL apoptotic death were down-regulated by pharmacological specific inhibitors of Ca2+/calmodulin/calcineurin and Ca2+-independent protein kinase C signaling pathways. These findings clarify the intracellular signaling pathways triggering FasL up-regulation and apoptosis in CTL upon sHLA-I/CD8 ligation and suggest that sHLA-I molecules can be proposed as therapeutic tools to modulate immune responses.
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PMID:Apoptosis of antigen-specific T lymphocytes upon the engagement of CD8 by soluble HLA class I molecules is Fas ligand/Fas mediated: evidence for the involvement of p56lck, calcium calmodulin kinase II, and Calcium-independent protein kinase C signaling pathways and for NF-kappaB and NF-AT nuclear translocation. 1630 29

B cell activating transcription factor (BATF) belongs to the activator protein-1 (AP-1) superfamily of basic leucine zipper transcription factors and forms heterodimers with Jun that possess minimal transcriptional activity. Mice carrying a p56(lck)HA-BATF transgene were created to observe the effects of constitutive expression of this well-characterized AP-1 inhibitor on T cell proliferation. Consistent with the role of AP-1 in promoting the proliferation of many cell types, BATF-transgenic thymocytes proliferate poorly in vitro when stimulated with anti-CD3epsilon and anti-CD28 antibodies or with Concanavalin A. However, when BATF-transgenic thymocytes were stimulated using a standard treatment of PMA and ionomycin, proliferation is normal. The responsiveness to PMA and ionomycin can be attributed to the dramatic disappearance of the hemagglutinin antigen (HA)-tagged BATF protein which is a PKC-dependent process caused by the down-regulation of the p56(lck) proximal promoter coupled with the rapid turnover of the HA-BATF protein. These studies describe conditions of T cell stimulation that negatively influence transcription of the widely used p56(lck) proximal promoter expression cassette. In addition, the unique circumstances of this regulation were exploited to demonstrate that inhibition of AP-1 activity by BATF exerts a direct, and reversible, effect on T cell proliferation in vitro.
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PMID:Direct manipulation of activator protein-1 controls thymocyte proliferation in vitro. 1638 Sep 65


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