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)

Thrombin stimulation of the T leukemic cell line Jurkat induced a transient increase in [Ca2+]i. Proteolytic activity of the enzyme was required for this effect since diisopropyl fluorophosphate-thrombin failed to increase [Ca2+]i. Furthermore, hirudin and anti-thrombin III inhibited the thrombin-induced [Ca2+]i rise in Jurkat T cells. A synthetic thrombin receptor agonist peptide (TRP) of 7 residues (SFLLRNP) was found to be as effective as thrombin for [Ca2+]i mobilization, and both agonists induced Ca2+ release exclusively from internal stores. Thrombin stimulated tyrosine phosphorylation of several proteins of molecular mass 40, 42, 70, 120, and 130 kDa. There was a good correlation between thrombin-induced tyrosine phosphorylation of the latter three proteins and Ca2+ mobilization. Thrombin and TRP also caused translocation of protein kinase C from the cytosol to the plasma membrane. As a likely consequence of these events, thrombin activated the nuclear factor NF-kB. Several cell lines of hematopoietic origin including the leukemic T cell line HPB.ALL and the erythroleukemic cell line K562 were responsive to thrombin, whereas others such as THP1, a myelomonocytic cell line, and BL2, a Burkitt lymphoma were refractory to thrombin or TRP stimulation. The magnitude of the thrombin response in the different cell types paralleled the expression of the thrombin receptor mRNA. We found that activation of Jurkat T cells by a combination of phytohemagglutinin and phorbol 12-myristate 13-acetate led to a dramatic inhibition of thrombin receptor mRNA expression and to a concomitant loss of the thrombin response. Finally, we demonstrate that thrombin and TRP enhanced CD69 expression and interleukin 2 production induced by T cell receptor cross-linking in both Jurkat T cells and peripheral blood lymphocytes. These findings highlight the role of thrombin as a potential regulator of T lymphocyte activation.
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PMID:Thrombin and thrombin receptor agonist peptide induce early events of T cell activation and synergize with TCR cross-linking for CD69 expression and interleukin 2 production. 751 Jun 89

The regulation by protein kinase C (PKC) of TCR-mediated changes in phosphoinositide metabolism and intracellular calcium ([Ca2+]i) was investigated in HPB-ALL T cells. Low concentrations (< 1 microgram/ml) of the anti-CD3 OKT3 mAb triggered large calcium signals but not detectable increase in D-myo-inositol 1,4,5-trisophate (IP3) production. CD3-CD4 coligation amplified the calcium signal twofold, compared with CD3 cross-linking alone, but this protocol also did not stimulate IP3 production. At higher OKT3 concentrations (> 2.5 micrograms/ml), IP3 production was detected but was not inhibited by activating PKC with phorbol ester. In contrast, PKC activation caused a marked inhibition (53 to 64%) of the CD3- or CD3-CD4-triggered calcium signals, but had only a small inhibitory effect (20 to 30%) on the release of intracellular Ca2+. PKC activation also inhibited by 47% calcium signals triggered by thapsigargin, an inhibition that was completely reversed by addition of the specific PKC inhibitor RO 31-8220 (1 microM). Addition of 1 microM RO 31-8220 caused a twofold stimulation of CD3-induced calcium signals. This effect was not mediated at the level of Ca2+ influx, because RO 31-8220 did not significantly increase thapsigargin-triggered calcium signals. However, RO 31-8220 did slightly increase the CD3-induced release of intracellular Ca2+, suggesting that amplification of Ca2+ influx may be secondary to increased release of Ca2+ from intracellular stores. Our results indicate that PKC regulates TCR-mediated changes in [Ca2+]i in HPB-ALL T cells by two distinct mechanisms. First, PKC activation causes a marked inhibition of Ca2+ influx by a mechanism independent of changes in IP3 production, possibly involving inhibition of ion channels. Second, PKC activity causes a small inhibition of intracellular Ca2+ release, most likely by promoting Ca2+ sequestration.
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PMID:Protein kinase C activation inhibits TCR-mediated calcium influx but not inositol trisphosphate production in HPB-ALL T cells. 782 90

Video microscopy and digital imaging were used to quantitatively analyze lymphocyte adhesion and formation of pseudopodia on the extracellular matrix protein fibronectin (FN). A morphology kinetics assay comparing pseudopodial extension values over a 24-h period showed that HPB-ALL T leukemic cells undergo a wave of morphologic change, returning to a round shape after 8 h. Using anti-alpha 4 and anti-alpha 5 mAbs and a panel of cell types that are single or double positive for expression of the alpha 4/beta 1 and alpha 5/beta 1 FN binding integrins, it was determined that cell adhesion to FN was influenced by both beta 1-integrins, whereas alpha 4/beta 1 was found to be the major FN receptor mediating pseudopodia extension. The protein kinase inhibitor staurosporine, the protein kinase C inhibitors calphostin C and chelerythrine, and the protein tyrosine kinase inhibitor herbimycin A blocked pseudopodial extension in HPB-ALL cells. In contrast, two cAMP-dependent protein kinase inhibitors H8 and H89 did not inhibit. Inhibitors of phospholipase A2, lipoxygenases, and cyclooxygenases could block formation of pseudopodia, yet had little or no effect on cell adhesion to FN. The preincubation of cells with arachidonic acid could prevent the inhibition mediated by the reversible phospholipase A2 inhibitor cibacron blue. We conclude that the formation of lymphocyte pseudopodia in response to FN can utilize the adhesive and signaling activities of the alpha 4/beta 1-integrin and the enzymatic activities of protein kinases and phospholipases.
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PMID:Regulation of lymphocyte pseudopodia formation by triggering the integrin alpha 4/beta 1. 786 87

The immunosuppressive synthetic methylated polycyclic aromatic hydrocarbon (PAH), 7,12-dimethylbenz[a]anthracene (DMBA), has been shown to cause both an immediate and a sustained elevation of free intracellular calcium (Ca2+) in human T cells. In the present studies, a series of anthracene- and pyrene-based PAHs were tested for rapid (3 min) and sustained (4 hr) Ca2+ mobilization in the HPB-ALL human T cell line measured by flow cytometry using Fluo-3 as a Ca2+ indicator. Immunosuppressive PAHs produced a sustained Ca2+ elevation for at least 4 hr, while weakly immunosuppressive PAHs caused only a transient increase in Ca2+. The immunosuppressive PAHs, DMBA, benzo[a]pyrene, dibenz[a,h]anthracene, and 9,10-dimethylanthracene, produced a sustained increase in intracellular Ca2+ in HPB-ALL cells. Those PAHs with moderate to minimal immunosuppressive properties (i.e., dibenz[a,c]anthracene, benz[a]anthracene, benzo[e]pyrene, and anthracene) produced small and transient Ca2+ mobilization responses in HPB-ALL cells. It appeared that methylation of anthracene at the 9,10-positions increased the duration of Ca2+ mobilization, whereas the addition of a benzene group in the "a" position was associated with a transient increase in Ca2+ levels. Genistein, a protein tyrosine kinase (PTK) inhibitor, partially inhibited the rapid and sustained PAH-induced Ca2+ mobilization responses, while the protein kinase C (PKC) inhibitors, staurosporine and calphostin C, had essentially no effect on PAH-induced Ca2+ elevation. It appears that the action of PAHs on PTKs is important in the rapid Ca2+ response of human T cells. However, additional biochemical mechanisms appear to be responsible for the sustained elevation of Ca2+ produced by PAHs in T cells. The results of these studies demonstrate that persistent elevation of intracellular Ca2+ by PAHs correlates with their known immunosuppressive properties.
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PMID:Persistence of calcium elevation in the HPB-ALL human T cell line correlates with immunosuppressive properties of polycyclic aromatic hydrocarbons. 804 70

In HPB-ALL T-cells the p59fyn tyrosine kinase is regulated by the CD45 phosphotyrosine phosphatase and plays a critical role in coupling the T cell receptor (TCR) to the generation of intracellular signals which include diacylglycerol (DAG) production and protein kinase C activation. The aim of this study was to determine the phospholipid pools from which the DAG is generated and to identify which phospholipase activities are regulated by the TCR. When CD45+ cells were pre-labeled with [3H]arachidonic acid, CD3-antigen cross-linking stimulated negligible increases in both [3H]DAG and [3H]phosphatidic acid (PA). However, CD3 monoclonal antibody (mAb) induced an increase of 300% in [3H]PA when the cells were permeabilized with streptolysin-O, and this correlated with increased levels of protein tyrosine phosphorylation. Stimulation of [3H]PA production upon CD3 cross-linking was 77% lower in permeabilized CD45- cells than in CD45+ cells, consistent with the reduced activity of p59fyn in CD45- cells. The stimulated production of PA was not mediated by activation of phospholipase D (PLD), although the presence of a G-protein-regulated PLD activity was established. The CD3-induced increase in total inositol phosphates (InsP) in permeabilized cells was similar to the stimulated production of [3H]PA production in both CD45+ and CD45- cells. Dose-response curves for InsP and PA production triggered by CD3 mAb were super-imposable and the production of InsP and PA over a range of Ca2+ concentrations was comparable. Differential labeling of phospholipids with 3H-labeled fatty acids revealed that CD3-induced PA production reflected incorporation of label into the phosphatidylinositol pool. Our data suggest that in HPB-ALL cells the production of DAG following CD3-antigen cross-linking can be fully accounted for by the selective coupling of the TCR to breakdown of phosphatidylinositol-(4,5)-bisphosphate as the result of phospholipase C gamma 1 activation. This event correlates with the activity of the CD45-regulated TCR-associated tyrosine kinase, p59fyn.
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PMID:Selective coupling of the T cell antigen receptor to phosphoinositide-derived diacylglycerol production in HPB-ALL T cells correlates with CD45-regulated p59fyn activity. 822 75

Activation of protein kinase C results in phosphorylation of a 19-kDa protein termed 19K. Isolation and sequence analysis of a cDNA encoding the 19K protein revealed that this protein has been studied in other systems under different names. The name oncoprotein 18 (Op18) has been proposed on the basis of a postulated up-regulation in neoplastic cells. In the present report we adopt the designation Op18 for the 19K protein, and quantify this phosphoprotein in a series of leukemia/lymphoma cell lines, a panel of non-transformed cells and some terminally differentiated cell types. For this purpose we have developed reagents allowing quantitative Western-blot analysis, and quantification of Op18 on the single cell level by flow cytometric analysis. The data demonstrates a pronounced up-regulation of the Op18 protein in most leukemia/lymphoma cell lines. The HPB-ALL cell line provided the most extreme case and expressed 7 x 10(6) Op18 molecules/cell, which compares with 0.65 x 10(6) Op18 molecules/cell in non-transformed lymphoblastoid cells. The expression of Op18 appears to be restricted to cell types with proliferative potential, but it is clear from our results that up-regulation of Op18 is uncoupled from cellular proliferation. Moreover, by employing an Epstein-Barr virus based shuttle vector, we expressed Op18 cDNA in lymphoblastoid cells. This resulted in a three to fourfold up-regulation of Op18 that did not have any detectable consequences for cell-surface phenotype or cell size. However, increased expression of Op18 resulted in a partial inhibition of cell proliferation. Taken altogether, the results suggest that up-regulation Op18 levels in leukemia/lymphoma cells are strongly associated with, but not a direct cause of tumour progression.
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PMID:Quantitative analysis of the expression and regulation of an activation-regulated phosphoprotein (oncoprotein 18) in normal and neoplastic cells. 846 35

In order to investigate the functional similarities of the high affinity receptor for IgE (Fc epsilon RI) and the T cell receptor for antigen, we have developed a high efficiency polyethylene glycol-mediated fusion method to make somatic hybrids between cells from a mast cell line (RBL-2H3) and cells from T lymphoma cell lines (Jurkat and HPB-ALL). Using flow cytometry to select for the heterologously fused cells, we demonstrated that aggregation of the T cell receptor results in the efficient secretion of [3H]5-hydroxytryptamine from RBL cell-derived granules. In addition, both receptors mediate Ca2+ mobilization in the hybrid cells that is insensitive to inhibition by the protein kinase C activator phorbol-12-myristoyl-13-acetate (PMA). In contrast, Ca2+ mobilization caused by aggregation of Fc epsilon RI in the parent RBL cells is completely inhibited by PMA. The results indicate that these two different receptors for foreign antigen can substitute for each other to trigger responses in the hybrid cells that are unique to each cell type. The methodology employed has general utility for studying signal transduction mediated by mammalian cell surface receptors.
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PMID:Fc epsilon RI and the T cell receptor for antigen activate similar signalling pathways in T cell-RBL cell hybrids. 849 25

The involvement of the early signaling messengers, inositol tris-phosphate (IP3), intracellular calcium, [Ca2+]i, and protein kinase C (PKC), in angiotensin II (AII)-induced fluid phase endocytosis was investigated in human brain capillary and microvascular endothelial cells (HCEC). ALL (0.01-10 microM) stimulated the uptake of Lucifer yellow CH, an inert dye used as a marker for fluid phase endocytosis, in HCEC by 50-230%. AII also triggered a fast accumulation of IP3 and a rapid increase in [Ca2+]i in cells loaded with the Ca(2+)-responsive fluorescent dye fura-2. The prompt AII-induced [Ca2+]i spike was not affected by incubating HCEC in Ca(2+)-free medium containing 2 mM EGTA or by pretreating the cultures with the Ca2+ channel blockers, methoxyverapamil (D600; 50 microM), nickel (1 mM), or lanthanum (1 mM), suggesting that the activation of AII receptors on HCEC triggers the release of Ca2+ from intracellular stores. The AII-triggered increases in IP3, [Ca2+]i, and Lucifer yellow uptake were inhibited by the nonselective AII receptor antagonist, Sar1, Val5, Ala8-AII (SVA-AII), and by the phospholipase C (PLC) inhibitors, neomycin and U-73122. By contrast, the protein kinase C (PKC) inhibitors, staurosporine and calphostin C, failed to affect any of these AII-induced events. This study demonstrates that increased fluid phase endocytotosis induced by AII in human brain capillary endothelium, an event thought to be linked to the observed increases in blood-brain barrier permeability in acute hypertension, is likely dependent on PLC-mediated changes in [Ca2+]i and independent of PKC.
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PMID:Angiotensin II-induced fluid phase endocytosis in human cerebromicrovascular endothelial cells is regulated by the inositol-phosphate signaling pathway. 895 95

Protein kinase C (PKC) has been implicated in signaling induced by diverse sets of stimuli regulating growth, differentiation, and apoptosis. The present study focused on the fate of PKC isotype proteins during Fas-mediated apoptosis of human leukemic cell lines. Among the PKC isotypes expressed in different cell types, such as Jurkat, HPB-ALL, U937, and HL60, all the nPKC isotypes including nPKCdelta, nPKC epsilon, and nPKCtheta, but not cPKC alpha and betaII and aPKCzeta (n, c, and a represent novel, conventional and atypical, respectively), showed limited proteolytic cleavage during Fas-mediated apoptosis. The limited proteolysis of nPKC isotypes means the disappearance of the intact protein band concomitant with the appearance of two fragments, most likely containing the kinase and regulatory domains, in contrast to the so-called down-regulation known for both cPKC and nPKC isotypes following exposure to stimuli such as 12-O-tetradecanoyl-phorbol 13-acetate (TPA). The time course of Fas-mediated apoptosis in Jurkat cells parallels that of the activation of a 32-kDa cysteine protease (CPP32)-like protease and also closely parallels the proteolytic cleavage of nPKC isotypes. A peptide inhibitor of the CPP32-like protease, Ac-DEVD-CHO, blocked the proteolytic cleavage of nPKC isotypes as well as apoptosis mediated by Fas. Transfection of recombinant protein coding for the catalytic fragment of nPKCdelta to COS1 cells resulted in the apoptotic morphology of cells and nuclei. The effect of TPA on apoptosis depends on the cell type. TPA significantly suppressed Fas-mediated apoptosis in Jurkat, whereas TPA alone caused apoptosis in HPB-ALL, U937, and HL60, only slight apoptosis in Jurkat. The proteolytic fragmentation of nPKC isotypes again closely correlated with the degree of apoptosis even in apoptosis induced by TPA. Separation of TPA-treated cells into apoptotic and non-apoptotic differentiating cells revealed that the proteolytic fragmentation of nPKC isotypes occurs only in apoptotic cells and, in adherent differentiating cells, nPKC isotypes as well as cPKC alpha were down-regulated without the generation of nPKC fragments. These results are consistent with the idea that nPKC isotypes meet two different fates, down-regulation and proteolytic cleavage generating kinase and regulatory fragments, and that the proteolytic cleavage of nPKC isotypes is a step in the signaling pathway involved in Fas-mediated and TPA-induced apoptosis.
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PMID:The proteolytic cleavage of protein kinase C isotypes, which generates kinase and regulatory fragments, correlates with Fas-mediated and 12-O-tetradecanoyl-phorbol-13-acetate-induced apoptosis. 943 85

Calphostin C is a potent inhibitor of protein kinase C and can induce Ca2+-dependent apoptosis in human ALL cells. Further development of calphostin C will require detailed pharmacodynamic studies in preclinical animal models. Therefore, we established a sensitive and accurate high-performance liquid chromatography (HPLC)-based quantitative detection method for the measurement of calphostin C levels in plasma. Extraction of calphostin C from plasma was performed by precipitation of plasma protein using acetonitrile and an aliquot of extracted supernatant was injected onto a Hewlett-Packard HPLC system constituting a 250x4 mm LiChrospher 100, RP-18 (5 microm) in conjunction with a 4x4 mm LiChrospher 100, RP-18 guard column (5 microm). The eluted compounds were detected by diode array detection set at a wavelength of 479 nm. Acetonitrile-water containing 0.1% trifluoroacetic acid and 0.1% triethylamine (70:30, v/v) was used as the mobile phase. The average extraction recovery from plasma was 97.3%. Good linearity (r>0.999) was observed throughout the concentration range of 0.05-40 microM for calphostin C in 50 microl of plasma. Intra- and inter-assay variabilities were less than 6% in plasma. The lowest detection limit of calphostin C in 50 microl plasma was 0.02 microM at a signal-to-noise ratio of approximately 3. The availability of this assay will now permit detailed pharmacodynamic and pharmacokinetic studies of calphostin C in vivo.
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PMID:Quantitative high-performance liquid chromatography-based detection method for calphostin C, a naturally occurring perylenequinone with potent antileukemic activity. 1020 68


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