共查询到20条相似文献,搜索用时 8 毫秒
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The purpose of the present study was to examine protein kinase C (PKC) isotype expression in T lymphoblasts derived from peripheral blood and the T leukaemic cell Jurkat. Using antisera reactive with PKC alpha, beta 1, and beta 2 and gamma, it was observed that T cells expressed two PKC isotypes, PKC alpha and beta 1. No PKC gamma was detected in T lymphocytes. In lymphoblasts, high levels of PKC beta compared to PKC alpha were found whereas Jurkat cells expressed high levels of alpha compared to PKC beta. Differences in the calcium sensitivity of phorbol ester-induced phosphorylation were observed in Jurkat and T lymphoblasts which correlated with the relative levels of PKC alpha and beta isotypes expressed by the cells. 相似文献
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A D Beyers C Hanekom A Rheeder A F Strachan M W Wooten A E Nel 《Journal of immunology (Baltimore, Md. : 1950)》1988,141(10):3463-3470
Protein kinase C (PKC) regulates numerous T cell functions and is present in abundance in normal human T cells and certain T cell lines. Although crude Triton X-100 soluble material obtained from T cell pellets contains minimal PKC activity, DEAE chromatography revealed that 12 to 37% of cellular PKC was membrane associated, probably due to removal of an inhibitor through column chromatography. As in other tissues, PKC from lymphoid tissue was phospholipid and Ca2+ dependent and diolein reduced the Ca2+ requirements for enzyme activity. Hydroxylapatite chromatography revealed that T cells possess two major peaks of PKC activity. Although, the enzyme in these peaks had similar m.w. and identical iso-electric mobility, the proteins differed with respect to their autophosphorylation sites and immunoreactivity toward an isoform specific antibody. Furthermore, differences in their activities in the presence of phospholipid, diolein, and limiting amounts of Ca2+ imply that these isoforms may be differentially activated. We discuss optimal conditions for activation of PKC and its isoforms for study of T lymphocyte cellular function. 相似文献
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G R Guy J Gordon L Walker R H Michell G Brown 《Biochemical and biophysical research communications》1986,135(1):146-153
G0 human tonsillar B-lymphocytes were stimulated to divide by the polyclonal mitogen Staphylococcus Aureus Cowan strain 1 (SAC) and by the combined use of 12-O-tetradecanoyl phorbol-13-acetate (TPA) and the calcium ionophore ionomycin. The activities of protein kinase C, which requires Ca++ and phospholipid as co-factors, and a proteolytically cleaved form of this enzyme (protein kinase M), which is independent of calcium and phospholipid control, were determined in soluble and particulate fractions obtained from activated B cells. Treatment of G0 B cells with SAC or TPA together with ionomycin caused redistribution of protein kinase C from the soluble to the particulate fraction where the 80,000-Dalton protein kinase C was cleaved to give rise to a 50,000-Dalton form of the kinase which was also found in the cytoplasm. These data suggest that redistribution and proteolytic cleavage of protein kinase C are key signal transduction events in B cell mitogenesis. 相似文献
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Protein kinase C (PKC) is encoded by a complex of a gene family, and its multiple isoforms are expressed in various mammalian tissues. The objective of this study was to investigate the expression and localization of a PKC theta isoform in mouse testis. PKC theta displays the highest homology to PKC delta, lacks the Ca2+-binding C2 domain and, thus, belongs to the subfamily of Ca2+-independent PKC enzymes which also includes the delta, epsilon, zeta and eta isoforms. We analyzed the PKC theta mRNA and protein by Northern blotting, in situ hybridization, and immunohistochemistry. In testes of normal mice, signals of PKC theta isoform expression were detected specifically in the interstitial cells of testes. The expression of PKC theta isoform was also detected in testes of germ cell-deficient W/W(v) mice. These results suggest that PKC theta isoform has the specific biological functions in the interstitial cells of testis. 相似文献
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Joel M. Depper Warren J. Leonard Cynthia L. Drogula Martin Krnke Thomas A. Waldmann Warner C. Greene 《Journal of cellular biochemistry》1985,27(3):267-276
Resting human T lymphocytes do not express receptors for interleukin-2, but expression is rapidly induced by exposure to PHA. After maximal expression 2-3 days after stimulation, a progressive decline in receptor number is observed. Receptor expression can be augmented by reexposure to PHA. In this study we show that activators of protein kinase C including phorbol diester, phospholipase C, and the diacylglycerol congener diC8 also increase IL-2 receptor expression. Moreover, 5-azacytidine, which inhibits cytosine methyltransferase, and hydroxyurea, which inhibits ribonucleotide reductase, also increased receptor number. These studies demonstrate that IL-2 receptor expression can be altered in vitro, and that IL-2 receptor number, in combination with IL-2 secretion, may contribute to the regulation of IL-2-dependent immune responses. 相似文献
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Murugappan S Tuluc F Dorsam RT Shankar H Kunapuli SP 《The Journal of biological chemistry》2004,279(4):2360-2367
Several platelet agonists, including thrombin, collagen, and thromboxane A(2), cause dense granule release independently of thromboxane generation. Because protein kinase C (PKC) isoforms are implicated in platelet secretion, we investigated the role of individual PKC isoforms in platelet dense granule release. PKCdelta was phosphorylated in a time-dependent manner that coincided with dense granule release in response to protease-activated receptor-activating peptides SFLLRN and AYPGKF in human platelets. Only agonists that caused platelet dense granule secretion activated PKCdelta. SFLLRN- or AYPGKF-induced dense granule release and PKCdelta phosphorylation occurred at the same respective agonist concentration. Furthermore, AYPGKF and SFLLRN-induced dense granule release was blocked by rottlerin, a PKCdelta selective inhibitor. In contrast, convulxin-induced dense granule secretion was potentiated by rottlerin but was abolished by Go6976, a classical PKC isoform inhibitor. However, SFLLRN-induced dense granule release was unaffected in the presence of Go6976. Finally, rottlerin did not affect SFLLRN-induced platelet aggregation, even in the presence of dimethyl-BAPTA, indicating that PKCdelta has no role in platelet fibrinogen receptor activation. We conclude that PKCdelta and the classical PKC isoforms play a differential role in platelet dense granule release mediated by protease-activated receptors and glycoprotein VI. Furthermore, PKCdelta plays a positive role in protease-activated receptor-mediated dense granule secretion, whereas it functions as a negative regulator downstream of glycoprotein VI signaling. 相似文献
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Schreiber KL Paquet L Allen BG Rindt H 《American journal of physiology. Heart and circulatory physiology》2001,281(5):H2062-H2071
The expression of protein kinase C (PKC) isoforms in the developing murine ventricle was studied using Western blotting, assays of PKC activity, and immunoprecipitations. The abundance of two Ca2+-dependent isoforms, PKCalpha and PKCbetaII, as well as two Ca2+-independent isoforms, PKCdelta and PKCepsilon, decreased during postnatal development to <15% of the levels detected at embryonic day 18. The analysis of the subcellular distribution of the four isoforms showed that PKCdelta and PKCepsilon were associated preferentially with the particulate fraction in fetal ventricles, indicating a high intrinsic activation state of these isoforms at this developmental time point. The expression of PKCalpha in cardiomyocytes underwent a developmental change. Although preferentially expressed in neonatal cardiomyocytes, this isoform was downregulated in adult cardiomyocytes. In fast-performance liquid chromatography-purified ventricular extracts, the majority of PKC activity was Ca2+-independent in both fetal and adult ventricles. Immunoprecipitation assays indicated that PKCdelta and PKCepsilon were responsible for the majority of the Ca2+-independent activity. These studies indicate a prominent role for Ca2+-independent PKC isoforms in the mouse heart. 相似文献
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Protein kinase C (PKC) isoforms are key mediators in hormone, growth factor, and neurotransmitter triggered pathways of cell activation (Nishizuka: Science 233:305-312, 1986; Nature 334:661-665, 1988). Stimulation of kinase activity by diacylglycerol and calcium often leads to translocation of PKC from the cytosol to a particulate fraction (Kraft and Anderson: Nature 301:621-623, 1983). The beta isoform of PKC is translocated and degraded much more rapidly than the alpha isoform in phorbolester-stimulated rat basophilic leukemia (RBL) cells (Huang et al.: J. Biol. Chem. 264:4238-4243, 1989). We report here immunofluorescence evidence that the distributions of PKC alpha and beta are strikingly different in antigen-activated RBL cells. PKC beta associates with perinuclear filaments and filaments that extend from the perinuclear area to the cell periphery whereas PKC alpha concentrates in regions of the cell periphery. This distribution of PKC beta is distinctly different from that of actin filaments and microtubules as determined by phalloidin staining and by anti-tubulin antibody labeling. In contrast, the staining patterns obtained with antibodies to PKC beta and to the intermediate filament protein vimentin are almost identical, indicating that PKC beta associates with vimentin filaments. These bundles of 100 A filaments may provide docking sites for interactions of PKC beta with its substrates and thus confer specificity to the actions of this isoform. 相似文献
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Jiang K Apostolatos AH Ghansah T Watson JE Vickers T Cooper DR Epling-Burnette PK Patel NA 《Biochemistry》2008,47(2):787-797
Protein kinase C (PKC) delta plays an important role in cellular proliferation and apoptosis where it is involved in the caspase-3 mediated apoptotic pathway. Cleavage of PKCdeltaI by caspase-3 releases a catalytically active C-terminal fragment that is sufficient to induce apoptosis. In this paper, we identified a novel human PKCdelta isozyme, PKCdeltaVIII (Genbank accession number DQ516383) in human teratocarcinoma (NT2) cells that differentiate into hNT neurons upon retinoic acid (RA) treatment. Expression of PKCdeltaVIII was confirmed by real-time RT-PCR analysis, and we observed that after an initial peak at 24 h following RA treatment, its expression gradually declined with prolonged RA treatment. PKCdeltaVIII is generated via the utilization of an alternative 5' splice site, and this results in an insertion of 31 amino acids in the caspase-3 recognition sequence DMQD. The function of PKCdeltaVIII was examined by subcloning it into an expression vector and raising an antibody specific to PKCdeltaVIII. Using in vivo and in vitro assays, we demonstrated that PKCdeltaVIII is resistant to caspase-3 cleavage. Next, we sought to determine the role of PKCdeltaVIII in apoptosis in NT2 cells. Overexpression of PKCdeltaVIII and knockdown using PKCdeltaVIII siRNA suggest an antiapoptotic function for the PKCdeltaVIII isozyme. We demonstrate that antisense oligonucleotides (ASO) directed toward the 5' splice site I promote the expression of the PKCdeltaVIII isozyme. Our results indicated that ASO mediated PKCdeltaVIII expression rescued NT2 cells from etoposide-induced apoptosis. We conclude that the novel human PKCdeltaVIII splice variant functions as an antiapoptotic protein in NT2 cells. 相似文献
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Matsumura M Tanaka N Kuroki T Ichihashi M Ohba M 《Biochemical and biophysical research communications》2003,303(1):350-356
Protein kinase C (PKC) fulfills a central role in the decision of cell fate in keratinocytes. Both PKC delta and PKC eta induce growth inhibition and differentiation of normal human keratinocytes (NHK). Here we show that PKC delta and PKC eta play opposite roles in UVB-induced apoptosis in NHK. PKC delta enhanced UVB-induced caspase-3 activity, while overexpression of PKC eta reduced it. In keeping with these observations, the dominant negative mutant of PKC delta significantly inhibited the activation of caspase-3, whereas dominant negative PKC eta increased it in a dose (MOI)-dependent manner. Unlike PKC delta, cleavage and translocation to mitochondria of PKC eta were not observed, resulting in no detection of cytochorome c release. Furthermore, UV-induced activation of p38 MAP kinase, which suppressed the caspase-3 activity in NHK, was blocked by dominant negative PKC eta. These findings suggest that PKC eta negatively regulates UV-induced apoptosis through its localization, resistance to cleavage, and the p38 MAPK pathway. 相似文献
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The eta isoform of protein kinase C is localized on rough endoplasmic reticulum. 总被引:3,自引:1,他引:2
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K Chida H Sagara Y Suzuki A Murakami S Osada S Ohno K Hirosawa T Kuroki 《Molecular and cellular biology》1994,14(6):3782-3790
The eta isoform of protein kinase C, isolated from a cDNA library of mouse skin, has unique tissue and cellular distributions. It is predominantly expressed in epithelia of the skin, digestive tract, and respiratory tract in close association with epithelial differentiation. We report here that this isoform is localized on the rough endoplasmic reticulum in transiently expressing COS1 cells and constitutively expressing keratinocytes. By the use of polyclonal antibodies raised against peptides of the diverse D1 and D2/D3 regions, we found that immunofluorescent signals were strongest in the cytoplasm around the nucleus and became weaker toward the peripheral cytoplasm. Under immunoelectron microscopic examination, electron-dense signals were located on the rough endoplasmic reticulum and on the outer nuclear membrane which is continuous with the endoplasmic reticulum membrane. However, no signals were detected in the nucleus, inner nuclear membrane, smooth endoplasmic reticulum, Golgi apparatus, mitochondria, or plasma membrane. Treatment of the cells in situ with detergents suggested association of the isoform of protein kinase C with intracellular structures. By immunoblotting, a distinct single band with an M(r) of 80,000 was detected in whole-cell lysate and in rough microsomal and crude nuclear fractions, all of which contain outer nuclear membrane and/or rough endoplasmic reticulum. We further demonstrated the absence of a nuclear localization signal in the pseudosubstrate sequence. The present observation is not consistent with the report of Greif et al. (H. Greif, J. Ben-Chaim, T. Shimon, E. Bechor, H. Eldar, and E. Livneh, Mol. Cell. Biol. 12:1304-1311, 1992). 相似文献
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Microgravity modifies protein kinase C isoform translocation in the human monocytic cell line U937 and human peripheral blood T-cells 总被引:4,自引:0,他引:4
Individual protein kinase C (PKC) isoforms fulfill distinct roles in the regulation of the commitment to differentiation, cell cycle arrest, and apoptosis in both monocytes and T-cells. The human monocyte like cell line U937 and T-cells were exposed to microgravity, during spaceflight and the translocation (a critical step in PKC signaling) of individual isoforms to cell particulate fraction examined. PKC activating phorbol esters induced a rapid translocation of several PKC isoforms to the particulate fraction of U937 monocytes under terrestrial gravity (1 g) conditions in the laboratory. In microgravity, the translocation of PKC beta II, delta, and epsilon in response to phorbol esters was reduced in microgravity compared to 1 g, but was enhanced in weak hypergravity (1.4 g). All isoforms showed a net increase in particulate PKC following phorbol ester stimulation, except PKC delta which showed a net decrease in microgravity. In T-cells, phorbol ester induced translocation of PKC delta was reduced in microgravity, compared to 1 g, while PKC beta II translocation was not significantly different at the two g-levels. These data show that microgravity differentially alters the translocation of individual PKC isoforms in monocytes and T-cells, thus providing a partial explanation for the modifications previously observed in the activation of these cell types under microgravity. 相似文献
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P Basta M B Strickland W Holmes C R Loomis L M Ballas D J Burns 《Biochimica et biophysica acta》1992,1132(2):154-160
Two human homologues of protein kinase C-epsilon (E1 and E2) were isolated from two distinct cDNA libraries. Sequence comparisons to PKC-epsilon cDNAs from several species indicated that each of these human epsilon clones contained cloning artifacts. Thus, a composite PKC-epsilon (E3) clone was derived from clones E1 and E2. Human PKC-epsilon (E3) has an overall sequence identity of 90-92% at the nucleotide level compared to the previously characterized mouse, rat and rabbit clones. At the amino acid level, the deduced human epsilon sequence shows a 98-99% identity with the mouse, rat and rabbit sequences. Expression of the human PKC-epsilon clone in Sf9 cells confirmed that the recombinant protein displayed protein kinase C activity and phorbol ester binding activity. The recombinant protein was also recognized by two distinct epsilon-specific polyclonal antibodies. 相似文献
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P D Phatak C H Packman M A Lichtman 《Journal of immunology (Baltimore, Md. : 1950)》1988,141(9):2929-2934
We studied the effect of activators and inhibitors of protein kinase C on actin conformation in human blood lymphocytes by flow cytometry and gel electrophoresis. PMA, 1-oleyl-2-acetyl-glycerol, and mezerein, activators of protein kinase C, caused an increase in lymphocyte F-actin within 2 to 5 min. After stimulation with PMA, lymphocytes formed pseudopods containing an increased concentration of F-actin and had an increase of actin in the Triton-insoluble cytoskeletal fraction. Sphingosine and H-7, inhibitors of protein kinase C activation, inhibited the increase in F-actin induced by PMA. The increase in F-actin in response to PMA was striking in Th and Ts lymphocytes (2- to 3-fold increase), but B lymphocytes had only a slight increase (1.15-fold). Thus, activation of protein kinase C modulates actin conformation specifically in T lymphocytes. 相似文献
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Purification and characterization of the zeta isoform of protein kinase C from bovine kidney. 总被引:8,自引:0,他引:8
The zeta isoform of protein kinase C (PKC zeta) was purified to near homogeneity from the cytosolic fraction of bovine kidney by successive chromatography on DEAE-Sephacel, heparin-Sepharose, phenyl-5PW, hydroxyapatite, and Mono Q. The purified enzyme had a molecular mass of 78 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein was recognized by an antibody raised against a synthetic oligopeptide corresponding to the deduced amino acid sequence of rat PKC zeta. The enzymatic properties of PKC zeta were examined and compared with conventional protein kinase C purified from rat brain. The activity of PKC zeta was stimulated by phospholipid but was unaffected by phorbol ester, diacylglycerol, or Ca2+. PKC zeta did not bind phorbol ester, and autophosphorylation was not affected by phorbol ester. Unsaturated fatty acid activated PKC zeta, but this activation was neither additive nor synergistic with phospholipid. These results indicate that regulation of PKC zeta is distinct from that of other isoforms and suggest that hormone-stimulated increases in diacylglycerol and Ca2+ do not activate this isoform in cells. It is possible that PKC zeta belongs to another enzyme family, in which regulation is by a different mechanism from that for other isoforms of protein kinase C. 相似文献
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Role for zinc in a cellular response mediated by protein kinase C in human B lymphocytes 总被引:1,自引:0,他引:1
Recent studies have suggested a role for Zn2+, distinct from that of Ca2+, in the subcellular distribution and activation of protein kinase C (PKC). Here we show that Zn2+ is required for a cellular response mediated by PKC, the rapid loss of expression of a human B cell receptor MER, detected by rosetting with mouse erythrocytes. Zn2+, in the presence of the Zn2+ ionophore pyrithione, caused rapid inhibition of MER rosetting at concentrations which induce the translocation and activation of PKC. This required cellular uptake of Zn2+ and was blocked by 1,10-phenanthroline and TPEN which chelate Zn2+ but not Ca2+. Gold, a metal with similar properties, also induced translocation of PKC and inhibition of MER. By contrast, Ca2+ ionophores A23187 and ionomycin, which induce a different pathway of translocation of PKC, had no effect on MER. Phenanthroline and TPEN also blocked the inhibition of MER induced by the PKC activators phorbol ester and sodium fluoride, suggesting that endogenous cellular Zn2+ is required. We propose that some cellular actions of PKC require a Zn(2+)-dependent event and that these may be a target for gold during chrysotherapy in rheumatoid arthritis. 相似文献