Differential localization of protein kinase C delta by phorbol esters and related compounds using a fusion protein with green fluorescent protein.
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Enzyme localization often plays a controlling role in determining its activity and specificity. Protein kinase C (PKC) has long been known to translocate in response to physiological stimuli as well as to exogenous ligands such as the phorbol esters. We report here that different phorbol derivatives and related ligands, selected for differences in chemical structure and profile of biological activity, induce distinct patterns of redistribution of PKC delta. Localization of a PKC delta-green fluorescent protein (GFP) fusion construct was monitored in living Chinese hamster ovary cells as a function of ligand, concentration, and time using confocal laser scanning microscopy. delta-PKC-GFP was expressed predominantly in the cytoplasm, with some in the nucleus and perinuclear region. Phorbol 12-myristate 13-acetate (PMA) induced plasma membrane translocation followed by slower nuclear membrane translocation. As the concentration of PMA increased, the proportion of nuclear to plasma membrane localization increased markedly. In contrast to PMA, bryostatin 1, a unique activator of PKC that induces a subset of PMA-mediated responses while antagonizing others, at all doses induced almost exclusively nuclear membrane translocation. Like PMA, the complete tumor promoter 12-deoxyphorbol 13-tetradecanoate induced plasma membrane and slower nuclear membrane translocation, whereas the inhibitor of tumor promotion 12-deoxyphorbol 13-phenylacetate, which differs only in its side chain, induced a distinctive distribution of PKC delta-GFP. Finally, the novel constrained diacylglycerol derivative B8-DL-B8 induced a slow Golgi localization. We speculate that differential control of PKC delta localization may provide an interesting strategy for producing ligands with differential biological consequences. (+info)
Expression in yeast and tobacco of plant cDNAs encoding acyl CoA:diacylglycerol acyltransferase.
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During the course of a search for cDNAs encoding plant sterol acyltransferases, an expressed sequence tag clone presenting substantial identity with yeast and animal acyl CoA:cholesterol acyltransferases was used to screen cDNA libraries from Arabidopsis and tobacco. This resulted in the isolation of two full-length cDNAs encoding proteins of 520 and 532 amino acids, respectively. Attempts to complement the yeast double-mutant are1 are2 defective in acyl CoA:cholesterol acyltransferase were unsuccessful, showing that neither gene encodes acyl CoA:cholesterol acyltransferase. Their deduced amino acid sequences were then shown to have 40 and 38% identity, respectively, with a murine acyl CoA:diacylglycerol acyltransferase and their expression in are1 are2 or wild-type yeast resulted in a strong increase in the incorporation of oleyl CoA into triacylglycerols. Incorporation was 2-3 times higher in microsomes from yeast transformed with these plant cDNAs than in yeast transformed with the void vector, clearly showing that these cDNAs encode acyl CoA:diacylglycerol acyltransferases. Moreover, during the preparation of microsomes from the Arabidopsis DGAT-transformed yeast, a floating layer was observed on top of the 100 000 g supernatant. This fraction was enriched in triacylglycerols and exhibited strong acyl CoA:diacylglycerol acyltransferase activity, whereas almost no activity was detected in the corresponding clear fraction from the control yeast. Thanks to the use of this active fraction and dihexanoylglycerol as a substrate, the de novo synthesis of 1,2-dihexanoyl 3-oleyl glycerol by AtDGAT could be demonstrated. Transformation of tobacco with AtDGAT was also performed. Analysis of 19 primary transformants allowed detection, in several individuals, of a marked increase (up to seven times) of triacylglycerol content which correlated with the AtDGAT mRNA expression. Furthermore, light-microscopy observations of leaf epidermis cells, stained with a lipid-specific dye, showed the presence of lipid droplets in the cells of triacylglycerol-overproducer plants, thus illustrating the potential application of acyl CoA:diacylglycerol acyltransferase-transformed plants. (+info)
Inhibitory mechanism of aloe single component (alprogen) on mediator release in guinea pig lung mast cells activated with specific antigen-antibody reactions.
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We previously reported that the glycoprotein extracted from aloe strongly inhibited the mediator releases caused by the activation of guinea pig lung mast cells. Therefore, this study aimed to purify a single component that has an antiallergic effect from crude aloe extract and then to assess the effects of aloe single component (alprogen) on the mechanism of mediator releases caused by the mast cell activation. We purified aloe extracts by using various columns. We also purified mast cells from guinea pig lung tissues by using enzyme digestion, rough and discontinuous density Percoll gradient. Mast cells were sensitized with IgG(1) (anti-ovalbumin) and challenged with ovalbumin. Histamine was assayed by using a fluorometric analyzer and leukotrienes by radioimmunoassay. [Ca(2+)](i) level was analyzed by using a confocal laser scanning microscope. Protein kinase activity was determined by the protein phosphorylated with [gamma-(32)P]ATP. The phospholipase D activity was assessed by the labeled phosphatidylalcohol. The amount of mass 1,2-diacylglycerol (DAG) was measured by the [(3)H]DAG produced when prelabeled with [(3)H]myristic acid. Phospholipase A(2) activity was determined by measuring the lyso-phosphatidylcholine released from the labeled phospholipids. Alprogen significantly decreased histamine and leukotriene releases and blocked completely Ca(2+) influx during mast cell activation. The protein kinase C and phospholipase D activities were decreased by alprogen in dose-dependent manner. Alprogen inhibited mass DAG formation and the phospholipase A(2) activity during mast cell activation. The data suggest that alprogen purified from aloe inhibits multiple signals as well as blocking Ca(2+) influx caused by mast cells activated with specific antigen-antibody reactions and that then the inhibition of histamine and leukotriene release follows. (+info)
Hybrid transgenic mice reveal in vivo specificity of G protein-coupled receptor kinases in the heart.
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G protein-coupled receptor kinases (GRKs) phosphorylate activated G protein-coupled receptors, including alpha(1B)-adrenergic receptors (ARs), resulting in desensitization. In vivo analysis of GRK substrate selectivity has been limited. Therefore, we generated hybrid transgenic mice with myocardium-targeted overexpression of 1 of 3 GRKs expressed in the heart (GRK2 [commonly known as the beta-AR kinase 1], GRK3, or GRK5) with concomitant cardiac expression of a constitutively activated mutant (CAM) or wild-type alpha(1B)AR. Transgenic mice with cardiac CAMalpha(1B)AR overexpression had enhanced myocardial alpha(1)AR signaling and elevated heart-to-body weight ratios with ventricular atrial natriuretic factor expression denoting myocardial hypertrophy. Transgenic mouse hearts overexpressing only GRK2, GRK3, or GRK5 had no hypertrophy. In hybrid transgenic mice, enhanced in vivo signaling through CAMalpha(1B)ARs, as measured by myocardial diacylglycerol content, was attenuated by concomitant overexpression of GRK3 but not GRK2 or GRK5. CAMalpha(1B)AR-induced hypertrophy and ventricular atrial natriuretic factor expression were significantly attenuated with either concurrent GRK3 or GRK5 overexpression. Similar GRK selectivity was seen in hybrid transgenic mice with wild-type alpha(1B)AR overexpression concurrently with a GRK. GRK2 overexpression was without effect on any in vivo CAM or wild-type alpha(1B)AR cardiac phenotype, which is in contrast to previously reported in vitro findings. Furthermore, endogenous myocardial alpha(1)AR mitogen-activated protein kinase signaling in single-GRK transgenic mice also exhibited selectivity, as GRK3 and GRK5 desensitized in vivo alpha(1)AR mitogen-activated protein kinase responses that were unaffected by GRK2 overexpression. Thus, these results demonstrate that GRKs differentially interact with alpha(1B)ARs in vivo such that GRK3 desensitizes all alpha(1B)AR signaling, whereas GRK5 has partial effects and, most interestingly, GRK2 has no effect on in vivo alpha(1B)AR signaling in the heart. (+info)
Hyperosmolality induces activation of cPKC and nPKC, a requirement for ERK1/2 activation in NIH/3T3 cells.
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Protein kinase C (PKC) has been reported to be associated with the activation of extracellular signal-regulated kinase (ERK) by hyperosmolality. However, it is unclear whether hyperosmolality induces PKC activation and which PKC isoforms are involved in ERK activation. In this study, we demonstrate that NaCl increases total PKC activity and induces PKCalpha, PKCdelta, and PKCepsilon translocation from the cytosol to the membrane in NIH/3T3 cells, suggesting that hyperosmotic stress activates conventional PKC (cPKC) and novel PKC (nPKC). Further studies show that NaCl-inducible ERK1 and ERK2 (ERK1/2) activation is a consequence of cPKC and nPKC activation, because either downregulation with 12-O-tetradecanoylphorbol 13-acetate or selective inhibition of cPKC and nPKC by GF-109203X and rottlerin largely inhibited the stimulation of ERK1/2 phosphorylation by NaCl. In addition, we show that NaCl increases diacylglycerol (DAG) levels and that a phospholipase C (PLC) inhibitor, U-73122, inhibits NaCl-induced ERK1/2 phosphorylation. These results, together, suggest that a hyperosmotic NaCl-induced signaling pathway that leads to activation of ERK1/2 may sequentially involve PLC activation, DAG release, and cPKC and nPKC activation. (+info)
Activity of the phosphatidylcholine biosynthetic pathway modulates the distribution of fatty acids into glycerolipids in proliferating cells.
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PtdCho accumulation is a periodic, S phase-specific event that is modulated in part by cell cycle-dependent fluctuations in CTP:phosphocholine cytidylyltransferase (CCT) activity. A supply of fatty acids is essential to generate the diacylglycerol (DG) precursors for phosphatidylcholine (PtdCho) biosynthesis but it is not known whether the DG supply is also coupled to the cell cycle. Although the rate of fatty acid synthesis in a macrophage cell line was dramatically stimulated in response to the growth factor, CSF-1, it was not regulated by the cell cycle. Increased fatty acid synthesis correlated with elevated acetyl-CoA carboxylase (ACC) and fatty acid synthase (FAS) steady-state mRNA levels. Cellular fatty acid synthesis was essential for membrane PL synthesis. Cerulenin inhibition of endogenous fatty acid synthesis also inhibited PtdCho synthesis, which was not relieved by exogenous fatty acids. Inhibition of CCT activity by the addition of lysophosphatidylcholine (lysoPtdCho) or temperature-shift of a conditionally defective CCT diverted newly synthesized DG to the TG pool where it accumulated. Enforced expression of CCT stimulated PtdCho biosynthesis and reduced TG synthesis. Thus, the cellular DG supply did not regulate PtdCho biosynthesis and CCT activity governs the partitioning of DG into either the PL or TG pools, thereby controlling both PtdCho and TG biosynthesis. (+info)
Inhibition of phosphatidylserine synthesis during Jurkat T cell activation. The phosphatase inhibitor, sodium ortho-vanadate bypasses the CD3/T cell receptor-induced second messenger signaling pathway.
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Sodium ortho-vanadate (Na3VO4), an inhibitor of protein tyrosine phosphatase, induces a rapid (15 min) and strong inhibition of phosphatidylserine synthesis with an IC50 = 100 microM. The mode of action of Na3VO4 was compared to that of CD3 mAbs. It was found that Na3VO4 bypasses the major CD3-induced T cell activation signals including protein tyrosine phosphorylation, p56lck activation and the generation of second messengers including inositol phosphates and its subsequent Ca2+ mobilization as well as diacylglycerol production. These facts were confirmed by using a panel of Jurkat clones that differs by the expression of either tyrosine kinases involved in the CD3-induced T cell activation pathway such as p56lck, p72syk and ZAP-70 or some cell surface receptors such as the CD3/TCR complex or the CD45 phosphatase. (+info)
Signalling pathway for histamine activation of non-selective cation channels in equine tracheal myocytes.
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1. The signalling pathway underlying histamine activation of non-selective cation channels was investigated in single equine tracheal myocytes. Application of histamine (100 microM) activated the transient calcium-activated chloride current (ICl(Ca)) and sustained, low amplitude non-selective cation current (ICat). The H1 receptor antagonist pyrilamine (10 microM) blocked activation of ICl(Ca) and ICat. Simultaneous application of histamine (100 microM) and caffeine (8 mM) during H1 receptor blockade activated ICl(Ca), but not ICat. Neither the H2 receptor antagonist cimetidine (20 microM) nor the H3 receptor antagonist thioperamide (20 microM) prevented activation of ICl(Ca) and ICat. 2. Intracellular dialysis of anti-Galphai/Galphao antibodies completely blocked activation of ICat by histamine, whereas ICl(Ca) was not affected. By contrast, anti-Galphaq/Galpha11 antibodies greatly inhibited ICl(Ca), but did not alter activation of ICat. 3. 1-Oleoyl-2-acetyl-sn-glycerol (OAG, 20-100 microM) did not induce any current or affect currents activated by histamine or methacholine (mACH). Simultaneous application of OAG and caffeine activated ICl(Ca), but not ICat, indicating that a rise in [Ca2+]i and stimulation of diacylglycerol-sensitive protein kinase C (PKC) is not sufficient to activate ICat. The phospholipase C inhibitor U73122 (2 microM) blocked histamine activation of ICl(Ca) and ICat, but simultaneous exposure of myocytes to histamine and caffeine restored both ICl(Ca) and ICat in the presence of U73122. 4. Histamine and mACH activated currents with equivalent I-V relationships. The currents activated by these agonists were not additive; following activation of ICat by mACH, histamine failed to induce an additional membrane current. Similarly, mACH did not induce an additional current after full activation of ICat by histamine. 5. We conclude that H1 histamine receptors activate ICat through coupling to Gi/Go proteins. Activation of ICat also requires intracellular calcium release, mediated by H1 receptors coupling to Gq/G11 proteins. This coupling is analogous to the activation of ICat by co-stimulation of M2 and M3 receptors. (+info)