EC:2.7.11.13 - FACTA Search

Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Sphingosine inhibits protein kinase C activity in vitro and has been used to implicate this enzyme in signal transduction and cell function. We report that sphingosine directly inhibits phospholipases A2 and D. Sphingosine inhibits Ca(2+)-dependent phospholipases A2 from Naja naja, porcine pancreas, Crotalus adamanteus, human disc and neutrophil in a dose-dependent manner with IC50 values ranging from 5-40 microM using [1-14C]oleate-labelled autoclaved E. coli (20 microM) as substrate. Inhibition is comparable using the same concentrations (20 microM) of [1-14C]oleate-labelled C. albicans or E. coli, or aqueous dispersions of 1-acyl-2-[1-14C]linoleoylglycerophosphoethanolamine or -choline. Sphinganine and stearylamine are as inhibitory as sphingosine; monoolein is less inhibitory (IC50 = 70 microM), while octylamine, N-acetylsphingosine, sphingomyelin and ceramide have no effect. Inhibition is relieved by increasing concentrations of substrate phospholipid. The molar ratio of sphingosine to phospholipid required for 50% inhibition ranges from 0.5 to 1.0 with 2-100 microM E. coli phospholipid. In contrast, sphingosine has a biphasic effect on the hydrolysis of E. coli by S. chromofuscus phospholipase D; concentrations less than or equal to 25 microM stimulate activity while concentrations greater than 25 microM are inhibitory. Addition of Triton X-100 eliminates both the stimulatory and inhibitory effects of sphingosine on phospholipase D activity.
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PMID:Sphingolipid metabolism and signal transduction: inhibition of in vitro phospholipase activity by sphingosine. 150 2

Pulmonary artery (PA) smooth muscle cell (SMC) proliferation occurs with hypoxic pulmonary hypertension in vivo. However, proliferation of cultured PA SMC to hypoxia has not been demonstrated, and thus the mechanism by which these cells respond to hypoxia is unknown. Because protein kinase C (PKC) plays a role in intracellular transduction of proliferative signals, we asked whether PKC activation 1) causes proliferation of bovine PA SMC and 2) is important in PA SMC proliferative response to hypoxia. By measuring [3H]thymidine incorporation and cell counts, we found that quiescent PA SMC from four different cows proliferated with the PKC activator, phorbol 12-myristate 13-acetate (PMA), in a concentration-dependent manner. The proliferation was blocked with a PKC inhibitor, dihydrosphingosine, or by downregulating SMC PKC. We tested whether "priming" PA SMC by PKC activation was required for in vitro SMC proliferative response to hypoxia. Each SMC population was treated with PMA and then exposed for 24 h to 20, 10, 7, 3 or 0% O2. These cells proliferated with hypoxia reaching a peak response at 3% O2. The magnitude of the response to PMA and hypoxia was different for each cell population tested. No hypoxic proliferation occurred in control cells (no PMA). Dihydrosphingosine blocked the hypoxic response to the same extent that it inhibited the initial PMA conditioning stimulus. PKC-downregulated PA SMC did not proliferate to PMA or to subsequent hypoxia. The hypoxic response was not due to a reduction in O2 radical-mediated antiproliferative effect; rather, the PMA-primed cells seemed to "acquire" the ability to directly sense hypoxia and proliferate. In summary, PKC activation caused proliferation of PA SMC in vitro and allowed an additional proliferative response to hypoxia. Activation of PKC may be a requisite step for PA SMC to respond directly to hypoxia.
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PMID:Protein kinase C activation allows pulmonary artery smooth muscle cells to proliferate to hypoxia. 199 57

Long-chain bases are potent inhibitors of protein kinase C and cellular processes mediated by this enzyme. However, when added to cells they usually cause some degree of growth inhibition and cytotoxicity and it is unclear whether this reflects inhibition of protein kinase C or nonspecific detergent effects of these amphipathic compounds. This study examined the effects of sphinganine on Chinese hamster ovary (CHO) cells to gain more insight into these possibilities. Sphinganine concentrations between 0.75 and 4 microM resulted in a combination of growth inhibition and cytotoxicity that correlated with protein kinase C inhibition by five criteria: (1) the effective concentrations were comparable to those for protein kinase C inhibition in vitro and in other intact cells; (2) the structural specificity for the long-chain base moiety paralleled the potency of protein kinase C inhibition; (3) sphinganine blocked changes in protein phosphorylation patterns that occurred in response to phorbol 12-myristate 13-acetate (and vice versa); whereas (4) a mutant cell line that exhibited increased resistance to sphinganine cytotoxicity lacked both phorbol ester- and sphinganine-induced phosphorylation changes and differed somewhat in the behavior of protein kinase C assayed in vitro; and (5) sphinganine did not appear to be acting as a detergent (except at higher concentrations) nor as a lysosomotrophic agent. While the complexity of this cellular behavior mandates caution in interpreting these results, they suggest that the cytotoxicity and growth inhibition may be a consequence of protein kinase C inhibition.
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PMID:Characteristics of the growth inhibition and cytotoxicity of long-chain (sphingoid) bases for Chinese hamster ovary cells: evidence for an involvement of protein kinase C. 229 38

Conditions were developed to prolong the ability of sphinganine, a potent inhibitor of protein kinase C, to block the phorbol ester-induced adherence of HL-60 cells beyond 24 h. The loss of inhibition after this time seen previously (A.H. Merrill, Jr., A.M. Sereni, V.L. Stevens, Y.A. Hannun, R.M. Bell, and J.M. Kinkade, Jr., J. Biol. Chem., 261: 12610-12615, 1986), which appeared to be due to metabolism of this long-chain base, was overcome by supplying sphinganine daily. After 4 days, phorbol myristate acetate-induced adherence was inhibited approximately 50% by sphinganine. Sphinganine significantly decreased the expression of nonspecific esterase induced by phorbol myristate acetate in the nonadherent cells, indicating that other aspects of maturation besides adherence were blocked. The effects of daily sphinganine treatments on the monocytic differentiation induced by 1 alpha-25-dihydroxyvitamin D3 or ganglioside GM3 were also investigated. The increases in nonspecific esterase expression, nitroblue tetrazolium reduction, and morphological maturation caused by either agent were unaffected by the long-chain base. In addition, the changes in several cell surface antigens caused by 1 alpha,25-dihydroxyvitamin D3 were unaltered by sphinganine. Although phorbol esters, 1 alpha,25-dihydroxyvitamin D3, and ganglioside GM3 all induce the maturation of HL-60 cells along the monocytic lineage, the finding that sphinganine only affected the differentiation initiated by phorbol esters, in which protein kinase C clearly is a major regulator, suggests that this enzyme does not play a major role in these other pathways of differentiation.
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PMID:Differential effects of long-chain (sphingoid) bases on the monocytic differentiation of human leukemia (HL-60) cells induced by phorbol esters, 1 alpha, 25-dihydroxyvitamin D3, or ganglioside GM3. 272 Jun 76

Sphinganine has been proposed to be a specific inhibitor of protein kinase C. In the present study we have evaluated whether sphinganine is a convenient tool to probe for the role of protein kinase C in neutrophil function. Human neutrophils were loaded with the fluorescent probe quin2 and then tested in parallel for cytosolic free Ca2+, [Ca2+]i, membrane potential changes, O2- production, and exocytosis of primary granules (containing beta-glucuronidase) in response to various stimuli. In addition to inhibiting O2- production and exocytosis in a dose-dependent manner, sphinganine also blocked formyl-methionyl-leucyl-phenylalanine-induced [Ca2+]i, transients. Furthermore, sphinganine inhibited exocytosis elicited by the calcium ionophore ionomycin. Although sphinganine blocked O2- production due to phorbol 12-myristate 13-acetate, the most striking finding was that the drug rendered the cells leaky. Thus, at similar concentrations as those inhibiting cellular functions, sphinganine was shown to lead to cell permeabilization, as assessed by release of quin2 and cytoplasmic markers into the extracellular medium, and changes in plasma membrane potential. We conclude, therefore, that sphinganine does not appear to be a suitable compound for the evaluation of the involvement of protein kinase C in neutrophil activation.
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PMID:Nonselective inhibition of neutrophil functions by sphinganine. 303 65

The effects of long-chain (sphingoid) bases on the phorbol ester-dependent differentiation of HL-60 cells were investigated since these molecules are potent inhibitors of protein kinase C (Hannun, Y. A., Loomis, C. R., Merrill, A. H., Jr., and Bell, R. M. (1986) J. Biol. Chem. 261, 12604-12609). After 24 h, low concentrations of sphinganine (1-5 microM blocked both cell adherence and the inhibition of growth in response to phorbol 12-myristate 13-acetate, as measured by cell number and acid phosphatase activity. Sphinganine and sphingosine decreased adherence by 50% at 1-3 microM; other long-chain bases were effective in parallel to their inhibition of protein kinase C. Sphinganine decreased the binding of [3H]phorbol dibutyrate by the phorbol receptor of HL-60 cells, protein kinase C, and inhibited the response of HL-60 cells to dioctanoylglycerol, a cell permeable activator of this enzyme. Long-chain base uptake by HL-60 cells was demonstrated with [3-3H]sphinganine and within 1-3 days much had been converted to ceramides. By day 3, most of the cells had recovered the ability to adhere and exhibited macrophage characteristics, whereas cells in suspension did not differentiate. The level of free sphinganine in HL-60 cells was determined to be 12.3 +/- 1.2 pmol/10(6) cells. These results establish that sphingoid bases inhibit protein kinase C in HL-60 cells and may function physiologically as negative effectors of this enzyme.
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PMID:Inhibition of phorbol ester-dependent differentiation of human promyelocytic leukemic (HL-60) cells by sphinganine and other long-chain bases. 346 89

The cytokine-mediated stimulation of sphingomyelin (SM) metabolism is emerging as an important signal transduction pathway via the generation of ceramide and sphingosine, products which have been shown to affect a wide variety of biological processes. Because SM-mediated signal transduction is initiated via the hydrolysis of an integral membrane phospholipid by a phospholipase C-like enzyme (sphingomyelinase) to yield lipids which modulate protein kinase C activity, the SM and phosphatidylinositol (PI) signaling pathways share certain similarities. The present study was undertaken to examine the potential for interplay between SM and PI turnover by testing the effects of sphingosine, sphingosine-1-phosphate, and ceramide on PI turnover. In dermal fibroblasts, sphingosine stimulated a rapid dose-dependent hydrolysis of PI, yielding inositol 1,4,5-triphosphate, followed by increased levels of intracellular calcium. Sphingosine-induced inositol phosphate (IP) accumulation was observed between 5 and 30 microM sphingosine with a maximal accumulation of 2.7-fold over control levels. Enhanced IP formation was measured as early as 5 s following sphingosine treatment and IP levels remained elevated for more than 60 min. Intracellular calcium mobilization accompanied the dose-dependent accumulation of IPs in response to sphingosine, although this effect was not apparent until after a 30-40-s lag period. Interestingly, sphingosine-1-phosphate stimulated a more rapid release of intracellular Ca2+ than sphingosine, but it had no effect on PI turnover. DL-threo-Dihydrosphingosine, a competitive inhibitor of sphingosine kinase, stimulates both PI turnover and Ca2+ flux, but does not block the action of sphingosine relative to those two processes. Ceramide (added as C2-ceramide), N-stearylamine, and stearoyl-D-sphingosine did not affect PI turnover or Ca2+ mobilization. Pretreatment of intact cells with pertussis toxin partially inhibited sphingosine-mediated IP accumulation, suggesting a role for guanine nucleotide binding protein(s) (G protein) in sphingosine-stimulated PI turnover. Furthermore, guanosine 5'-O-(3-thiotriphosphate) stimulated, whereas guanosine 5'-O-(2-thiodiphosphate) inhibited, sphingosine-induced IP accumulation in permeabilized cells. Collectively, these data suggest that sphingosine enhances PI turnover by stimulating phospholipase C activity, and the activation of this process may be modulated by G protein interactions. Thus, the regulation of PI turnover and Ca2+ mobilization by sphingosine may represent another mechanism by which sphingosine modulates cell function and that these effects can be distinguished from those of ceramide.
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PMID:Sphingosine-mediated phosphatidylinositol metabolism and calcium mobilization. 811 27

To separate the role of changes in parathyroid diacylglycerol (DG) from other effects of extracellular calcium, we studied the effect of inhibition of DG metabolism on PTH secretion and cellular DG content in acutely dispersed bovine parathyroid cells. R 59 022, an inhibitor of DG kinase, increased cellular DG, but significantly decreased PTH secretion. Particulate protein kinase C (PKC) activity decreased in bovine parathyroid cells incubated at high extracellular calcium or in the presence of R 59 022, which is the opposite of what was observed in the presence of the phorbol ester, phorbol myristate acetate. Sphinganine, a normal cellular product that is a known inhibitor of PKC, significantly inhibited PTH secretion at low extracellular calcium, but had no significant effect at normal or high extracellular calcium. We then measured sphingosine in bovine parathyroid cells incubated with high extracellular calcium or R 59 022. Both conditions were associated with significant elevations of cellular sphingosine. These studies suggest that inhibition of PTH secretion and PKC activity by enhanced cellular DG may result from the activation of an inhibitory second messenger pathway involving the sphingoid lipids.
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PMID:Relationship among cellular diacylglycerol, sphingosine formation, protein kinase C activity, and parathyroid hormone secretion from dispersed bovine parathyroid cells. 864 Dec 1

We previously reported that prostaglandin (PG)E1 and PGF2alpha induce the synthesis of interleukin-6 (IL-6) via activation of protein kinase (PK)A and PKC, respectively, in osteoblast-like MC3T3-E1 cells. In addition, we have shown that basic fibroblast growth factor (bFGF) elicits IL-6 synthesis through intracellular Ca2+ mobilization in these cells and that tumor necrosis factor-alpha (TNF) induces IL-6 synthesis through sphingosine 1-phosphate produced by sphingomyelin hydrolysis. In the present study, among sphingomyelin metabolites, we examined the effect of sphingosine on IL-6 synthesis induced by various agonists in MC3T3-E1 cells. Sphingosine inhibited the IL-6 synthesis induced by PGF2alpha or 12-O-tetradecanoylphorbol-13-acetate, an activator of PKC. Sphingosine suppressed the PGE1-induced IL-6 synthesis. The IL-6 synthesis induced by cholera toxin, forskolin, or dibutyryl cAMP was inhibited by sphingosine. Sphingosine inhibited the IL-6 synthesis induced by bFGF or A23187. However, sphingosine did not affect the IL-6 synthesis induced by interleukin-1. On the contrary, sphingosine enhanced the TNF-induced IL-6 synthesis. DL-threo-Dihydrosphingosine, an inhibitor of sphingosine kinase, reduced the enhancement by sphingosine as well as the TNF-effect. These results indicate that sphingosine modulates the IL-6 synthesis stimulated by various agonists in osteoblasts.
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PMID:Sphingosine modulates interleukin-6 synthesis in osteoblasts. 970 71

Fumonisin B(1), a potent inhibitor of ceramide synthase, leads to accumulation of sphinganine, and later sphingosine, in vivo and in vitro. Fumonisin B(1) modulates the activity of protein kinase C (PKC), however, which metabolite of disrupted sphingolipid metabolism is involved, has not been ascertained. In the present study, we evaluated the modulation of PKC by sphingolipid bases and their metabolites using exogenous sphingolipid analogues in porcine renal epithelial (LLC-PK(1)) cells. In preliminary studies we found that fumonisin B(1) (1 microM) selectively and transiently activated PKCalpha, whereas fumonisin B(1) concentrations of 1-50 microM at 48 h repressed PKC-alpha, -delta, - epsilon and -zeta isoforms in a concentration-dependent manner. Addition of exogenous sphinganine-1-phosphate (1 microM for 5 min) alone stimulated cytosolic to membrane translocation of PKCalpha. Co-exposure of fumonisin B(1) with N,N-dimethylsphingosine, an inhibitor of sphingosine/sphinganine kinase, prevented the effects of fumonisin B(1) on PKCalpha. Sphinganine, sphingosine, sphingosine-1-phosphate and ceramide (all at 1 microM) added exogenously, did not alter PKCalpha cytosolic to membrane translocation at 5 min. Fumonisin B(1) (10 microM), sphinganine, sphingosine and ceramide (1 microM each) significantly repressed PKC-alpha and -delta isoforms at 48 h, whereas all the exogenously added sphingolipids significantly repressed PKC- epsilon and zeta similar to fumonisin B(1). Co-exposure of myriocin with fumonisin B(1) prevented the delayed inhibitory effects of fumonisin B(1) on PKC isoforms in LLC-PK(1) cells. This study demonstrated that selective and transient activation of PKCalpha may be due to the fumonisin B(1)-induced accumulation of the bioactive sphinganine-1-phosphate, whereas the long-term repression of PKC isoforms may be predominantly due to the accumulation of sphinganine or its metabolite, and to a lesser extent sphingosine or its metabolite in LLC-PK(1) cells. These findings suggest that the direct or indirect modulation of PKC by these sphingolipids is involved at least in part in the action of fumonisin B(1).
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PMID:Sphingoid bases and their phosphates: transient activation and delayed repression of protein kinase C isoforms and their possible involvement in fumonisin B1 cytotoxicity. 1269 12

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