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ABSTRACT: Protein kinase C (PKC) isoenzymes are essential components of cell signaling. In this study, we investigated the regulation of PKC-alpha in murine B16 amelanotic melanoma (B16a) cells by the monohydroxy fatty acids 12(S)-hydroxyeicosatetraenoic acid [12(S)-HETE] and 13(S)-hydroxyoctadecadienoic acid [13(S)-HODE]. 12(S)-HETE induced a translocation of PKC-alpha to the plasma membrane and focal adhesion plaques, leading to enhanced adhesion of B16a cells to the matrix protein fibronectin. However, 13(S)-HODE inhibited these 12(S)-HETE effects on PKC-alpha. A receptor-mediated mechanism of action for 12(S)-HETE and 13(S)-HODE is supported by the following findings. First, 12(S)-HETE triggered a rapid increase in cellular levels of diacylglycerol and inositol trisphosphate in B16a cells. 13(S)-HODE blocked the 12(S)-HETE-induced bursts of both second messengers. Second, the 12(S)-HETE-increased adhesion of B16a cells to fibronectin was sensitive to inhibition by a phospholipase C inhibitor and pertussis toxin. Finally, a high-affinity binding site (Kd = 1 nM) for 12(S)-HETE was detected in B16a cells, and binding of 12(S)-HETE to B16a cells was effectively inhibited by 13(S)-HODE (IC50 = 4 nM). In summary, our data provide evidence that regulation of PKC-alpha by 12(S)-HETE and 13(S)-HODE may be through a guanine nucleotide-binding protein-linked receptor-mediated hydrolysis of inositol phospholipids.
Proceedings of the National Academy of Sciences 10/1995; 92(20):9323-7. · 9.68 Impact Factor
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ABSTRACT: In addition to serving as the precursor to a plethora of eicosanoids and other bioactive molecules, arachidonate may function as a bona fide second messenger. A number of studies have documented the ability of arachidonate to regulate the function of multiple targets in vitro systems. This has been particularly well established and studied with the activation of protein kinase C by arachidonate in a mechanism distinct from activation by diacylglycerol. In cells, arachidonate induces a number of activities, many of which may be independent of further metabolism to eicosanoids; suggesting possible direct action of arachidonate. This review summarizes the current state of knowledge on the possible second messenger function of arachidonate with specific emphasis on the regulation of protein kinase C.
Cellular Signalling 04/1995; 7(3):171-84. · 4.06 Impact Factor
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ABSTRACT: A novel lipid-dependent protein kinase in human platelets was partially purified and characterized. This enzyme was calcium-independent and was selective for phosphatidic acid as a cofactor/activator with initial activation observed at approximately 2 mol % and peak activity achieved at 4 mol % phosphatidic acid. In the presence of phosphatidylserine, enzyme activation was observed with concentrations of phosphatidic acid as low as 0.5 mol % with peak activity at 2 mol %. Other anionic phospholipids also activated the enzyme but to a lesser extent and with less potency. Enzyme activity was independent of diacylglycerol or phorbol esters and the enzyme did not bind [3H]phorbol dibutyrate. In a soluble protein kinase assay, the enzyme was activated by cis-unsaturated fatty acids with maximum activation occurring at 5-10 microM sodium oleate. Western blot analysis showed that this enzyme did not cross-react immunologically with antibodies raised against the currently identified isoenzymes of protein kinase C. A number of additional biochemical criteria distinguished this enzyme from known isoenzymes of protein kinase C. These biochemical and immunologic data define a novel lipid-dependent protein kinase in human platelets. The role of this enzyme in signal transduction as a phosphatidic acid-activated enzyme and as a possible target for cis-unsaturated fatty acids is discussed.
Journal of Biological Chemistry 05/1994; 269(13):9729-35. · 4.77 Impact Factor
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ABSTRACT: Na/K-ATPase in renal epithelium is expressed at the basolateral surface and thus is critical for vectorial solute transport. One potential mode of regulation of Na/K-ATPase involves the intracellular effector protein kinase C (PKC). In kidney cell lines, activation of PKC by the phorbol ester phorbol 12,13-dibutyrate (PDBu) (1 microM) inhibited Na/K-ATPase transport activity in OK cells (Vmax decreased 42%; p < 0.02), but not in LLC-PK1 cells. By immunoblot, both cell types expressed detectable levels of PKC alpha and PKC sigma. In response to PDBu, PKC alpha translocated from the cytosol to the membrane fractions of both cell lines. Phorbol ester treatment increased incorporation of 32PO4 in multiple substrates in both cell types, but a approximately 109-kDa substrate with neutral pI was detected only in the OK cell. Anti-LEAVE, directed against a highly conserved sequence in the H4-H5 loop of all known alpha isoforms of Na/K-ATPase, recognized a approximately 109-kDa membrane protein from both cell lines. Anti-LEAVE also identified a protein that comigrated with the large phosphoprotein which was only present in OK cells. Following 32PO4 loading and PDBu treatment, anti-LEAVE immunoprecipitated a approximately 109-kDa phosphoprotein in OK but not LLC-PK1 cells. These data support the notion that PKC is capable of phosphorylating the alpha subunit and inhibiting Na/K-ATPase transport activity in intact renal cells. Furthermore, they suggest that some forms of Na/K-ATPase in the kidney are not susceptible to PKC phosphorylation and that this heterogeneity may contribute to response diversity.
Journal of Biological Chemistry 07/1993; 268(21):15958-64. · 4.77 Impact Factor
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ABSTRACT: The mechanisms involved in regulating the selective expression of protein kinase C (PKC) isoenzymes are poorly understood. Two human B lymphoblastoid cell lines, IM-9 and BJA-B, exhibited differential expression of the two alternatively spliced products of the PKC beta gene, PKC beta I and beta II. The IM-9 cell line expressed 3-4-fold more PKC beta II protein than the BJA-B cell line, whereas the BJA-B cell line expressed 2-3-fold more PKC beta I protein. This differential expression was found to be regulated at the mRNA level. Comparison of PKC beta I and beta II messages in poly(A)+ mRNA and total cellular RNA revealed that selective polyadenylation was not involved. The messages for PKC beta I and beta II had comparable half-lives in both cell lines, ruling out differential message stability. In addition, similar ratios of PKC beta I and beta II messages in cytosolic and nuclear fractions suggested that differential mRNA transport was not involved. In the IM-9 cell line, the predominance of mature PKC beta II message as well as that of a larger message spliced to PKC beta II provided evidence that the differential expression of PKC beta II was regulated at the level of mRNA splicing. In the BJA-B cell line, equal amounts of mature PKC beta I and beta II message and the absence of the larger message suggested that the splicing of the PKC beta gene product can be regulated to produce altered ratios of PKC beta I and beta II. Implications of these studies on the differential expression of PKC isoenzymes and their roles in biology are discussed.
Journal of Biological Chemistry 06/1993; 268(14):10627-35. · 4.77 Impact Factor
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ABSTRACT: Cis-unsaturated fatty acids activate soluble protein kinase C (PKC) in vitro and in intact platelets. The following studies were conducted to determine the effects of oleate on individual isoenzymes of PKC in human platelets. Human platelets were found to contain predominantly PKC alpha, beta I, beta II, and delta with minor immunoreactivity for PKC epsilon, zeta, and eta. In intact platelets, sodium oleate caused a time-dependent redistribution of PKC alpha, beta II, and delta from cytosol to membrane fractions with little effects on PKC beta I. On the other hand, PMA and thrombin induced translocation of all four isoenzymes of PKC. In vitro, oleate partially activated (50% of Vmax) purified calcium-dependent PKC (alpha, beta I, and beta II) with an EC50 of 50 microM whereas it fully activated (100% of Vmax) purified calcium-independent PKC (predominantly delta) with an EC50 of 5 microM. The selective effects of oleate on PKC isoenzymes were investigated in platelet cytosol which contains endogenous PKC and its physiologic substrates. Under these conditions, oleate potently activated calcium-independent PKC causing the phosphorylation of the 40-kDa substrate. Activation of calcium-dependent isoforms occurred only at higher concentrations of oleate. Thus, oleate activates multiple isoenzymes of PKC with predominant effects on calcium-independent PKC.
Journal of Biological Chemistry 04/1993; 268(7):5063-8. · 4.77 Impact Factor
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ABSTRACT: The multidrug resistance (MDR) phenotype induces cross-resistance to many chemotherapeutic agents in cancer cells. Protein kinase C (PKC) has been implicated in the regulation of the MDR phenotype. In order to determine the role of specific PKC isoenzymes in regulating the MDR phenotype, the expression and activity of PKC isoenzymes in the human breast cancer cell line, MCF-7-WT, and an MDR subline, MCF-7-MDR, were examined. The MDR phenotype was associated with a 10-fold increase in calcium-dependent PKC activity as well as a 10-fold decrease in calcium-independent activity was due to a selective increase in the activity was due to a selective increase in the expression of PKC alpha as determined by Western blot analysis and hydroxylapatite chromatography. This increase in expression of PKC alpha was regulated at the message level as demonstrated by Northern blot analysis. The decrease in calcium-independent activity was caused by a decrease in the expression of PCK delta and epsilon. The significance of the increase in PKC alpha expression was then demonstrated by a commensurate 11-fold increase in the basal and stimulated phosphorylation of the myristolated alanine-rich C kinase substrate. Phosphorylation of P-glycoprotein, the cellular mediator of the MDR phenotype, was increased > 20-fold in the unstimulated MCF-7-MDR cell line and its phosphorylation was further increased 2-fold in response to phorbol 12-myristate 13-acetate. These changes paralleled the increases in P-glycoprotein pump function and the MDR phenotype underscoring the role for PKC alpha in regulating P-glycoprotein phosphorylation and function.
Journal of Biological Chemistry 01/1993; 268(1):658-64. · 4.77 Impact Factor
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ABSTRACT: Sphingosine displays multiple biochemical and biological effects, in particular inhibition and activation of protein kinases. To determine the predominant interaction of sphingosine with cellular kinases, the effects of sphingosine on endogenous protein phosphorylation in Jurkat T lymphoblastic cells were investigated in vitro. Sphingosine was found to cause prominent phosphorylation of a number of cytosolic proteins ranging in molecular mass from 18 to 165 kDa. Phosphorylation was calcium-independent. Phosphorylation of substrates was increased in response to concentrations of sphingosine as low as 10 microM and peaked at concentrations of 20-200 microM. Multiple lines of evidence suggested that sphingosine activated more than one protein kinase: 1) the concentration dependence on sphingosine differed from substrate to substrate, 2) phosphorylation of one group of substrates required ATP as the phosphate donor, whereas a second group showed no preference between ATP and GTP, and 3) phosphorylation of some substrates was inhibited by heparin, whereas other substrates were resistant. Activation of these kinases demonstrated a very specific requirement for D-erythro-sphingoid bases. DL-erythro-dihydrosphingosine was partially active, whereas DL-threo-dihydrosphingosine was not. Other related molecules such as stearylamine, sphingomyelin, and C2-ceramide were not active. Sphingosine-activated kinase(s) were distinct from protein kinase C, cyclic nucleotide-activated kinases, and calcium-dependent kinases. These observations demonstrate the existence of multiple sphingosine-activated protein kinases with high specificity for D-erythro-sphingosine, suggesting physiologic regulation of protein phosphorylation by sphingosine.
Journal of Biological Chemistry 08/1992; 267(21):15246-51. · 4.77 Impact Factor
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ABSTRACT: Polyclonal isoenzyme-specific antisera were developed against four calcium-independent protein kinase C (PKC) isoenzymes (delta, epsilon, epsilon', and zeta) as well as the calcium-dependent isoforms (alpha, beta I, beta II, and gamma). These antisera showed high specificities, high titers, and high binding affinities (3-370 nM) for the peptide antigens to which they were raised. Each antiserum detected a species of the predicted molecular weight by Western blot that could be blocked with the immunizing peptide. PKC was sequentially purified from rat brain, and the calcium-dependent forms were finally resolved by hydroxyapatite chromatography. Peak I reacted exclusively with antisera to PKC gamma, peak II with PKC beta I and -beta II, and peak III with PKC alpha. These same fractions, however, were devoid of immunoreactivity for the calcium-independent isoenzymes. The PKC isoenzymes demonstrated a distinctive tissue distribution when evaluated by Western blot and immunocytochemistry. PCK delta was present in brain, heart, spleen, lung, liver, ovary, pancreas, and adrenal tissues. PKC epsilon was present in brain, kidney, and pancreas, whereas PKC epsilon' was present predominantly in brain. PKC zeta was present in most tissues, particularly the lung, brain, and liver. Both PKC delta and PKC zeta showed some heterogeneity of size among the different tissues. PKC alpha was present in all organs and tissues examined. PKC beta I and -beta II were present in greatest amount in brain and spleen. Although the brain contained the most PKC gamma immunoreactivity, some immunostaining was also seen in adrenal tissue. These studies provide the first evidence of selective organ and tissue distributions of the calcium-independent PKC isoenzymes.
The Journal of Cell Biology 05/1992; 117(1):121-33. · 10.26 Impact Factor
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ABSTRACT: Sodium oleate is able to activate soluble protein kinase C (Murakami, K., Chan, S. Y., and Routtenberg, A. (1986) J. Biol. Chem. 261, 15424-15429) but is unable to activate membrane-bound enzyme (El Touny, S., Khan, W., and Hannun, Y. (1990) J. Biol. Chem. 265, 16437-16443). Because physiologic interactions of fatty acids with protein kinase C occur in the presence of membranes, the following studies were conducted to evaluate the effects of surfaces (detergent micelles or platelet membranes) on the activation of protein kinase C by oleate. At concentrations at or above the critical micellar concentration (CMC) of Triton X-100, oleate was present primarily in Triton X-100/oleate-mixed micelles, as determined by gel permeation chromatography and equilibrium dialysis binding studies. At concentrations slightly below the CMC for Triton X-100, the presence of oleate caused the formation of a limited number of mixed micelles. Studies of the dose-dependent activation of purified platelet protein kinase C by sodium oleate in the presence of different concentrations of Triton X-100 indicated that only unbound oleate was able to activate protein kinase C. Platelet protein kinase C was resolved into two major isoenzymes (types II (beta) and III (alpha)) which displayed nearly identical interaction with oleate. Activation of protein kinase C by oleate in a physiologic setting employing platelet substrates and endogenous platelet protein kinase C was investigated. Oleate potently activated protein kinase C in the cytosolic compartment. In platelet homogenates as well as in a reconstituted platelet cytosol and membrane system, the dose dependence of protein kinase C on oleate showed a significant shift to the right. Approximately 30% of oleate was associated with platelet cytosol and 70% was associated with platelet membranes. Partitioning of oleate into the two platelet compartments showed little change with pH, temperature, or duration of incubation. When corrected for free oleate concentration, activation of protein kinase C by oleate showed identical dose dependence in cytosol and homogenate. Arachidonate, a potential physiologic activator of protein kinase C, showed similar behavior as oleate although only 30% of arachidonate partitioned into platelet membranes with the majority of arachidonate (70%) remaining in the cytosolic fraction.(ABSTRACT TRUNCATED AT 400 WORDS)
Journal of Biological Chemistry 03/1992; 267(6):3605-12. · 4.77 Impact Factor
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ABSTRACT: Using isoenzyme-specific antisera, protein kinase C (PKC) alpha and PKC delta were detected in total liver homogenate and in isolated nuclei. PKC beta I, beta II, epsilon, epsilon', and zeta were not detected. During liver regeneration, nuclear PKC alpha levels decreased while PKC delta levels increased. These studies demonstrate, for the first time, the presence of a calcium-independent PKC isoenzyme in liver nuclei and suggest that PKC alpha and PKC delta may have different roles in liver regeneration and cell proliferation.
Biochemical and Biophysical Research Communications 03/1992; 182(3):1333-9. · 2.48 Impact Factor
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ABSTRACT: Sphingosine is a naturally occurring long-chain amino diol with potent inhibitory activity against protein kinase C in vitro and in cell systems. The use of sphingosine as a pharmacological tool to probe the activity of protein kinase C has been hampered by its amphiphilicity, possible contamination of its commercial preparations, and the existence of other targets for its action. To address these problems, high-purity D-erythro-sphingosine was prepared and employed to develop an approach for the use of sphingosine as a pharmacological agent. The addition of synthetic D-erythro-sphingosine to intact human platelets resulted in quick uptake and preferential partitioning into the particulate fraction. It was rapidly metabolized by intact platelets, 60% being degraded within 1 min after addition. Sphingosine was found to be a potent inhibitor of gamma-thrombin-induced aggregation and secretion of washed human platelets. Multiple criteria indicated that this effect is probably mediated through the inhibition of protein kinase C: (1) sphingosine inhibited protein kinase C activity in intact platelets with a similar dose/response to its inhibition of platelet aggregation and secretion; (2) sphingosine inhibited phorbol binding to intact platelets under identical conditions and with a similar dose-dependence; (3) exogenous dioctanoylglycerol overcame sphingosine's inhibition of platelet activation. The effectiveness of sphingosine in inhibiting platelet activation was primarily determined by the ratio of sphingosine to total number of platelets. These data are discussed in relation to a general approach for the use of sphingosine and other parameters for determining biological activities of protein kinase C.
Biochemical Journal 10/1991; 278 ( Pt 2):387-92. · 4.90 Impact Factor
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ABSTRACT: Sphingosine has been shown to be a potent and specific inhibitor of protein kinase C in vitro and in cell systems including human platelets. Questions have been raised as to the validity of commercial sphingosine as a protein kinase C inhibitor and whether sphingosine or N,N-dimethylsphingosine is the active species. In the present study, we compared the effects of synthetic D-erythro-sphingosine, N,N-dimethylsphingosine and commercial sphingosine on purified protein kinase C in vitro and washed human platelets. These three compounds were found to be of high purity and well-defined structure based on [1H]NMR, FAB-mass Spectrometry, and TLC analysis. Both synthetic D-erythro-sphingosine and commercial sphingosine inhibited protein kinase C in vitro using vesicle as well as mixed micellar assays. N,N-dimethylsphingosine also significantly inhibited purified protein kinase C in vitro. Both preparations of sphingosine inhibited phosphorylation for 40 kD protein, a known substrate of protein kinase C in platelets. Similarly both sphingosine preparations inhibited aggregation and secretion of human platelets induced by 8 nM gamma-thrombin. These results indicate that sphingosine from commercial source, synthetic sphingosine and N,N-dimethylsphingosine are equipotent in inhibiting protein kinase C. These studies also validate the utility of sphingosine as a phamarcologic inhibitor of protein kinase C in vitro and in cell systems.
Biochemical and Biophysical Research Communications 11/1990; 172(2):683-91. · 2.48 Impact Factor
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Prostaglandins Leukotrienes and Essential Fatty Acids 52(2-3):129-35. · 3.37 Impact Factor
