Ceramide 1-phosphate, a novel phospholipid in human leukemia (HL-60) cells. Synthesis via ceramide from sphingomyelin

Prior studies demonstrated that conversion of sphingomyelin to ceramide via sphingomyelinase action resulted in the generation of free sphingoid bases and inactivation of protein kinase C in human leukemia (HL-60) cells (Kolesnick, R. N. (1989) J. Biol. Chem. 264, 7617-7623). The present studies define the novel phospholipid ceramide 1-phosphate in these cells and present evidence for formation of this compound by preferential utilization of ceramide derived from spingomyelin. A ceramide 1-phosphate standard, prepared enzymatically via diacylglycerol kinase, was utilized for localization. In cells labeled to equilibrium with 32Pi to label the head group of the molecule, the basal ceramide 1-phosphate level was 30 +/- 2 pmol/10(6) cells. Generation of ceramide via the use of exogenous sphingomyelinase resulted in time- and concentration-dependent formation of ceramide 1-phosphate. As little as 3.8 x 10(-5) units/ml was effective and a 3-fold increase was observed with a maximal concentration of 3.8 x 10(-2) units/ml; ED50 approximately 2 x 10(-4) units/ml. This effect was observed by 5 min and maximal at 30 min. Similarly, in cells labeled with [3H]serine to probe the sphingoid base backbone, the basal level of ceramide 1-phosphate was 39 +/- 5 pmol/10(6) and increased 2.5-fold with sphingomyelinase; ED 50 approximately 5 x 10(-5) units/ml. To determine the source of the phosphate moiety, studies were performed with cells short term labeled with 32Pi and resuspended in medium without radiolabel. Under these conditions, sphingomyelin was virtually unlabeled. Nevertheless, sphingomyelin (3.8 x 10(-2) units/ml) induced a 12-fold increase in radiolabel incorporation, suggesting ceramide 1-phosphate formation occurred via ceramide phosphorylation. This event appeared specific for ceramide derived from sphingomyelin since ceramide from glycosphingolipids was not converted to ceramide 1-phosphate. In sum, these studies demonstrate the novel phospholipid ceramide 1-phosphate in HL-60 cells and suggest the possibility that a path exists from sphingomyelin to ceramide 1-phosphate via the phosphorylation of ceramide.     

Ceramide is a competitive inhibitor of diacylglycerol kinase in vitro and in intact human leukemia (HL-60) cells.

Prior studies demonstrated that ceramide was phosphorylated by a novel Ca(2+)-dependent kinase distinct from diacylglycerol (DG) kinase in human myelogenous leukemia (HL-60) cells (Kolesnick, R. N., and Hemer, M. R. (1990) J. Biol. Chem. 265, 10900-10904). The present studies were initiated to determine whether mammalian DG kinase purified to homogeneity possessed phosphotransferase activity toward ceramide. A high molecular weight rat brain DG kinase demonstrated Mg(2+)-(but not Ca(2+)-) dependent DG kinase activity and did not phosphorylate ceramide in the presence of either cation. In contrast, ceramide served as a competitive inhibitor with an inhibition constant (Ki) 2-6-fold greater than the Km for DG. Inhibition was noncompetitive with respect to ATP and Mg2+. A cell-permeable ceramide, N-octanoyl sphingosine (C8-cer), was used to study effects of ceramide on DG kinase in intact HL-60 cells. C8-cer induced dose- and time-dependent increases in cellular DG levels. As little as 1 microM C8-cer increased DG from a basal level of 103 to 177 pmol.10(6) cells-1, and a maximal 2.9-fold elevation to 292 pmol.10(6) cells-1 occurred with 10 microM C8-cer. DG elevation was detected after 1 min, maximal by 7.5 min, and sustained for 30 min. The DG elevation was accompanied by a reduction in 32P incorporation in phosphatidic acid in cells short term-labeled with [32P]orthophosphoric acid, consistent with inhibition of DG kinase. In contrast, a similar elevation in the DG level induced by exogenous phospholipase C increased 32P incorporation into phosphatidic acid. C8-cer was not metabolized to sphingomyelin, indicating that DG was not generated through the phosphatidylcholine:ceramide cholinephosphotransferase reaction. DG elevation after C8-cer or phospholipase C treatment was sufficient to redistribute protein kinase C from cytosol to membrane. These findings provide evidence that ceramide may serve as a competitive inhibitor of DG kinase.     

Role of ceramide as a lipid mediator of 1 alpha,25-dihydroxyvitamin D3-induced HL-60 cell differentiation

The treatment of HL-60 myelocytic leukemia cells with 1 alpha,25-dihydroxyvitamin D3 (1,25-(OH)2D3) resulted in the activation of a neutral sphingomyelinase and in sphingomyelin turnover (Okazaki, T., Bell, R., and Hannun, Y. (1989) J. Biol. Chem. 264, 19076-19080). In this paper, the effects of 1,25-(OH)2D3 on the product of sphingomyelin hydrolysis, ceramide, and the possible function of ceramide as a lipid mediator of the effects of 1,25-(OH)2D3 on HL-60 cell differentiation were investigated. Treatment of HL-60 cells with 1,25-(OH)2D3 resulted in a time- and dose-dependent increase in ceramide mass levels. Ceramide levels peaked at 2 h following treatment of HL-60 cells with 100 nM 1,25-(OH)2D3 with an increase of 41% over base line. The mass of generated ceramide (13 +/- 2 pmol/nmol of phospholipid) agreed with the mass of hydrolyzed sphingomyelin (17 +/- 4 pmol/nmol of phospholipid). Cell-permeable ceramides with shorter N-acyl chains induced HL-60 cell differentiation at subthreshold concentrations of 1,25-(OH)2D3. Higher concentrations of cell-permeable ceramides potently induced HL-60 cell differentiation independent of 1,25-(OH)2D3. A 2-h exposure of HL-60 cells to N-acetyl-sphingosine was sufficient to cause differentiation. Morphologically, N-acetylsphingosine caused a similar monocytic differentiation of HL-60 cells as did 1,25-(OH)2D3. Exogenous ceramide was further metabolized to sphingomyelin and other sphingolipids, but no conversion to sphingosine was detected. Moreover, sphingosine and its analogs failed to affect monocytic differentiation of HL-60 cells in response to subthreshold 1,25-(OH)2D3, indicating that the effect of ceramide was independent of sphingosine generation. These studies demonstrate that ceramide is a lipid mediator that may transduce the action of 1,25-(OH)2D3 on HL-60 cell differentiation.     

Sphingomyelin turnover induced by vitamin D3 in HL-60 cells. Role in cell differentiation

Sphingolipid metabolism was examined in human promyelocytic leukemia HL-60 cells. Differentiation of HL-60 cells with 1 alpha, 25-dihydroxyvitamin D3 (vitamin D3; 100 nM) was accompanied by sphingomyelin turnover. Maximum turnover of [3H]choline-labeled sphingomyelin occurred 2 h following vitamin D3 treatment, with sphingomyelin levels decreasing to 77 +/- 6% of control and returning to base-line levels by 4 h. Ceramide and phosphorylcholine were concomitantly generated. Ceramide mass levels increased by 55% at 2 h following vitamin D3 treatment and returned to base-line levels by 4 h. The amount of phosphorylcholine produced equaled the amount of sphingomyelin hydrolyzed, suggesting the involvement of a sphingomyelinase. Vitamin D3 treatment resulted in a 90% increase in the activity of a neutral sphingomyelinase from HL-60 cells. The inferred role of sphingomyelin hydrolysis in the induction of cell differentiation was investigated using an exogenous sphingomyelinase. When a bacterial sphingomyelinase was added at concentrations that caused a similar degree of sphingomyelin hydrolysis as 100 nM vitamin D3, it enhanced the ability of subthreshold levels of vitamin D3 to induce HL-60 cell differentiation. This study demonstrates the existence of a "sphingomyelin cycle" in human cells. Such sphingolipid cycles (Hannun, Y., and Bell, R. (1989) Science 243, 500-507) may function in a signal transduction pathway and in cellular differentiation.     

The role of sphingomyelin cycle metabolites in transduction of signals of cell proliferation, differentiation and death

Sphingomyelin cycle metabolites ceramide, sphingosine and sphingosine 1-phosphate play an important role in cell proliferation, differentiation, reception, oncogenesis and apoptosis. Ceramide is an intracellular second messenger for apoptosis activating proteases and specific phosphatases. Sphingosine is an endogenous inhibitor of protein kinase C and has an inhibitory effect on many cell functions depending on the activity of this enzyme. On the other hand, sphingosine can activate other kinases depending on the concentration, cell type and nature of a stimulus and release Ca2+ from intracellular stores thereby regulating cell proliferation. Sphingosine induces apoptosis and its level is increased in cells as a result of action of apoptotic inducers. A phosphorylated product of sphingosine, sphingosine 1-phosphate, mediates the mitogenic signal, induces Ca2+ mobilization and protects cells from apoptosis resulting from elevation of ceramide. The quantitative levels of sphingomyelin metabolites in the cell determine the dynamic balance between the apoptotic and mitogenic signals.     

Evidence for a high activity of sphingomyelin biosynthesis by phosphocholine transfer from phosphatidylcholine to ceramides in lung lamellar bodies

Biosynthesis of sphingomyelin from ceramides was investigated in lung subcellular fractions by incubating a lyophilized mixture of albumin and subcellular fraction (0.1-0.2 mg of protein) coated with [acyl-14C]-ceramide and phosphatidyl[methyl-3H]choline in Tris-buffer. The lamellar-body-rich fraction exhibited the highest specific activity for sphingomyelin biosynthesis measured by 14C incorporation into sphingomyelins or by [3H]phosphocholine transfer from phosphatidylcholines. Plasma membranes formed the next most active fraction, followed by the 'smooth' and, then, the 'rough' endoplasmic reticulum. Sphingomyelin biosynthesis by lamellar bodies was optimum at pH 7.4 and was inhibited by sphingomyelins formed. Slight inhibitory effects were also observed with Mn2+, Ca2+ and lysophosphatidylcholine. Phosphocholine transfer from CDPcholine was not observed under the reaction conditions employed. Ceramide conversion and phosphocholine transfer increased with ceramide concentration to reach a maximum at about 0.06 mM. The highest conversion rate was observed when 18:1 ceramide was used as an acceptor. When 1-palmitoyl-2-oleoylphosphatidylcholine was the phosphocholine donor, the overall biosynthesis of sphingomyelin was much higher than when using dipalmitoylphosphatidylcholine. These results suggest the possible involvement of the studied reaction in the control of the degree of saturation of the surfactant phosphatidylcholine.     

Regulation and traffic of ceramide 1-phosphate produced by ceramide kinase: comparative analysis to glucosylceramide and sphingomyelin

Ceramide 1-phosphate (C1P) has been characterized as a sphingolipid that participates in cell signaling. Although C1P synthesis is thought to occur via phosphorylation of ceramide by ceramide kinase (CerK), the processes that regulate C1P formation and fate remain largely unknown. In this study we analyzed bone marrow-derived macrophages (BMDM) from CerK-null mice (Cerk(-/-)) and found significant levels of C1P, suggesting that previously unrecognized pathways may also lead to C1P formation. After these experiments we used an overexpression system, BMDM from Cerk(-/-) mice, and short-chain fluorescent ceramides to trace CerK-dependent formation of C1P. Because the ceramide analogs can also be converted to glucosylceramide (GlcCer) and sphingomyelin (SM), they allowed us to directly compare all three metabolites. We found that C1P produced by CerK is turned over rapidly when serum is removed or upon calcium chelation, whereas GlcCer and SM are stable under these conditions. We further demonstrated that ceramide must be transported to the Golgi complex to be phosphorylated by CerK. Inhibition of the ceramide transfer protein slowed down SM formation without decreasing C1P, suggesting an alternate route of ceramide transport. Other experiments indicated that, like GlcCer and SM, C1P traffics along the secretory pathway to reach the plasma membrane. Furthermore, in BMDM C1P was secreted more readily than was GlcCer or SM. Altogether, our results indicate that CerK is essential to C1P formation via phosphorylation of Cer, providing the first insights into mechanisms underlying ceramide access to CerK and C1P trafficking as well as clarifying C1P as a signaling entity.     

Increase of ceramide in adriamycin-induced HL-60 cell apoptosis: detection by a novel anti-ceramide antibody

We recently raised an IgM class of monoclonal antibody (Ab) for ceramide (NHCER-2), and examined its specificity and sensitivity. Enzyme-linked immunosorbent assay (ELISA) and thin-layer chromatography (TLC) showed that NHCER-2 recognized ceramides but not other sphingolipids such as sphingosine, sphinganine, sphingomyelin, sphingosine-1-phosphate, ceramide-1-phosphate, glucosylceramide and cerebroside. In addition, N-hexanoyl, N-octanoyl and N-palmitoylsphingosine were detected by NHCER-2, but N-acetylsphingosine and dihydroceramide were not. Densities of ceramide detected by NHCER-2 were proportional to the amounts of ceramide standard up to 250 ng. When various concentrations of adriamycin (ADR) was added to induce apoptosis, the amounts of ceramide detected by NHCER-2 time- and dose-dependently increased in apoptosis-sensitive HL-60 cells as well as by DGK assay, but not in apoptosis-resistant HL-60/ADR cells. After cell fractionation, ceramide levels judged not only by diacylglycerol kinase (DGK) assay but also by NHCER-2 were shown to increase in the microsomal and the nuclear fraction in apoptosis-sensitive cells, but not in apoptosis-resistant cells. Moreover, absolute amounts of ceramide determined by NHCER-2 were well correlated with those by DGK assay. These results suggest that increase of ceramide in the nuclear fraction as well as in the microsomal fraction may play a role in ADR-induced apoptosis and that a novel anti-ceramide Ab NHCER-2 could be beneficial to investigate changes of ceramide content in the cells.     

Sphingolipid-mediated apoptotic signaling pathways

Various sphingolipids are being viewed as bioactive molecules and/or second messengers. Among them, ceramide (or N-acylsphingosine) and sphingosine generally behave as pro-apoptotic mediators. Indeed, ceramide mediates the death signal initiated by numerous stress agents which either stimulate its de novo synthesis or activate sphingomyelinases that release ceramide from sphingomyelin. For instance, the early generation of ceramide promoted by TNF is mediated by a neutral sphingomyelinase the activity of which is regulated by the FAN adaptor protein, thereby controlling caspase activation and the cell death programme. In addition, the activity of this neutral sphingomyelinase is negatively modulated by caveolin, a major constituent of some membrane microdomains. The enzyme sphingosine kinase also plays a crucial role in apoptosis signalling by regulating the intracellular levels of two sphingolipids having opposite effects, namely the pro-apoptotic sphingosine and the anti-apoptotic sphingosine 1-phosphate molecule. Ceramide and sphingosine metabolism therefore appears as a pivotal regulatory pathway in the determination of cell fate.     

Subtype-specific translocation of the delta subtype of protein kinase C and its activation by tyrosine phosphorylation induced by ceramide in HeLa cells

We investigated the functional roles of ceramide, an intracellular lipid mediator, in cell signaling pathways by monitoring the intracellular movement of protein kinase C (PKC) subtypes fused to green fluorescent protein (GFP) in HeLa living cells. C(2)-ceramide but not C(2)-dihydroceramide induced translocation of delta PKC-GFP to the Golgi complex, while alpha PKC- and zeta PKC-GFP did not respond to ceramide. The Golgi-associated delta PKC-GFP induced by ceramide was further translocated to the plasma membrane by phorbol ester treatment. Ceramide itself accumulated to the Golgi complex where delta PKC was translocated by ceramide. Gamma interferon also induced the delta PKC-specific translocation from the cytoplasm to the Golgi complex via the activation of Janus kinase and Mg(2+)-dependent neutral sphingomyelinase. Photobleaching studies showed that ceramide does not evoke tight binding of delta PKC-GFP to the Golgi complex but induces the continuous association and dissociation of delta PKC with the Golgi complex. Ceramide inhibited the kinase activity of delta PKC-GFP in the presence of phosphatidylserine and diolein in vitro, while the kinase activity of delta PKC-GFP immunoprecipitated from ceramide-treated cells was increased. The immunoprecipitated delta PKC-GFP was tyrosine phosphorylated after ceramide treatment. Tyrosine kinase inhibitor abolished the ceramide-induced activation and tyrosine phosphorylation of delta PKC-GFP. These results suggested that gamma interferon stimulation followed by ceramide generation through Mg(2+)-dependent sphingomyelinase induced delta PKC-specific translocation to the Golgi complex and that translocation results in delta PKC activation through tyrosine phosphorylation of the enzyme.     

Manganese-phospholipid-stimulated protein kinase activity of human leukemic cells

A protein kinase system with unusual characteristics was noted in extracts of HL-60 cells using endogenous proteins as substrates. This system exhibited a cation preference for manganese at an optimal concentration of 0.5 mM. Moderate activity was detectable with magnesium at an optimal concentration of 5.0 mM, but calcium was inactive. Activity was markedly stimulated by phospholipid, with phosphatidylglycerol and phosphatidylinositol exhibiting greater activity than phosphatidylserine. In isolated plasma membranes, the major substrate of this system was a 73-kDa protein, while cytoplasmic extracts exhibited larger amounts of a 42-kDa substrate. 73-kDa phosphorylating activity of membranes was comparably active at 0 and 31 degrees C, although in cytosol activity was greater at 31 degrees C. No 73-kDa protein phosphorylation was observed in the presence of Ca2+, Mg2+, and phosphatidylserine. Phosphoamino acid analysis of the 73-kD band revealed phosphothreonine and phosphoserine. The 42-kDa substrate was distinguishable from actin by two-dimensional gel electrophoresis, which disclosed that both major substrates were highly basic in the isoelectric focusing dimension. Protamine and histones (H2B greater than H1 greater than H3) exhibited phospholipid-stimulated phosphorylation in the presence of Mn2+, but phosvitin, casein, and vinculin were not substrates. High levels of phosphorylative activity involving the 73-kDa substrate were noted in nuclear extracts. Complex patterns of increase of this activity were noted in both cytosol and nuclear extracts following induction of differentiation with dimethyl sulfoxide, retinoic acid, or phorbol 12-myristate 13-acetate. This study thus demonstrated the presence of a previously undescribed type of protein kinase activity which exhibited alterations during leukemic cellular differentiation.     

Identification of sphingomyelin turnover as an effector mechanism for the action of tumor necrosis factor alpha and gamma-interferon. Specific role in cell differentiation.

The biochemical signaling mechanisms involved in transducing the effects of tumor necrosis factor alpha (TNF alpha) and gamma-interferon (gamma-IFN) on leukemia cell differentiation are poorly defined. Recent studies established the existence of a sphingomyelin cycle that operates in response to the action of vitamin D3 on HL-60 cells and that may transduce the effects of vitamin D3 on cell differentiation (Okazaki, T., Bell, R., and Hannun, Y. (1989) J. Biol. Chem. 264, 19076-19080). The effects of TNF alpha and gamma-IFN on sphingomyelin turnover were determined, and the specificity and role of sphingomyelin hydrolysis in HL-60 human promyelocytic leukemia cells with 20% hydrolysis of sphingomyelin at 15 min, 40% hydrolysis at 30-60 min, and return to base line at 2 h. The hydrolyzed sphingomyelin (18 pmol/nmol total phospholipid) was accompanied by the concomitant generation of ceramide (11.2 pmol/nmol total phospholipid). gamma-IFN also caused reversible hydrolysis of sphingomyelin with onset at 1 h and peak effect at 2 h. This sphingomyelin cycle appeared to be specific to the monocytic pathway of HL-60 differentiation, since it was not activated by retinoic acid or dibutyryl cAMP, inducers of granulocytic differentiation, nor with phorbol myristate acetate, an inducer of macrophage-like differentiation. Addition of synthetic ceramide or bacterial sphingomyelinase induced monocytic differentiation of HL-60 cells. Cell-permeable ceramide also caused prompt down-regulation of mRNA for the c-myc protooncogene. The time course of c-myc down-regulation was consistent with the action of ceramide as the mediator of TNF alpha action. These results suggest that sphingomyelin turnover may be an important signaling mechanism transducing the actions of TNF alpha and gamma-IFN with specific function in cell differentiation.     

The activity of sphingomyelin cycle enzymes and concentration of sphingomyelin degradation products in rat liver in dynamics of acute toxic hepatitis

Activity of key enzymes of the sphingomyelin cycle and the content of its components (sphingomyelin, ceramide and sphingosine-1-phosphate) have been studied in livers of rats in dynamics of acute toxic hepatitis induced by subcutaneous administration of oil solution of CCl₄. Sphingomyelinase activity significantly increased already on early terms and remained increased over the whole period of observation. Ceramidase activity insignificantly differed from the control level. The levels of sphingomyelin and sphingosine-1-phosphate did not undergo marked changes while ceramide content significantly increased. The balance between liver content of ceramide (proapoptotic) and sphingosine-1-phosphate (the antiapoptotic factor) was shifted towards ceramide over the whole observation period. In sphingomyelin molecules there was a significant decrease in the content of the fatty acids C_18:1 and C_22:2, while in ceramide molecules and sphingosine-1-phosphate only the fatty acid C_22:2 changed. In spite of a significant decrease in the content of some unsaturated fatty acids, calculated unsaturation coefficients of the fatty acid component of the sphingomyelin cycle metabolites insignificantly differed from control. Taking into consideration literature data our results suggest involvement of ceramide-mediated apoptosis in the pathogenesis of acute toxic hepatitis. Elimination of damaged hepatocytes permits realization of repair processes and promotes optimization of cellular community in the liver.     

A conserved cysteine motif is critical for rice ceramide kinase activity and function

Background

Ceramide kinase (CERK) is a key regulator of cell survival in dicotyledonous plants and animals. Much less is known about the roles of CERK and ceramides in mediating cellular processes in monocot plants. Here, we report the characterization of a ceramide kinase, OsCERK, from rice (Oryza sativa spp. Japonica cv. Nipponbare) and investigate the effects of ceramides on rice cell viability.

Principal Findings

OsCERK can complement the Arabidopsis CERK mutant acd5. Recombinant OsCERK has ceramide kinase activity with Michaelis-Menten kinetics and optimal activity at 7.0 pH and 40°C. Mg²⁺ activates OsCERK in a concentration-dependent manner. Importantly, a CXXXCXXC motif, conserved in all ceramide kinases and important for the activity of the human enzyme, is critical for OsCERK enzyme activity and in planta function. In a rice protoplast system, inhibition of CERK leads to cell death and the ratio of added ceramide and ceramide-1-phosphate, CERK''s substrate and product, respectively, influences cell survival. Ceramide-induced rice cell death has apoptotic features and is an active process that requires both de novo protein synthesis and phosphorylation, respectively. Finally, mitochondria membrane potential loss previously associated with ceramide-induced cell death in Arabidopsis was also found in rice, but it occurred with different timing.

Conclusions

OsCERK is a bona fide ceramide kinase with a functionally and evolutionarily conserved Cys-rich motif that plays an important role in modulating cell fate in plants. The vital function of the conserved motif in both human and rice CERKs suggests that the biochemical mechanism of CERKs is similar in animals and plants. Furthermore, ceramides induce cell death with similar features in monocot and dicot plants.     

A fluorescent plate reader assay for ceramide kinase

Ceramide kinase and its product ceramide 1-phosphate have been implicated in cellular proliferation and survival, activation of cytosolic phospholipase A(2), mast cell degranulation, and phagocytosis. Current assays for ceramide kinase activity employ [(32)P]ATP, with separation of labeled product from excess ATP by organic extraction and thin-layer chromatography. We have developed a fluorescent plate reader assay for ceramide kinase that uses commercially available C6-NBD ceramide (N-{6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]hexanoyl}-D-erythro-sphingosine). Our assay is based on the differential partitioning of substrate and product following a single chloroform/methanol extraction. The product, which partitions into the aqueous phase at physiological pH, is quantitated directly in a plate reader. The substrate may be delivered using either fatty acid-free albumin or detergent/lipid mixed micelles, and we have found that the use of albumin rather than detergent micelles allows one to detect lipid interactions with the enzyme that might otherwise go unnoticed. Our method is useful for assaying ceramide kinase activity both in vitro and in cultured cells, and it offers several advantages over the conventional assay, including greater speed, the ability to run a larger number of assay replicates at one time, and the elimination of environmental and safety issues associated with the use of radioactive materials.     

Interorganelle trafficking of ceramide is regulated by phosphorylation-dependent cooperativity between the PH and START domains of CERT

The synthesis and transport of lipids are essential events for membrane biogenesis. However, little is known about how intracellular trafficking of lipids is regulated. Ceramide is synthesized at the endoplasmic reticulum (ER) and transported by the ceramide transfer protein CERT to the Golgi apparatus, where it is converted to sphingomyelin. CERT has a phosphoinositide-binding pleckstrin homology (PH) domain for Golgi-targeting and a lipid transfer START domain for intermembrane transfer of ceramide. We here show that CERT receives multiple phosphorylations at a serine-repeat motif, a possibe site for casein kinase I, and that the phosphorylation down-regulates the ER-to-Golgi transport of ceramide. In vitro assays show that the phosphorylation induces an autoinhibitory interaction between the PH and START domains and consequently inactivates both the phosphoinositide binding and ceramide transfer activities of CERT. Loss of sphingomyelin and cholesterol from cells causes dephosphorylation of CERT to activate it. The cooperative control of functionally distinct domains of CERT is a novel molecular event to regulate the intracellular trafficking of ceramide.     

Disparate effects of activators of protein kinase C on HL-60 promyelocytic leukemia cell differentiation

In previously published studies (Kreutter, D., Caldwell, A. B., and Morin, M. J. (1985) J. Biol. Chem. 260, 5979-5984), we demonstrated that the activation of the calcium- and phospholipid-dependent protein kinase C by phorbol esters was dissociable from the induction of monocytic differentiation by these agents in HL-60 promyelocytic leukemia cells. We have now compared the effects of two related diterpenes (mezerein and 12-O-tetradecanoylphorbol-13-acetate) and two cell-permeable diacylglycerols (1-oleoyl-2-acetoylglycerol and 1,2-dioctanoylglycerol) on the induction of differentiation in HL-60 cells. Each of these agents activated protein kinase C in vitro and stimulated the phosphorylation of a number of identical proteins in intact HL-60 cells. Exposure to either of the diterpenes at nanomolar concentrations resulted in an inhibition of cell growth and the induction of qualitatively distinct types of monocytic maturation in HL-60 cells. Conversely, neither of the two diacylglycerols was found to be a potent or efficacious inducer of differentiation, as measured by increases in cell adhesion, nonspecific esterase activity, or phagocytosis, even at growth-inhibitory concentrations. However, concurrent exposure of HL-60 cells to both 1,2-dioctanoylglycerol and the calcium ionophore A23187, at concentrations which were without maturational activity when used separately, resulted in measurable increases in both protein phosphorylation and in the fraction of cells expressing a differentiated phenotype. Taken together, these results suggest that specific biochemical effects associated with 12-O-tetradecanoylphorbol-13-acetate, in addition to the activation of protein kinase C, may be important determinants for the induction of leukemia cell differentiation.     

Synaptic vesicle ceramide kinase. A calcium-stimulated lipid kinase that co-purifies with brain synaptic vesicles

Much current work on the mechanism of neurosecretion has focused on proteins specific to neural secretory vesicles (synaptic vesicles). We report a calcium-stimulated lipid kinase that co-purifies with rat brain synaptic vesicles. This enzyme activity is found only in membrane fractions that contain synaptic vesicle markers. Based on identification of the lipid product as ceramide 1-phosphate and on the finding that ceramide kinase activity co-purifies with synaptic vesicles, the enzyme is proposed to be a ceramide kinase. Kinase activity is stimulated by micromolar concentrations of calcium. Calcium increases the apparent Vmax of the reaction with little effect on the Km for ceramide. The vesicular localization of this enzyme, the requirement for ATP, and the stimulation of enzyme activity by micromolar calcium suggest that ceramide phosphorylation may be associated with neurotransmitter release.