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We report the identification and characterization of a novel lipid kinase that phosphorylates multiple substrates. This enzyme, which we term MuLK for multi-substrate lipid kinase, does not belong to a previously described lipid kinase family. MuLK has orthologs in many organisms and is broadly expressed in human tissues. Although predicted to be a soluble protein, MuLK co-fractionates with membranes and localizes to an internal membrane compartment. Recombinant MuLK phosphorylates diacylglycerol, ceramide, and 1-acylglycerol but not sphingosine. Although its affinity for diacylglycerol and ceramide are similar, MuLK exhibits a higher V(max) toward diacylglycerol in vitro, consistent with it acting primarily as a diacylglycerol kinase. MuLK activity was inhibited by sphingosine and enhanced by cardiolipin. It was stimulated by calcium when magnesium concentrations were low and inhibited by calcium when magnesium concentrations were high. The effects of charged lipids and cations on MuLK activity in vitro suggest that its activity in vivo is tightly regulated by cellular conditions.

The serum-derived phospholipid growth factor, lysophosphatidate (LPA), activates cells through the EDG family of G protein-coupled receptors. The present study investigated mechanisms by which dephosphorylation of exogenous LPA by lipid phosphate phosphatase-1 (LPP-1) controls cell signaling. Overexpressing LPP-1 decreased the net specific cell association of LPA with Rat2 fibroblasts by approximately 50% at 37 degrees C when less than 10% of LPA was dephosphorylated. This attenuated cell activation as indicated by diminished responses, including cAMP, Ca(2+), activation of phospholipase D and ERK, DNA synthesis, and cell division. Conversely, decreasing LPP-1 expression increased net LPA association, ERK stimulation, and DNA synthesis. Whereas changing LPP-1 expression did not alter the apparent K(d) and B(max) for LPA binding at 4 degrees C, increasing Ca(2+) from 0 to 50 micrometer increased the K(d) from 40 to 900 nm. Decreasing extracellular Ca(2+) from 1.8 mm to 10 micrometer increased LPA binding by 20-fold, shifting the threshold for ERK activation to the nanomolar range. Hence the Ca(2+) dependence of the apparent K(d) values explains the long-standing discrepancy of why micromolar LPA is often needed to activate cells at physiological Ca(2+) levels. In addition, the work demonstrates that LPP-1 can regulate specific LPA association with cells without significantly depleting bulk LPA concentrations in the extracellular medium. This identifies a novel mechanism for controlling EDG-2 receptor activation.

Mammalian lipid phosphate phosphatases (LPPs, or Type 2 phosphatidate phosphohydrolases) constitute a family of enzymes that belongs to a phosphatase superfamily. The LPPs dephosphorylate a variety of bioactive lipid phosphates including phosphatidate, lysophosphatidate, sphingosine 1-phosphate, and ceramide 1-phosphate. Mouse LPP-1 was stably expressed in rat2 fibroblasts to determine its structural and functional properties. Transduced cells showed increased dephosphorylation of exogenous lysophosphatidate. This result is compatible with mutational studies that show the active site of LPP-1 to be located on the external surface of the plasma membrane. Elevated LPP-1 activity attenuated the ability of lysophosphatidate to stimulate mitogen-activated protein kinase (ERK1 and 2) activities and DNA synthesis. It is concluded that one function of LPP-1 is to dephosphorylate exogenous lysophosphatidate, thereby attenuating cell signaling through endothelial cell differentiation gene (EDG) receptors.

This article describes the regulation of cell signaling by lipid phosphate phosphatases (LPPs) that control the conversion of bioactive lipid phosphates to their dephosphorylated counterparts. A structural model of the LPPs, that were previously called Type 2 phosphatidate phosphatases, is described. LPPs are characterized by having no Mg(2+) requirement and their insensitivity to inhibition by N-ethylmaleimide. The LPPs have six putative transmembrane domains and three highly conserved domains that define a phosphatase superfamily. The conserved domains are juxtaposed to the proposed membrane spanning domains such that they probably form the active sites of the phosphatases. It is predicted that the active sites of the LPPs are exposed at the cell surface or on the luminal surface of intracellular organelles, such as Golgi or the endoplasmic reticulum, depending where various LPPs are expressed. LPPs could attenuate cell activation by dephosphorylating bioactive lipid phosphate esters such as phosphatidate, lysophosphatidate, sphingosine 1-phosphate and ceramide 1-phosphate. In so doing, the LPPs could generate alternative signals from diacylglycerol, sphingosine and ceramide. The LPPs might help to modulate cell signaling by the phospholipase D pathway. For example, phosphatidate generated within the cell by phospholipase D could be converted by an LPP to diacylglycerol. This should change the relative balance of signaling by these two lipids. Another possible function of the LPPs relates to the secretion of lysophosphatidate and sphingosine 1-phosphate by activated platelets and other cells. These exogenous lipids activate phospholipid growth factor receptors on the surface of cells. LPP activities could attenuate cell activation by lysophosphatidate and sphingosine 1-phosphate through their respective receptors.

Lipid phosphate phosphohydrolase (LPP)-1 cDNA was cloned from a rat liver cDNA library. It codes for a 32-kDa protein that shares 87 and 82% amino acid sequence identities with putative products of murine and human LPP-1 cDNAs, respectively. Membrane fractions of rat2 fibroblasts that stably expressed mouse or rat LPP-1 exhibited 3.1-3. 6-fold higher specific activities for phosphatidate dephosphorylation compared with vector controls. Increases in the dephosphorylation of lysophosphatidate, ceramide 1-phosphate, sphingosine 1-phosphate and diacylglycerol pyrophosphate were similar to those for phosphatidate. Rat2 fibroblasts expressing mouse LPP-1 cDNA showed 1.6-2.3-fold increases in the hydrolysis of exogenous lysophosphatidate, phosphatidate and ceramide 1-phosphate compared with vector control cells. Recombinant LPP-1 was located partially in plasma membranes with its C-terminus on the cytosolic surface. Lysophosphatidate dephosphorylation was inhibited by extracellular Ca2+ and this inhibition was diminished by extracellular Mg2+. Changing intracellular Ca2+ concentrations did not alter exogenous lysophosphatidate dephosphorylation significantly. Permeabilized fibroblasts showed relatively little latency for the dephosphorylation of exogenous lysophosphatidate. LPP-1 expression decreased the activation of mitogen-activated protein kinase and DNA synthesis by exogenous lysophosphatidate. The product of LPP-1 cDNA is concluded to act partly to degrade exogenous lysophosphatidate and thereby regulate its effects on cell signalling.

Recent studies indicate that the metabolism of diacylglycerol pyrophosphate (DGPP) is involved in a novel lipid signaling pathway. DGPP phosphatases (DGPP phosphohydrolase) from Saccharomyces cerevisiae and Escherichia coli catalyze the dephosphorylation of DGPP to yield phosphatidate (PA) and then catalyze the dephosphorylation of PA to yield diacylglycerol. We demonstrated that the Mg2+-independent form of PA phosphatase (PA phosphohydrolase, PAP2) purified from rat liver catalyzed the dephosphorylation of DGPP. This reaction was Mg2+-independent, insensitive to inhibition by N-ethylmaleimide and bromoenol lactone, and inhibited by Mn2+ ions. PAP2 exhibited a high affinity for DGPP (Km = 0.04 mol %). The specificity constant (Vmax/Km) for DGPP was 1. 3-fold higher than that of PA. DGPP inhibited the ability of PAP2 to dephosphorylate PA, and PA inhibited the dephosphorylation of DGPP. Like rat liver PAP2, the Mg2+-independent PA phosphatase activity of DGPP phosphatase purified from S. cerevisiae was inhibited by lyso-PA, sphingosine 1-phosphate, and ceramide 1-phosphate. Mouse PAP2 showed homology to DGPP phosphatases from S. cerevisiae and E. coli, especially in localized regions that constitute a novel phosphatase sequence motif. Collectively, our work indicated that rat liver PAP2 is a member of a phosphatase family that includes DGPP phosphatases from S. cerevisiae and E. coli. We propose a model in which the phosphatase activities of rat liver PAP2 and the DGPP phosphatase of S. cerevisiae regulate the cellular levels of DGPP, PA, and diacylglycerol.

A Mg2+-independent phosphatidate phosphohydrolase was purified from rat liver plasma membranes in two distinct forms, an anionic protein and a cationic protein. Both forms of the enzyme dephosphorylated phosphatidate, ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate. When assayed at a constant molar ratio of lipid to Triton X-100 of 1:500, the apparent Km values of the anionic phosphohydrolase for the lipid substrates was 3.5, 1.9, 0.4, and 4.0 microM, respectively. The relative catalytic efficiency of the enzyme for phosphatidate, ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate was 0.16, 0.14, 0.48, and 0.04 liter (min x mg)-1, respectively. The hydrolysis of phosphatidate was inhibited competitively by ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate. The Ki(app) values were 5.5, 5.9, and 4.0 microM, respectively. The hydrolysis of phosphatidate by the phosphohydrolase conformed to a surface dilution kinetic model. It is concluded that the enzyme is a lipid phosphomonoesterase that could modify the balance of phosphatidate, ceramide 1-phosphate, lysophosphatidate, and sphingosine 1-phosphate relative to diacylglycerol, ceramide, monoacylglycerol, and sphingosine, respectively. The enzyme could thus play an important role in regulating cell activation and signal transduction.

A Mg(2+)-independent and N-ethylmaleimide-insensitive phosphatidate phosphohydrolase (PAP-2) has been identified in the plasma membrane of cells and it has been purified. The enzyme is a multi-functional phosphohydrolase that can dephosphorylate phosphatidate, lysophosphatidate, sphingosine 1-phosphate and ceramide 1-phosphate and these substrates are competitive inhibitors of the reaction. The action of PAP-2 could terminate signalling by these bioactive lipids and at the same time generates compounds such as diacylglycerol, sphingosine and ceramide which are also potent signalling molecules. In relation to phosphatidate metabolism, sphingosine (or sphingosine 1-phosphate) stimulates phospholipase D and thus the formation of phosphatidate. At the same time sphingosine inhibits PAP-2 activity thus further increasing phosphatidate concentrations. By contrast, ceramides inhibit the activation of phospholipase D by a wide variety of agonists and increase the dephosphorylation of phosphatidate, lysophosphatidate, sphingosine 1-phosphate and ceramide 1-phosphate. These actions demonstrate "cross-talk' between the glycerolipid and sphingolipid signalling pathways and the involvement of PAP-2 in modifying the balance of the bioactive lipids generated by these pathways during cell activation.

Hydrolysis of phosphatidylcholine via receptor-mediated stimulation of phospholipase D produces phosphatidate that can be converted to lysophosphatidate and diacylglycerol. Diacylglycerol is an activator of protein kinase C, whereas phosphatidate and lysophosphatidate stimulate tyrosine kinases and activate the Ras-Raf-mitogen-activated protein kinase pathway. These three lipids can stimulate cell division. Conversely, activation of sphingomyelinase by agonists (e.g., tumor necrosis factor-alpha) causes ceramide production that inhibits cell division and produces apoptosis. If ceramides are metabolized to sphingosine and sphingosine 1-phosphate, then these lipids can stimulate phospholipase D and are also mitogenic. By contrast, ceramides inhibit the activation of phospholipase D by decreasing its interaction with the G-proteins, ARF and Rho, which are necessary for its activation. In whole cells, ceramides also stimulate the degradation of phosphatidate, lysophosphatidate, ceramide 1-phosphate, and sphingosine 1-phosphate through a multifunctional phosphohydrolase (the Mg(2+)-independent phosphatidate phosphohydrolase), whereas sphingosine inhibits phosphatidate phosphohydrolase. Tumor necrosis factor-alpha causes insulin resistance, which may be partly explained by ceramide production. Cell-permeable ceramides decrease insulin-stimulated glucose uptake in 3T3-L1 adipocytes after 2-24 h, whereas they stimulate basal glucose uptake. These effects do not depend on decreased tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 or the interaction of insulin receptor substrate-1 with phosphatidylinositol 3-kinase. They appear to rely on the differential effects of ceramides on the translocation of GLUT1-and GLUT4-containing vesicles. It is concluded that there is a significant interaction and "cross-talk" between the sphingolipid and glycerolipid pathways that modifies signal transduction to control vesicle movement, cell division, and cell death.

C2- and C6-ceramides (N-acetylsphingosine and N-hexanoylsphingosine, respectively) abolished the stimulation of DNA synthesis by sphingosine 1-phosphate in rat fibroblasts. This inhibition by ceramide was partially prevented by insulin. C2-ceramide did not alter the stimulation of DNA synthesis by insulin and decreased the sphingosine-induced stimulation by only 16%. The ceramides did not significantly modify the actions of sphingosine or sphingosine 1-phosphate in decreasing cAMP concentrations. C2- and C6-ceramides blocked the activation of phospholipase D by sphingosine 1-phosphate, and this inhibition was not affected by insulin. Okadaic acid decreased the activation of phospholipase D by sphingosine 1-phosphate and did not reverse the inhibitory effect of C2-ceramide on this activation. Therefore, this effect of C2-ceramide is unlikely to involve the stimulation of phosphoprotein phosphatase activity. Sphingosine did not activate phospholipase D activity significantly after 10 min. C2-ceramide stimulated the conversion of exogenous [3H]sphingosine 1-phosphate to sphingosine and ceramide in fibroblasts. Ceramides can inhibit some effects of sphingosine 1-phosphate by stimulating its degradation via a phosphohydrolase that also hydrolyzes phosphatidate. Furthermore, C2- and C6-ceramides stimulated ceramide production from endogenous lipids, and this could propagate the intracellular signal. This work demonstrates that controlling the production of ceramide versus sphingosine and sphingosine 1-phosphate after sphingomyelinase activation could have profound effects on signal transduction.

An N-ethylmaleimide-insensitive phosphatidate phosphohydrolase, which also hydrolyzes lysophosphatidate, was isolated from the plasma membranes of rat liver. The specific activity of an anionic form of the enzyme (53 kDa, pI < 4) was increased 2700-fold. A cationic form of enzyme (51 kDa, pI = 9) was purified to homogeneity, but the -fold purification was low because the activity of the highly purified enzyme was unstable. Immunoprecipitating antibodies raised against the homogeneous protein confirmed the identity of the cationic protein as the phosphohydrolase and were used to identify the anionic enzyme. Both forms are integral membrane glycoproteins that were converted to 28-kDa proteins upon treatment with N-glycanase F. Treatment of the anionic form with neuraminidase allowed it to be purified in the same manner as the cationic enzyme and yielded an immunoreactive protein with a molecular mass identical to the cationic protein. Thus, the two ionic forms most likely represent different sialated states of protein. An immunoreactive 51-53-kDa protein was detected in rat liver, heart, kidney, skeletal muscle, testis, and brain. Little immunoreactive 51-53-kDa protein was detected in rat thymus, spleen, adipose, or lung tissue. This work provides the tools for determining the regulation and function of the phosphatidate phosphohydrolase in signal transduction and cell activation.

The activity of N-ethylmaleimide-insensitive phosphatidate phosphohydrolase (PAP-2) was characterized in control, ras-transformed, and tyrosine kinase-(fps) transformed rat fibroblasts. PAP-2 was assayed in two different ways: 1) within its natural membrane using liposomes of phosphatidate and 2) in the presence of sufficient Triton X-100 to solubilize PAP-2, and to form mixed micelles with the phosphatidate. Harvesting the fibroblasts in medium containing orthovanadate and Zn2+ gave up to 3-fold higher PAP-2 activities when measured in the absence, but not in the presence, of Triton X-100. PAP-2-specific activities from both assays increased in the control fibroblasts as the cells reached confluence. Both specific activities were lower in the oncogenically transformed fibroblasts than in controls at all cell densities tested. The specific activities of PAP-2 did not increase with time in culture in transformed cells which continued to divide. The relative increase in activity of phospholipase D after stimulation with serum or phorbol myristate acetate was lower in the transformed fibroblasts compared to control cells. This indicates a coordinated decrease in the phospholipase D/phosphatidate phosphohydrolase pathway at the level of both enzymes in ras and fps transformed fibroblasts. The ratio of the production of diacylglycerol relative to phosphatidate, after stimulation with serum, or phorbol ester, was lower in both transformed fibroblasts relative to the controls. This is compatible with the decreased specific activity of PAP-2 and indicates functional significance for the differences in PAP-2 activity in regulating the balance between the two mitogenic lipids, phosphatidate and diacylglycerol. Control of PAP-2 activity could be an important factor in regulating appropriate signals for cell division.

A photoactivatable radioiodinated fatty acid analogue, 3-[125I]iodo-4-azido-N-hexadecylsalicylamide (125I-AHS) has been synthesized and used to investigate the involvement of cellular lipid carriers in hepatic fatty acid utilization. Photoactivation of Hep G2 internalized 125I-AHS revealed that several cellular proteins were crosslinked with the radiolabeled fatty acid analogue. Three predominant proteins in the membrane fraction of the cell with molecular masses 17, 50 and 127 kDa were crosslinked with the lipid analogue, as determined using autoradiography after SDS-PAGE. Three other proteins in the soluble fraction of the cell, with molecular masses 14, 24 and 35 kDa, were also labeled in situ. In contrast to the other labeled proteins, the fatty acid analogue accumulated on the cytoplasmic 14 kDa protein in a time and temperature dependent fashion. The in situ-labeled 14 kDa protein was identified from primary rat hepatocytes as the liver fatty acid binding protein by partial purification and its ability to be immunoprecipitated with immunospecific L-FABP antiserum. Collectively the results indicate that fatty acids traverse the plasma membrane and are bound cytoplasmically by the liver fatty acid binding protein, as well as other proteins in the cell. This represents the first demonstration in intact hepatocytes that the liver fatty acid binding protein participates in the process of intracellular fatty acid trafficking, and supports a model in which cytoplasmic lipid carriers solubilize fatty acids as a step in their metabolic utilization.

Differentiating 3T3-L1 cells have been used to investigate the process of fatty acid uptake, its cellular specificity, and the involvement of cytoplasmic carrier proteins. The profile of fatty acid uptake in both differentiated and undifferentiated cells was biphasic, consisting of an initial rapid phase (0-20 s) followed by a second slower phase (60-480 s). In both cell types the initial phase of fatty acid (FA) uptake was temperature-insensitive whereas the rate of uptake during the second phase decreased 4-fold when measurements were made at 4 degrees C. The rate of [9,10-3H]oleate uptake in 3T3-L1 adipocytes was 10-fold greater than in the fibroblastic precursor cells. The acquisition of a differentially expressed cytoplasmic fatty acid binding protein (adipocyte lipid binding protein (ALBP] occurs coincident with the increased ability of these cells to take up FAs. Uptake experiments with 3-[125I]iodo-4-azido-N-hexadecylsalicylamide demonstrated that this photoactivatable FA analogue accumulated intracellularly in a time-, temperature-, and cell-specific fashion. Moreover, when 3T3-L1 adipocytes were presented with 3-[125I]iodo-4-azido-N-hexadecylsalicylamide and then irradiated, a single cytoplasmic 15-kDa protein was labeled. The in situ-labeled 15-kDa protein was identified as ALBP by its ability to be immunoprecipitated with anti-ALBP antisera. Taken together these results indicate that fatty acids traverse the plasma membrane and are bound by ALBP in the cytoplasmic compartment. It is likely that lipid uptake in other cell systems, such as liver, heart, intestine, and nerve tissue, proceeds by a similar process and that this represents a general mechanism for cell-specific FA uptake and utilization.

Opiate systems in the brain are thought to play a major, though not exclusive, role in the regulation of intake. The rough correspondence of feeding and pineal activity rhythms in the rat offers the possibility that the pineal may also modulate ingestive behavior. In these studies we measured the possibility that pineal manipulations would influence feeding responses to opiate agonists and the antagonist naloxone. Male rats received one of four treatments (or a corresponding control treatment): pinealectomy, removal of the superior cervical ganglia (SCG), transection of the optic nerves or chronic melatonin treatment (1 mg/kg daily). Pinealectomy and melatonin treatment reduced intake during the first half of the dark period, and removal of the SCG reduced intake during the second half of the light period. The most striking effect was seen after optic nerve transection, which reduced nocturnal and increased diurnal intake. Pinealectomy, but no other manipulation, caused a slight decrease in sensitivity to the inhibitory effects of naloxone on intake. None of the treatments affected daytime feeding responses to morphine, ketocyclazocine, or butorphanol. These results suggest that the pineal gland has a minimal role in modulating the opioid regulation of food intake.