Enzymatic analysis of lipid phosphate phosphatases

Lipid phosphate monoesters including phosphatidic acid, lysophosphatidic acid, sphingosine 1-phosphate and ceramide 1-phosphate are intermediates in phosho- and sphingo-lipid biosynthesis and also play important roles in intra- and extra-cellular signaling. Dephosphorylation of these lipids terminates their signaling actions and, in some cases, generates products with additional biological activities or metabolic fates. The key enzymes responsible for dephosphorylation of these lipid phosphate substrates are collectively termed lipid phosphate phosphatases (LPPs). They are integral membrane enzymes with a core domain of six transmembrane spanning alpha-helices linked by extramembrane loops. LPPs are oriented in the membrane with their N- and C-termini facing the cytoplasm. LPPs exhibit isoform and cell specific localization patterns being variably distributed between endomembrane compartments (primarily the endoplasmic reticulum and Golgi apparatus) and the plasma membrane. The active site of these enzymes is formed from residues within two of the extramembrane loops and faces the lumen of endomembrane compartments or, when localized to the plasma membrane, towards, the extracellular space. Biochemical, pharmacological, cell biological and genetic studies identify roles for LPPs in both intracellular lipid metabolism and the regulation of both intra- and extra-cellular signaling pathways that control cell growth, survival and migration. This article describes procedures for the expression of LPPs in insect and mammalian cells and their analysis by SDS-PAGE and Western blotting. The most straightforward way to determine LPP activity is to measure release of the substrate phosphate group. We described methods for the synthesis and purification of [(32)P]-labeled LPP substrates. We describe the use of both radiolabeled and fluorescent lipid substrates for the detection, quantitation and analysis of the enzymatic activities of the LPPs measured using intact or broken cell preparations as the source of enzyme.     

Lipid phosphate phosphatases regulate signal transduction through glycerolipids and sphingolipids

Lipid phosphate esters including lysophosphatidate (LPA), phosphatidate (PA), sphingosine 1-phosphate (S1P) and ceramide 1-phosphate (C1P) are bioactive in mammalian cells and serve as mediators of signal transduction. LPA and S1P are present in biological fluids and activate cells through stimulation of their respective G-protein-coupled receptors, LPA(1-3) and S1P(1-5). LPA stimulates fibroblast division and is important in wound repair. It is also active in maintaining the growth of ovarian cancers. S1P stimulates chemotaxis, proliferation and differentiation of vascular endothelial and smooth muscle cells and is an important participant in the angiogenic response and neovessel maturation. PA and C1P are believed to act primarily inside the cell where they facilitate vesicle transport. The lipid phosphates are substrates for a family of lipid phosphate phosphatases (LPPs) that dramatically alter the signaling balance between the phosphate esters and their dephosphorylated products. In the case of PA, S1P and C1P, the products are diacylglycerol (DAG), sphingosine and ceramide, respectively. These latter lipids are also bioactive and, thus, the LPPs change signals that the cell receives. The LPPs are integral membrane proteins that act both inside and outside the cell. The "ecto-activity" of the LPPs regulates the circulating and locally effective concentrations of LPA and S1P. Conversely, the internal activity controls the relative accumulation of PA or C1P in response to stimulation by various agonists thereby affecting cell signaling downstream of EDG and other receptors. This article will review the various LPPs and discuss how these enzymes could regulate signal transduction by lipid mediators.     

Lipid phosphate phosphatases and their roles in mammalian physiology and pathology

Lipid phosphate phosphatases (LPPs) are a group of enzymes that belong to a phosphatase/phosphotransferase family. Mammalian LPPs consist of three isoforms: LPP1, LPP2, and LPP3. They share highly conserved catalytic domains and catalyze the dephosphorylation of a variety of lipid phosphates, including phosphatidate, lysophosphatidate (LPA), sphingosine 1-phosphate (S1P), ceramide 1-phosphate, and diacylglycerol pyrophosphate. LPPs are integral membrane proteins, which are localized on plasma membranes with the active site on the outer leaflet. This enables the LPPs to degrade extracellular LPA and S1P, thereby attenuating their effects on the activation of surface receptors. LPP3 also exhibits noncatalytic effects at the cell surface. LPP expression on internal membranes, such as endoplasmic reticulum and Golgi, facilitates the metabolism of internal lipid phosphates, presumably on the luminal surface of these organelles. This action probably explains the signaling effects of the LPPs, which occur downstream of receptor activation. The three isoforms of LPPs show distinct and nonredundant effects in several physiological and pathological processes including embryo development, vascular function, and tumor progression. This review is intended to present an up-to-date understanding of the physiological and pathological consequences of changing the activities of the different LPPs, especially in relation to cell signaling by LPA and S1P.     

Phosphatidate degradation: Phosphatidate phosphatases (lipins) and lipid phosphate phosphatases

Three lipid phosphate phosphatases (LPPs) regulate cell signaling by modifying the concentrations of a variety of lipid phosphates versus their dephosphorylated products. In particular, the LPPs are normally considered to regulate signaling by the phospholipase D (PLD) pathway by converting phosphatidate (PA) to diacylglycerol (DAG). LPP activities do modulate the accumulations of PA and DAG following PLD activation, but this could also involve an effect upstream of PLD activation. The active sites of the LPPs are on the exterior surface of plasma membranes, or on the luminal surface of internal membranes. Consequently, the actions of the LPPs in metabolizing PA formed by PLD1 or PLD2 should depend on the access of this substrate to the active site of the LPPs. Alternatively, PA generated on the cytosolic surface of membranes should be readily accessible to the family of specific phosphatidate phosphatases, namely the lipins. Presently, there is only indirect evidence for the lipins participating in cell signaling following PLD activation. So far, we know relatively little about how individual LPPs and specific phosphatidate phosphatases (lipins) modulate cell signaling through controlling the turnover of bioactive lipids that are formed after PLD activation.     

Sphingosine-1-phosphate and lipid phosphohydrolases

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that acts as both an extracellular ligand for the endothelial differentiation gene-1 (EDG-1) G-protein coupled receptor (GPCR) family and as an intracellular messenger. Cellular levels of S1P are low and tightly regulated in a spatial-temporal manner not only by sphingosine kinase (SPHK) but also by degradation catalyzed by S1P lyase, specific S1P phosphohydrolases, and by general lipid phosphate phosphohydrolases (LPPs). LPPs are characterized as magnesium-independent, insensitive to inhibition by N-ethylmaleimide (NEM) and possessing broad substrate specificity with a variety of phosphorylated lipids, including S1P, phosphatidic acid (PA), and lysophosphatidic acid (LPA). LPPs contain three highly conserved domains that define a phosphohydrolase superfamily. Recently, several specific S1P phosphohydrolases have been identified in yeast and mammalian cells. Phylogenetic and biochemical analyses indicate that these enzymes constitute a new subset of the LPP family. As further evidence, S1P phosphohydrolases exhibit high specificity for phosphorylated sphingoid bases. Enforced expression of S1P phosphohydrolase alters the cellular levels of sphingolipid metabolites in yeast and mammalian cells, increasing sphingosine and ceramide, bioactive sphingolipids that often have opposing biological actions to S1P. By regulating the cellular ratio between ceramide/sphingosine and S1P, S1P phosphohydrolase is poised to be a critical factor in cell survival/cell death decisions. Indeed, expression of S1P phosphohydrolase in mammalian cells increases apoptosis, whereas deletion of S1P phosphohydrolases in yeast correlates with resistance to heat stress. In this review, we discuss the role of phosphohydrolases in the metabolism of S1P and how turnover of S1P can regulate sphingolipid metabolites signaling.     

Lipid phosphate phosphatases and related proteins: signaling functions in development, cell division, and cancer

Lipid phosphates initiate key signaling cascades in cell activation. Lysophosphatidate (LPA) and sphingosine 1-phosphate (S1P) are produced by activated platelets. LPA is also formed from circulating lysophosphatidylcholine by autotaxin, a protein involved tumor progression and metastasis. Extracellular LPA and S1P stimulate families of G-protein coupled receptors that elicit diverse responses. LPA is involved in wound repair and tumor growth. Exogenous S1P is a potent stimulator of angiogenesis, a process vital in development, tissue repair and the growth of aggressive tumors. Inside the cell, phosphatidate (PA), ceramide 1-phosphate (C1P), LPA, and S1P act as signaling molecules with distinct functions including the stimulation of cell division, cytoskeletal rearrangement, Ca(2+) transients, and membrane movement. These observations imply that phosphatases that degrade lipid phosphates on the cell surface, or inside the cell, regulate cell signaling under physiological and pathological conditions. This occurs through attenuation of signaling by the lipid phosphates and by the production of bioactive products (diacylglycerol, ceramide, and sphingosine). Three lipid phosphate phosphatases (LPPs) and a splice variant dephosphorylate LPA, PA, CIP, and S1P. Two S1P phosphatases (SPPs) act specifically on S1P. In addition, there is family of four LPP-related proteins (LPRs, or plasticity-related genes, PRGs). PRG-1 expression in neurons has been reported to increase extracellular LPA breakdown and attenuate LPA-induced axonal retraction. It is unclear whether the LRPs dephosphorylate LPA directly, stimulate LPP activity, or bind LPA and S1P. Also, the importance of extra- versus intra-cellular actions of the LPPs and SPPs, and the individual roles of different isoforms is not firmly established. Understanding the functions and regulation of the LPPs, SPPs and related proteins will hopefully contribute to interventions to correct dysfunctions in conditions such as wound repair, inflammation, angiogenesis, tumor growth, and metastasis.     

Lipid phosphate phosphatase-2 activity regulates S-phase entry of the cell cycle in Rat2 fibroblasts

Lipid phosphates are potent mediators of cell signaling and control processes including development, cell migration and division, blood vessel formation, wound repair, and tumor progression. Lipid phosphate phosphatases (LPPs) regulate the dephosphorylation of lipid phosphates, thus modulating their signals and producing new bioactive compounds both at the cell surface and in intracellular compartments. Knock-down of endogenous LPP2 in fibroblasts delayed cyclin A accumulation and entry into S-phase of the cell cycle. Conversely, overexpression of LPP2, but not a catalytically inactive mutant, caused premature S-phase entry, accompanied by premature cyclin A accumulation. At high passage, many LPP2 overexpressing cells arrested in G(2)/M and the rate of proliferation declined severely. This was accompanied by changes in proteins and lipids characteristic of senescence. Additionally, arrested LPP2 cells contained decreased lysophosphatidate concentrations and increased ceramide. These effects of LPP2 activity were not reproduced by overexpression or knock-down of LPP1 or LPP3. This work identifies a novel and specific role for LPP2 activity and bioactive lipids in regulating cell cycle progression.     

Structural organization of mammalian lipid phosphate phosphatases: implications for signal transduction

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 Mg²⁺ 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.     

Structural organization of mammalian lipid phosphate phosphatases: implications for signal transduction.

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.     

Regulation of lysophosphatidate signaling by autotaxin and lipid phosphate phosphatases with respect to tumor progression, angiogenesis, metastasis and chemo-resistance

Evidence from clinical, animal and cell culture studies demonstrates that increased autotaxin (ATX) expression is responsible for enhancing tumor progression, cell migration, metastases, angiogenesis and chemo-resistance. These effects depend mainly on the rapid formation of lysophosphatidate (LPA) by ATX. Circulating LPA has a half-life of about 3 min in mice and it is degraded by the ecto-activities of lipid phosphate phosphatases (LPPs). These enzymes also hydrolyze extracellular sphingosine 1-phosphate (S1P), a potent signal for cell division, survival and angiogenesis. Many aggressive tumor cells express high ATX levels and low LPP activities. This favors the formation of locally high LPA and S1P concentrations. Furthermore, LPPs attenuate signaling downstream of the activation of G-protein coupled receptors and receptor tyrosine kinases. Therefore, we propose that the low expression of LPPs in many tumor cells makes them hypersensitive to growth promoting and survival signals that are provided by LPA, S1P, platelet-derived growth factor (PDGF) and epidermal growth factor (EGF). One of the key signaling pathways in this respect appears to be activation of phospholipase D (PLD) and phosphatidate (PA) production. This is required for the transactivations of the EGFR and PDGFR and also for LPA-induced cell migration. PA also increases the activities of ERK, mTOR, myc and sphingosine kinase-1 (SK-1), which provide individual signals for cells division, survival, chemo-resistance and angiogenesis. This review focuses on the balance of signaling by bioactive lipids including LPA, phosphatidylinositol 3,4,5-trisphosphate, PA and S1P versus the action of ceramides. We will discuss how these lipid mediators interact to produce an aggressive neoplastic phenotype.     

Lipid phosphate phosphatases regulate lysophosphatidic acid production and signaling in platelets: studies using chemical inhibitors of lipid phosphate phosphatase activity

Blood platelets play an essential role in ischemic heart disease and stroke contributing to acute thrombotic events by release of potent inflammatory agents within the vasculature. Lysophosphatidic acid (LPA) is a bioactive lipid mediator produced by platelets and found in the blood and atherosclerotic plaques. LPA receptors on platelets, leukocytes, endothelial cells, and smooth muscle cells regulate growth, differentiation, survival, motility, and contractile activity. Definition of the opposing pathways of synthesis and degradation that control extracellular LPA levels is critical to understanding how LPA bioactivity is regulated. We show that intact platelets and platelet membranes actively dephosphorylate LPA and identify the major enzyme responsible as lipid phosphate phosphatase 1 (LPP1). Localization of LPP1 to the platelet surface is increased by exposure to LPA. A novel receptor-inactive sn-3-substituted difluoromethylenephosphonate analog of phosphatidic acid that is a potent competitive inhibitor of LPP1 activity potentiates platelet aggregation and shape change responses to LPA and amplifies LPA production by agonist-stimulated platelets. Our results identify LPP1 as a pivotal regulator of LPA signaling in the cardiovascular system. These findings are consistent with genetic and cell biological evidence implicating LPPs as negative regulators of lysophospholipid signaling and suggest that the mechanisms involve both attenuation of lysophospholipid actions at cell surface receptors and opposition of lysophospholipid production.     

Plasticity-related genes (PRGs/LRPs): a brain-specific class of lysophospholipid-modifying proteins

Recently, a set of five brain-specifically expressed membrane proteins, which define a novel subclass of the lipid phosphate phosphatases (LPP-)superfamily, has been identified, namely plasticity-related genes (PRGs/LRPs). The primary known significance of these genes is their involvement in regeneration processes and attenuation of effects induced by lysophosphatidic acid (LPA). LPA is key player in lysophospholipids, a hydrophilic group of lipids that have been recognized as important signaling molecules. It is a lipid mediator with a wide variety of biological actions, such as cell proliferation, migration and survival. Its extracellular effects are mediated through five distinct G-protein-coupled receptors (LPA(1-5)) and LPA therefore activates multiple signal transduction pathways. LPA signaling has been implicated in diverse processes, such as wound healing, brain development, vascular remodeling and tumor progression. LPA levels are controlled by enzymes that synthesize or degrade LPA and, thus, these enzymes also regulate many aspects of signaling transduction. Three LPPs and a splice variant have been demonstrated as deactivating LPA. Studies of PRGs indicate that this group of proteins may in fact serve as controllers of LPA and therefore opening the door to new therapeutic approaches.     

Recent advances in the chemistry and biology of anti-inflammatory and specialized pro-resolving mediators biosynthesized from n-3 docosapentaenoic acid

Several novel oxygenated polyunsaturated lipid mediators biosynthesized from n-3 docosapentaenoic acid were recently isolated from murine inflammatory exudates and human primary cells. These compounds belong to a distinct family of specialized pro-resolving mediators, and display potent in vivo anti-inflammatory and pro-resolution effects. The endogenously formed specialized pro-resolving mediators have attracted a great interest as lead compounds in drug discovery programs towards the development of new classes of drugs that dampen inflammation without interfering with the immune response. Detailed information on the chemical structures, cellular functions and distinct biosynthetic pathways of specialized pro-resolving lipid mediators is a central aspect of these biological actions. Herein, the isolation, structural elucidation, biosynthetic pathways, total synthesis and bioactions of the n-3 docosapentaenoic acid derived mediators PD1_{n-3 DPA} and MaR1_{n-3 DPA} are discussed. In addition, a brief discussion of a novel family of mediators derived from n-3 docosapentaenoic acid, termed 13-series resolvins is included.     

Tetracyclines increase lipid phosphate phosphatase expression on plasma membranes and turnover of plasma lysophosphatidate

Extracellular lysophosphatidate and sphingosine 1-phosphate (S1P) are important bioactive lipids, which signal through G-protein-coupled receptors to stimulate cell growth and survival. The lysophosphatidate and S1P signals are terminated partly by degradation through three broad-specificity lipid phosphate phosphatases (LPPs) on the cell surface. Significantly, the expression of LPP1 and LPP3 is decreased in many cancers, and this increases the impact of lysophosphatidate and S1P signaling. However, relatively little is known about the physiological or pharmacological regulation of the expression of the different LPPs. We now show that treating several malignant and nonmalignant cell lines with 1 μg/ml tetracycline, doxycycline, or minocycline significantly increased the extracellular degradation of lysophosphatidate. S1P degradation was also increased in cells that expressed high LPP3 activity. These results depended on an increase in the stabilities of the three LPPs and increased expression on the plasma membrane. We tested the physiological significance of these results and showed that treating rats with doxycycline accelerated the clearance of lysophosphatidate, but not S1P, from the circulation. However, administering 100 mg/kg/day doxycycline to mice decreased plasma concentrations of lysophosphatidate and S1P. This study demonstrates a completely new property of tetracyclines in increasing the plasma membrane expression of the LPPs.     

Lipid phosphate phosphatases form homo- and hetero-oligomers: catalytic competency, subcellular distribution and function

Lipid phosphate phosphatases (LPP1-LPP3) have been topographically modelled as monomers (molecular mass of 31-36 kDa) composed of six transmembrane domains and with the catalytic site facing the extracellular side of the plasma membrane or the luminal side of intracellular membranes. The catalytic motif has three conserved domains, termed C1, C2 and C3. The C1 domain may be involved in substrate recognition, whereas C2 and C3 domains appear to participate in the catalytic dephosphorylation of the substrate. We have obtained three lines of evidence to demonstrate that LPPs exist as functional oligomers. First, we have used recombinant expression and immunoprecipitation analysis to demonstrate that LPP1, LPP2 and LPP3 form both homo- and hetero-oligomers. Secondly, large LPP oligomeric complexes that are catalytically active were isolated using gel-exclusion chromatography. Thirdly, we demonstrate that catalytically deficient guinea-pig FLAG-tagged H223L LPP1 mutant can form an oligomer with wild-type LPP1 and that wild-type LPP1 activity is preserved in the oligomer. These findings suggest that, in an oligomeric arrangement, the catalytic site of the wild-type LPP can function independently of the catalytic site of the mutant LPP. Finally, we demonstrate that endogenous LPP2 and LPP3 form homo- and hetero-oligomers, which differ in their subcellular localization and which may confer differing spatial regulation of phosphatidic acid and sphingosine 1-phosphate signalling.     

Recombinant bovine neurokinin-2 receptor stably expressed in Chinese hamster ovary cells couples to multiple signal transduction pathways.

Neurokinins are a family of neuropeptides with widespread distribution mediating a broad spectrum of physiological actions through three distinct receptor subtypes: NK-1, NK-2, and NK-3. We investigated some of the second messenger and cellular processes under control by the recombinant bovine NK-2 receptor stably expressed in Chinese hamster ovary cells. In this system the NK-2 receptor displays its expected pharmacological characteristics, and the physiological agonist neurokinin A stimulates several cellular responses. These include 1) transient inositol 1,4,5-trisphosphate (IP3) formation and Ca2+ mobilization, 2) increased out put of arachidonic acid and prostaglandin E2 (PGE2), 3) enhanced cyclic AMP (cAMP) generation, 4) increased de novo DNA synthesis, and 5) an induction of the "immediate early" genes c-fos and c-jun. Although NK-2 receptor-mediated IP3 formation involves activation of a pertussis toxin-insensitive G-protein, increased cAMP production is largely a secondary response and can be at least partially attributed to autocrine stimulation by endogenously generated eicosanoids, particularly PGE2. This is the first demonstration that a single recombinant neurokinin receptor subtype can regulate, either directly or indirectly, multiple signal transduction pathways and suggests several potential important mediators of neurokinin actions under physiological conditions.