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.     

Role of type 2C protein phosphatases in growth regulation and in cellular stress signaling

A number of interesting features, phenotypes, and potential clinical applications have recently been ascribed to the type 2C family of protein phosphatases. Thus far, 16 different PP2C genes have been identified in the human genome, encoding (by means of alternative splicing) for at least 22 different isozymes. Virtually ever since their discovery, type 2C phosphatases have been predominantly linked to cell growth and to cellular stress signaling. Here, we provide an overview of the involvement of type 2C phosphatases in these two processes, and we show that four of them (PP2Calpha, PP2Cbeta, ILKAP, and PHLPP) can be expected to function as tumor suppressor proteins, and one as an oncoprotein (PP2Cdelta /Wip1). In addition, we demonstrate that in virtually all cases in which they have been linked to the stress response, PP2Cs act as inhibitors of cellular stress signaling. Based on the vast amount of experimental evidence obtained thus far, it therefore seems justified to conclude that type 2C protein phosphatases are important physiological regulators of cell growth and of cellular stress signaling.     

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.     

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.     

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.     

Plastidic phosphatidic acid phosphatases identified in a distinct subfamily of lipid phosphate phosphatases with prokaryotic origin

Plastidic phosphatidic acid phosphatase (PAP) dephosphorylates phosphatidic acid to yield diacylglycerol, which is a precursor for galactolipids, a primary and indispensable component of photosynthetic membranes. Despite its functional importance, the molecular characteristics and phylogenetic origin of plastidic PAP were unknown because no potential homologs have been found. Here, we report the isolation and characterization of plastidic PAPs in Arabidopsis that belong to a distinct lipid phosphate phosphatase (LPP) subfamily with prokaryotic origin. Because no homolog of mammalian LPP was found in cyanobacteria, we sought an LPP ortholog in a more primitive organism, Chlorobium tepidum, and its homologs in cyanobacteria. Arabidopsis had five homologs of cyanobacterial LPP, three of which (LPP gamma, LPP epsilon 1, and LPP epsilon 2) localized to chloroplasts. Complementation of yeast Delta dpp1 Delta lpp1 Delta pah1 by plastidic LPPs rescued the relevant phenotype in vitro and in vivo, suggesting that they function as PAPs. Of the three LPPs, LPP gamma activity best resembled the native activity. The three plastidic LPPs were differentially expressed both in green and nongreen tissues, with LPP gamma expressed the highest in shoots. A knock-out mutant for LPP gamma could not be obtained, although a lpp epsilon 1 lpp epsilon 2 double knock-out showed no significant changes in lipid composition. However, lpp gamma homozygous mutant was isolated only under ectopic overexpression of LPP gamma, suggesting that loss of LPP gamma may cause lethal effect on plant viability. Thus, in Arabidopsis, there are three isoforms of plastidic PAP that belong to a distinct subfamily of LPP, and LPP gamma may be the primary plastidic PAP.     

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.     

Phosphatidylinositol transfer proteins and cellular nanoreactors for lipid signaling

Membrane lipids function as structural molecules, reservoirs for second messengers, membrane platforms that scaffold protein assembly and regulators of enzymes and ion channels. Such diverse lipid functions contribute substantially to cellular mechanisms for fine-tuning membrane-signaling events. Meaningful coordination of these events requires exquisite spatial and temporal control of lipid metabolism and organization, and reliable mechanisms for specifically coupling these parameters to dedicated physiological processes. Recent studies suggest such integration is linked to the action of phosphatidylinositol transfer proteins that operate at the interface of the metabolism, trafficking and organization of specific lipids.     

Lipid phosphate phosphatases 1 and 3 are localized in distinct lipid rafts

Lipid phosphate phosphatases (LPPs), integral membrane proteins with six transmembrane domains, dephosphorylate a variety of extracellular lipid phosphates. Although LPP3 is already known to bind to Triton X-100-insoluble rafts, we here report that LPP1 is also associated with lipid rafts distinct from those harboring LPP3. We found that LPP1 was Triton X-100-soluble, but CHAPS-insoluble in LNCaP cells endogenously expressing LPP1 and several LPP1 cDNA-transfected cells including NIH3T3 fibroblasts. In addition to the non-ionic detergent insolubility, LPP1 further possessed several properties formulated for raft-localizing proteins as follows: first, the CHAPS-insolubility was resistant to the actin-disrupting drug cytochalasin D; second, the CHAPS-insoluble LPP1 floated in an Optiprep density gradient; third, the CHAPS insolubility of LPP1 was lost by cholesterol depletion; and finally, the subcellular distribution pattern of LPP1 exclusively overlapped with that of a raft marker, cholera toxin B subunit. Interestingly, confocal microscopic analysis showed that LPP1 was distributed to membrane compartments distinct from those of LPP3. Analysis using various LPP1/LPP3 chimeras revealed that their first extracellular regions determine the different Triton X-100 solubilities. These results indicate that LPP1 and LPP3 are distributed in distinct lipid rafts that may provide unique microenvironments defining their non-redundant physiological functions.     

The protein phosphatases involved in cellular regulation. Identification of the inhibitor-2 phosphatases in rabbit skeletal muscle

Inhibitor-2, purified by an improved procedure, was used to identify protein phosphatases capable of catalysing its dephosphorylation. The results showed that, under our experimental conditions, protein phosphatases-1, 2A and 2B were the only significant protein phosphatases in rabbit skeletal muscle extracts acting on this substrate. Protein phosphatases-1 and 2A accounted for all the inhibitor-2 phosphatase activity in the absence of Ca2+ (resting muscle), and the potential importance of these enzymes in vivo is discussed. Protein phosphatase-2B, a Ca2+-calmodulin-dependent enzyme, could account for up to 30% of the inhibitor-2 phosphatase activity in contracting muscle. The Km of protein phosphatase-1 for inhibitor-2 (40 nM) was 100-fold lower than the Km for phosphorylase a (4.8 microM). This finding, coupled with the failure of inhibitor-2 to inhibit its own dephosphorylation, suggests that inhibitor-2 is dephosphorylated at one of the two sites on protein phosphatase-1 involved in preventing the dephosphorylation of other substrates. The dephosphorylation of inhibitor-2 by protein phosphatase-1 was also unaffected by inhibitor-1, suggesting that the phosphorylation state of inhibitor-2 is unlikely to be controlled by cyclic AMP in vivo.     

The protein phosphatases involved in cellular regulation. 2. Glycogen metabolism

The nature of protein phosphatases that are active against the phosphorylated proteins of glycogen metabolism was investigated in rabbit skeletal muscle and liver. Six 32P-labelled substrates corresponding to the major phosphorylation sites on glycogen phosphorylase, phosphorylase kinase, glycogen synthase and inhibitor-1 were used in these studies. The results showed that the four protein phosphatases defined in the preceding paper, namely protein phosphatases-1, 2A, 2B and 2C [Ingebritsen, T. S. and Cohen, P. (1983) Eur. J. Biochem. 132, 255-261] were the only significant enzymes acting on these substrates. The four enzymes can be conveniently separated and identified by a combination of ion-exchange chromatography and gel filtration and by the use of specific inhibitors. Three species of protein phosphatase-2A were resolved on DEAE-cellulose, termed protein phosphatases-2Ao (0.12 M NaCl), 2A1 (0.2 M NaCl) and 2A2 (0.28 M NaCl) that had apparent molecular weights of 210000, 210000 and 150000 respectively. Protein phosphatase-2Ao was a completely inactive enzyme whose activity was only expressed after dissociation to a 34000-Mr(app) catalytic subunit by freezing and thawing in 0.2 M 2-mercaptoethanol. This treatment also dissociated protein phosphatases 2A1 and 2A2 to more active 34000-Mr(app) catalytic subunits. The catalytic subunits derived from protein phosphatases-2Ao, 2A1 and 2A2 possessed identical substrate specificities, preferentially dephosphorylated the alpha-subunit of phosphorylase kinase, were unaffected by inhibitor-1 and inhibitor-2 and were inhibited by similar concentrations of ATP. The properties of protein phosphatases-2A1 and 2A2 were very similar to those of the catalytic subunits, except that they were less sensitive to inhibition by ATP. Protein phosphatase-2B was eluted from DEAE-cellulose in the same fraction as protein phosphatase-2Ao. These activities were resolved by gel filtration, the Mr(app) of protein phosphatase-2B being 98000. Protein phosphatase-2B was completely inhibited by 100 microM trifluoperazine, which did not affect the activity of protein phosphatase-2Ao or any other protein phosphatase. Freezing and thawing in 0.2 M 2-mercaptoethanol resulted in partial inactivation of protein phosphatase-2B. Protein phosphatase-2C was eluted from DEAE-cellulose at the leading edge of the peak of protein phosphatase-2A1. These activities were completely resolved by gel filtration, since the Mr(app) of protein phosphatase-2C was 46000. Two forms of protein phosphatase-1 can be identified by chromatography on DEAE-cellulose, namely protein phosphatase-1 itself and the Mg X ATP-dependent protein phosphatase. Both these species were eluted at 0.16 M NaCl just ahead of protein phosphatases-2C and 2A1. These enzymes did not interfere with measurements of type-2 protein phosphatases, since it was possible to block their activity with inhibitor-2...     

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.     

Phosphoinositide lipid phosphatases: natural regulators of phosphoinositide 3-kinase signaling in T lymphocytes

The phosphoinositide 3-kinase signaling pathway has been implicated in a range of T lymphocyte cellular functions, particularly growth, proliferation, cytokine secretion, and survival. Dysregulation of phosphoinositide 3-kinase-dependent signaling and function in leukocytes, including B and T lymphocytes, has been implicated in many inflammatory and autoimmune diseases. As befits a pivotal signaling cascade, several mechanisms exist to ensure that the pathway is tightly regulated. This minireview focuses on two lipid phosphatases, viz. the 3'-phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10) and SHIP (Src homology 2 domain-containing inositol-5-phosphatase). We discuss their role in regulating T lymphocyte signaling as well their potential as future therapeutic targets.     

Lysophosphatidic acid-induced mitogenesis is regulated by lipid phosphate phosphatases and is Edg-receptor independent

Lysophosphatidic acid (LPA) is an extracellular signaling mediator with a broad range of cellular responses. Three G-protein-coupled receptors (Edg-2, -4, and -7) have been identified as receptors for LPA. In this study, the ectophosphatase lipid phosphate phosphatase 1 (LPP1) has been shown to down-regulate LPA-mediated mitogenesis. Furthermore, using degradation-resistant phosphonate analogs of LPA and stereoselective agonists of the Edg receptors we have demonstrated that the mitogenic and platelet aggregation responses to LPA are independent of Edg-2, -4, and -7. Specifically, we found that LPA degradation is insufficient to account for the decrease in LPA potency in mitogenic assays, and the stereoselectivity observed at the Edg receptors is not reflected in mitogenesis. Additionally, RH7777 cells, which are devoid of Edg-2, -4, and -7 receptor mRNA, have a mitogenic response to LPA and LPA analogs. Finally, we have determined that the ligand selectivity of the platelet aggregation response is consistent with that of mitogenesis, but not with Edg-2, -4, and -7.     

Lysophosphatidic acid and sphingosine 1-phosphate biology: the role of lipid phosphate phosphatases

The biological actions of the lysolipid agonists sphingosine 1-phosphate and lysophosphatidic acid, in addition to other bioactive lipid phosphates such as phosphatidic acid and ceramide 1-phosphate, can be influenced by a family of lipid phosphate phosphatases (LPP), including LPP1, LPP2, LPP3, the Drosophila homologues Wunen (Wun) and Wunen2 (Wun2) and sphingosine 1-phosphate phosphatases 1 and 2 (SPP1, SPP2). This review describes the characteristic of these enzymes and their potential physiological roles in regulating intracellular and extracellular actions and amounts of these lipids in addition to the involvement of these phosphatases in development.     

The protein phosphatases involved in cellular regulation. 1. Classification and substrate specificities

The protein phosphatase activities involved in regulating the major pathways of intermediary metabolism can be explained by only four enzymes which can be conveniently divided into two classes, type-1 and type-2. Type-1 protein phosphatases dephosphorylate the beta-subunit of phosphorylase kinase and are potently inhibited by two thermostable proteins termed inhibitor-1 and inhibitor-2, whereas type-2 protein phosphatases preferentially dephosphorylate the alpha-subunit of phosphorylase kinase and are insensitive to inhibitor-1 and inhibitor-2. The substrate specificities of the four enzymes, namely protein phosphatase-1 (type-1) and protein phosphatases 2A, 2B and 2C (type-2) have been investigated. Eight different protein kinases were used to phosphorylate 13 different substrate proteins on a minimum of 20 different serine and threonine residues. These substrates include proteins involved in the regulation of glycogen metabolism, glycolysis, fatty acid synthesis, cholesterol synthesis, protein synthesis and muscle contraction. The studies demonstrate that protein phosphatase-1 and protein phosphatase 2A have very broad substrate specificities. The major differences, apart from the site specificity for phosphorylase kinase, are the much higher myosin light chain phosphatase and ATP-citrate lyase phosphatase activities of protein phosphatase-2A. Protein phosphatase-2C (an Mg2+-dependent enzyme) also has a broad specificity, but can be distinguished from protein phosphatase-2A by its extremely low phosphorylase phosphatase and histone H1 phosphatase activities, and its slow dephosphorylation of sites (3a + 3b + 3c) on glycogen synthase relative to site-2 of glycogen synthase. It has extremely high hydroxymethylglutaryl-CoA (HMG-CoA) reductase phosphatase and HMG-CoA reductase kinase phosphatase activity. Protein phosphatase-2B (a Ca2+-calmodulin-dependent enzyme) is the most specific phosphatase and only dephosphorylated three of the substrates (the alpha-subunit of phosphorylase kinase, inhibitor-1 and myosin light chains) at a significant rate. It is specifically inhibited by the phenathiazine drug, trifluoperazine. Examination of the amino acid sequences around each phosphorylation site does not support the idea that protein phosphatase specificity is determined by the primary structure in the immediate vicinity of the phosphorylation site.     

Identification of two lipid phosphatases that regulate sphingosine-1-phosphate cellular uptake and recycling

Sphingosine-1-phosphate (S1P) is a sphingolipid metabolite that serves as a potent extracellular signaling molecule. Metabolic regulation of extracellular S1P levels impacts key cellular activities through altered S1P receptor signaling. Although the pathway through which S1P is degraded within the cell and thereby eliminated from reuse has been previously described, the mechanism used for S1P cellular uptake and the subsequent recycling of its sphingoid base into the sphingolipid synthesis pathway is not completely understood. To identify the genes within this S1P uptake and recycling pathway, we performed a genome-wide CRISPR/Cas9 KO screen using a positive-selection scheme with Shiga toxin, which binds a cell-surface glycosphingolipid receptor, globotriaosylceramide (Gb3), and causes lethality upon internalization. The screen was performed in HeLa cells with their sphingolipid de novo pathway disabled so that Gb3 cell-surface expression was dependent on salvage of the sphingoid base of S1P taken up from the medium. The screen identified a suite of genes necessary for S1P uptake and the recycling of its sphingoid base to synthesize Gb3, including two lipid phosphatases, PLPP3 (phospholipid phosphatase 3) and SGPP1 (S1P phosphatase 1). The results delineate a pathway in which plasma membrane–bound PLPP3 dephosphorylates extracellular S1P to sphingosine, which then enters cells and is rephosphorylated to S1P by the sphingosine kinases. This rephosphorylation step is important to regenerate intracellular S1P as a branch-point substrate that can be routed either for dephosphorylation to salvage sphingosine for recycling into complex sphingolipid synthesis or for degradation to remove it from the sphingolipid synthesis pathway.