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Vishwakarma S, Joshi D, Pandey R, Das S, Mukhopadhyay S, Rai R, Singhal R, Kapoor N, Kumar A. Downregulation of Lipid Phosphate Phosphatase 3 Correlates With Tumor-Infiltrating Immune Cells in Oral Cancer. Cureus 2022; 14:e23553. [PMID: 35494957 PMCID: PMC9045791 DOI: 10.7759/cureus.23553] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/27/2022] [Indexed: 11/27/2022] Open
Abstract
Background Sphingosine-1-phosphate (S1P) is a potent oncogenic lipid. Intracellular levels of S1P are tightly regulated by eight S1P-metabolizing enzymes. S1P synthesis is catalyzed by two sphingosine kinases, i.e., sphingosine kinase 1 (SphK1) and sphingosine kinase 2 (SphK2). Five lipid phosphatases (two S1P phosphatases and lipid phosphate phosphatases (LPPs) 1, 2, and 3) reversibly convert S1P back to sphingosine. Previously, we have determined the mRNA expression profile of eight S1P-metabolizing enzymes in tumor tissues and adjacent normal tissues from oral squamous cell carcinoma (OSCC) patients. Except for SphK1, the role of S1P-metabolizing enzymes in OSCC has been poorly studied. Methods We have determined the protein expression of four S1P-metabolizing enzymes (SphK1, SphK2, sphingosine-1-phosphate phosphatase 1 (SGPP1), and lipid phosphate phosphatase 3 (LPP3)) by immunohistochemistry (IHC) in tumor tissues of 46 OSCC patients. Six subjects with non-dysplastic oral mucosa were also included in the study. The immunoreactivity score (IRS) was calculated for each protein in every subject. Further, we determined the associations of expression of S1P-metabolizing enzymes with clinicopathological features of OSCC patients. Results We demonstrate the low IRS for SphK2 and LPP3 in OSCC tumors. Importantly, expression of SphK2 and LPP3 was downregulated in malignant epithelial cells compared to non-malignant mucosa. Further, LPP3 expression negatively correlated with tumor‑node‑metastasis (TNM) staging of patients (r = −0.307, p = 0.043). Importantly, expression of LPP3 in tumors was found to be an independent predictor of perinodal extension (b = −0.440, p = 0.009), lymphovascular invasion (b = −0.614, p < 0.001), lymph node ratio (b = 0.336, p = 0.039), and TNM staging (b = −0.364, p = 0.030). Conclusion Taken together, our data show that expression of SphK2 and LPP3 is decreased compared to normal mucosa. Thus, the S1P signaling pathway could represent a potential therapeutic target.
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Tang X, Brindley DN. Lipid Phosphate Phosphatases and Cancer. Biomolecules 2020; 10:biom10091263. [PMID: 32887262 PMCID: PMC7564803 DOI: 10.3390/biom10091263] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2020] [Revised: 08/28/2020] [Accepted: 08/30/2020] [Indexed: 12/22/2022] Open
Abstract
Lipid phosphate phosphatases (LPPs) are a group of three enzymes (LPP1–3) that belong to a phospholipid phosphatase (PLPP) family. The LPPs dephosphorylate a wide spectrum of bioactive lipid phosphates, among which lysophosphatidate (LPA) and sphingosine 1-phosphate (S1P) are two important extracellular signaling molecules. The LPPs are integral membrane proteins, which are localized on plasma membranes and intracellular membranes, including the endoplasmic reticulum and Golgi network. LPPs regulate signaling transduction in cancer cells and demonstrate different effects in cancer progression through the breakdown of extracellular LPA and S1P and other intracellular substrates. This review is intended to summarize an up-to-date understanding about the functions of LPPs in cancers.
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Affiliation(s)
- Xiaoyun Tang
- Department of Biochemistry, University of Alberta, Edmonton, AB T6G 2S2, Canada;
- Cancer Research Institute of Northern Alberta, University of Alberta, Edmonton, AB T6G 2E1, Canada
| | - David N. Brindley
- Department of Biochemistry, University of Alberta, Edmonton, AB T6G 2S2, Canada;
- Cancer Research Institute of Northern Alberta, University of Alberta, Edmonton, AB T6G 2E1, Canada
- Correspondence:
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Roy J, Dibaeinia P, Fan TM, Sinha S, Das A. Global analysis of osteosarcoma lipidomes reveal altered lipid profiles in metastatic versus nonmetastatic cells. J Lipid Res 2018; 60:375-387. [PMID: 30504231 DOI: 10.1194/jlr.m088559] [Citation(s) in RCA: 32] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/23/2018] [Revised: 11/21/2018] [Indexed: 12/15/2022] Open
Abstract
Osteosarcoma (OS) is the most common form of primary bone cancer in humans. The early detection and subsequent control of metastasis has been challenging in OS. Lipids are important constituents of cells that maintain structural integrity that can be converted into lipid-signaling molecules and are reprogrammed in cancerous states. Here, we investigate the global lipidomic differences in metastatic (143B) and nonmetastatic (HOS) human OS cells as compared with normal fetal osteoblast cells (FOB) using lipidomics. We detect 15 distinct lipid classes in all three cell lines that included over 1,000 lipid species across various classes including phospholipids, sphingolipids and ceramides, glycolipids, and cholesterol. We identify a key class of lipids, diacylglycerols, which are overexpressed in metastatic OS cells as compared with their nonmetastatic or nontumorigenic counterparts. As a proof of concept, we show that blocking diacylglycerol synthesis reduces cellular viability and reduces cell migration in metastatic OS cells. Thus, the differentially regulated lipids identified in this study might aid in biomarker discovery, and the synthesis and metabolism of specific lipids could serve as future targets for therapeutic development.
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Affiliation(s)
- Jahnabi Roy
- Department of Chemistry, University of Illinois Urbana-Champaign, Urbana, IL 61802
| | - Payam Dibaeinia
- Department of Computer Science, University of Illinois Urbana-Champaign, Urbana, IL 61802
| | - Timothy M Fan
- Department of Veterinary Clinical Medicine, University of Illinois Urbana-Champaign, Urbana, IL 61802
| | - Saurabh Sinha
- Department of Computer Science, University of Illinois Urbana-Champaign, Urbana, IL 61802.,Neuroscience Program and Department of Bioengineering, Institute of Genomic Biology, University of Illinois Urbana-Champaign, Urbana, IL 61802
| | - Aditi Das
- Department of Comparative Biosciences, University of Illinois Urbana-Champaign, Urbana, IL 61802 .,Department of Biochemistry, University of Illinois Urbana-Champaign, Urbana, IL 61802.,Beckman Institute for Advanced Science, Division of Nutritional Sciences, Neuroscience Program, University of Illinois Urbana-Champaign, Urbana, IL 61802
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Yang F, Chen GX. Production of extracellular lysophosphatidic acid in the regulation of adipocyte functions and liver fibrosis. World J Gastroenterol 2018; 24:4132-4151. [PMID: 30271079 PMCID: PMC6158478 DOI: 10.3748/wjg.v24.i36.4132] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/24/2018] [Revised: 04/24/2018] [Accepted: 05/06/2018] [Indexed: 02/06/2023] Open
Abstract
Lysophosphatidic acid (LPA), a glycerophospholipid, consists of a glycerol backbone connected to a phosphate head group and an acyl chain linked to sn-1 or sn-2 position. In the circulation, LPA is in sub-millimolar range and mainly derived from hydrolysis of lysophosphatidylcholine, a process mediated by lysophospholipase D activity in proteins such as autotaxin (ATX). Intracellular and extracellular LPAs act as bioactive lipid mediators with diverse functions in almost every mammalian cell type. The binding of LPA to its receptors LPA1-6 activates multiple cellular processes such as migration, proliferation and survival. The production of LPA and activation of LPA receptor signaling pathways in the events of physiology and pathophysiology have attracted the interest of researchers. Results from studies using transgenic and gene knockout animals with alterations of ATX and LPA receptors genes, have revealed the roles of LPA signaling pathways in metabolic active tissues and organs. The present review was aimed to summarize recent progresses in the studies of extracellular and intracellular LPA production pathways. This includes the functional, structural and biochemical properties of ATX and LPA receptors. The potential roles of LPA production and LPA receptor signaling pathways in obesity, insulin resistance and liver fibrosis are also discussed.
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Affiliation(s)
- Fang Yang
- School of Laboratory Medicine, Hubei University of Chinese Medicine, Wuhan 430065, Hubei Province, China
| | - Guo-Xun Chen
- Department of Nutrition, University of Tennessee at Knoxville, Knoxville, TN 37996, United States
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Brindley DN, Lin FT, Tigyi GJ. Role of the autotaxin-lysophosphatidate axis in cancer resistance to chemotherapy and radiotherapy. BIOCHIMICA ET BIOPHYSICA ACTA 2013; 1831:74-85. [PMID: 22954454 PMCID: PMC3584168 DOI: 10.1016/j.bbalip.2012.08.015] [Citation(s) in RCA: 104] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/03/2012] [Revised: 08/20/2012] [Accepted: 08/21/2012] [Indexed: 02/01/2023]
Abstract
High expression of autotaxin in cancers is often associated with increased tumor progression, angiogenesis and metastasis. This is explained mainly since autotaxin produces the lipid growth factor, lysophosphatidate (LPA), which stimulates cell division, survival and migration. It has recently become evident that these signaling effects of LPA also produce resistance to chemotherapy and radiation-induced cell death. This results especially from the stimulation of LPA(2) receptors, which depletes the cell of Siva-1, a pro-apoptotic signaling protein and stimulates prosurvival kinase pathways through a mechanism mediated via TRIP-6. LPA signaling also increases the formation of sphingosine 1-phosphate, a pro-survival lipid. At the same time, LPA decreases the accumulation of ceramides, which are used in radiation therapy and by many chemotherapeutic agents to stimulate apoptosis. The signaling actions of extracellular LPA are terminated by its dephosphorylation by a family of lipid phosphate phosphatases (LPP) that act as ecto-enzymes. In addition, lipid phosphate phoshatase-1 attenuates signaling downstream of the activation of both LPA receptors and receptor tyrosine kinases. This makes many cancer cells hypersensitive to the action of various growth factors since they often express low LPP1/3 activity. Increasing our understanding of the complicated signaling pathways that are used by LPA to stimulate cell survival should identify new therapeutic targets that can be exploited to increase the efficacy of chemo- and radio-therapy. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.
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Affiliation(s)
- David N Brindley
- Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada.
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Kok BPC, Venkatraman G, Capatos D, Brindley DN. Unlike two peas in a pod: lipid phosphate phosphatases and phosphatidate phosphatases. Chem Rev 2012; 112:5121-46. [PMID: 22742522 DOI: 10.1021/cr200433m] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Affiliation(s)
- Bernard P C Kok
- Signal Transduction Research Group, Department of Biochemistry, School of Translational Medicine, University of Alberta, Edmonton, Alberta T6G 2S2, Canada
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Bekele RT, Brindley DN. Role of autotaxin and lysophosphatidate in cancer progression and resistance to chemotherapy and radiotherapy. ACTA ACUST UNITED AC 2012. [DOI: 10.2217/clp.12.30] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
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Senda K, Koizumi K, Prangsaengtong O, Minami T, Suzuki S, Takasaki I, Tabuchi Y, Sakurai H, Doki Y, Misaki T, Saiki I. Inducible capillary formation in lymphatic endothelial cells by blocking lipid phosphate phosphatase-3 activity. Lymphat Res Biol 2009; 7:69-74. [PMID: 19473074 DOI: 10.1089/lrb.2009.0005] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Lymphangiogenesis plays critical roles under normal and/or pathological conditions; however, the molecular contributors to this event were unknown until recently. In the present study, we first employed gene chip analysis and confirmed that lipid phosphate phosphatase-3 (LPP3) expression was increased until capillary formation in the conditionally immortalized rat lymphatic endothelial cell line. Signaling responses occur when several lipids induce acute biological functions; further, lipid phosphate phosphatases (LPPs) control their functions via dephosphorylation; however, there is no report on the association between LPP3 and lymphangiogenesis. siRNA-targeted LPP3 significantly increased capillary formation of human lymphatic endothelial cells; in contrast, it decreased cell adhesion to the basement membrane matrix. Furthermore, the inducible effect of the LPP inhibitor on capillary formation was observed. For the first time, we report that LPP3 abolishes accelerated abnormal lymphangiogenesis. Blocking LPP3 activities may aid in the development of novel therapy for lymph vessel defects.
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Affiliation(s)
- Kazutaka Senda
- Department of Surgery (I), Faculty of Medicine, University of Toyama, Toyama, Japan
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Tomsig JL, Snyder AH, Berdyshev EV, Skobeleva A, Mataya C, Natarajan V, Brindley DN, Lynch KR. Lipid phosphate phosphohydrolase type 1 (LPP1) degrades extracellular lysophosphatidic acid in vivo. Biochem J 2009; 419:611-8. [PMID: 19215222 PMCID: PMC2677185 DOI: 10.1042/bj20081888] [Citation(s) in RCA: 102] [Impact Index Per Article: 6.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
LPA (lysophosphatidic acid) is a lipid mediator that stimulates cell proliferation and growth, and is involved in physiological and pathological processes such as wound healing, platelet activation, angiogenesis and the growth of tumours. Therefore defining the mechanisms of LPA production and degradation are of interest in understanding the regulation of these processes. Extracellular LPA synthesis is relatively well understood, whereas the mechanisms of its degradation are not. One route of LPA degradation is dephosphorylation. A candidate enzyme is the integral membrane exophosphatase LPP1 (lipid phosphate phosphohydrolase type 1). In the present paper, we report the development of a mouse wherein the LPP1 gene (Ppap2a) was disrupted. The homozygous mice, which are phenotypically unremarkable, generally lack Ppap2a mRNA, and multiple tissues exhibit a substantial (35-95%) reduction in LPA phosphatase activity. Compared with wild-type littermates, Ppap2a(tr/tr) animals have increased levels of plasma LPA, and LPA injected intravenously is metabolized at a 4-fold lower rate. Our results demonstrate that LPA is rapidly metabolized in the bloodstream and that LPP1 is an important determinant of this turnover. These results indicate that LPP1 is a catabolic enzyme for LPA in vivo.
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Affiliation(s)
- Jose L. Tomsig
- Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908
| | - Ashley H. Snyder
- Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908
| | - Evgeny V. Berdyshev
- Department of Medicine, Biological Sciences Division, University of Chicago, Chicago, Illinois 60637
| | - Anastasia Skobeleva
- Department of Medicine, Biological Sciences Division, University of Chicago, Chicago, Illinois 60637
| | - Chifundo Mataya
- Department of Biochemistry, University of Alberta, Edmonton, T6G 2S2, Canada
| | - Viswanathan Natarajan
- Department of Medicine, Biological Sciences Division, University of Chicago, Chicago, Illinois 60637
| | - David N. Brindley
- Department of Biochemistry, University of Alberta, Edmonton, T6G 2S2, Canada
| | - Kevin R. Lynch
- Department of Pharmacology, University of Virginia, Charlottesville, Virginia 22908
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Zhao Y, Natarajan V. Lysophosphatidic acid signaling in airway epithelium: role in airway inflammation and remodeling. Cell Signal 2009; 21:367-77. [PMID: 18996473 PMCID: PMC2660380 DOI: 10.1016/j.cellsig.2008.10.010] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/10/2008] [Revised: 09/27/2008] [Accepted: 10/21/2008] [Indexed: 02/07/2023]
Abstract
Lysophosphatidic acid (LPA), a potent bioactive phospholipid, induces diverse cellular responses, including cell proliferation, migration, and cytokine release. LPA can be generated intracellularly and extracellularly through multiple synthetic pathways by action of various enzymes, such as phospholipase A(1/2) (PLA(1/2)), phospholipase D (PLD), acylglycerol kinase (AGK), and lysophospholipase D (lysoPLD). Metabolism of LPA is regulated by a family of lipid phosphate phosphatases (LPPs). Significant amounts of LPA have been detected in various biological fluids, including serum, saliva, and bronchoalveolar lavage fluid (BALF). The most significant effects of LPA appear to be through activation of the G-protein-coupled receptors (GPCRs), termed LPA(1-6). LPA regulates gene expression through activation of several transcriptional factors, such as nuclear factor-kappaB (NF-kappaB), AP-1, and C/EBPbeta. In addition to GPCRs, cross-talk between LPA receptors and receptor tyrosine kinases (RTKs) partly regulates LPA-induced intracellular signaling and cellular responses. Airway epithelial cells participate in innate immunity through the release of cytokines, chemokines, lipid mediators, other inflammatory mediators and an increase in barrier function in response to a variety of inhaled stimuli. Expression of LPA receptors has been demonstrated in airway epithelial cells. This review summarizes our recent observations of the role of LPA/LPA-Rs in regulation of airway epithelium, especially in relation to the secretion of pro- and anti-inflammatory mediators and regulation of airway barrier function.
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Affiliation(s)
- Yutong Zhao
- Department of Medicine, University of Chicago, Chicago, IL 60637, USA.
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Theofilopoulos S, Lykidis A, Leondaritis G, Mangoura D. Novel function of the human presqualene diphosphate phosphatase as a type II phosphatidate phosphatase in phosphatidylcholine and triacylglyceride biosynthesis pathways. Biochim Biophys Acta Mol Cell Biol Lipids 2008; 1781:731-42. [DOI: 10.1016/j.bbalip.2008.09.001] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2008] [Revised: 08/29/2008] [Accepted: 09/11/2008] [Indexed: 11/27/2022]
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Yamanaka M, Anada Y, Igarashi Y, Kihara A. A splicing isoform of LPP1, LPP1a, exhibits high phosphatase activity toward FTY720 phosphate. Biochem Biophys Res Commun 2008; 375:675-9. [PMID: 18755152 DOI: 10.1016/j.bbrc.2008.07.165] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/22/2008] [Accepted: 07/29/2008] [Indexed: 12/01/2022]
Abstract
The sphingolipid metabolite sphingosine 1-phosphate (S1P) plays an essential function in the egress of T cells from the thymus and secondary lymphoid organs. The novel immunomodulating agent FTY720 is phosphorylated in vivo to the functional form FTY720 phosphate (FTY720-P), which is structurally similar to S1P. FTY720-P inhibits the S1P-mediated T cell egress as an agonist of S1P receptors. FTY720-P is not stable in plasma and is dephosphorylated to FTY720. In the present study, we investigated activities toward FTY720-P of LPP family members (LPP1, LPP1a, LPP2, and LPP3), which exhibit broad substrate specificity. Of the four, LPP1a, the splicing isoform of LPP1, had the highest activity toward FTY720-P, and the highest affinity. Among blood-facing cells tested, only endothelial cells displayed high phosphatase activity for FTY720-P. Significant levels of LPP1a expression were found in endothelial cells, suggesting that LPP1a is important for the dephosphorylation of FTY720-P in plasma.
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Affiliation(s)
- Masao Yamanaka
- Laboratory of Biomembrane and Biofunctional Chemistry, Faculty of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo, Nishi 6-choume, Kita-ku, Sapporo 060-0812, Japan
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Lipid phosphate phosphatases form homo- and hetero-oligomers: catalytic competency, subcellular distribution and function. Biochem J 2008; 411:371-7. [PMID: 18215144 DOI: 10.1042/bj20071607] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
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.
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Pilquil C, Dewald J, Cherney A, Gorshkova I, Tigyi G, English D, Natarajan V, Brindley DN. Lipid phosphate phosphatase-1 regulates lysophosphatidate-induced fibroblast migration by controlling phospholipase D2-dependent phosphatidate generation. J Biol Chem 2006; 281:38418-29. [PMID: 17057224 DOI: 10.1074/jbc.m601670200] [Citation(s) in RCA: 51] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Lysophosphatidate (LPA) stimulates cell migration and division through a family of G-protein-coupled receptors. Lipid phosphate phosphatase-1 (LPP1) regulates the degradation of extracellular LPA as well as the intracellular accumulation of lipid phosphates. Here we show that increasing the catalytic activity of LPP1 decreased the pertussis toxin-sensitive stimulation of fibroblast migration by LPA and an LPA-receptor agonist that could not be dephosphorylated. Conversely, knockdown of endogenous LPP1 activity increased LPA-induced migration. However, LPP1 did not affect PDGF- or endothelin-induced migration of fibroblasts in Transwell chamber and "wound healing" assays. Thus, in addition to degrading exogenous LPA, LPP1 controls signaling downstream of LPA receptors. Consistent with this conclusion, LPP1 expression decreased phospholipase D (PLD) stimulation by LPA and PDGF, and phosphatidate accumulation. This LPP1 effect was upstream of PLD activation in addition to the possible metabolism of phosphatidate to diacylglycerol. PLD(2) activation was necessary for LPA-, but not PDGF-induced migration. Increased LPP1 expression also decreased the LPA-, but not the PDGF-induced activation of important proteins involved in fibroblast migration. These included decreased LPA-induced activation of ERK and Rho, and the basal activities of Rac and Cdc42. However, ERK and Rho activation were not downstream targets of LPA-induced PLD(2) activity. We conclude that the intracellular actions of LPP1 play important functions in regulating LPA-induced fibroblast migration through PLD2. LPP1 also controls PDGF-induced phosphatidate formation. These results shed new light on the roles of LPP1 in controlling wound healing and the growth and metastasis of tumors.
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Affiliation(s)
- Carlos Pilquil
- Signal Transduction Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2S2, Canada
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15
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Long J, Yokoyama K, Tigyi G, Pyne N, Pyne S. Lipid phosphate phosphatase-1 regulates lysophosphatidic acid- and platelet-derived-growth-factor-induced cell migration. Biochem J 2006; 394:495-500. [PMID: 16356167 PMCID: PMC1408680 DOI: 10.1042/bj20051674] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/17/2022]
Abstract
LPPs (lipid phosphate phosphatases) are members of a family of enzymes that catalyse the dephosphorylation of lipid phosphates. The only known form of regulation of this family of enzymes is via de novo expression of LPP isoforms in response to growth factors. In this respect, we evaluated the effect of moderate increases in the expression of recombinant LPP1 on signal transduction by both G-protein-coupled receptors and receptor tyrosine kinases. We present evidence for a novel role of LPP1 in reducing PDGF (platelet-derived growth factor)- and lysophosphatidic acid-induced migration of embryonic fibroblasts. We demonstrate that the overexpression of LPP1 inhibits cell migration by reducing the PDGF-induced activation of p42/p44 MAPK (mitogen-activated protein kinase). This appears to occur via a mechanism that involves the LPP1-induced down-regulation of typical PKC (protein kinase C) isoform(s), which are normally required for PDGF-induced activation of p42/p44 MAPK and migration. In this regard, DAG (diacylglycerol) levels are high and sustained in cells overexpressing LPP1, suggesting a dynamic interconversion of phosphatidic acid into DAG by LPP1. This may account for the effects of LPP1 on cell migration, as sustained DAG is known to down-regulate PKC isoforms in cells. Therefore the physiological changes in the expression levels of LPP1 might represent a heterologous desensitization mechanism for attenuating PKC-mediated signalling and regulation of cell migration.
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Affiliation(s)
- Jaclyn S. Long
- *Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, U.K
| | - Kazuaki Yokoyama
- †Department of Physiology, University of Tennessee Health Science Center Memphis, 894 Union Avenue, Memphis, TN, U.S.A
| | - Gabor Tigyi
- †Department of Physiology, University of Tennessee Health Science Center Memphis, 894 Union Avenue, Memphis, TN, U.S.A
| | - Nigel J. Pyne
- *Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, U.K
| | - Susan Pyne
- *Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, U.K
- To whom correspondence should be addressed (email )
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Long J, Darroch P, Wan K, Kong K, Ktistakis N, Pyne N, Pyne S. Regulation of cell survival by lipid phosphate phosphatases involves the modulation of intracellular phosphatidic acid and sphingosine 1-phosphate pools. Biochem J 2006; 391:25-32. [PMID: 15960610 PMCID: PMC1237135 DOI: 10.1042/bj20050342] [Citation(s) in RCA: 58] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/30/2023]
Abstract
We have shown previously that LPPs (lipid phosphate phosphatases) reduce the stimulation of the p42/p44 MAPK (p42/p44 mitogen-activated protein kinase) pathway by the GPCR (G-protein-coupled receptor) agonists S1P (sphingosine 1-phosphate) and LPA (lysophosphatidic acid) in serum-deprived HEK-293 cells [Alderton, Darroch, Sambi, McKie, Ahmed, N. J. Pyne and S. Pyne (2001) J. Biol. Chem. 276, 13452-13460]. In the present study, we now show that this can be blocked by pretreating HEK-293 cells with the caspase 3/7 inhibitor, Ac-DEVD-CHO [N-acetyl-Asp-Glu-Val-Asp-CHO (aldehyde)]. Therefore LPP2 and LPP3 appear to regulate the apoptotic status of serum-deprived HEK-293 cells. This was supported further by: (i) caspase 3/7-catalysed cleavage of PARP [poly(ADP-ribose) polymerase] was increased in serum-deprived LPP2-overexpressing compared with vector-transfected HEK-293 cells; and (ii) serum-deprived LPP2- and LPP3-overexpressing cells exhibited limited intranucleosomal DNA laddering, which was absent in vector-transfected cells. Moreover, LPP2 reduced basal intracellular phosphatidic acid levels, whereas LPP3 decreased intracellular S1P in serum-deprived HEK-293 cells. LPP2 and LPP3 are constitutively co-localized with SK1 (sphingosine kinase 1) in cytoplasmic vesicles in HEK-293 cells. Moreover, LPP2 but not LPP3 prevents SK1 from being recruited to a perinuclear compartment upon induction of PLD1 (phospholipase D1) in CHO (Chinese-hamster ovary) cells. Taken together, these data are consistent with an important role for LPP2 and LPP3 in regulating an intracellular pool of PA and S1P respectively, that may govern the apoptotic status of the cell upon serum deprivation.
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Affiliation(s)
- Jaclyn Long
- *Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, U.K
| | - Peter Darroch
- *Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, U.K
| | - Kah Fei Wan
- *Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, U.K
| | - Kok Choi Kong
- *Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, U.K
| | | | - Nigel J. Pyne
- *Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, U.K
| | - Susan Pyne
- *Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, U.K
- To whom correspondence should be addressed (email )
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17
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Abstract
Mammalian LPPs (lipid phosphate phosphatases) are integral membrane proteins that belong to a superfamily of lipid phosphatases/phosphotransferases. They have broad substrate specificity in vitro, dephosphorylating PA (phosphatidic acid), S1P (sphingosine 1-phosphate), LPA (lysophosphatidic acid) etc. Their physiological role may include the attenuation of S1P- and LPA-stimulated signalling by virtue of an ecto-activity (i.e. dephosphorylation of extracellular S1P and LPA), thereby limiting the activation of LPA- and S1P-specific G-protein-coupled receptors at the cell surface. However, our recent work suggests that an intracellular action of LPP2 and LPP3 may account for the reduced agonist-stimulated p42/p44 mitogen-activated protein kinase activation of HEK-293 (human embryonic kidney 293) cells. This may involve a reduction in the basal levels of PA and S1P respectively and the presence of an early apoptotic phenotype under conditions of stress (serum deprivation). Additionally, we describe a model whereby LPP2, but not LPP3, may be functionally linked to the phospholipase D1-derived PA-dependent recruitment of sphingosine kinase 1 to the perinuclear compartment. We also consider the potential regulatory mechanisms for LPPs, which may involve oligomerization. Lastly, we highlight many aspects of the LPP biology that remain to be fully defined.
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18
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Sigal Y, McDERMOTT M, Morris A. Integral membrane lipid phosphatases/phosphotransferases: common structure and diverse functions. Biochem J 2005; 387:281-93. [PMID: 15801912 PMCID: PMC1134956 DOI: 10.1042/bj20041771] [Citation(s) in RCA: 150] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
Phospholipids and sphingolipids play critical roles in signal transduction, intracellular membrane trafficking, and control of cell growth and survival. We discuss recent progress in the identification and characterization of a family of integral membrane proteins with central roles in bioactive lipid metabolism and signalling. These five groups of homologous proteins, which we collectively term LPTs (lipid phosphatases/phosphotransferases), are characterized by a core domain containing six transmembrane-spanning alpha-helices connected by extramembrane loops, two of which interact to form the catalytic site. LPT family members are localized to all major membrane compartments of the cell. The transmembrane topology of these proteins places their active site facing the lumen of endomembrane compartments or the extracellular face of the plasma membrane. Sequence conservation between the active site of the LPPs (lipid phosphate phosphatases), SPPs (sphingosine phosphate phosphatases) and the recently identified SMSs (sphingomyelin synthases) with vanadium-dependent fungal oxidases provides a framework for understanding their common catalytic mechanism. LPPs hydrolyse LPA (lysophosphatidic acid), S1P (sphingosine 1-phosphate) and structurally-related substrates. Although LPPs can dephosphorylate intracellularly generated substrates to control intracellular lipid metabolism and signalling, their best understood function is to regulate cell surface receptor-mediated signalling by LPA and S1P by inactivating these lipids at the plasma membrane or in the extracellular space. SPPs are intracellularly localized S1P-selective phosphatases, with key roles in the pathways of sphingolipid metabolism linked to control of cell growth and survival. The SMS enzymes catalyse the interconversion of phosphatidylcholine and ceramide with sphingomyelin and diacylglycerol, suggesting a pivotal role in both housekeeping lipid synthesis and regulation of bioactive lipid mediators. The remaining members of the LPT family, the LPR/PRGs (lipid phosphatase-related proteins/plasticity-related genes) and CSS2s (type 2 candidate sphingomyelin synthases), are presently much less well studied. These two groups include proteins that lack critical amino acids within the catalytic site, and could therefore not use the conserved LPT reaction mechanism to catalyse lipid phosphatase or phosphotransferase reactions. In this review, we discuss recent ideas about their possible biological activities and functions, which appear to involve regulation of cellular morphology and, possibly, lipid metabolism and signalling in the nuclear envelope.
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Affiliation(s)
- Yury J. Sigal
- Department of Cell and Developmental Biology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090, U.S.A
| | - Mark I. McDERMOTT
- Department of Cell and Developmental Biology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090, U.S.A
| | - Andrew J. Morris
- Department of Cell and Developmental Biology and Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7090, U.S.A
- To whom correspondence should be addressed (email )
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19
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Brindley DN. Lipid phosphate phosphatases and related proteins: signaling functions in development, cell division, and cancer. J Cell Biochem 2005; 92:900-12. [PMID: 15258914 DOI: 10.1002/jcb.20126] [Citation(s) in RCA: 198] [Impact Index Per Article: 9.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Abstract
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.
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Affiliation(s)
- David N Brindley
- Signal Transduction Research Group, Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2S2.
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20
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Pyne S, Kong KC, Darroch PI. Lysophosphatidic acid and sphingosine 1-phosphate biology: the role of lipid phosphate phosphatases. Semin Cell Dev Biol 2005; 15:491-501. [PMID: 15271294 DOI: 10.1016/j.semcdb.2004.05.007] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
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.
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Affiliation(s)
- Susan Pyne
- Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Research, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, Scotland, UK.
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21
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Affiliation(s)
- Gabor Tigyi
- Department of Physiology, The University of Tennessee Health Science Center, Memphis, TN 38163, USA.
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22
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Bernard R, Joseph P, Guiseppi A, Chippaux M, Denizot F. YtsCD and YwoA, two independent systems that confer bacitracin resistance toBacillus subtilis. FEMS Microbiol Lett 2003; 228:93-7. [PMID: 14612242 DOI: 10.1016/s0378-1097(03)00738-9] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/27/2022] Open
Abstract
The Bacillus subtilis yts, yxd and yvc gene clusters encode a putative ABC transporter and a functionally coupled two-component system. When tested for their sensitivity towards a series of antibiotics, null yts mutants were found to be sensitive to bacitracin. Real-time polymerase chain reaction (PCR) experiments demonstrated that the presence of bacitracin in the growth medium strongly stimulates the expression of the ytsCD genes encoding the ABC transporter and that this stimulation strictly depends on the YtsA response regulator. The ywoA gene encodes a protein known to confer some resistance to bacitracin on the bacterium. When it was mutated in a null yts background, the ywoA yts double mutant was found to be five times more sensitive than the yts one. We propose that (i) the YtsCD ABC transporter exports the bacitracin; (ii) YwoA, the protein that contains an acidPPc (PAP2 or PgpB) domain, is not part of an ABC transporter but competes with bacitracin for the dephosphorylation of the C55-isoprenyl pyrophosphate (IPP); (iii) the two resistance mechanisms are independent and complementary.
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Affiliation(s)
- Remi Bernard
- Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et Microbiologie, CNRS, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France
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23
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Burnett C, Howard K. Fly and mammalian lipid phosphate phosphatase isoforms differ in activity both in vitro and in vivo. EMBO Rep 2003; 4:793-9. [PMID: 12856002 PMCID: PMC1326340 DOI: 10.1038/sj.embor.embor900] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/14/2003] [Revised: 06/10/2003] [Accepted: 06/11/2003] [Indexed: 11/08/2022] Open
Abstract
Wunen (Wun), a homologue of a lipid phosphate phosphatase (LPP), has a crucial function in the migration and survival of primordial germ cells (PGCs) during Drosophila embryogenesis. Past work has indicated that the LPP isoforms may show functional redundancy in certain systems, and that they have broad-range lipid phosphatase activities in vitro, with little apparent specificity between them. We show here that there are marked differences in biochemical activity between fly Wun and mammalian LPPs, with Wun having a narrower activity range than has been reported for the mammalian LPPs. Furthermore, although it is active on a range of substrates in vitro, mouse Lpp1 has no activity on an endogenous Drosophila germ-cell-specific factor in vivo. Conversely, human LPP3 is active, resulting in aberrant migration and PGC death. These results show an absolute difference in bioactivity among LPP isoforms for the first time in a model organism and may point towards an underlying signalling system that is conserved between flies and humans.
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Affiliation(s)
- Camilla Burnett
- MRC Laboratory for Molecular Cell Biology, and
Department of Physiology, University College London, Gower
Street, London WC1E 6BT, UK
| | - Ken Howard
- MRC Laboratory for Molecular Cell Biology, and
Department of Physiology, University College London, Gower
Street, London WC1E 6BT, UK
- Tel: +44 20 7679 2248; Fax: +44 20 7679 7805;
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24
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Nanjundan M, Possmayer F. Pulmonary phosphatidic acid phosphatase and lipid phosphate phosphohydrolase. Am J Physiol Lung Cell Mol Physiol 2003; 284:L1-23. [PMID: 12471011 DOI: 10.1152/ajplung.00029.2002] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/22/2022] Open
Abstract
The lung contains two distinct forms of phosphatidic acid phosphatase (PAP). PAP1 is a cytosolic enzyme that is activated through fatty acid-induced translocation to the endoplasmic reticulum, where it converts phosphatidic acid (PA) to diacylglycerol (DAG) for the biosynthesis of phospholipids and neutral lipids. PAP1 is Mg(2+) dependent and sulfhydryl reagent sensitive. PAP2 is a six-transmembrane-domain integral protein localized to the plasma membrane. Because PAP2 degrades sphingosine-1-phosphate (S1P) and ceramide-1-phosphate in addition to PA and lyso-PA, it has been renamed lipid phosphate phosphohydrolase (LPP). LPP is Mg(2+) independent and sulfhydryl reagent insensitive. This review describes LPP isoforms found in the lung and their location in signaling platforms (rafts/caveolae). Pulmonary LPPs likely function in the phospholipase D pathway, thereby controlling surfactant secretion. Through lowering the levels of lyso-PA and S1P, which serve as agonists for endothelial differentiation gene receptors, LPPs regulate cell division, differentiation, apoptosis, and mobility. LPP activity could also influence transdifferentiation of alveolar type II to type I cells. It is considered likely that these lipid phosphohydrolases have critical roles in lung morphogenesis and in acute lung injury and repair.
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Affiliation(s)
- Meera Nanjundan
- Department of Obstetrics and Gynaecology, Canadian Institutes of Health Research Group in Fetal and Neonatal Health and Development, The University of Western Ontario, 339 Windermere Road, London, Ontario, Canada N6A 5A5
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25
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Nanjundan M, Possmayer F. Molecular cloning and expression of pulmonary lipid phosphate phosphohydrolases. Am J Physiol Lung Cell Mol Physiol 2001; 281:L1484-93. [PMID: 11704545 DOI: 10.1152/ajplung.2001.281.6.l1484] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022] Open
Abstract
Pulmonary lipid phosphate phosphohydrolase (LPP) was shown previously to hydrolyze phosphatidic acid and lysophosphatidic acid in purified rat lung plasma membranes. To better investigate the nature of pulmonary LPP isoforms and their role in the lung, LPPs were cloned by RT-PCR from both adult rat lung and type II cell RNA. The RT-PCR generated LPP1 (849 bp), up to three LPP1 variants, and LPP3 (936 bp) cDNAs. The three LPP1 variants include LPP1a (852 bp) and two novel isoforms, LPP1b (697 bp) and LPP1c (1004 bp). The pulmonary LPP1 and LPP3 isoforms are essentially identical to the previously cloned rat liver and intestinal LPPs, respectively, and the LPP1a isoform has 80% sequence identity to the human homolog. The LPP2 isoform was not detected in lung by RT-PCR. Northern analyses revealed that the mRNAs for LPP1 and LPP3 increase in fetal rat lung in late gestation to day 1 after birth. These mRNAs decrease somewhat during the neonatal period but increase slightly during postnatal development. Expression of LPP1, LPP1a, and LPP3 cDNAs in HEK 293 cells established that they encode functional LPP. In contrast, the novel isoforms LPP1b and LPP1c contain frameshifts that would result in premature termination, producing putative catalytically inactive polypeptides of 30 and 76 amino acids, respectively. Further investigation of the LPP1b isoform revealed that it was present across a variety of tissues, although at lower levels than LPP1/1a. Transient mammalian expression of LPP1b failed to increase phosphatidate phosphohydrolase activity in HEK 293 cells.
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Affiliation(s)
- M Nanjundan
- Medical Research Council Group in Fetal and Neonatal Health and Development, Department of Biochemistry, University of Western Ontario, London, Ontario, Canada N6A 5A5
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26
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Loring JF, Wen X, Lee JM, Seilhamer J, Somogyi R. A gene expression profile of Alzheimer's disease. DNA Cell Biol 2001; 20:683-95. [PMID: 11788046 DOI: 10.1089/10445490152717541] [Citation(s) in RCA: 231] [Impact Index Per Article: 9.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Postmortem analysis of brains of patients with Alzheimer's disease (AD) has led to diverse theories about the causes of the pathology, suggesting that this complex disease involves multiple physiological changes. In an effort to better understand the variety and integration of these changes, we generated a gene expression profile for AD brain. Comparing affected and unaffected brain regions in nine controls and six AD cases, we showed that 118 of the 7050 sequences on a broadly representative cDNA microarray were differentially expressed in the amygdala and cingulate cortex, two regions affected early in the disease. The identity of these genes suggests the most prominent upregulated physiological correlates of pathology involve chronic inflammation, cell adhesion, cell proliferation, and protein synthesis (31 upregulated genes). Conversely, downregulated correlates of pathology involve signal transduction, energy metabolism, stress response, synaptic vesicle synthesis and function, calcium binding, and cytoskeleton (87 downregulated genes). The results support several separate theories of the causes of AD pathology, as well as add to the list of genes associated with AD. In addition, approximately 10 genes of unknown function were found to correlate with the pathology.
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Affiliation(s)
- J F Loring
- Department of Life Sciences, Incyte Genomics, Inc., Palo Alto, California 94304, USA.
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27
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Alderton F, Darroch P, Sambi B, McKie A, Ahmed IS, Pyne N, Pyne S. G-protein-coupled receptor stimulation of the p42/p44 mitogen-activated protein kinase pathway is attenuated by lipid phosphate phosphatases 1, 1a, and 2 in human embryonic kidney 293 cells. J Biol Chem 2001; 276:13452-60. [PMID: 11278307 DOI: 10.1074/jbc.m006582200] [Citation(s) in RCA: 66] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidic acid bind to G-protein-coupled receptors to stimulate intracellular signaling in mammalian cells. Lipid phosphate phosphatases (1, 1a, 2, and 3) are a group of enzymes that catalyze de-phosphorylation of these lipid agonists. It has been proposed that the lipid phosphate phosphatases exhibit ecto activity that may function to limit bioavailability of these lipid agonists at their receptors. In this study, we show that the stimulation of the p42/p44 mitogen-activated protein kinase pathway by sphingosine 1-phosphate, lysophosphatidic acid, and phosphatidic acid, all of which bind to G(i/o)-coupled receptors, is substantially reduced in human embyronic kidney 293 cells transfected with lipid phosphate phosphatases 1, 1a, and 2 but not 3. This was correlated with reduced basal intracellular phosphatidic acid and not ecto lipid phosphate phosphatase activity. These findings were supported by results showing that lipid phosphate phosphatases 1, 1a, and 2 also abrogate the stimulation of p42/p44 mitogen-activated protein kinase by thrombin, a peptide G(i/o)-coupled receptor agonist whose bioavailability at its receptor is not subject to regulation by the phosphatases. Furthermore, the lipid phosphate phosphatases have no effect on the stimulation of p42/p44 mitogen-activated protein kinase by other agents that do not use G-proteins to signal, such as serum factors and phorbol ester. Therefore, these findings show that the lipid phosphate phosphatases 1, 1a, and 2 may function to perturb G-protein-coupled receptor signaling per se rather than limiting bioavailability of lipid agonists at their respective receptors.
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Affiliation(s)
- F Alderton
- Department of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor St., Glasgow, G4 0NR, United Kingdom
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28
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Porkka KP, Visakorpi T. Detection of differentially expressed genes in prostate cancer by combining suppression subtractive hybridization and cDNA library array. J Pathol 2001; 193:73-9. [PMID: 11169518 DOI: 10.1002/1096-9896(2000)9999:9999<::aid-path751>3.0.co;2-y] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
The molecular mechanisms underlying the development and progression of prostate cancer have remained poorly understood. The identification of differentially expressed genes has been used as a tool to recognize genes that are involved in disease processes. In this study we combined suppression subtractive hybridization (SSH) and cDNA array hybridization to identify genes whose expression is decreased in prostate cancer. cDNA from benign prostatic hyperplasia (BPH) was subtracted with cDNA from the prostate cancer cell line PC-3 and 386 of the subtracted clones were arrayed onto a nylon filter membrane. The differential gene expression was then verified by hybridizing the filter with radioactively labelled first-strand cDNA preparations from BPH, PC-3, four other cancer cell lines, and a normal prostate epithelial cell line (PrEC). In order to validate SSH and cDNA array hybridization, the enrichment of clones in the subtraction, as well as the sensitivity and linearity of array hybridization, was first evaluated. The array hydridization results were confirmed by northern analysis and selected clones were sequenced. Altogether, several known genes, such as prostate-specific antigen (PSA), human glandular kallikrein 2 (hK2), phosphatidic acid phosphatase type 2a (PAP2a), alpha-tropomyosin, and insulin-like growth factor binding protein 7 (IGFBP-7), as well as an anonymous transcript (EST), were found to be expressed less in PC-3 than in BPH. Further studies on the significance of these genes in the development of prostate cancer are now warranted.
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Affiliation(s)
- K P Porkka
- Laboratory of Cancer Genetics, Institute of Medical Technology, University of Tampere and Tampere University Hospital, Tampere, Finland
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29
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Roberts RZ, Morris AJ. Role of phosphatidic acid phosphatase 2a in uptake of extracellular lipid phosphate mediators. BIOCHIMICA ET BIOPHYSICA ACTA 2000; 1487:33-49. [PMID: 10962286 DOI: 10.1016/s1388-1981(00)00081-0] [Citation(s) in RCA: 28] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 10/18/2022]
Abstract
Phosphatidic acid (PA) phosphatase 2a (PAP2a) is an integral membrane glycoprotein that hydrolyzes a number of structurally related lipid phosphate substrates when presented in mixed phospholipid and detergent micelles. The physiological substrate specificity and functions of this enzyme are unclear. Using reconstitution studies we demonstrate that PAP2a hydrolyses both PA and LysoPA substrates in a lipid bilayer. To investigate the activity of PAP2a against cellular substrates we generated HEK293 cell variants stably overexpressing the enzyme. Although one of these lines exhibited a 27-fold increase in PAP2 activity measured in vitro, levels of PA were not significantly reduced in comparison with control cells. Cell surface labeling and activity measurements demonstrate that a portion of the enzyme was localized to the cell surface. Pagano and Longmuir (J. Biol. Chem. 260 (1985) 1909) described the rapid uptake of PA by cultured cells, but the mechanisms and proteins involved were not identified. We found that overexpression of PAP2a was accompanied by a 2.1-fold increase in uptake of a fluorescent PA analog but that uptake of other phospholipids and diacylglycerols was unaltered. The increase in lipid uptake was completely dependent on PAP activity and unaffected by endocytosis inhibitors. Our results indicate that PAP2a is a cell surface enzyme that plays an active role in the hydrolysis and uptake of lipids from the extracellular space.
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Affiliation(s)
- R Z Roberts
- Department of Pharmacological Sciences, Institute for Cell and Developmental Biology, Stony Brook Health Sciences Center, Stony Brook, NY, USA
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30
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Zhang N, Sundberg JP, Gridley T. Mice mutant for Ppap2c, a homolog of the germ cell migration regulator wunen, are viable and fertile. Genesis 2000; 27:137-40. [PMID: 10992322 DOI: 10.1002/1526-968x(200008)27:4<137::aid-gene10>3.0.co;2-4] [Citation(s) in RCA: 57] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/09/2022]
Abstract
Phosphatidic acid phosphatases (PAPs) catalyze the conversion of phosphatidic acid to diacylglycerol and inorganic phosphate and have been postulated to function both in lipid biosynthesis and in cellular signal transduction. In Drosophila melanogaster, the Type 2 phosphatidic acid phosphatase protein encoded by the wunen gene, negatively regulates primordial germ cell migration. We recently described the cloning and characterization of the mouse Ppap2c gene, which encodes the Type 2 phosphatidic acid phosphatase Pap2c (Zhang et al., Genomics 63:142-144). To analyze the in vivo role of the Ppap2c gene we constructed a null mutation by gene targeting. Ppap2c(-/-) homozygous mutant mice were viable, fertile, and exhibited no obvious phenotypic defects. These data demonstrate that the Ppap2c gene is not essential for embryonic development or fertility in mice.
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Affiliation(s)
- N Zhang
- The Jackson Laboratory, Bar Harbor, Maine 04609, USA
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31
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Leung DW, Tompkins CK, White T. Characterization of two spliced variants of human phosphatidic acid phosphatase cDNAs that are differentially expressed in normal and tumor cells. ADVANCES IN EXPERIMENTAL MEDICINE AND BIOLOGY 2000; 469:639-46. [PMID: 10667393 DOI: 10.1007/978-1-4615-4793-8_92] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/15/2023]
Affiliation(s)
- D W Leung
- Molecular Biology Department, Cell Therapeutics, Inc., Seattle, WA 98119, USA
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32
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Sciorra VA, Morris AJ. Sequential actions of phospholipase D and phosphatidic acid phosphohydrolase 2b generate diglyceride in mammalian cells. Mol Biol Cell 1999; 10:3863-76. [PMID: 10564277 PMCID: PMC25685 DOI: 10.1091/mbc.10.11.3863] [Citation(s) in RCA: 94] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/11/2022] Open
Abstract
Phosphatidylcholine (PC) is a major source of lipid-derived second messenger molecules that function as both intracellular and extracellular signals. PC-specific phospholipase D (PLD) and phosphatidic acid phosphohydrolase (PAP) are two pivotal enzymes in this signaling system, and they act in series to generate the biologically active lipids phosphatidic acid (PA) and diglyceride. The identity of the PAP enzyme involved in PLD-mediated signal transduction is unclear. We provide the first evidence for a functional role of a type 2 PAP, PAP2b, in the metabolism of PLD-generated PA. Our data indicate that PAP2b localizes to regions of the cell in which PC hydrolysis by PLD is taking place. Using a newly developed PAP2b-specific antibody, we have characterized the expression, posttranslational modification, and localization of endogenous PAP2b. Glycosylation and localization of PAP2b appear to be cell type and tissue specific. Biochemical fractionation and immunoprecipitation analyses revealed that PAP2b and PLD2 activities are present in caveolin-1-enriched detergent-resistant membrane microdomains. We found that PLD2 and PAP2b act sequentially to generate diglyceride within this specialized membrane compartment. The unique lipid composition of these membranes may provide a selective environment for the regulation and actions of enzymes involved in signaling through PC hydrolysis.
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Affiliation(s)
- V A Sciorra
- Department of Pharmacological Sciences and Institute for Cell and Developmental Biology, State University of New York, Stony Brook, New York 11794-8651, USA
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Waggoner DW, Xu J, Singh I, Jasinska R, Zhang QX, Brindley DN. Structural organization of mammalian lipid phosphate phosphatases: implications for signal transduction. BIOCHIMICA ET BIOPHYSICA ACTA 1999; 1439:299-316. [PMID: 10425403 DOI: 10.1016/s1388-1981(99)00102-x] [Citation(s) in RCA: 110] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/03/2023]
Abstract
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.
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Affiliation(s)
- D W Waggoner
- Department of Biochemistry (Signal Transduction Laboratories), Lipid and Lipoprotein Research Group, University of Alberta, 357 Heritage Medical Research Centre, Edmonton, Alberta T6G 2S2, Canada
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Tate RJ, Tolan D, Pyne S. Molecular cloning of magnesium-independent type 2 phosphatidic acid phosphatases from airway smooth muscle. Cell Signal 1999; 11:515-22. [PMID: 10405762 DOI: 10.1016/s0898-6568(99)00028-5] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
Members of the type 2 phosphatidic acid phosphatase (PAP2) family catalyse the dephosphorylation of phosphatidic acid (PA), lysophosphatidate and sphingosine 1-phosphate. Here, we demonstrate the presence of a Mg(2+)-independent and N-ethymaleimide-insensitive PAP2 activity in cultured guinea-pig airway smooth muscle (ASM) cells. Two PAP2 cDNAs of 923 and 926 base pairs were identified and subsequently cloned from these cells. The ORF of the 923 base pair cDNA encoded a protein of 285 amino acids (Mr = 32.1 kDa), which had 94% homology with human PAP2a (hPAP2a) and which probably represents a guinea-pig specific PAP2a (gpPAP2a1). The ORF of the 926 base pair cDNA encoded a protein of 286 amino acids (Mr = 32.1 kDa) which had 84% and 91% homology with hPAP2a and gpPAP2a1, respectively. This protein, termed gpPAP2a2, has two regions (aa 21-33 and 51-74) of marked divergence and altered hydrophobicity compared with hPAP2a and gpPAP2a1. This occurs in the predicted first and second transmembrane domains and at the extremes of the first outer loop. Other significant differences between gpPAP2a1/2 and hPAP2a, hPAP2b and hPAP2c occur at the cytoplasmic C-terminal. Transient expression of gpPAP2a2 in Cos-7 cells resulted in an approx. 4-fold increase in Mg(2+)-independent PAP activity, thereby confirming that gpPAP2a2 is another catalytically active member of an extended PAP2 family.
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Affiliation(s)
- R J Tate
- Strathclyde Institute for Biomedical Sciences, University of Strathclyde, Glasgow, UK
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Abstract
Phosphatidic acid phosphatase (PAP) converts phosphatidic acid to diacylglycerol, thus regulating the de novo synthesis of glycerolipids and also signal transduction mediated by phospholipase D. We initially succeeded in the cDNA cloning of the mouse 35 kDa PAP bound to plasma membranes (type 2 enzyme). This work subsequently led us to the identification of two human PAP isozymes designated 2a and 2b. A third human PAP isozyme (2c) has also been described. The cloned enzymes are, in common, N-glycosylated and possess six transmembrane domains. The transmembrane dispositions of these enzymes are predicted and the catalytic sites are tentatively located in the 2nd and 3rd extracellular loops, thus suggesting that the type 2 PAPs may act as ecto-enzymes dephosphorylating exogenous substrates. Furthermore, the type 2 PAPs have been proposed to belong to a novel phosphatase superfamily consisting of a number of soluble and membrane-bound enzymes. In vitro enzyme assays show that the type 2 PAPs can dephosphorylate lyso-phosphatidate, ceramide-1-phosphate, sphingosine-1-phosphate and diacylglycerol pyrophosphate. Although the physiological implications of such a broad substrate specificity need to be further investigated, the type 2 PAPs appear to metabolize a wide range of lipid mediators derived from both glycero- and sphingolipids.
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Affiliation(s)
- H Kanoh
- Department of Biochemistry, Sapporo Medical University School of Medicine, Japan.
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Horrobin DF, Bennett CN. New gene targets related to schizophrenia and other psychiatric disorders: enzymes, binding proteins and transport proteins involved in phospholipid and fatty acid metabolism. Prostaglandins Leukot Essent Fatty Acids 1999; 60:141-67. [PMID: 10359017 DOI: 10.1054/plef.1999.0027] [Citation(s) in RCA: 61] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 11/18/2022]
Abstract
Phospholipids make up about 60% of the brain's dry weight. In spite of this, phospholipid metabolism has received relatively little attention from those seeking genetic factors involved in psychiatric and neurological disorders. However, there is now increasing evidence from many quarters that abnormal phospholipid and related fatty acid metabolism may contribute to illnesses such as schizophrenia, bipolar disorder, depression and attention deficit hyperactivity disorder. To date the possible specific proteins and genes involved have been relatively ill-defined. This paper reviews the main pathways of phospholipid metabolism, emphasizing the roles of phospholipases of the A2 and C series in signal transduction processes. It identifies some likely protein candidates for involvement in psychiatric and neurological disorders. It also reviews the chromosomal locations of regions likely to be involved in these disorders, and relates these to the known locations of genes directly or indirectly involved in phospholipid and fatty acid metabolism.
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Affiliation(s)
- D F Horrobin
- Laxdale Research, Kings Park House, Laurelhill Business Park, Stirling, UK
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Affiliation(s)
- D N Brindley
- Signal Transduction Laboratories, Lipid and Lipoprotein Research Group, and Department of Biochemistry, University of Alberta, Edmonton, Alberta T6G 2S2, Canada.
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