FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2132801119> ?p ?o ?g. }

• W2132801119 endingPage "8927" @default.
• W2132801119 startingPage "8920" @default.
• W2132801119 abstract "In the present study we have characterized mammalian sphingosine-1-phosphate phosphohydrolase (SPP1), an enzyme that specifically dephosphorylates sphingosine 1-phosphate (S1P) and which differs from previously described lipid phosphate phosphohydrolases. Based on sequence homology to murine SPP1, we cloned the human homolog. Transfection of human embryonic kidney 293 and Chinese hamster ovary cells with murine or human SPP1 resulted in marked increases in SPP1 activity in membrane fractions that were used to examine its enzymological properties. Unlike other known type 2 lipid phosphate phosphohydrolases (LPPs), but similar to the yeast orthologs, mammalian SPP1s are highly specific toward long chain sphingoid base phosphates and degrade S1P, dihydro-S1P, and phyto-S1P. SPP1 exhibited apparent Michaelis-Menten kinetics with S1P as substrate with an apparent Km of 38.5 μmand optimum activity at pH 7.5. Similar to other LPPs, SPP1 activity was also independent of any cation requirements, including Mg2+, and was not inhibited by EDTA but was markedly inhibited by NaF and Zn2+. However, SPP1 has some significantly different enzymological properties than the LPPs: the aliphatic cation propanolol, which is an effective inhibitor of type 1 phosphatidate phosphohydrolase activities and is only modestly effective as an inhibitor of LPPs, is a potent inhibitor of SPP1; the activity was partially sensitive to N-ethylmaleimide but not to the thioreactive compound iodoacetamide; and importantly, low concentrations of Triton X-100 and other non-ionic detergents were strongly inhibitory. Thus, in agreement with Cluster analysis which shows that outside of the consensus motif there is very little homology between SPP1s and the other type 2 lipid phosphohydrolases, SPP1s are significantly different and divergent from the mammalian LPPs. In the present study we have characterized mammalian sphingosine-1-phosphate phosphohydrolase (SPP1), an enzyme that specifically dephosphorylates sphingosine 1-phosphate (S1P) and which differs from previously described lipid phosphate phosphohydrolases. Based on sequence homology to murine SPP1, we cloned the human homolog. Transfection of human embryonic kidney 293 and Chinese hamster ovary cells with murine or human SPP1 resulted in marked increases in SPP1 activity in membrane fractions that were used to examine its enzymological properties. Unlike other known type 2 lipid phosphate phosphohydrolases (LPPs), but similar to the yeast orthologs, mammalian SPP1s are highly specific toward long chain sphingoid base phosphates and degrade S1P, dihydro-S1P, and phyto-S1P. SPP1 exhibited apparent Michaelis-Menten kinetics with S1P as substrate with an apparent Km of 38.5 μmand optimum activity at pH 7.5. Similar to other LPPs, SPP1 activity was also independent of any cation requirements, including Mg2+, and was not inhibited by EDTA but was markedly inhibited by NaF and Zn2+. However, SPP1 has some significantly different enzymological properties than the LPPs: the aliphatic cation propanolol, which is an effective inhibitor of type 1 phosphatidate phosphohydrolase activities and is only modestly effective as an inhibitor of LPPs, is a potent inhibitor of SPP1; the activity was partially sensitive to N-ethylmaleimide but not to the thioreactive compound iodoacetamide; and importantly, low concentrations of Triton X-100 and other non-ionic detergents were strongly inhibitory. Thus, in agreement with Cluster analysis which shows that outside of the consensus motif there is very little homology between SPP1s and the other type 2 lipid phosphohydrolases, SPP1s are significantly different and divergent from the mammalian LPPs. sphingosine 1-phosphate phosphatidate phosphohydrolase N-ethylmaleimide lipid phosphate phosphohydrolase S1P phosphohydrolase 1 murine SPP1 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid expressed sequence tag human SPP1 human embryonic kidney bovine serum albumin 4-morpholineethanesulfonic acid Sphingosine 1-phosphate (S1P)1 is a sphingolipid metabolite that plays important roles in the regulation of diverse biological processes (for review, see Refs. 1Pyne S. Pyne N.J. Biochem. J. 2000; 349: 385-402Crossref PubMed Scopus (661) Google Scholar, 2Spiegel S. Milstien S. Biochim. Biophys. Acta. 2000; 1484: 107-116Crossref PubMed Scopus (186) Google Scholar, 3Goetzl E.J. Prostaglandins. 2001; 64: 11-20Crossref PubMed Scopus (72) Google Scholar, 4Hla T. Prostaglandins. 2001; 64: 135-142Crossref PubMed Scopus (118) Google Scholar). Interest in S1P has increased recently with the discovery that it is the ligand for specific G protein-coupled receptors, known as the EDG-1 or S1PR family (1Pyne S. Pyne N.J. Biochem. J. 2000; 349: 385-402Crossref PubMed Scopus (661) Google Scholar, 2Spiegel S. Milstien S. Biochim. Biophys. Acta. 2000; 1484: 107-116Crossref PubMed Scopus (186) Google Scholar, 3Goetzl E.J. Prostaglandins. 2001; 64: 11-20Crossref PubMed Scopus (72) Google Scholar, 4Hla T. Prostaglandins. 2001; 64: 135-142Crossref PubMed Scopus (118) Google Scholar). To date, there are five members, EDG-1 (S1P1), EDG-3 (S1P3), EDG-5 (S1P2), EDG-6 (S1P4), and EDG-8 (S1P5). These receptors are expressed ubiquitously and couple to diverse G proteins and thus regulate different biological processes depending on the pattern of expression of the receptors themselves and the various G proteins (1Pyne S. Pyne N.J. Biochem. J. 2000; 349: 385-402Crossref PubMed Scopus (661) Google Scholar, 2Spiegel S. Milstien S. Biochim. Biophys. Acta. 2000; 1484: 107-116Crossref PubMed Scopus (186) Google Scholar, 3Goetzl E.J. Prostaglandins. 2001; 64: 11-20Crossref PubMed Scopus (72) Google Scholar, 4Hla T. Prostaglandins. 2001; 64: 135-142Crossref PubMed Scopus (118) Google Scholar). There is also abundant evidence that S1P can function as a second messenger important for regulation of calcium homeostasis (5Mattie M. Brooker G. Spiegel S. J. Biol. Chem. 1994; 269: 3181-3188Abstract Full Text PDF PubMed Google Scholar, 6Meyer zu Heringdorf D. Lass H. Alemany R. Laser K.T. Neumann E. Zhang C. Schmidt M. Rauen U. Jakobs K.H. van Koppen C.J. EMBO J. 1998; 17: 2830-2837Crossref PubMed Scopus (202) Google Scholar, 7Alemany R. Sichelschmidt B. Meyer zu Heringdorf D.M. Lass H. van Koppen C.J. Jakobs K.H. Mol. Pharmacol. 2000; 58: 491-497Crossref PubMed Scopus (38) Google Scholar, 8van Koppen C.J. Meyer zu Heringdorf D. Alemany R. Jakobs K.H. Life Sci. 2001; 68: 2535-2540Crossref PubMed Scopus (56) Google Scholar) and suppression of apoptosis (9Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1341) Google Scholar, 10Morita Y. Perez G.I. Paris F. Miranda S.R. Ehleiter D. Haimovitz-Friedman A. Fuks Z. Xie Z. Reed J.C. Schuchman E.H. Kolesnick R.N. Tilly J.L. Nature Med. 2000; 6: 1109-1114Crossref PubMed Scopus (508) Google Scholar, 11Edsall L.C. Cuvillier O. Twitty S. Spiegel S. Milstien S. J. Neurochem. 2001; 76: 1573-1584Crossref PubMed Scopus (170) Google Scholar, 12Spiegel S. Milstien S. FEBS Lett. 2000; 476: 55-67Crossref PubMed Scopus (238) Google Scholar). However, the intracellular targets of S1P have not yet been identified. Moreover, it has recently been shown that S1P generated intracellularly by platelet-derived growth factor stimulation of sphingosine kinase can activate EDG-1 and that this event is critical for directional migration toward platelet-derived growth factor (13Hobson J.P. Rosenfeldt H.M. Barak L.S. Olivera A. Poulton S. Caron M.G. Milstien S. Spiegel S. Science. 2001; 291: 1800-1803Crossref PubMed Scopus (383) Google Scholar). Cellular levels of S1P are low and regulated by the balance between synthesis by sphingosine kinase-catalyzed phosphorylation of sphingosine and degradation by an endoplasmic S1P lyase and also by ill defined phosphohydrolase activities. Several mammalian lipid phosphate phosphohydrolases have been characterized previously (14Brindley D.N. Waggoner D.W. J. Biol. Chem. 1998; 273: 24281-24284Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 15Stukey J. Carman G.M. Protein Sci. 1997; 6: 469-472Crossref PubMed Scopus (221) Google Scholar). Type 1 phosphatidate phosphohydrolase (PAP) is sensitive to inhibition by sulfhydryl reagents (most notably N-ethylmaleimide (NEM)) and displays a requirement for Mg2+. In contrast, the type 2 lipid phosphate phosphohydrolases (LPPs) are magnesium-independent, membrane-associated phosphatases that share sequence conservation and NEM insensitivity (14Brindley D.N. Waggoner D.W. J. Biol. Chem. 1998; 273: 24281-24284Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). In mammals, there are three isoforms of type 2 LPPs, known as LPP1/PAP2a, LPP3/PAP2b, and LPP2/PAP2c (14Brindley D.N. Waggoner D.W. J. Biol. Chem. 1998; 273: 24281-24284Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 16Roberts R. Sciorra V.A. Morris A.J. J. Biol. Chem. 1998; 273: 22059-22067Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 17Alderton F. Darroch P. Sambi B. McKie A. Ahmed I.S. Pyne N. Pyne S. J. Biol. Chem. 2001; 276: 13452-13460Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Although specific biological roles for these LPPs have not yet been established, wunen, the Drosophila gene that encodes a homolog of PAP2c and PAP2a, negatively regulates primordial germ cell migration (18Zhang N. Zhang J. Purcell K.J. Cheng Y. Howard K. Nature. 1997; 385: 64-67Crossref PubMed Scopus (175) Google Scholar). However, PAP2c homozygous null mice were viable, fertile, had no obvious phenotypic defects and thus were not informative (19Zhang N. Sundberg J.P. Gridley T. Genesis. 2000; 27: 137-140Crossref PubMed Scopus (64) Google Scholar). The mammalian LPPs do not appear to be specific phosphatases and are active with various related phosphorylated lipids including phosphatidic acid, lysophosphatidic acid, ceramide 1-phosphate, and diacyglycerol pyrophosphate, as well as with S1P (14Brindley D.N. Waggoner D.W. J. Biol. Chem. 1998; 273: 24281-24284Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Recently, two genes were identified in Saccharomyces cerevisiae, LBP1/YSR2/LCB3 and LBP2/YSR3, which encode specific sphingoid base phosphate phosphatases that regulate the levels of phosphorylated sphingoid bases and ceramide (20Mandala S.M. Thornton R. Tu Z. Kurtz M.B. Nickels J. Broach J. Menzeleev R. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 150-155Crossref PubMed Scopus (235) Google Scholar, 21Mao C. Wadleigh M. Jenkins G.M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 1997; 272: 28690-28694Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 22Qie L. Nagiec M.M. Baltisberger J.A. Lester R.L. Dickson R.C. J. Biol. Chem. 1997; 272: 16110-16117Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). These S1P phosphatases have the conserved sequence motif present in all LPPs and thus belong to the same family. On the basis of sequence homology withLBP1, we recently cloned the first mammalian homolog, murine S1P phosphatase-1 (mSPP1) (23Mandala S.M. Thornton R. Galve-Roperh I. Poulton S. Peterson C. Olivera A. Bergstrom J. Kurtz M.B. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7859-7864Crossref PubMed Scopus (174) Google Scholar). This hydrophobic enzyme degraded S1P, but not lysophosphatidic acid, phosphatidic acid, or ceramide 1-phosphate and may regulate the dynamic balance between sphingolipid metabolite levels in mammalian cells and consequently influence cell fate (23Mandala S.M. Thornton R. Galve-Roperh I. Poulton S. Peterson C. Olivera A. Bergstrom J. Kurtz M.B. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7859-7864Crossref PubMed Scopus (174) Google Scholar). In this study, we have cloned the human homolog of mSPP1. Extensive characterization of the enzymological properties of mammalian SPP1s revealed that although like LPPs, they are magnesium-independent, mammalian SPP1s and LPPs have significant differences, suggesting that they are evolutionarily divergent. [γ-32P]ATP (3,000 Ci/mmol) was purchased from Amersham Biosciences, Inc.d-Erythro-[3-3H]sphingosine (30 Ci/mmol) came from PerkinElmer Life Sciences. [4,5-3H]Dihydrosphingosine was prepared as described (24Mandala S.M. Thornton R.A. Rosenbach M. Milligan J. Garcia-Calvo M. Bull H.G. Kurtz M.B. J. Biol. Chem. 1997; 272: 32709-32714Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). d-Erythrosphingosine,d-erythrodihydrosphingosine, phytosphingosine, S1P, and dihydro-S1P were obtained from Biomol Research Laboratory, Inc. (Plymouth Meeting, PA). Aprotinin, bovine serum albumin, leupeptin, phenylmethylsulfonyl fluoride, propranolol, NEM, β-glycerophosphate, Triton X-100, and CHAPS were purchased from Sigma. Bradford protein assay reagent was from Bio-Rad. Silica Gel G60 thin layer chromatography plates were from EM Science (Gibbstown, NJ). Octyl β-glucopyranoside was from Calbiochem. Monoclonal antibodies against c-Myc were from Zymed Laboratories Inc. (San Francisco). Serum and medium were obtained from Biofluids, Inc. (Rockville, MD). G418 was from Invitrogen (Carlsbad, CA). Rabbit antibodies were raised to a keyhole limpet hemocyanin-coupled peptide that is unique to mammalian SPP1 (CTKDIIRWPRPASPP). All other reagents were of reagent grade. Human ESTs with high homology to mSPP1 were identified in BLAST searches, and the sequence information was used to design gene-specific primers. The 3′-end of hSPP1 was amplified from human placental cDNA (CLONTECH) using primers 5′-GGAAGTGGTGCTGGAATTGCATG and 5′-GCCTCCCATGTTCAACATCATGG, and an 830-bp BamHI-EcoRI fragment was isolated from the cloned PCR product. For the 5′-end of hSPP1, a 1,222-bpSalI-BamHI fragment was isolated from gsc:00005190 DNA and cloned with the 3′-end into pcDNA3.1zeo. Human embryonic kidney cells (HEK 293, ATCC CRL-1573) were grown in high glucose Dulbecco's modified Eagle's medium containing 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mml-glutamine supplemented with 10% fetal bovine serum (25Olivera A. Kohama T. Edsall L.C. Nava V. Cuvillier O. Poulton S. Spiegel S. J. Cell Biol. 1999; 147: 545-558Crossref PubMed Scopus (461) Google Scholar). Chinese hamster ovary cells were grown in Iscove's, 1× HT supplement, 2 mmglutamine, and 10% fetal bovine serum. Cells were transfected using LipofectAMINE Plus (Invitrogen) according to the manufacturer's instructions. Stable transfectants containing either pcDNA3.1 or pcDNA3.1-cMyc-mSPP1 plasmids were selected in medium containing 1 g/liter G418. Cells were washed twice in ice-cold phosphate-buffered saline. 200 μl of buffer A (100 mm HEPES (pH 7.5) containing 10 mm EDTA, 1 mm dithiothreitol, and 10 μg/ml each leupeptin, aprotinin, and soybean trypsin inhibitor) was added to each well, and cells were scraped on ice. Cells were freeze-thawed seven times, and the disrupted cell suspension was centrifuged at 1500 × g for 5 min to remove unbroken cells and cell debris. The cell lysate was then centrifuged at 100,000 × g for 1 h to obtain cytosol and total membrane fractions. The total membrane fraction was resuspended in 200 μl of buffer A and protein concentration determined with the Bradford reagent. 32P-Labeled S1P, dihydro-S1P, and phyto-S1P were prepared by incubating sphingosine, dihydrosphingosine, and phytosphingosine, respectively, with [32P]ATP and recombinant sphingosine kinase 1 as described previously (26Van Brocklyn J.R. Spiegel S. Methods Enzymol. 2000; 312: 401-416Crossref PubMed Google Scholar). Briefly, phosphorylation reactions were carried out using 1 ml of sphingosine kinase buffer (0.1 m Tris-HCl (pH 7.4), 1 mm2-mercaptoethanol, 1 mm EDTA, 10 mmMgCl2, 1 mm NaVO3, 15 mm NaF, 10 μg/ml leupeptin/aprotinin, 1 mmphenylmethylsulfonyl fluoride, 0.5 mm 4-deoxypyridoxine, 40 mm β-glycerophosphate, and 20% glycerol) containing 50 μm sphingoid base, 100 μg of protein from recombinant kinase extracts (26Van Brocklyn J.R. Spiegel S. Methods Enzymol. 2000; 312: 401-416Crossref PubMed Google Scholar), and 300 μCi of [γ-32P]ATP (3,000 Ci/mmol). After 1 h at 37 °C, the reaction was stopped by the addition of 20 μl of 1 n HCl.32P-Labeled long chain sphingoid base was then extracted into the organic phase by the addition of 1.6 ml of CHCl3/MeOH/concentrated HCl (100/200/1), 1 ml of 2m KCl, and 1 ml of CHCl3, then isolated by basic extraction into the aqueous phase by the addition of 2 ml of MeOH, 1 ml of CHCl3, 2 ml of 2 m KCl, and 100 μl of NH4OH. The aqueous phase was transferred to a new tube, and 3 ml of CHCl3 and 200 μl of concentrated HCl were added to extract 32P-labeled products back to the organic phase. 3H-Labeled S1P and dihydro-S1P were prepared by incubating [3H]sphingosine or [3H]dihydrosphingosine with ATP and yeast sphingosine kinase as described previously (27Im D.S. Heise C.E. Ancellin N. O'Dowd B.F. Shei G.J. Heavens R.P. Rigby M.R. Hla T. Mandala S. McAllister G. George S.R. Lynch K.R. J. Biol. Chem. 2000; 275: 14281-14286Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). SPP1 activity using [32P]S1P as substrate was determined as described previously with minor modifications (23Mandala S.M. Thornton R. Galve-Roperh I. Poulton S. Peterson C. Olivera A. Bergstrom J. Kurtz M.B. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7859-7864Crossref PubMed Scopus (174) Google Scholar). Briefly, SPP1 activity was determined at 37 °C by adding 32P-labeled S1P (10 μm, 100,000 cpm, 0.3% BSA complex) to membrane fractions (4 μg) in 200 μl of buffer A incubated at 37 °C. Reactions were stopped after 30 min by the addition of 2.5 volumes of water-saturated butan-1-ol and 1.5 volumes of 1.5 m KCl. The upper phases were then extracted three times with 2.5 volumes of 1.5 m KCl. The remaining labeled S1P was measured by liquid scintillation counting of the radioactivity in the upper phases. Analysis of the reaction products by TLC on Silica Gel G60 with methanol/chloroform/acetone/acetic acid/water (10:4:3:2:1, v/v) as solvent confirmed the specificity of the reactions. SPP1 activity is expressed as nmol of S1P degraded/min/mg of protein. In some experiments, SPP1 activity using [3H]S1P or [3H]dihydro-S1P as substrate was determined as described previously (20Mandala S.M. Thornton R. Tu Z. Kurtz M.B. Nickels J. Broach J. Menzeleev R. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 150-155Crossref PubMed Scopus (235) Google Scholar). In brief, phosphatase activity was measured in 200 μl containing 50 mm KPO4 (pH 7.2), 0.02% tergitol Nonidet P-40, 2 μm [3H]S1P or [3H]dihydro-S1P (40,000 cpm), 2 mmsemicarbazide, and 1.5 μg of membrane protein. After a 60-min incubation at 37 °C, the assay was terminated with 200 μl of 7m NH4OH. One ml of chloroform:methanol (3:2) was added, and 50 μl of the chloroform layer was counted by liquid scintillation. Similar results were obtained by measurement of disappearance of labeled S1P or by formation of labeled sphingosine. S1P in methanol was transferred to a test tube and dried under a stream of nitrogen. Triton X-100/S1P-mixed micelles were prepared by adding Triton X-100 to the tubes and vortexing. The S1P concentration in Triton X-100/phospholipid-mixed micelles did not exceed 15 mol% to ensure that the structure of the micelles was similar to that of pure Triton X-100 (28Furneisen J.M. Carman G.M. Biochim. Biophys. Acta. 2000; 1484: 71-82Crossref PubMed Scopus (37) Google Scholar). For investigations of the dependence of enzyme activity on the surface concentration of S1P, the substrate concentration was held constant at 50 μm, and the detergent concentration was varied. Equal amounts of protein were separated on 10% SDS-PAGE and electroblotted onto nitrocellulose membranes for 1 h at 100 V and 4 °C. Blots were blocked in 5% nonfat dry milk in TBS containing 0.1% Tween 20 (TBST) for 2 h at room temperature and probed with anti c-Myc monoclonal antibody or with anti-mSPP1 polyclonal antibody (1763), in the same buffer. After washing three times with TBST, blots were incubated with secondary antibodies for 1 h at room temperature. Protein bands were visualized by enhanced chemiluminescence using Super Signal (Pierce). Previously, it has been found that endogenous LPP and SPP1 activity of HEK 293 cells was very low (17Alderton F. Darroch P. Sambi B. McKie A. Ahmed I.S. Pyne N. Pyne S. J. Biol. Chem. 2001; 276: 13452-13460Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 23Mandala S.M. Thornton R. Galve-Roperh I. Poulton S. Peterson C. Olivera A. Bergstrom J. Kurtz M.B. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7859-7864Crossref PubMed Scopus (174) Google Scholar), making these cells suitable for overexpression studies. HEK 293 cells stably transfected with mSPP1 showed a marked increase in SPP1 activity compared with vector-transfected cells. In agreement with previous results (23Mandala S.M. Thornton R. Galve-Roperh I. Poulton S. Peterson C. Olivera A. Bergstrom J. Kurtz M.B. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7859-7864Crossref PubMed Scopus (174) Google Scholar), SPP1 activity was exclusively in the membrane fraction, and vector-transfected HEK 293 cells had low levels of activity (Fig. 1 A). Additional evidence demonstrating the expression of c-Myc-SPP1 in membrane fractions was obtained by Western blot analysis with an antibody that recognizes an internal peptide (CTKDIIRWPRPASPP). As shown in Fig.2, a band with an apparent molecular mass of 47 kDa was specifically immunostained in mSPP1-transfected cell lysates and was absent in vector-transfected cells. Immunocomplex formation was blocked by the internal peptide (Fig. 2), demonstrating the specificity of this antibody. The molecular mass of 47 kDa is in agreement with the value calculated from the predicted amino acid composition containing the c-Myc tag (23Mandala S.M. Thornton R. Galve-Roperh I. Poulton S. Peterson C. Olivera A. Bergstrom J. Kurtz M.B. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7859-7864Crossref PubMed Scopus (174) Google Scholar). As expected, this band was also detected with anti-c-Myc monoclonal antibody. It should be noted that the presence of the c-Myc epitope tag had no significant effect on activity or membrane distribution of the recombinant protein (data not shown).Figure 2Immunoblot analysis of mSPP1 expression.Membrane and cytosolic fractions (40 μg of proteins) from HEK 293 cells transfected with vector (lanes 1, 3,5, 7, 9, and 11) or c-Myc-mSPP1 (lanes 2, 4, 6,8, 10, 12) were resolved by SDS-PAGE and then immunoblotted with a monoclonal c-Myc antibody (Ab) or a polyclonal antibody 1763 directed against a specific internal peptide. Where indicated, the peptide CTKDIIRWPRPASPP was preincubated with the antibody prior to immunoblot analysis.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In agreement with our previous study using mSPP1 from transiently transfected cells (23Mandala S.M. Thornton R. Galve-Roperh I. Poulton S. Peterson C. Olivera A. Bergstrom J. Kurtz M.B. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7859-7864Crossref PubMed Scopus (174) Google Scholar), membranes isolated from HEK 293 cells stably overexpressing mSPP1 dephosphorylate S1P but not ceramide 1-phosphate, lysophosphatidic acid, or phosphatidic acid (data not shown). In contrast, LPP1, LPP2, and LPP3 are broad specificity lipid phosphate phosphatases that readily catalyze the dephosphorylation of all of these structurally different lipid phosphates (14Brindley D.N. Waggoner D.W. J. Biol. Chem. 1998; 273: 24281-24284Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 16Roberts R. Sciorra V.A. Morris A.J. J. Biol. Chem. 1998; 273: 22059-22067Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 17Alderton F. Darroch P. Sambi B. McKie A. Ahmed I.S. Pyne N. Pyne S. J. Biol. Chem. 2001; 276: 13452-13460Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). SPP1 activity in membrane fractions determined with a concentration of 50 μm S1P was 5-fold greater (26.85 ± 3.1 nmol/min/mg) than in untransfected cells (5.05 ± 0.6 nmol/min/mg) and thus provides an enriched source of this enzyme for further characterization. S1P degradation increased linearly with the incubation time for the first 30 min of the reaction, then gradually decreased (Fig. 1 A). mSPP1 activity was linear with increasing protein concentration (Fig. 1 B), and a concentration of 4 μg was used for further characterization. Typical Michaelis-Menten kinetics were observed with recombinant mSPP1 (Fig.3 A). The apparentKm and V max values for S1P were calculated from a Lineweaver-Burk plot to be 38.5 μm and 36.4 nmol/min/mg, respectively. The S1P phosphatase activity exhibited a broad pH optimum with maximum activity between pH 6 and 7.5 (Fig. 3 B), and the activity decreased markedly at lower or higher pH. Thus, the activity was routinely measured at pH 7.5. To identify human homologs of mSPP1, we searched the hEST data base and found several ESTs (gb:AA375349, gb:AA376229, gb:AA133909, and gsc:00005190) with high sequence homology to mSPP1. The full-length cDNA encoding hSPP1 was constructed from rapid amplification of cDNA ends products and fragments isolated from these EST clones. The open reading frame encodes a protein with 441 amino acids and 76% identity and 81% similarity to mSPP1 at the amino acid level (Fig. 4). HEK 293 and Chinese hamster ovary cells were transfected with expression vectors containing hSPP1 to confirm whether it encodes abona fide SPP1. 48 h after transfection with hSPP1, S1P phosphatase activity increased 2–3-fold in HEK 293 cells and 10–20-fold in Chinese hamster ovary cells relative to vector-transfected controls (Fig.5 A). Because, in addition to S1P, eukaryotic cells also contain dihydro-S1P and phyto-S1P (29Spiegel S. Merrill Jr., A.H. FASEB J. 1996; 10: 1388-1397Crossref PubMed Scopus (644) Google Scholar), it was of interest to examine whether these phosphorylated sphingolipids were also substrates for mSPP1. Dihydro-S1P, which has a structure identical to that of S1P except for the lack of thetrans-4,5 double bond, is hydrolyzed by mSPP1 and hSPP1 (Fig. 5, A and B). Phyto-S1P, which has the same structure as dihydro-S1P but contains an additional hydroxyl group at the 4 position, was dephosphorylated by mSPP1 to the same extent as dihydro-S1P. Although membranes from vector-transfected cells have some phosphohydrolase activity with these phosphorylated sphingoid bases, it was much lower than membranes from cells overexpressing mSPP1, and the rates of dephosphorylation were the same for these three substrates (Fig. 5, A and B). Because both mSPP1 and hSPP1 have the conserved residues within the catalytic domains present in all type 2 LPPs (30Carman G.M. Deems R.A. Dennis E.A. J. Biol. Chem. 1995; 270: 18711-18714Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar), which are characterized as Mg2+-independent and NEM-insensitive (14Brindley D.N. Waggoner D.W. J. Biol. Chem. 1998; 273: 24281-24284Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 16Roberts R. Sciorra V.A. Morris A.J. J. Biol. Chem. 1998; 273: 22059-22067Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 17Alderton F. Darroch P. Sambi B. McKie A. Ahmed I.S. Pyne N. Pyne S. J. Biol. Chem. 2001; 276: 13452-13460Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar), it was of interest to compare the properties of SPP1 with those of the known LPPs. To examine the dependence on metal ions, recombinant mSPP1 activity was first measured in the presence of the metal chelator EDTA. Rather than inhibiting, low concentrations of EDTA slightly stimulated SPP1 activity, and increasing the concentration up to 10 mmhad no further effects on the activity (Fig.6 A), suggesting that divalent cations are not required for activity of this phosphatase. Moreover, similar to LPPs, SPP1 activity was completely independent of added divalent cations, including magnesium and calcium (Fig. 6 B) but was slightly inhibited by Mn2+ (Fig. 6 C). Thus, like other type 2 lipid phosphohydrolases, SPP1 activity is also independent of any cation requirements. Zn2+ was used previously as an effective inhibitor of all three type 2 LPPs. SPP1 appears to be sensitive to Zn2+ (Fig. 6 D). Of note, membrane fractions from vector-transfected HEK 293 cells contain a phosphatase activity that is markedly stimulated by Zn2+. The effect of various inhibitors of other type 2 lipid phosphatases on SPP1 activity was then examined using 10 μm S1P, a concentration below the apparent Km, to observe more readily the inhibitory or stimulatory effects on enzyme activity. SPP1 activity was inhibited markedly by NaF, a general phosphatase inhibitor, in a dose-dependent manner, with 75% inhibition at a concentration of 5 mm (Fig.7 A). The pentacoordinate vanadate cofactor resembles the transition state structure of phosphate and has been used extensively as a phosphotyrosine phosphatase inhibitor. Sodium orthovanadate also strongly inhibited SPP1 (Fig.7 A). Although the aliphatic cation propanolol, a β-adrenergic receptor antagonist, is an effective inhibitor of type 1 PAP activities and only modestly inhibits LPPs (16Roberts R. Sciorra V.A. Morris A.J. J. Biol. Chem. 1998; 273: 22059-22067Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 28Furneisen J.M. Carman G.M. Biochim. Biophys. Acta. 2000; 1484: 71-82Crossref PubMed Scopus (37) Google Scholar, 31Jamal Z. Martin A. Munoz A.G. Brindley D.N. J. Biol. Chem. 1991; 266: 2988-2996Abstract Full Text PDF PubMed Google Scholar), it is a fairly potent inhibitor of SPP1 (Fig. 7 B). In contrast, ATP, pyrophosphate, phosphate monoesters such as β−glycerol phosphate, or tartrate, an inhibitor of lysosomal acid phosphatase (32Brightwell R. Tappel A.L. Arch. Biochem. Biophys. 1968; 124: 333-343Crossref PubMed Scopus (69) Google Scholar), had no significant effects on S1P phosphatase activity (Fig. 7, Cand D). NEM is an alkylating reagent that also reacts with sulfhydryl groups and has been used to differentiate the Mg2+-dependent and Mg2+-independent lipid phosphatases from yeast and mammalian cells (14Brindley D.N. Waggoner D.W. J. Biol. Chem. 1998; 273: 24281-24284Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar). Surprisingly, unlike the yeast homolog, which is NEM-insensitive (20Mandala S.M. Thornton R. Tu Z. Kurtz M.B. Nickels J. Broach J. Menzeleev R. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 150-155Crossref PubMed Scopus (235) Google Scholar), we found that NEM inhibited mSPP1 in a dose-dependent manner. A maximum inhibition of 40% was achieved at a concentration of 0.5–1 mm (Fig. 8 A). The inhibitory effect of NEM was prevented by coincubation with dithiothreitol (Fig. 8 A). Thus, unlike other Mg2+-independent lipid phosphatase enzymes from yeast and mammalian cells, the mammalian SPP1 should be considered to be sensitive to NEM, suggesting that this enzyme may differ in structure and/or reaction mechanism. However, the thioreactive compound iodoacetamide, even when preincubated with the enzyme at a concentration of 10 mm, had no significant effect on enzyme activity (Fig. 8 B), indicating that NEM may inhibit mSPP1 by alkylation of non-sulfhydryl residues. Interestingly, NEM also potently inhibited the phosphatidate phosphatase, diacylglycerol pyrophosphate phosphatase, and lysophosphatidic acid phosphatase activities of the LPP1-encoded lipid phosphatase from S. cerevisiae (28Furneisen J.M. Carman G.M. Biochim. Biophys. Acta. 2000; 1484: 71-82Crossref PubMed Scopus (37) Google Scholar). Hence, as suggested by Furneisen and Carman (28Furneisen J.M. Carman G.M. Biochim. Biophys. Acta. 2000; 1484: 71-82Crossref PubMed Scopus (37) Google Scholar), the criteria of NEM sensitivity used to classify the Mg2+-independent phosphatidate phosphatase enzymes should be modified (28Furneisen J.M. Carman G.M. Biochim. Biophys. Acta. 2000; 1484: 71-82Crossref PubMed Scopus (37) Google Scholar). The LPPs have little or no activity with lipid phosphates as substrates in the absence of detergents (14Brindley D.N. Waggoner D.W. J. Biol. Chem. 1998; 273: 24281-24284Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 16Roberts R. Sciorra V.A. Morris A.J. J. Biol. Chem. 1998; 273: 22059-22067Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar, 17Alderton F. Darroch P. Sambi B. McKie A. Ahmed I.S. Pyne N. Pyne S. J. Biol. Chem. 2001; 276: 13452-13460Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 28Furneisen J.M. Carman G.M. Biochim. Biophys. Acta. 2000; 1484: 71-82Crossref PubMed Scopus (37) Google Scholar, 33Jasinska R. Zhang Q.X. Pilquil C. Singh I. Xu J. Dewald J. Dillon D.A. Berthiaume L.G. Carman G.M. Waggoner D.W. Brindley D.N. Biochem. J. 1999; 340: 677-686Crossref PubMed Scopus (129) Google Scholar). In contrast, mSPP1 has significant activity toward S1P and dihydro-S1P in the absence of any detergent, when either of these lipid phosphates is presented to the enzyme even without any carrier or as BSA complexes. Studies of substrate specificities and kinetic analyses of the other lipid phosphate phosphohydrolases have usually been carried out in the presence of a non-ionic detergent such as Triton X-100 (28Furneisen J.M. Carman G.M. Biochim. Biophys. Acta. 2000; 1484: 71-82Crossref PubMed Scopus (37) Google Scholar, 33Jasinska R. Zhang Q.X. Pilquil C. Singh I. Xu J. Dewald J. Dillon D.A. Berthiaume L.G. Carman G.M. Waggoner D.W. Brindley D.N. Biochem. J. 1999; 340: 677-686Crossref PubMed Scopus (129) Google Scholar). Surprisingly, when mSPP1-catalyzed hydrolysis of S1P was measured in the presence of increasing amounts of Triton X-100, the ability of mSPP1 to degrade S1P was strongly inhibited by concentrations of Triton X-100 above 0.2 mm (Fig.9 A), which is the critical micelle concentration of this detergent (aggregation no. 100–155), a result indicative of surface dilution effects (30Carman G.M. Deems R.A. Dennis E.A. J. Biol. Chem. 1995; 270: 18711-18714Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). We then attempted to determine whether this was a specific effect of Triton X-100 or whether other detergents were similarly inhibitory. The non-ionic detergent β-octyl glucoside (CMC 20–25 mm, aggregation no. 84), inhibited mSPP1, albeit with slightly less potency than Triton X-100, whereas the zwitterionic detergent CHAPS (CMC 6–10 mm, aggregation no. 4–14) inhibited the activity to a lesser extent. The potencies of these detergents to inhibit SPP1 activity correlated with their critical micelle concentrations and aggregation numbers, again indicative of surface dilution effects. However, phosphohydrolase activity with membranes from vector-transfected HEK 293 cells was 1–2 nmol/min/mg and was not influenced by these detergents. Previously, a surface dilution kinetic model has been employed usefully to examine kinetic properties of substrates presented as Triton X-100 micelles for kinetic analysis of yeast and mammalian LPPs (30Carman G.M. Deems R.A. Dennis E.A. J. Biol. Chem. 1995; 270: 18711-18714Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). Substrates are presented to the enzymes in micellar form with detergents with high aggregation numbers, and thus the surface concentration of substrate can be varied without compromising the physical state of the micelles. In this type of analysis, the enzyme activity depends on both bulk and surface concentrations of substrate because enzyme binding to the micelle interface precedes substrate binding and catalysis. It has been suggested that in this mixed micellar condition, the enzyme is in an environment similar to the physiological surface of membranes (30Carman G.M. Deems R.A. Dennis E.A. J. Biol. Chem. 1995; 270: 18711-18714Abstract Full Text Full Text PDF PubMed Scopus (269) Google Scholar). We thus attempted to examine the surface dilution effect on the activity of mSPP1 when S1P was presented in Triton X-100 micelles. SPP1 activity was measured at a fixed concentration of S1P (50 μm), and the mol% was varied by increasing the concentration of Triton X-100. Higher S1P concentrations are needed in this case to form uniform micelles of the substrate. As expected from the surface dilution model, at Triton X-100 concentrations below 1 mm, there was a severe reduction in SPP1 activity. However, it was not possible to derive kinetic constants from this analysis because Triton X-100 dramatically stimulated endogenous SPP1 activity in this micellar assay in parallel with its effect on the overexpressed SPP1 activity (Fig. 9 C). These results further substantiate the notion that SPP1 is affected differently by detergents than the type 2 LPPs. It is important for many types of immunoprecipitation experiments to be able to measure SPP1 activity in the presence of a stabilizing detergent. Because SPP1 is strongly inhibited by detergents with high aggregation numbers, we also examined the effect of Tween 20, a weak detergent that only forms very small micelles. SPP1 activity was even slightly stimulated by low concentrations of Tween 20, which makes it a suitable detergent for SPP1 assays (Fig. 9 D). In summary, in this paper we report the cloning of hSPP1, the human homolog of mSPP1. Based on EST sequences, hSPP1 has been localized on chromosome 14q22.1-q22 (UniGene Cluster Hs.24678, URL: www.ncbi.nlm.nih.gov/UniGene/clust.cgi?ORG=Hs&CID = 24678). In Blast searches, the most closely related proteins to mammalian SPP1 were sphingoid base phosphatases from S. cerevisiae (yLBP1 and yLBP2) and two uncharacterized genes fromSchizosaccharomyces pombe and Arabidopsis thaliana, followed by bacterial phosphatases. Cluster analysis of the protein sequences places mSPP1 and hSPP1 off the same branch point with the yeast S1Pases, and divergent from the LPP subfamily, which includes mammalian LPPs, Drosophila Wunen proteins, and yeast DPP1 and LPP1 (Fig. 10). Thus, mammalian SPP1s belong to a conserved family of genes which is distinct from other known type 2 LPPs, both in sequence and in biochemical properties." @default.
• W2132801119 created "2016-06-24" @default.
• W2132801119 creator A5011102538 @default.
• W2132801119 creator A5014072958 @default.
• W2132801119 creator A5064310295 @default.
• W2132801119 creator A5079153351 @default.
• W2132801119 creator A5080902194 @default.
• W2132801119 creator A5082600065 @default.
• W2132801119 date "2002-03-01" @default.
• W2132801119 modified "2023-10-18" @default.
• W2132801119 title "Characterization of Murine Sphingosine-1-phosphate Phosphohydrolase" @default.
• W2132801119 cites W117936949 @default.
• W2132801119 cites W1553049210 @default.
• W2132801119 cites W1570743730 @default.
• W2132801119 cites W1595397037 @default.
• W2132801119 cites W1613312511 @default.
• W2132801119 cites W1843461492 @default.
• W2132801119 cites W1929157658 @default.
• W2132801119 cites W1969653816 @default.
• W2132801119 cites W1975671269 @default.
• W2132801119 cites W1977998332 @default.
• W2132801119 cites W1981531462 @default.
• W2132801119 cites W1989492043 @default.
• W2132801119 cites W1989975786 @default.
• W2132801119 cites W1996200711 @default.
• W2132801119 cites W2005960599 @default.
• W2132801119 cites W2010370015 @default.
• W2132801119 cites W2013063326 @default.
• W2132801119 cites W2013264233 @default.
• W2132801119 cites W2018175015 @default.
• W2132801119 cites W2022589671 @default.
• W2132801119 cites W2030063540 @default.
• W2132801119 cites W2032122936 @default.
• W2132801119 cites W2032620622 @default.
• W2132801119 cites W2057229979 @default.
• W2132801119 cites W2057323682 @default.
• W2132801119 cites W2057680304 @default.
• W2132801119 cites W2061574494 @default.
• W2132801119 cites W2066321464 @default.
• W2132801119 cites W2069496353 @default.
• W2132801119 cites W2075468442 @default.
• W2132801119 cites W2079930884 @default.
• W2132801119 cites W2084172135 @default.
• W2132801119 cites W2094336459 @default.
• W2132801119 cites W2124925407 @default.
• W2132801119 cites W2141921458 @default.
• W2132801119 cites W2155487600 @default.
• W2132801119 cites W2160241210 @default.
• W2132801119 cites W4240595053 @default.
• W2132801119 doi "https://doi.org/10.1074/jbc.m109968200" @default.
• W2132801119 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/11756451" @default.
• W2132801119 hasPublicationYear "2002" @default.
• W2132801119 type Work @default.
• W2132801119 sameAs 2132801119 @default.
• W2132801119 citedByCount "84" @default.
• W2132801119 countsByYear W21328011192012 @default.
• W2132801119 countsByYear W21328011192013 @default.
• W2132801119 countsByYear W21328011192014 @default.
• W2132801119 countsByYear W21328011192016 @default.
• W2132801119 countsByYear W21328011192017 @default.
• W2132801119 countsByYear W21328011192018 @default.
• W2132801119 countsByYear W21328011192019 @default.
• W2132801119 countsByYear W21328011192020 @default.
• W2132801119 countsByYear W21328011192021 @default.
• W2132801119 countsByYear W21328011192022 @default.
• W2132801119 crossrefType "journal-article" @default.
• W2132801119 hasAuthorship W2132801119A5011102538 @default.
• W2132801119 hasAuthorship W2132801119A5014072958 @default.
• W2132801119 hasAuthorship W2132801119A5064310295 @default.
• W2132801119 hasAuthorship W2132801119A5079153351 @default.
• W2132801119 hasAuthorship W2132801119A5080902194 @default.
• W2132801119 hasAuthorship W2132801119A5082600065 @default.
• W2132801119 hasBestOaLocation W21328011191 @default.
• W2132801119 hasConcept C170493617 @default.
• W2132801119 hasConcept C185592680 @default.
• W2132801119 hasConcept C2776596873 @default.
• W2132801119 hasConcept C2777132085 @default.
• W2132801119 hasConcept C2778703144 @default.
• W2132801119 hasConcept C55493867 @default.
• W2132801119 hasConcept C86803240 @default.
• W2132801119 hasConceptScore W2132801119C170493617 @default.
• W2132801119 hasConceptScore W2132801119C185592680 @default.
• W2132801119 hasConceptScore W2132801119C2776596873 @default.
• W2132801119 hasConceptScore W2132801119C2777132085 @default.
• W2132801119 hasConceptScore W2132801119C2778703144 @default.
• W2132801119 hasConceptScore W2132801119C55493867 @default.
• W2132801119 hasConceptScore W2132801119C86803240 @default.
• W2132801119 hasIssue "11" @default.
• W2132801119 hasLocation W21328011191 @default.
• W2132801119 hasOpenAccess W2132801119 @default.
• W2132801119 hasPrimaryLocation W21328011191 @default.
• W2132801119 hasRelatedWork W1991575691 @default.
• W2132801119 hasRelatedWork W1999170927 @default.
• W2132801119 hasRelatedWork W2009993180 @default.
• W2132801119 hasRelatedWork W2037514664 @default.
• W2132801119 hasRelatedWork W2090181054 @default.
• W2132801119 hasRelatedWork W2118746823 @default.
• W2132801119 hasRelatedWork W2125499958 @default.

• next