Phosphorylation of the Immunomodulatory Drug FTY720 by Sphingosine Kinases
2003; Elsevier BV; Volume: 278; Issue: 48 Linguagem: Inglês
10.1074/jbc.m307687200
ISSN1083-351X
AutoresAndreas Billich, Frédéric Bornancin, Piroska Dévay, Diana Mechtcheriakova, Nicole Urtz, Thomas Baumruker,
Tópico(s)PI3K/AKT/mTOR signaling in cancer
ResumoThe immunomodulatory drug FTY720 is phosphorylated in vivo, and the resulting FTY720 phosphate as a ligand for sphingosine-1-phosphate receptors is responsible for the unique biological effects of the compound. So far, phosphorylation of FTY720 by murine sphingosine kinase (SPHK) 1a had been documented. We found that, while FTY720 is also phosphorylated by human SPHK1, the human type 2 isoform phosphorylates the drug 30-fold more efficiently, because of a lower Km of FTY720 for SPHK2. Similarly, murine SPHK2 was more efficient than SPHK1a. Among splice variants of the human SPHKs, an N-terminally extended SPHK2 isoform was even more active than SPHK2 itself. Further SPHK superfamily members, namely ceramide kinase and a "SPHK-like" protein, failed to phosphorylate sphingosine and FTY720. Thus, only SPHK1 and 2 appear to be capable of phosphorylating FTY720. Using selective assay conditions, SPHK1 and 2 activities in murine tissues were measured. While activity of SPHK2 toward sphingosine was generally lower than of SPHK1, FTY720 phosphorylation was higher under conditions favoring SPHK2. In human endothelial cells, while activity of SPHK1 toward sphingosine was 2-fold higher than of SPHK2, FTY720 phosphorylation was 7-fold faster under SPHK2 assay conditions. Finally, FTY720 was poorly phosphorylated in human blood as compared with rodent blood, in line with the low activity of SPHK1 and in particular of SPHK2 in human blood. To conclude, both SPHK1 and 2 are capable of phosphorylating FTY720, but SPHK2 is quantitatively more important than SPHK1. The immunomodulatory drug FTY720 is phosphorylated in vivo, and the resulting FTY720 phosphate as a ligand for sphingosine-1-phosphate receptors is responsible for the unique biological effects of the compound. So far, phosphorylation of FTY720 by murine sphingosine kinase (SPHK) 1a had been documented. We found that, while FTY720 is also phosphorylated by human SPHK1, the human type 2 isoform phosphorylates the drug 30-fold more efficiently, because of a lower Km of FTY720 for SPHK2. Similarly, murine SPHK2 was more efficient than SPHK1a. Among splice variants of the human SPHKs, an N-terminally extended SPHK2 isoform was even more active than SPHK2 itself. Further SPHK superfamily members, namely ceramide kinase and a "SPHK-like" protein, failed to phosphorylate sphingosine and FTY720. Thus, only SPHK1 and 2 appear to be capable of phosphorylating FTY720. Using selective assay conditions, SPHK1 and 2 activities in murine tissues were measured. While activity of SPHK2 toward sphingosine was generally lower than of SPHK1, FTY720 phosphorylation was higher under conditions favoring SPHK2. In human endothelial cells, while activity of SPHK1 toward sphingosine was 2-fold higher than of SPHK2, FTY720 phosphorylation was 7-fold faster under SPHK2 assay conditions. Finally, FTY720 was poorly phosphorylated in human blood as compared with rodent blood, in line with the low activity of SPHK1 and in particular of SPHK2 in human blood. To conclude, both SPHK1 and 2 are capable of phosphorylating FTY720, but SPHK2 is quantitatively more important than SPHK1. FTY720 is an immunomodulatory drug, which is highly efficacious in models of transplantation and of autoimmune diseases (1Brinkmann V. Pinschewer D.D. Feng L. Chen S. Transplantation. 2001; 72: 764-769Crossref PubMed Scopus (156) Google Scholar). It was recently found to be effective in kidney transplantation in humans (2Brinkmann V. Lynch K.R. Curr. Opin. Immunol. 2002; 14: 569-575Crossref PubMed Scopus (253) Google Scholar). FTY720 elicits a lymphopenia resulting from the reversible redistribution of lymphocytes from the circulation to secondary lymphoid organs, without leading to general immunosuppression (3Pinschewer D.D. Ochsenbein A.F. Odermatt B. Brinkmann V. Hengartner H. Zinkernagel R.M. J. Immunol. 2000; 164: 5761-5770Crossref PubMed Scopus (334) Google Scholar, 4Chiba K. Yanagawa Y. Masubuchi Y. Kataoka H. Kawaguchi T. Ohtsuki M. Hoshino Y. J. Immunol. 1998; 160: 5037-5044PubMed Google Scholar). Conversion of FTY720 to its monophosphate appears to be essential for the effects of the drug on lymphocyte homing, since FTY720 phosphate acts as an agonist at four of the five G-protein-coupled receptors for sphingosine-1-phosphate (S1P) 1The abbreviations used are: S1Psphingosine-1-phosphateAAL2-amino-4-(4-heptyloxyphenyl)-2-methylbutan-1-olCERKceramide kinaseDGKdiacylglycerol kinaseFCSfetal calf serumGFPgreen fluorescent proteinEGFPenhanced GFPhuSPHKhuman sphingosine kinaseHUVEChuman umbilical vein endothelial cellsmuSPHKmurine sphingosine kinaseRACErapid amplification of cDNA endsRefSeqNCBI Reference Sequence ProjectSPHKsphingosine kinaseTLCthin-layer chromatographyMOPS4-morpholinepropanesulfonic acid.1The abbreviations used are: S1Psphingosine-1-phosphateAAL2-amino-4-(4-heptyloxyphenyl)-2-methylbutan-1-olCERKceramide kinaseDGKdiacylglycerol kinaseFCSfetal calf serumGFPgreen fluorescent proteinEGFPenhanced GFPhuSPHKhuman sphingosine kinaseHUVEChuman umbilical vein endothelial cellsmuSPHKmurine sphingosine kinaseRACErapid amplification of cDNA endsRefSeqNCBI Reference Sequence ProjectSPHKsphingosine kinaseTLCthin-layer chromatographyMOPS4-morpholinepropanesulfonic acid. (5Brinkmann V. Davis M.D. Heise C.E. Albert R. Cottens S. Hof R. Bruns C. Prieschl E. Baumruker T. Hiestand P. Foster C.A. Zollinger M. Lynch K.R. J. Biol. Chem. 2002; 277: 21453-21457Abstract Full Text Full Text PDF PubMed Scopus (1302) Google Scholar, 6Mandala S. Hajdu R. Bergstrom J. Quackenbush E. Xie J. Milligan J. Thornton R. Shei G.J. Card D. Keohane C. Rosenbach M. Hale J. Lynch C.L. Rupprecht K. Parsons W. Rosen H. Science. 2002; 296: 346-349Crossref PubMed Scopus (1419) Google Scholar); it is assumed that at least one S1P receptor is critical to the lymphopenic response induced by FTY720 treatment (2Brinkmann V. Lynch K.R. Curr. Opin. Immunol. 2002; 14: 569-575Crossref PubMed Scopus (253) Google Scholar). More recently, FTY720 was found to stimulate multidrug transporter-dependent T-cell chemotaxis to lymph nodes (7Honig S.M. Fu S. Mao X. Yopp A. Gunn M.D. Randolph G.J Bromberg J.S. J. Clin. Invest. 2003; 111: 627-637Crossref PubMed Scopus (120) Google Scholar); in this instance, FTY720 phosphate as the active metabolite is hypothesized to be responsible for stimulation of efflux activity of the lipid transporter Abcb1.FTY720 has been reported to be phosphorylated ex vivo by rodent lymphoid tissues (5Brinkmann V. Davis M.D. Heise C.E. Albert R. Cottens S. Hof R. Bruns C. Prieschl E. Baumruker T. Hiestand P. Foster C.A. Zollinger M. Lynch K.R. J. Biol. Chem. 2002; 277: 21453-21457Abstract Full Text Full Text PDF PubMed Scopus (1302) Google Scholar) and whole blood of several species (6Mandala S. Hajdu R. Bergstrom J. Quackenbush E. Xie J. Milligan J. Thornton R. Shei G.J. Card D. Keohane C. Rosenbach M. Hale J. Lynch C.L. Rupprecht K. Parsons W. Rosen H. Science. 2002; 296: 346-349Crossref PubMed Scopus (1419) Google Scholar), and is rapidly phosphorylated in vivo (5Brinkmann V. Davis M.D. Heise C.E. Albert R. Cottens S. Hof R. Bruns C. Prieschl E. Baumruker T. Hiestand P. Foster C.A. Zollinger M. Lynch K.R. J. Biol. Chem. 2002; 277: 21453-21457Abstract Full Text Full Text PDF PubMed Scopus (1302) Google Scholar, 6Mandala S. Hajdu R. Bergstrom J. Quackenbush E. Xie J. Milligan J. Thornton R. Shei G.J. Card D. Keohane C. Rosenbach M. Hale J. Lynch C.L. Rupprecht K. Parsons W. Rosen H. Science. 2002; 296: 346-349Crossref PubMed Scopus (1419) Google Scholar). After oral application of FTY720 to rats, the blood levels of the monophosphate exceeded those of the parent compound 2–4 fold (5Brinkmann V. Davis M.D. Heise C.E. Albert R. Cottens S. Hof R. Bruns C. Prieschl E. Baumruker T. Hiestand P. Foster C.A. Zollinger M. Lynch K.R. J. Biol. Chem. 2002; 277: 21453-21457Abstract Full Text Full Text PDF PubMed Scopus (1302) Google Scholar). FTY720 was shown to be a substrate for recombinant murine sphingosine kinase 1a (muSPHK1a) (5Brinkmann V. Davis M.D. Heise C.E. Albert R. Cottens S. Hof R. Bruns C. Prieschl E. Baumruker T. Hiestand P. Foster C.A. Zollinger M. Lynch K.R. J. Biol. Chem. 2002; 277: 21453-21457Abstract Full Text Full Text PDF PubMed Scopus (1302) Google Scholar). Studies with chiral analogs of FTY720 (namely the R- and S-enantiomers of 2-amino-4-(4-heptyloxyphenyl)-2-methylbutan-1-ol (AAL) (5Brinkmann V. Davis M.D. Heise C.E. Albert R. Cottens S. Hof R. Bruns C. Prieschl E. Baumruker T. Hiestand P. Foster C.A. Zollinger M. Lynch K.R. J. Biol. Chem. 2002; 277: 21453-21457Abstract Full Text Full Text PDF PubMed Scopus (1302) Google Scholar)) showed (i) that muSPHK1a catalyzed phosphorylation of AAL(R) but not AAL(S), (ii) that phosphorylation of AAL(R) was essential for binding at S1P receptors in vitro, and (iii) that only AAL(R), as the isomer which gets phosphorylated, was therapeutically effective in experimental autoimmune encephalomyelitis in rats (5Brinkmann V. Davis M.D. Heise C.E. Albert R. Cottens S. Hof R. Bruns C. Prieschl E. Baumruker T. Hiestand P. Foster C.A. Zollinger M. Lynch K.R. J. Biol. Chem. 2002; 277: 21453-21457Abstract Full Text Full Text PDF PubMed Scopus (1302) Google Scholar). It was concluded that phosphorylation of FTY720 by SPHK type 1 is essential for its biological activity.More recently, a second SPHK isoform has been identified and the mouse and human type 2 enzymes have been cloned (8Liu H. Sugiura M. Nava V.E. Edsall L.C. Kono K. Poulton S. Milstien S. Kohama T. Spiegel S. J. Biol. Chem. 2002; 275: 19513-19520Abstract Full Text Full Text PDF Scopus (558) Google Scholar); human SPHK (huSPHK) type 2 shares conserved domains with huSPHK1 (about 80% similarity) but carries additional amino acids in the center and at the N terminus, making it a larger protein (383 versus 618 amino acids for huSPHK1 and 2, respectively). Distinct kinetic differences have been described for the two enzymes (8Liu H. Sugiura M. Nava V.E. Edsall L.C. Kono K. Poulton S. Milstien S. Kohama T. Spiegel S. J. Biol. Chem. 2002; 275: 19513-19520Abstract Full Text Full Text PDF Scopus (558) Google Scholar). Expression of SPHK1 was reported to be highest in lung and spleen, and of SPHK2 in liver and heart (8Liu H. Sugiura M. Nava V.E. Edsall L.C. Kono K. Poulton S. Milstien S. Kohama T. Spiegel S. J. Biol. Chem. 2002; 275: 19513-19520Abstract Full Text Full Text PDF Scopus (558) Google Scholar, 9Kohama T. Olivera A. Edsall L. Nagiec M.M. Dickson R. Spiegel S. J. Biol. Chem. 1998; 273: 23722-237228Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar). SPHKs have received considerable interest because their natural reaction product S1P has been recognized as a signaling molecule with both intra- and extracellular actions (recently reviewed in Refs. 10Spiegel S. Milstien S. Nat. Rev. Mol. Cell. Biol. 2003; 4: 397-407Crossref PubMed Scopus (1734) Google Scholar and 11Hla T. Pharmacol. Res. 2003; 47: 401-407Crossref PubMed Scopus (239) Google Scholar). Presently, a clear cut functional difference between the two enzymes SPHK1 and 2 has not been described, and most studies so far measured total SPHK activity increases upon stimulation of cells; only recently, selective antisense DNA and siRNA tools have been utilized (12Melendez A.J. Khaw A.K. J. Biol. Chem. 2002; 277: 17255-17262Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 13Shu X. Wu W. Mosteller R.D. Broek D. Mol. Cell. Biol. 2002; 22: 7758-7768Crossref PubMed Scopus (242) Google Scholar).Here we studied the phosphorylation of FTY720 by recombinant SPHK1 and 2 in vitro and by the endogeneous enzymes in murine tissues and in human endothelial cells. We conclude that, while both enzymes are contributing to FTY720 phosphorylation in vivo, SPHK2 is quantitatively the more important enzyme in producing the active form FTY720 phosphate. Furthermore, we determined the tissue distribution of SPHK1 and 2 enzyme activities in the mouse, to supplement previous studies on total SPHK activity in rats (14Gijsbers S. Van der Hoeven G. Van Veldhoven P.P. Biochim. Biophys. Acta. 2001; 1532: 37-50Crossref PubMed Scopus (33) Google Scholar).EXPERIMENTAL PROCEDURESMaterials—S1P and d-erythro-sphingosine were from Biomol Research Laboratory Inc. (Plymouth Meeting, PA). N-octanoyl ceramide was from Sigma. [γ-32P]ATP (3000 Ci/mmol) and [3H]sphingosine (23.5 Ci/mmol) was purchased from Amersham Biosciences. FTY720 and its monophosphate, [3H]FTY720 (50.1 Ci/mmol) and the R- and S-enantiomers of 2-amino-4-(4-heptyloxyphenyl)-2-methylbutan-1-ol (AAL) were synthesized at Novartis Pharma.Cell Culture—HEK293 cells were obtained from ATCC (Manassas, VA; CRL-1573) and cultured in Dulbecco's modified Eagle's medium/10% fetal calf serum (FCS) at 37 °C/5% CO2. Human umbilical vein endothelial cells (HUVECs) were isolated and cultured in human endothelial-serum free medium (Invitrogen) supplemented with 10% FCS (Invitrogen), 250 μg/ml dibutyryl cyclic AMP (Sigma), 10 μg/ml epidermal growth factor (BD Biosciences), and 1 μg/ml hydrocortisone (Sigma) at 37 °C, 5% CO2. These cells were used in passages 2 to 5; experiments were performed in collagen-coated plates.Cloning of Human Lipid Kinases and Variants—SPHK1 was amplified by PCR from human fetal cDNA (Clontech) using EXL DNA Polymerase (Stratagene, La Jolla, CA) using primers 5′-GCATTAGAATTCATGGATCCAGCGGGCGGCCCC and 5′-TAACGCGTCGACTCATAAGGGCTCTTCTGGCGGTGGCATCTG, which include terminal EcoRI and SalI restriction sites, respectively. Using these sites the amplified fragment was cloned into the expression vector pEGFP-C2 (Clontech) in-frame with an N-terminal EGFP fusion part (EGFP is a red-shifted variant of the wild-type green fluorescent protein, GFP); the amino acid sequence of the insert was identical to the SPHK1 protein sequence in SwissProt (Q9NYA1; AAG01980 in GenPept).In addition, two SPHK1 variants, one 14 and the other 86 amino acid longer at the N terminus (designated SPHK1+14 and SPHK1+86, accession numbers NP_068807 (in the NCBI Reference Sequence Project data base (RefSeq)) and AAH14439 (GenPept), respectively) were retrieved from the IMAGE Consortium Mammalian Gene Collection. They were amplified with Pfu Ultra (Stratagene) using the respective forward primers 5′CACCATGGATCCAGTGGTCGGTTGCGGACG for SPHK1+14 and 5′-CACCATGTCCGCTCAAG TTCTGGGATTTTTACG for SPHK1+86, and 5′-TCATAAGGGCTCTTCTGGCGGTGGCATCTG as reverse primer. The amplified DNA fragments were purified and cloned in pENTR/S.D./d-TOPO (Invitrogen). For expression, constructs were transferred to pcDNA6.2-DEST (native constructs) or pcDNA-DEST53 (to get N-terminally fused GFP constructs) by recombination using the LR clonase (Invitrogen).SPHK2 was amplified using primers 5′-GCATTAGAATTCATGGCCCCGCCCCCACCGCCACTGGCT and 5′-TAACGCGTCGACTCAGGGCTCCCGCCCCGGGCAGCCAGG and cloned into pEGFP-C2. The sequence obtained was identical to that published by Liu et al. (14Gijsbers S. Van der Hoeven G. Van Veldhoven P.P. Biochim. Biophys. Acta. 2001; 1532: 37-50Crossref PubMed Scopus (33) Google Scholar) (GenPept: AAF74124; SwissProt: Q9NRA0-2). A cloned variant with 36 additional amino acids at the N terminus (SPHK2 + 36; GenPept™: AAH06161) was obtained from the IMAGE Consortium Mammalian Gene Collection. The DNA insert was amplified with Pfu Ultra and the primers 5′-CACCATGAATGGACACCTTGAAGCAGAGG and 5′-TCAGGGCTCCCGCCCCGGGC, then purified, cloned in pENTR/S.D./d-TOPO, and finally transferred to the expression vectors pcDNA6.2-DEST or pcDNA-DEST53 as described above.A cDNA featuring similarity to SPHKs (RefSeq: NP_060708; designated here as "SPHK-like") was retrieved from an in house cDNA library and subcloned in pENTR/S.D./d-TOPO after amplification with Pfu Ultra and the following primer set: 5′-CACCATGACGGTGTTCTTTAAAACGC and 5′-TCACTGGGTGGGGCTTGTGAGCATC. An N-terminal EGFP-SPHK-like fusion construct was also prepared by PCR, cloned in pENTR/S.D./d-TOPO and transferred for expression to pcDNA6.2-DEST.Ceramide kinase (CERK) cDNA was obtained in two steps: a 5′-RACE (rapid amplification of cDNA ends) PCR was performed on a human leukemia Marathon cDNA library (Clontech) using Advantage GC polymerase (Clontech) and the primers 5′-CCATCCTAATACGACTCACTATAGGGC and 5′-AGCAGCGATGTCCTAACCGTAAACG. The full coding sequence was obtained by 5′ extension of the longest clone in successive PCR rounds using Pfu Ultra with the forward primers 247–315, 198–266, 149–217, and 127–168, and the same reverse primer 1757–1778 for all reactions (numbers correspond to sequence positions of GenBank™ accession number AB079066). A final PCR product encoding the complete ORF of CERK (identical to AB079066 (15Sugiura M. Kono K. Liu H. Shimizugawa T. Minekura H. Spiegel S. Kohama T. J. Biol. Chem. 2002; 277: 23294-23300Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar)) was obtained using 5′-CACCATGGGGGCGACGGGGGCGGCG and 5′-TCAGCTGTGTGAGTCT G GCTTCG and cloned in pENTR/S.D./ d-TOPO. An N-terminal EGFP-CERK fusion construct was also prepared by PCR and cloned in pENTR/S.D./d-TOPO. For expression, constructs were transferred to pcDNA6.2-DEST.Cloning of Murine Sphingosine Kinases—MuSPHK1a and 2 were amplified from total RNA of murine lung and from a mouse liver cDNA (Stratagene), respectively. Both PCR products were cloned for expression into pEGFP-C2 as described above for huSPHK1. The amino acid sequence of the insert was identical to the muSPHK1 and 2 protein sequences in SwissProt (O88885 and Q9JIA7, respectively).Search of the Human Genome for SPHK Homologues—We used BLAST to search the PIR-NREF (pir.georgetown.edu/pirwww/search/pirnref.shtml), the databases of NCBI (www.ncbi.nlm.nih.gov/entrez/query.fcgi) and of Celera Discovery System (cds.premier.celera.com/cds/login.cfm) for homologues of SPHK1 and SPHK2. Each hit was used in a subsequent search as a query. Sequences detected by both SPHKs and by all hits reflexively were considered as members of the group of SPHK homologues.Expression of Proteins in HEK293 Cells—HEK293 cells were seeded at 1 × 106 cells/well in 6-well plates. After 24 h, cells were transfected with 4 μg of vector alone or with vectors containing kinase constructs using calcium phosphate (16Schenborn E.T. Goiffon V. Methods Mol. Biol. 2000; 130: 135-145PubMed Google Scholar). Cells were incubated for 24 h (sphingosine kinases) or 48 h (SPHK-like protein and ceramide kinase) following transfection. Cells were washed with ice-cold phosphate-buffered saline and then scraped into 100 μl of ice-cold SPHK cell lysis buffer (20 mm Tris-HCl, pH 7.4, 20% glycerol, 1 mm β-mercaptoethanol, 1 mm EDTA, 1 mm sodium orthovanadate, 15 mm NaF, 40 mm β-glycerophosphate, 0.5 mm deoxpyridoxine, and complete™ protease inhibitors (Roche Applied Science), similar to conditions described in (9Kohama T. Olivera A. Edsall L. Nagiec M.M. Dickson R. Spiegel S. J. Biol. Chem. 1998; 273: 23722-237228Abstract Full Text Full Text PDF PubMed Scopus (469) Google Scholar)). Following two freeze/thaw cycles, membranes were centrifuged off (100,000 × g, 60 min, 4 °C) and the supernatant was harvested. In some instances, pellets were re-suspended in lysis buffer for testing. In the case of CERK, the lysis buffer (20 mm MOPS, pH 7.2, 2 mm EGTA, 1 mm dithiothreitol, complete™ protease inhibitors, 10% glycerol; as described in Ref. 5Brinkmann V. Davis M.D. Heise C.E. Albert R. Cottens S. Hof R. Bruns C. Prieschl E. Baumruker T. Hiestand P. Foster C.A. Zollinger M. Lynch K.R. J. Biol. Chem. 2002; 277: 21453-21457Abstract Full Text Full Text PDF PubMed Scopus (1302) Google Scholar) was used alternatively.Protein Analysis—Protein concentration in cell lysates were determined using Bradford reagent (BioRad), with bovine serum albumin as reference. In the case of EGFP fusion proteins, lysates were measured in a fluorescence plate reader (BMG, Offenburg, Germany) at λex = 485 nm and λem = 538 nm to obtain relative fluorescence intensities of the samples. Furthermore, expression of EGFP fusion proteins was monitored by Western blotting using anti-GFP antibodies (ab290; Abcam Ltd., Cambridge, UK).Assay of SPHKs in HEK293 Cell Lysates—Routinely, SPHK activity was measured in total volumes of 100 μl with 20 μm sphingosine or FTY720 (prepared as a complex with bovine serum albumin, as described for sphingosine in Ref. 17Olivera A. Barlow K.D. Spiegel S. Methods Enzymol. 2000; 311: 215-223Crossref PubMed Scopus (70) Google Scholar), 1 mm of ATP, and 2 μCi of [γ-32P]ATP as substrates in buffer U (50 mm Hepes, pH 7.4, 15 mm MgCl2, 0.005% Triton X-100, 10 mm KCl, 10 mm NaF, and 1.5 mm semicarbazide); NaF and semicarbazide were included as inhibitors of S1P phosphatase and lyase (14Gijsbers S. Van der Hoeven G. Van Veldhoven P.P. Biochim. Biophys. Acta. 2001; 1532: 37-50Crossref PubMed Scopus (33) Google Scholar), respectively, in order to prevent degradation of formed S1P. Reactions were started by addition of protein samples (usually 10 μl/assay). In the case of SPHK1 and 2 and their variants, the cytosolic fraction of the cell lysates was tested; in the case of SPHK-like protein and CERK the membrane fraction was tested in addition. Incubations routinely were carried out for 30 min at 30 °C, but were extended to up to 4 h for SPHK-like protein and CERK. Reactions were stopped by adding 150 μl of 1.33 m KCl in 2.66% HCl, 200 μl of methanol, and 300 μl of chloroform. Samples were mixed, centrifuged, and 200 μl of the organic layer was removed and evaporated in vacuo. The residue was taken up in 25 μl chloroform and applied to a silica gel G-60 thin-layer chromatography (TLC) plates (Merck). TLCs were developed with 1-butanol/acetic acid: water 3:1:1 (v/v) as the mobile phase. Radiolabeled S1P and FTY720 phosphate were visualized and quantified using a Molecular Dynamics Storm PhosphorImager (Sunnyvale, CA) and identified by co-migration with unlabelled standards, which were visualized using vanillin/sulfuric acid reagent (18Stahl E. Thin-layer Chromatography. Springer Verlag, New York1969: 904Google Scholar). For further confirmation of identity, an alternative mobile phase was used for TLC, namely CHCl3/MeOH/H2O/NH3 200:150:29:1.For some experiments, the buffer was changed either to 50 mm Hepes, pH 7.4, 0.5% Triton, 15 mm MgCl2, 10% glycerol, 10 mm NaF, 1.5 mm semicarbazide (buffer 1, favoring SPHK1 activity) or 50 mm Hepes, pH 7.4, 15 mm MgCl2, 1 m KCl, 10% glycerol, 10 mm NaF, 1.5 mm semicarbazide (buffer 2, favoring SPHK2 activity). Furthermore, in the case of SPHK-like protein and of CERK, substrate concentration was increased to up to 100 μm.Kinetic Measurements with SPHKs—Assays were set up as described above for the routine assay. The 30-min incubation time was found to be in the quasilinear phase of the assay. Concentrations of sphingosine and FTY720 were varied as indicated in the figures. In order to calculate nmol of phosphorylated product from the fluorescence units returned by the PhosphorImager, calibration curves with known amounts of radiolabeled product were established. Enzyme activity was calculated as nmol of product/min/mg of total protein. Kinetic parameters were calculated from the data using the program Grafit (Erithacus Software, Horley, UK).Assay of Ceramide Kinase in HEK293 Cell Lysates—This enzyme was assayed as described by Sugiura et al. (5Brinkmann V. Davis M.D. Heise C.E. Albert R. Cottens S. Hof R. Bruns C. Prieschl E. Baumruker T. Hiestand P. Foster C.A. Zollinger M. Lynch K.R. J. Biol. Chem. 2002; 277: 21453-21457Abstract Full Text Full Text PDF PubMed Scopus (1302) Google Scholar), using N-octanoyl-sphingosine (C8-ceramide) as the substrate.Analysis of muSPHK mRNA Levels in Mouse Tissues—Tissue distribution studies were performed by real-time PCR using reverse-transcribed cDNA samples from a mouse total RNA panel (for brain, kidney, liver, lung, spleen; from BALB/c (8–12 week old) mice; Clontech, Palo Alto, CA). For whole blood and lymph nodes total RNA was isolated using Trizol reagent according to the protocol of the manufacturer (Invitrogen). SPHK PCR primers were designed using the GCG Wisconsin Package (Accelrys Inc.). Primers for SPHK1 were designed to detect both SPHK1a and 1b. The primer for β-actin (Actb) was previously described on www.thelabrat.com/protocols. The testing of primer specificity included melting curve analysis and agarose gel electrophoresis of the PCR products. For PCR reaction 5 μl of a 1:10 diluted cDNA sample was subjected to PCR analysis using primer sets (Table I) specific for the muSPHK1, muSPHK2 and Actb as an endogenous housekeeping control gene. Reactions were performed in a total volume of 25 μl including SYBR Green PCR master mix as supplied by PE Applied Biosystems (Foster City, CA): 1× SYBR Green PCR buffer, 3 mm MgCl2, 0.2 mm dATP/dCTP/dGTP, 0.4 mm dUTP, 0.6 U AmpliTaq Gold, 0.25 units of AmpEraseUNG (to prevent carry-over of previous PCR products). Amplification cycles were as follows: 2 min at 50 °C (treatment with AmpEraseUNG), 10 min at 95 °C (activation of Ampli-Taq Gold) and 40 cycles of 15 s at 95 °C followed by 1 min at 60 °C for product amplification. Incubation and on-line detection of PCR products were carried out with optical 96-well plates in the ABI Prism 7700 Sequence Detection System from PE Applied Biosystems. Each PCR reaction was performed in duplicate. Based on melting curve analysis no primer-dimers were generated during the applied 40 real-time PCR amplification cycles. To determine real-time PCR efficiency for one cycle in the exponential phase, dilution series of one of the cDNA samples under investigation over four orders of magnitude were used for each primer pair to create a relative standard curve. The PCR efficiency (E) was then calculated from the slope of the standard curve based on the linear regression analysis (CT as the Y values versus log(serial dilutions) as the X values) using the formula E = 10–1/slope; R2 value shows linearity of the standard curve (perfect linearity: R2 = 1) (Table I). For relative quantification of target gene transcripts normalized to Actb and relative to a calibrator a mathematical model, the "Delta-delta CT method," developed by PE Applied Biosystems (PerkinElmer) was used. Thus, the calibrator (brain is designated as the calibrator in our calculations) has a value of 1, and all other quantities are expressed as an n-fold difference relative to the calibrator.Table IPrimers used for RT-PCRPrimer, accession number of geneSequence (5′-3′)RT-PCR efficiency (E) and linearity (R2)mSPHK1, AF068749F: ggcagtcatgtccggtgatgE = 1.894; R2 = 0.9985R: acagcagtgtgcagttgatgagmSPHK2, AF245448F: acagaaccatgcccgtgagE = 1.980; R2 = 0.9959R: aggtcaacaccgacaacctgActb, NM_007393F: aggtcatcactattggcaacgaE = 1.836; R2 = 0.9964R: cacttcatgatggaattgaatgtagtt Open table in a new tab Analysis of SPHK Activity in Mouse Tissues—Activity in mouse tissues was determined in analogy to procedures described by Gijsbers et al. (14Gijsbers S. Van der Hoeven G. Van Veldhoven P.P. Biochim. Biophys. Acta. 2001; 1532: 37-50Crossref PubMed Scopus (33) Google Scholar). Female Balb/c mice (obtained from Charles River, Sulzfeld, Germany) were killed, and their tissues were quickly dissected; heparinized whole blood was also obtained. The tissues from three animals each were pooled. Tissues were flushed with ice-cold PBS, weighed, and then homogenized in Potter-Elvejhem homogenizers in 3 volumes of ice-cold tissue lysis buffer (50 mm potassium phosphate buffer, pH 7.4, containing 1 mm EDTA, 1 mm dithiothreitol, and complete™ protease inhibitors). Samples were centrifuged at 100,000 × g at 4 °C for 60 min. Supernatants were harvested, and the pellet was resuspended in an equal volume of tissue lysis buffer. Samples were subjected to the routine SPHK assay as described above for HEK293 cell lysates, using either buffer 1 or 2. Incubation time was 30 min, which was found to be in the quasi-linear phase of the assay. Activity is reported as mU/g (nmol of phosphorylated product formation per min per g of tissue).Assay of Endogenous SPHKs Activity in HUVEC—The activity of SPHK1 and 2 in HUVEC lysates was measured as described for the HEK293 cells, but incubation time was extended to 2 h.Assay of SPHK Activity in Whole Blood—Heparinized blood was obtained from human donors, Balb/c mice, and Wistar rats (Charles River). The blood was diluted with 3 volumes of SPHK cell lysis buffer; following freeze/thawing and centrifugation supernatants were tested in the routine SPHK assay as described above, with sphingosine as the substrate and using either buffer 1 or 2. Alternatively, the phosphorylation of FTY720 was studied using intact blood cells: heparinized blood was diluted with 3 volumes of RPMI medium containing 10% FCS and incubated with [3H]FTY720 (0.2 μCi, 60 nm) at 37 °C for up to 18 h. Following incubation, samples were extracted with equal volumes of 1-butyl alcohol; the butyl alcohol phase was back-washed with an equal volume of water and evaporated in vacuo to dryness. Residues were taken up in 25 μl of butyl alcohol and subjected to TLC analysis as described for the routine SPHK assay. Radiolabeled products were visualized by autoradiography and quantified by scraping bands into scintillation mixture followed by counting. The percentage of conversion to the phosphate was calculated from the sum of radioactivity associated with the FTY720 and FTY720 phosphate spots.RESULTS AND DISCUSSIONKinetic Characterization of Sphingosine Kinases 1 and 2— We transiently expressed the human sphingosine kinases (huSPHK) type 1 and 2 in HEK293 cells. The proteins carried an N-terminal EGFP as fusion part, enabling their detection via fluorescence. SPHK activity in lysates of the transfected cells was measured, using the "unspecific" buffer U (see "Experimental Procedures") with low salt and Triton X-100 conc
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