Sphingosine-1-phosphate (SPP) is a novel lipid messenger that has dual function. Intracellularly it regulates proliferation and survival, and extracellularly, it is a ligand for the G protein-coupled receptor Edg-1. Based on peptide sequences obtained from purified rat kidney sphingosine kinase, the enzyme that regulates SPP levels, we report here the cloning, identification, and characterization of the first mammalian sphingosine kinases (murine SPHK1a and SPHK1b). Sequence analysis indicates that these are novel kinases, which are not similar to other known kinases, and that they are evolutionarily conserved. Comparison withSaccharomyces cerevisiae and Caenorhabditis elegans sphingosine kinase sequences shows that several blocks are highly conserved in all of these sequences. One of these blocks contains an invariant, positively charged motif, GGKGK, which may be part of the ATP binding site. From Northern blot analysis of multiple mouse tissues, we observed that expression was highest in adult lung and spleen, with barely detectable levels in skeletal muscle and liver. Human embryonic kidney cells and NIH 3T3 fibroblasts transiently transfected with either sphingosine kinase expression vectors had marked increases (more than 100-fold) in sphingosine kinase activity. The enzyme specifically phosphorylatedd-erythro-sphingosine and did not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide,d,l-threo-dihydrosphingosine orN, N-dimethylsphingosine. The latter two sphingolipids were competitive inhibitors of sphingosine kinase in the transfected cells as was previously found with the purified rat kidney enzyme. Transfected cells also had a marked increase in mass levels of SPP with a concomitant decrease in levels of sphingosine and, to a lesser extent, in ceramide levels. Our data suggest that sphingosine kinase is a prototypical member of a new class of lipid kinases. Cloning of sphingosine kinase is an important step in corroborating the intracellular role of SPP as a second messenger. Sphingosine-1-phosphate (SPP) is a novel lipid messenger that has dual function. Intracellularly it regulates proliferation and survival, and extracellularly, it is a ligand for the G protein-coupled receptor Edg-1. Based on peptide sequences obtained from purified rat kidney sphingosine kinase, the enzyme that regulates SPP levels, we report here the cloning, identification, and characterization of the first mammalian sphingosine kinases (murine SPHK1a and SPHK1b). Sequence analysis indicates that these are novel kinases, which are not similar to other known kinases, and that they are evolutionarily conserved. Comparison withSaccharomyces cerevisiae and Caenorhabditis elegans sphingosine kinase sequences shows that several blocks are highly conserved in all of these sequences. One of these blocks contains an invariant, positively charged motif, GGKGK, which may be part of the ATP binding site. From Northern blot analysis of multiple mouse tissues, we observed that expression was highest in adult lung and spleen, with barely detectable levels in skeletal muscle and liver. Human embryonic kidney cells and NIH 3T3 fibroblasts transiently transfected with either sphingosine kinase expression vectors had marked increases (more than 100-fold) in sphingosine kinase activity. The enzyme specifically phosphorylatedd-erythro-sphingosine and did not catalyze the phosphorylation of phosphatidylinositol, diacylglycerol, ceramide,d,l-threo-dihydrosphingosine orN, N-dimethylsphingosine. The latter two sphingolipids were competitive inhibitors of sphingosine kinase in the transfected cells as was previously found with the purified rat kidney enzyme. Transfected cells also had a marked increase in mass levels of SPP with a concomitant decrease in levels of sphingosine and, to a lesser extent, in ceramide levels. Our data suggest that sphingosine kinase is a prototypical member of a new class of lipid kinases. Cloning of sphingosine kinase is an important step in corroborating the intracellular role of SPP as a second messenger. sphingosine-1-phosphate high performance liquid chromatography. The sphingolipid metabolite, sphingosine-1-phosphate (SPP),1 is emerging as a prototype of a new class of lipid second messengers, which has both intracellular and extracellular actions (1Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (810) Google Scholar, 2Meyer 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, 3Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1337) Google Scholar, 4Lee M.-J. Van Brocklyn J.R. Thangada S. Liu C.H. Hand A.R. Menzeleev R. Spiegel S. Hla T. Science. 1998; 279: 1552-1555Crossref PubMed Scopus (881) Google Scholar). Ample evidence indicates that SPP can serve as an intracellular second messenger; SPP modulates intracellular pathways important for diverse biological processes including cell growth, survival, motility, and cytoskeletal changes (reviewed in Ref. 5Spiegel S. Foster D. Kolesnick R. Curr. Opin. Cell Biol. 1996; 8: 159-167Crossref PubMed Scopus (471) Google Scholar). Moreover, because SPP antagonizes apoptosis mediated by ceramide, a stress-induced sphingolipid metabolite (3Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1337) Google Scholar, 6Edsall L.C. Pirianov G.G. Spiegel S. J. Neurosci. 1997; 17: 6952-6960Crossref PubMed Google Scholar), we have proposed that the relative intracellular levels of these two sphingolipid metabolites is an important factor that determines whether cells will survive or die (3Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1337) Google Scholar). In support of this idea, it has recently been shown that unfertilized mouse oocytes exposed to the anticancer drug doxorubicin undergo ceramide-mediated apoptosis that is inhibited by SPP (7Perez G.I. Knudson C.M. Leykin L. Korsmeyer S.J. Tilly J.L. Nat. Med. 1997; 3: 1228-1232Crossref PubMed Scopus (307) Google Scholar). In addition, it seems that this ceramide/SPP rheostat is an evolutionarily conserved stress regulatory mechanism influencing growth and survival of yeast (8Mandala S. Thornton R. Tu Z. Kurtz M. Nickels J. Broach J. Menzeleev R. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 150-155Crossref PubMed Scopus (235) Google Scholar, 9Jenkins G.M. Richards A. Wahl T. Mao C. Obeid L. Hannun Y. J. Biol. Chem. 1997; 272: 32566-32572Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 10Dickson R.C. Nagiec E.E. Skrzypek M. Tillman P. Wells G.B. Lester R.L. J. Biol. Chem. 1997; 272: 30196-30200Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Recently, it has been shown that SPP, a serum borne lipid, is the ligand for the G protein-coupled endothelial-derived receptor-1 (EDG-1), which regulates morphogenetic differentiation of endothelial cells (4Lee M.-J. Van Brocklyn J.R. Thangada S. Liu C.H. Hand A.R. Menzeleev R. Spiegel S. Hla T. Science. 1998; 279: 1552-1555Crossref PubMed Scopus (881) Google Scholar). Taken together, these data suggest that SPP has dual actions (11Van Brocklyn J.R. Lee M.J. Menzeleev R. Olivera A. Edsall L. Cuvillier O. Dianne M.T. Coopman P.J.P. Thangada S. Hla T. Spiegel S. J. Cell Biol. 1998; 142: 229-240Crossref PubMed Scopus (445) Google Scholar).Various stimuli, including platelet-derived growth factor and serum (1Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (810) Google Scholar,12Bornfeldt K.E. Graves L.M. Raines E.W. Igarashi Y. Wayman G. Yamamura S. Yatomi Y. Sidhu J.S. Krebs E.G. Hakomori S. Ross R. J. Cell Biol. 1995; 130: 193-206Crossref PubMed Scopus (264) Google Scholar), nerve growth factor (6Edsall L.C. Pirianov G.G. Spiegel S. J. Neurosci. 1997; 17: 6952-6960Crossref PubMed Google Scholar, 13Rius R.A. Edsall L.C. Spiegel S. FEBS Lett. 1997; 417: 173-176Crossref PubMed Scopus (98) Google Scholar), activation of protein kinase C (14Mazurek N. Megidish T. Hakomori S.-I. Igarashi Y. Biochem. Biophys. Res. Comm. 1994; 198: 1-9Crossref PubMed Scopus (89) Google Scholar,15Buehrer B.M. Bardes E.S. Bell R.M. Biochim. Biophys. Acta. 1996; 1303: 233-242Crossref PubMed Scopus (79) Google Scholar), and cross-linking of FcεR1 and FcγR1 (16Melendez A. Floto R.A. Gillooly D.J. Harnett M.M. Allen J.M. J. Biol. Chem. 1998; 273: 9393-9402Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), increase cellular levels of SPP by activation of sphingosine kinase, the enzyme that catalyzes the phosphorylation of sphingosine. Competitive inhibitors of sphingosine kinase block formation of SPP and selectively inhibit cellular proliferation induced by platelet-derived growth factor and serum (1Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (810) Google Scholar, 17Rani C.S. Wang F. Fuior E. Berger A. Wu J. Sturgill T.W. Beitner-Johnson D. LeRoith D. Varticovski L. Spiegel S. J. Biol. Chem. 1997; 272: 10777-10783Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), the cytoprotective effects of protein kinase C (3Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1337) Google Scholar), and nerve growth factor (6Edsall L.C. Pirianov G.G. Spiegel S. J. Neurosci. 1997; 17: 6952-6960Crossref PubMed Google Scholar), as well as FcεRI- and FcγR1-mediated calcium signaling (16Melendez A. Floto R.A. Gillooly D.J. Harnett M.M. Allen J.M. J. Biol. Chem. 1998; 273: 9393-9402Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), further supporting a role for endogenous SPP in cell growth, survival, and calcium mobilization.Collectively, these results give new insights into the biological function of SPP and emphasize the importance of sphingosine kinase, the enzyme that regulates its formation. Recently, we have purified rat kidney sphingosine kinase 6 × 105-fold to apparent homogeneity (18Olivera A. Kohama T. Tu Z. Milstien S. Spiegel S. J. Biol. Chem. 1998; 273: 12576-12583Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Purified sphingosine kinase has an apparent molecular mass of approximately 49 kDa with K m values of 5 and 93 μm for sphingosine and ATP, respectively (18Olivera A. Kohama T. Tu Z. Milstien S. Spiegel S. J. Biol. Chem. 1998; 273: 12576-12583Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Based on peptide sequences, we report here the cloning and characterization of the first mammalian sphingosine kinases. The sphingolipid metabolite, sphingosine-1-phosphate (SPP),1 is emerging as a prototype of a new class of lipid second messengers, which has both intracellular and extracellular actions (1Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (810) Google Scholar, 2Meyer 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, 3Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1337) Google Scholar, 4Lee M.-J. Van Brocklyn J.R. Thangada S. Liu C.H. Hand A.R. Menzeleev R. Spiegel S. Hla T. Science. 1998; 279: 1552-1555Crossref PubMed Scopus (881) Google Scholar). Ample evidence indicates that SPP can serve as an intracellular second messenger; SPP modulates intracellular pathways important for diverse biological processes including cell growth, survival, motility, and cytoskeletal changes (reviewed in Ref. 5Spiegel S. Foster D. Kolesnick R. Curr. Opin. Cell Biol. 1996; 8: 159-167Crossref PubMed Scopus (471) Google Scholar). Moreover, because SPP antagonizes apoptosis mediated by ceramide, a stress-induced sphingolipid metabolite (3Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1337) Google Scholar, 6Edsall L.C. Pirianov G.G. Spiegel S. J. Neurosci. 1997; 17: 6952-6960Crossref PubMed Google Scholar), we have proposed that the relative intracellular levels of these two sphingolipid metabolites is an important factor that determines whether cells will survive or die (3Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1337) Google Scholar). In support of this idea, it has recently been shown that unfertilized mouse oocytes exposed to the anticancer drug doxorubicin undergo ceramide-mediated apoptosis that is inhibited by SPP (7Perez G.I. Knudson C.M. Leykin L. Korsmeyer S.J. Tilly J.L. Nat. Med. 1997; 3: 1228-1232Crossref PubMed Scopus (307) Google Scholar). In addition, it seems that this ceramide/SPP rheostat is an evolutionarily conserved stress regulatory mechanism influencing growth and survival of yeast (8Mandala S. Thornton R. Tu Z. Kurtz M. Nickels J. Broach J. Menzeleev R. Spiegel S. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 150-155Crossref PubMed Scopus (235) Google Scholar, 9Jenkins G.M. Richards A. Wahl T. Mao C. Obeid L. Hannun Y. J. Biol. Chem. 1997; 272: 32566-32572Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 10Dickson R.C. Nagiec E.E. Skrzypek M. Tillman P. Wells G.B. Lester R.L. J. Biol. Chem. 1997; 272: 30196-30200Abstract Full Text Full Text PDF PubMed Scopus (218) Google Scholar). Recently, it has been shown that SPP, a serum borne lipid, is the ligand for the G protein-coupled endothelial-derived receptor-1 (EDG-1), which regulates morphogenetic differentiation of endothelial cells (4Lee M.-J. Van Brocklyn J.R. Thangada S. Liu C.H. Hand A.R. Menzeleev R. Spiegel S. Hla T. Science. 1998; 279: 1552-1555Crossref PubMed Scopus (881) Google Scholar). Taken together, these data suggest that SPP has dual actions (11Van Brocklyn J.R. Lee M.J. Menzeleev R. Olivera A. Edsall L. Cuvillier O. Dianne M.T. Coopman P.J.P. Thangada S. Hla T. Spiegel S. J. Cell Biol. 1998; 142: 229-240Crossref PubMed Scopus (445) Google Scholar). Various stimuli, including platelet-derived growth factor and serum (1Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (810) Google Scholar,12Bornfeldt K.E. Graves L.M. Raines E.W. Igarashi Y. Wayman G. Yamamura S. Yatomi Y. Sidhu J.S. Krebs E.G. Hakomori S. Ross R. J. Cell Biol. 1995; 130: 193-206Crossref PubMed Scopus (264) Google Scholar), nerve growth factor (6Edsall L.C. Pirianov G.G. Spiegel S. J. Neurosci. 1997; 17: 6952-6960Crossref PubMed Google Scholar, 13Rius R.A. Edsall L.C. Spiegel S. FEBS Lett. 1997; 417: 173-176Crossref PubMed Scopus (98) Google Scholar), activation of protein kinase C (14Mazurek N. Megidish T. Hakomori S.-I. Igarashi Y. Biochem. Biophys. Res. Comm. 1994; 198: 1-9Crossref PubMed Scopus (89) Google Scholar,15Buehrer B.M. Bardes E.S. Bell R.M. Biochim. Biophys. Acta. 1996; 1303: 233-242Crossref PubMed Scopus (79) Google Scholar), and cross-linking of FcεR1 and FcγR1 (16Melendez A. Floto R.A. Gillooly D.J. Harnett M.M. Allen J.M. J. Biol. Chem. 1998; 273: 9393-9402Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), increase cellular levels of SPP by activation of sphingosine kinase, the enzyme that catalyzes the phosphorylation of sphingosine. Competitive inhibitors of sphingosine kinase block formation of SPP and selectively inhibit cellular proliferation induced by platelet-derived growth factor and serum (1Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (810) Google Scholar, 17Rani C.S. Wang F. Fuior E. Berger A. Wu J. Sturgill T.W. Beitner-Johnson D. LeRoith D. Varticovski L. Spiegel S. J. Biol. Chem. 1997; 272: 10777-10783Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar), the cytoprotective effects of protein kinase C (3Cuvillier O. Pirianov G. Kleuser B. Vanek P.G. Coso O.A. Gutkind S. Spiegel S. Nature. 1996; 381: 800-803Crossref PubMed Scopus (1337) Google Scholar), and nerve growth factor (6Edsall L.C. Pirianov G.G. Spiegel S. J. Neurosci. 1997; 17: 6952-6960Crossref PubMed Google Scholar), as well as FcεRI- and FcγR1-mediated calcium signaling (16Melendez A. Floto R.A. Gillooly D.J. Harnett M.M. Allen J.M. J. Biol. Chem. 1998; 273: 9393-9402Abstract Full Text Full Text PDF PubMed Scopus (166) Google Scholar), further supporting a role for endogenous SPP in cell growth, survival, and calcium mobilization. Collectively, these results give new insights into the biological function of SPP and emphasize the importance of sphingosine kinase, the enzyme that regulates its formation. Recently, we have purified rat kidney sphingosine kinase 6 × 105-fold to apparent homogeneity (18Olivera A. Kohama T. Tu Z. Milstien S. Spiegel S. J. Biol. Chem. 1998; 273: 12576-12583Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Purified sphingosine kinase has an apparent molecular mass of approximately 49 kDa with K m values of 5 and 93 μm for sphingosine and ATP, respectively (18Olivera A. Kohama T. Tu Z. Milstien S. Spiegel S. J. Biol. Chem. 1998; 273: 12576-12583Abstract Full Text Full Text PDF PubMed Scopus (201) Google Scholar). Based on peptide sequences, we report here the cloning and characterization of the first mammalian sphingosine kinases. We thank Drs. Alexander Yakovlev, James R. Van Brocklyn, Tom I. Bonner, Sheldon Milstien, and Masaaki Takahashi for helpful suggestions and Dr. Barry W. Cherney for assistance with Northern assays.