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• W2044138450 abstract "Sphingosine kinase is a highly conserved enzyme that catalyzes the synthesis of sphingosine 1-phosphate and reduces cellular levels of sphingosine and ceramide. Although ceramide is pro-apoptotic and sphingosine is generally growth-inhibitory, sphingosine 1-phosphate signaling promotes cell proliferation, survival, and migration. Sphingosine kinase is thus in a strategic position to regulate important cell fate decisions which may contribute to normal animal development. To facilitate studies examining the potential role of sphingosine kinase and long chain base metabolism in Drosophila development, we characterized two putative Drosophila sphingosine kinase genes, Sk1 and Sk2. Both genes functionally and biochemically complement a yeast sphingosine kinase mutant, express predominantly cytosolic activities, and are capable of phosphorylating a range of endogenous and non-endogenous sphingoid base substrates. The two genes demonstrate overlapping but distinct temporal and spatial expression patterns in the Drosophila embryo, and timing of expression is consistent with observed changes in long chain base levels throughout development. A null Sk2 transposon insertion mutant demonstrated elevated long chain base levels, impaired flight performance, and diminished ovulation. This is the first reported mutation of a sphingosine kinase in an animal model; the associated phenotypes indicate that Sk1 and Sk2 are not redundant in biological function and that sphingosine kinase is essential for diverse physiological functions in this organism. Sphingosine kinase is a highly conserved enzyme that catalyzes the synthesis of sphingosine 1-phosphate and reduces cellular levels of sphingosine and ceramide. Although ceramide is pro-apoptotic and sphingosine is generally growth-inhibitory, sphingosine 1-phosphate signaling promotes cell proliferation, survival, and migration. Sphingosine kinase is thus in a strategic position to regulate important cell fate decisions which may contribute to normal animal development. To facilitate studies examining the potential role of sphingosine kinase and long chain base metabolism in Drosophila development, we characterized two putative Drosophila sphingosine kinase genes, Sk1 and Sk2. Both genes functionally and biochemically complement a yeast sphingosine kinase mutant, express predominantly cytosolic activities, and are capable of phosphorylating a range of endogenous and non-endogenous sphingoid base substrates. The two genes demonstrate overlapping but distinct temporal and spatial expression patterns in the Drosophila embryo, and timing of expression is consistent with observed changes in long chain base levels throughout development. A null Sk2 transposon insertion mutant demonstrated elevated long chain base levels, impaired flight performance, and diminished ovulation. This is the first reported mutation of a sphingosine kinase in an animal model; the associated phenotypes indicate that Sk1 and Sk2 are not redundant in biological function and that sphingosine kinase is essential for diverse physiological functions in this organism. Sphingosine 1-phosphate (S1P) 1The abbreviations used are: S1P, sphingosine 1-phosphate; DHS-1P, dihydrosphingosine 1-phosphate; EST, expressed sequence tag; HPLC, high performance liquid chromatography; LCB, long chain sphingoid base; LCBP, phosphorylated long chain sphingoid base; PGC, primordial germ line cell; S1PP, sphingosine-1-phosphate phosphatase; SPL, sphingosine 1-phosphate lyase; SK, sphingosine kinase; MOPS, 4-morpholinepropanesulfonic acid; CS, Canton-S. is a bioactive sphingolipid metabolite that provides directional cues to migrating cells (1Spiegel S. English D. Milstien S. Trends Cell Biol. 2002; 12: 236-242Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar) and exerts proliferative and anti-apoptotic effects on many cell types (2Pyne S. Pyne N.J. Biochem. J. 2000; 349: 385-402Crossref PubMed Scopus (662) Google Scholar). S1P signals are transduced through both extracellular (receptor-mediated) and intracellular mechanisms. Intracellularly, S1P signaling affects cytoskeletal organization, calcium homeostasis, DNA synthesis, and the apoptotic machinery. S1P is generated by the phosphorylation of sphingosine, a reaction catalyzed by sphingosine kinase (SK). It is eliminated through dephosphorylation catalyzed by either S1P phosphatase (S1PP) or type 2 phosphatidate phosphohydrolases or through its irreversible degradation to a long chain aldehyde and ethanolamine phosphate, catalyzed by sphingosine 1-phosphate lyase (SPL) (Fig. 1). SK is a member of a growing class of lipid kinases, including diacylglycerol kinases and phosphatidylinositol 3-kinases, that participate in cell signaling. Many studies have implicated SK activation and S1P generation in mediating angiogenesis, tumorigenicity, metastasis, cell proliferation, motility, lymphocyte trafficking, endocytosis, and survival (reviewed in Refs. 3Duronio V. Scheid M.P. Ettinger S. Cell. Signal. 1998; 10: 233-239Crossref PubMed Scopus (186) Google Scholar, 4Topham M.K. Prescott S.M. J. Biol. Chem. 1999; 274: 11447-11450Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 5Corvera S. Traffic. 2001; 2: 859-866Crossref PubMed Scopus (53) Google Scholar, 6Kanoh H. Yamada K. Sakane F. J. Biochem. (Tokyo). 2002; 131: 629-633Crossref PubMed Scopus (117) Google Scholar, 7Martelli A.M. Bortul R. Tabellini G. Bareggi R. Manzoli L. Narducci P. Cocco L. Cell. Mol. Life Sci. 2002; 59: 1129-1137Crossref PubMed Scopus (45) Google Scholar, 8Maceyka M. Payne S.G. Milstien S. Spiegel S. Biochim. Biophys. Acta. 2002; 1585: 193-201Crossref PubMed Scopus (503) Google Scholar, 9Liu H. Chakravarty D. Maceyka M. Milstien S. Spiegel S. Prog. Nucleic Acids Res. Mol. Biol. 2002; 71: 493-511Crossref PubMed Google Scholar, 10Spiegel S. Milstien S. Nat. Rev. Mol. Cell. Biol. 2003; 4: 397-407Crossref PubMed Scopus (1768) Google Scholar). SK activation has been reported in response to many stimuli and regulates diverse functions such as heat stress response in yeast (11Lanterman M.M. Saba J.D. Biochem. J. 1998; 332: 525-531Crossref PubMed Scopus (60) Google Scholar) and stomatal closure in Arabidopsis (12Coursol S. Fan L. Le Stunff H. Speigel S. Gilroy S. Assmann S. Nature. 2003; 423: 651-654Crossref PubMed Scopus (285) Google Scholar). Thus, SK activation appears to be a widely employed mechanism for propagating mitogenic and survival signals in eukaryotes. Known downstream effectors of SK activation include ERK1/2 (13Vann L.R. Payne S.G. Edsall L.C. Twitty S. Spiegel S. Milstien S. J. Biol. Chem. 2002; 277: 12649-12656Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), NFκB (14Xia P. Wang L. Moretti P.A. Albanese N. Chai F. Pitson S.M. D'Andrea R.J. Gamble J.R. Vadas M.A. J. Biol. Chem. 2002; 277: 7996-8003Abstract Full Text Full Text PDF PubMed Scopus (263) Google Scholar), and RhoA/Rho kinase (15Bolz S.S. Vogel L. Sollinger D. Derwand R. Boer C. Pitson S.M. Spiegel S. Pohl U. Circulation. 2003; 108: 342-347Crossref PubMed Scopus (123) Google Scholar). SK was originally cloned in budding yeast (16Nagiec M.M. Skrzypek M. Nagiec E.E. Lester R.L. Dickson R.C. J. Biol. Chem. 1998; 273: 19437-19442Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), and members of the SK family have subsequently been identified in plant (17Nishiura H. Tamura K. Morimoto Y. Imai H. Biochem. Soc. Trans. 2000; 28: 747-748Crossref PubMed Google Scholar), mouse (18Kohama T. Olivera A. Edsall L. Nagiec M.M. Dickson R. Spiegel S. J. Biol. Chem. 1998; 273: 23722-23728Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar), rat (19Olivera A. Kohama T. Tu Z.X. Milstien S. Spiegel S. J. Biol. Chem. 1998; 273: 12576-12583Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar), and human (20Melendez A.J. Carlos-Dias E. Gosink M. Allen J.M. Takacs L. Gene (Amst.). 2000; 251: 19-26Crossref PubMed Scopus (126) Google Scholar). Each has a conserved lipid kinase catalytic domain that contains the ATP-binding site. SK enzymes thus far identified each possess five conserved domains, C1 through C5 (9Liu H. Chakravarty D. Maceyka M. Milstien S. Spiegel S. Prog. Nucleic Acids Res. Mol. Biol. 2002; 71: 493-511Crossref PubMed Google Scholar, 18Kohama T. Olivera A. Edsall L. Nagiec M.M. Dickson R. Spiegel S. J. Biol. Chem. 1998; 273: 23722-23728Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar) (Fig. 2A). These consensus sequences appear critical to the structural integrity of the proteins and probably enable substrate recognition, as they are altered in ceramide kinases (21Pitson S.M. Moretti P.A. Zebol J.R. Zareie R. Derian C.K. Darrow A.L. Qi J. D'Andrea R.J. Bagley C.J. Vadas M.A. Wattenberg B.W. J. Biol. Chem. 2002; 277: 49545-49553Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). The SK enzymes studied to date are capable of phosphorylating erythro-sphingosine, dihydrosphingosine, and phytosphingosine in an ATP-dependent fashion and are inhibited by a growing number of compounds, including the two most well tested in biological systems, dl-threo-dihydrosphingosine (22Buehrer B.M. Bell R.M. Adv. Lipid Res. 1993; 26: 59-67PubMed Google Scholar) and N,N-dimethylsphingosine (23Edsall L.C. Van Brocklyn J.R. Cuvillier O. Kleuser B. Spiegel S. Biochemistry. 1998; 37: 12892-12898Crossref PubMed Scopus (216) Google Scholar). SK activity is widely distributed in mouse and rat tissues and is primarily found in the cytosolic fraction (20Melendez A.J. Carlos-Dias E. Gosink M. Allen J.M. Takacs L. Gene (Amst.). 2000; 251: 19-26Crossref PubMed Scopus (126) Google Scholar, 24Nava V.E. Lacana E. Poulton S. Liu H. Sugiura M. Kono K. Milstien S. Kohama T. Spiegel S. FEBS Lett. 2000; 473: 81-84Crossref PubMed Scopus (89) Google Scholar, 25Murate T. Banno Y. T-Koizumi K. Watanabe K. Mori N. Wada A. Igarashi Y. Takagi A. Kojima T. Asano H. Akao Y. Yoshida S. Saito H. Nozawa Y. J. Histochem. Cytochem. 2001; 49: 845-855Crossref PubMed Scopus (53) Google Scholar). However, SK activity is also found to be associated with mitochondrial and microsomal fractions, and activated forms of SK are associated with the plasma membrane to which they have been demonstrated to translocate in several cell types (26Johnson K.R. Becker K.P. Facchinetti M.M. Hannun Y.A. Obeid L.M. J. Biol. Chem. 2002; 277: 35257-35262Abstract Full Text Full Text PDF PubMed Scopus (262) Google Scholar, 27Young K.W. Willets J.M. Parkinson M.J. Bartlett P. Spiegel S. Nahorski S.R. Challiss R.A. Cell Calcium. 2003; 33: 119-128Crossref PubMed Scopus (54) Google Scholar). Mammalian SK activity is encoded by at least two genes, Sphk1 (18Kohama T. Olivera A. Edsall L. Nagiec M.M. Dickson R. Spiegel S. J. Biol. Chem. 1998; 273: 23722-23728Abstract Full Text Full Text PDF PubMed Scopus (474) Google Scholar, 28Pitson S.M. D'Andrea R.J. Vandeleur L. Moretti P.A. Xia P. Gamble J.R. Vadas M.A. Wattenberg B.W. Biochem. J. 2000; 350: 429-441Crossref PubMed Scopus (166) Google Scholar) and Sphk2 (29Liu H. Sugiura M. Nava V.E. Edsall L.C. Kono K. Poulton S. Milstien S. Kohama T. Spiegel S. J. Biol. Chem. 2000; 275: 19513-19520Abstract Full Text Full Text PDF PubMed Scopus (566) Google Scholar). Sphk1 is regulated at both transcriptional and post-transcriptional (phosphorylation and translocation) levels, whereas regulation of Sphk2 has not been established (see Ref. 30Sukocheva O.A. Wang L. Albanese N. Pitson S.M. Vadas M.A. Xia P. Mol. Endocrinol. 2003; 17: 2002-2012Crossref PubMed Scopus (135) Google Scholar and reviewed in Ref. 31Olivera A. Spiegel S. Prostaglandins Other Lipid Mediat. 2001; 64: 123-134Crossref PubMed Scopus (146) Google Scholar). Sphk1 and -2 demonstrate distinct enzyme kinetics and temporal and spatial expression patterns. Additional functional distinctions between Sphk1 and Sphk2 are evident. For example, whereas Sphk1 enhances cell survival and proliferation, Sphk2 induces apoptosis through the release of cytochrome c and activation of caspase 3, potentially through a BH3 domain and Bcl-xL interactions (32Liu H. Toman R.E. Goparaju S. Maceyka M. Nava V.E. Sankala H. Payne S.G. Bektas M. Ishii I. Chun J. Milstien S. Spiegel S. J. Biol. Chem. 2003; 278: 40330-40336Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). Sphk2 also appears to be the enzyme that most efficiently phosphorylates the novel immune modulator FTY720 to the active drug (33Billich A. Bornancin F. Devay P. Mechtcheriakova D. Urtz N. Baumruker T. J. Biol. Chem. 2003; 278: 47408-47415Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). Cell fate must be precisely regulated during development. The formation of physiologically and morphologically normal tissues requires exquisitely choreographed developmental processes that involve proliferation, migration, and apoptosis. Because of the diverse cellular responses elicited by S1P, it is not surprising that genes involved in S1P signaling and metabolism have proven essential for the development of complex organisms (34Kupperman E. An S. Osborne N. Waldron S. Stainier D.Y. Nature. 2000; 406: 192-195Crossref PubMed Scopus (347) Google Scholar, 35Lee O.H. Kim Y.M. Lee Y.M. Moon E.J. Lee D.J. Kim J.H. Kim K.W. Kwon Y.G. Biochem. Biophys. Res. Commun. 1999; 264: 743-750Crossref PubMed Scopus (335) Google Scholar, 36Liu Y. Wada R. Yamashita T. Mi Y. Deng C.X. Hobson J.P. Rosenfeldt H.M. Nava V.E. Chae S.S. Lee M.J. Liu C.H. Hla T. Spiegel S. Proia R.L. J. Clin. Investig. 2000; 106: 951-961Crossref PubMed Scopus (996) Google Scholar, 37Herr D.R. Fyrst H. Phan V. Heinecke K. Georges R. Harris G.L. Saba J.D. Development. 2003; 130: 2443-2453Crossref PubMed Scopus (104) Google Scholar, 38Mendel J. Heinecke K. Fyrst H. Saba J.D. J. Biol. Chem. 2003; 278: 22341-22349Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). We reported previously (37Herr D.R. Fyrst H. Phan V. Heinecke K. Georges R. Harris G.L. Saba J.D. Development. 2003; 130: 2443-2453Crossref PubMed Scopus (104) Google Scholar) that both accumulation and depletion of sphingolipid intermediates can be achieved predictably in vivo through mutation of genes encoding sphingolipid metabolic enzymes in Drosophila melanogaster. Disrupting sphingolipid synthesis through mutation of serine acyltransferase (lace) depletes endogenous long chain bases (LCBs) and phosphorylated long chain bases (LCBPs) and results in embryonic lethality. Hypomorphic alleles are associated with morphological abnormalities of the eye, wing, antennae, and bristles (39Adachi-Yamada T. Gotoh T. Sugimura I. Tateno M. Nishida Y. Onuki T. Date H. Mol. Cell. Biol. 1999; 19: 7276-7286Crossref PubMed Scopus (88) Google Scholar). LCB and LCBP accumulation caused by disrupting SPL (Sply) and the degradative pathway results in a different complement of phenotypes including cell-specific increases in embryonic and post-embryonic apoptosis, defects in adult muscle patterning, and reduced fecundity and viability. The array of phenotypes associated with each of these single mutations, although distinct, can be ameliorated by introduction of a second mutation disrupting the other gene, resulting in the normalization of phosphorylated and unphosphorylated long chain base levels. However, because these initial studies involved mutations that altered the content of more than one sphingolipid species, additional genetic models are required to determine the precise roles of individual sphingolipid metabolites in mediating these effects. In this regard, a recent study has shown that the overexpression of ceramidase, which would be predicted to reduce intracellular concentrations of ceramide, rescues the degeneration of photoreceptor cells in Drosophila arrestin mutants (40Acharya U. Patel S. Koundakjian E. Nagashima K. Han X. Acharya J.K. Science. 2003; 299: 1740-1743Crossref PubMed Scopus (120) Google Scholar). In the work presented here, we have identified two genes that encode distinct SK isoforms in Drosophila and have characterized a null mutation of one of these genes, Sk2, that results in impaired flight performance and diminished fecundity. Cloning of Sk1 and Sk2—The D. melanogaster genomic database (www.fruitfly.org/) was searched for nucleotide sequences encoding SK genes using the mouse Sphk1a sequence (GenBank™ accession number AF068748). DNA homology searches were performed via the Berkeley Drosophila Genome Project web site using the BLAST search program. Expressed sequence tags (ESTs) were identified that correspond to two Drosophila genes, Sk1 (CG1747) and Sk2 (CG32484/CG2159). This nomenclature was established previously (41Dobrosotskaya I.Y. Seegmiller A.C. Brown M.S. Goldstein J.L. Rawson R.B. Science. 2002; 296: 879-883Crossref PubMed Scopus (261) Google Scholar) and does not imply a homologous relationship between Drosophila Sk1 and human Sphk1. The Sk1 open reading frame was amplified by PCR from EST RE64552 using the primer pair CG1747-5′ (5′-ATGACGGCCAACACAGGGAC-3′) and CG1747-3′ (5′-CTACTGCCCACTGGTGGTCA-3′) and introduced into the cloning vector pCR2.1-TOPO (Invitrogen). The Sk1 open reading frame was subsequently recloned into yeast shuttle vector, pYES2 (Invitrogen), at HindIII and NotI restriction sites. The Sk2 open reading frame was amplified by PCR from EST LD11247 using the primer pair CG2159-5′ (5′-TCCAAAGCTTATGAGCGAATCTCTTGATA-3′) and CG2159-3′ (5′-TGCTGGATTCTCTTCAGTAAGCTCCTCCT-3′) and cloned directly into pYES2 at HindIII and BamHI sites. The resulting galactose-inducible constructs were introduced into Saccharomyces cerevisiae strain JSK392 (Δlcb4 Δysr2 Δdpl1) (42Kim S. Fyrst H. Saba J. Genetics. 2000; 156: 1519-1529Crossref PubMed Google Scholar) using the lithium acetate method (1Spiegel S. English D. Milstien S. Trends Cell Biol. 2002; 12: 236-242Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). Functional Expression and Complementation of S. cerevisiae Mutants—Yeast strain JSK392 contains null alleles of endogenous SPL (DPL1), major S1PP (YSR2), and major SK (LCB4) genes (42Kim S. Fyrst H. Saba J. Genetics. 2000; 156: 1519-1529Crossref PubMed Google Scholar). This strain is viable and healthy in the absence of SK activity. However, when a functional SK is expressed in this background, the cells cannot grow due to massive accumulation of LCBPs. To evaluate the ability of Sk1 and Sk2 to functionally complement a yeast SK mutant, strains JSK392[Sk1-pYES2] and JSK392[Sk2-pYES2] were inoculated onto uracil- agar plates containing 2% galactose and control uracil- agar plates containing 2% glucose. Complementation was demonstrated by lack of growth in the presence of galactose. LCBP Measurements in Yeast Overexpressing Sk1 and Sk2—Endogenous yeast LCBPs were evaluated in yeast strains JSK398 (pGAL-vector [LEU2] in JSK392), JSK392(Sk1-pYES2). and JSK392(Sk2-pYES2) (42Kim S. Fyrst H. Saba J. Genetics. 2000; 156: 1519-1529Crossref PubMed Google Scholar) under induction and repression conditions. Colonies were picked from plates and grown overnight at 30 °C with shaking in synthetic complete medium lacking either leucine (SC-LEU) or uracil (SC-URA) in the presence of 2% glucose. One aliquot was resuspended at A600 = 0.2 in rich medium containing 2% glucose (YPGlu) and incubated at 30 °C with shaking (repression conditions). A second aliquot was washed three times in sterile water followed by a wash in rich media containing 2% galactose (YPGal), resuspended at A600 = 0.2 in YPGal, and incubated at 30 °C with shaking (induction conditions). Cells were harvested at 12 h, and lipids were extracted essentially as described (42Kim S. Fyrst H. Saba J. Genetics. 2000; 156: 1519-1529Crossref PubMed Google Scholar). The dried lipid extracts were resuspended in 2 ml of chloroform/methanol, 1:1 (v/v), and 0.5 ml of 1.5 m ammonium hydroxide in water was added while vortexing. A clear separation of the two phases was obtained following centrifugation at 1500 × g for 5 min. The aqueous phase was recovered, dried down, and resuspended in 0.5 ml of methanol/water, 1:1 (v/v), containing 0.1% acetic acid. Additional purification of the LCBPs in the aqueous phase was performed on a Strata C18-E column, as described previously (37Herr D.R. Fyrst H. Phan V. Heinecke K. Georges R. Harris G.L. Saba J.D. Development. 2003; 130: 2443-2453Crossref PubMed Scopus (104) Google Scholar), and the purified LCBPs were labeled with ortho-phthalaldehyde and quantified by high performance liquid chromatography (HPLC) as described previously (42Kim S. Fyrst H. Saba J. Genetics. 2000; 156: 1519-1529Crossref PubMed Google Scholar). SK Activity in Yeast Overexpressing Sk1 and Sk2—Yeast were grown essentially as described above except that overnight aliquots were resuspended at A600 = 1.0 in either YPGlu or YPGal and incubated at 30 °C with shaking for 5 h. Cell extracts were prepared by vortexing with glass beads as described previously, and protein concentration was determined by using the Bradford method (Bio-Rad) (11Lanterman M.M. Saba J.D. Biochem. J. 1998; 332: 525-531Crossref PubMed Scopus (60) Google Scholar). The substrate for the SK activity assay was prepared by drying down a mixture of LCBs and C16-lysophosphatidylcholine, 1:9 (mol/mol). Substrate was resuspended in 100 mm MOPS, pH 7.2, containing 5 mm 2-mercaptoethanol by tip sonication for 20 s. Aliquots of 25 μl of substrate mixture containing 10 nmol of LCBs were added into each sample tube followed by the addition of 50 μl of 100 mm MOPS, pH 7.2, containing 5 mm 2-mercaptoethanol and 15 mm magnesium chloride, and cell extract with the equivalent of 50 μg of protein. Samples were pre-incubated for 5 min at 37 °C, and the SK reaction was started by adding 25 μl of 10 mm ATP. Samples were incubated for 30 min, at which time 1 nmol of an internal LCBP standard was added to each sample. The SK reaction was stopped by adding 0.5 ml of 1.5 m ammonium hydroxide in water and 2 ml of chloroform/methanol, 2:1 (v/v). The aqueous phase was recovered and dried down, and the LCBPs were labeled with ortho-phthalaldehyde and quantified by HPLC as described (42Kim S. Fyrst H. Saba J. Genetics. 2000; 156: 1519-1529Crossref PubMed Google Scholar). In Vitro Kinase Assay—Embryos aged 12–24 h were homogenized in lysate buffer and centrifuged at 100,000 × g for 60 min at 4 °C to separate membrane and cytosolic fractions. The membrane fraction (pellet) was resuspended in lysis buffer by sonication. SK activity was determined by the formation of 32P-labeled C18-S1P essentially as described (11Lanterman M.M. Saba J.D. Biochem. J. 1998; 332: 525-531Crossref PubMed Scopus (60) Google Scholar). Relative signal intensity was determined using ImageQuant software. In Situ Hybridization—Hybridizations were performed using standard conditions (43Tautz D. Pfeifle C. Chromosoma. 1989; 98: 81-85Crossref PubMed Scopus (2090) Google Scholar). Briefly, whole embryos of all stages were fixed, hybridized to a digoxygenin-labeled riboprobe (Roche Applied Science catalog number 1 175 025) overnight at 45 °C, washed vigorously over 24 h, bound to an alkaline phosphatase-conjugated antibody (Roche Applied Science catalog number 1 201 085), and developed in a nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate solution (Roche Applied Science catalog number 1 175 041). When indicated, embryos were also incubated in rabbit α-VASA (1:10,000) (44Lasko P.F. Ashburner M. Genes Dev. 1990; 4: 905-921Crossref PubMed Scopus (297) Google Scholar) for 4 h and then in fluorescein-conjugated goat α-rabbit IgG (1:100) (Jackson ImmunoResearch, catalog number 111-095-003). Northern Analysis—10 μg of total RNA was electrophoresed on a denaturing 1% agarose gel, transferred to a nitrocellulose membrane, and UV cross-linked. 32P-Labeled cDNA probes were heat-denatured and hybridized to the membrane for 2 h in ExpressHyb solution (Clontech catalog number 8015-2). Real Time Quantitative PCR—Total RNA was isolated from Drosophila at each indicated stage using an RNeasy Protect Mini Kit (Qiagen catalog number 74124). 5 μg of each sample were DNase-treated and primed with oligo(dT) prior to cDNA synthesis with Moloney murine leukemia virus reverse transcriptase. Targets were amplified with iQ sybr green supermix (Bio-Rad catalog number 170-8880) on a Bio-Rad iCycler with the following primer pairs: RpL32-RT-L (5′-ATGCTAAGCTGTCGCACAAA-3′) and RpL32-RT-R (5′-GTTCGATCCGTAACCGATGT-3′); Sk1-RT-L (5′-TCACCACACATCCGCAGTAT-3′) and Sk1-RT-R (5′-AATCCATGCGTTCCATTAGC-3′); and Sk2-RT-L (5′-GACGGCACCATCTACCTGAT-3′) and Sk2-RT-R (5′-ATGATGCCGTGATTGTCGTA-3′). Relative gene expression was determined using the 2-ΔΔCT method (45Livak K.J. Schmittgen T.D. Methods. 2001; 25: 402-408Crossref PubMed Scopus (127155) Google Scholar). Drosophila Husbandry and Genetics—Flies were reared on standard yeast/sucrose media at 22 °C. Sk2KG05894 was a gift of the P-element Screen/Gene Disruption Project of the Bellen/Rubin/Spradling laboratories. Wild-type Canton-S (BL-1) was obtained from the Bloomington Drosophila Stock Center (Indiana University, Bloomington, IN). Sk253A was generated by introducing a stable source of transposase to Sk2KG05894 for a single generation and screening progeny for loss of the dominant marker (y+) carried on the P-element (SUPor-P). Precise excision was verified by PCR amplification and sequencing of the insertion site. Flight Analysis—2–7-Day-old flies were released in a top-lit, Plexiglas chamber and scored for flight performance as follows: upward flight = 3, horizontal flight = 2, downward flight = 1, no flight = 0. Mean flight performance was determined ± S.E. (n = 168–365). Quantitation of Endogenous Drosophila C14 and C16 Long Chain Bases (LCBs) in Sk2 Mutant and Canton-S Flies throughout Development—Endogenous Drosophila LCBs were extracted and analyzed as described (46Fyrst H. Herr D.R. Harris G.L. Saba J.D. J. Lipid Res. 2003; 45: 54-62Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Identification of Two Putative D. melanogaster Sphingosine Kinases—The peptide sequence of human Sphk1 (GenBank™ accession number NP_068807) was used in TBLASTN searches against all known Drosophila sequences. Two loci with substantial sequence similarity were identified (Fig. 2A). Both were annotated as predicted genes (47Misra S. Crosby M.A. Mungall C.J. Matthews B.B. Campbell K.S. Hradecky P. Huang Y. Kaminker J.S. Millburn G.H. Prochnik S.E. Smith C.D. Tupy J.L. Whitfied E.J. Bayraktaroglu L. Berman B.P. Bettencourt B.R. Celniker S.E. de Grey A.D. Drysdale R.A. Harris N.L. Richter J. Russo S. Schroeder A.J. Shu S.Q. Stapleton M. Yamada C. Ashburner M. Gelbart W.M. Rubin G.M. Lewis S.E. Genome Biol. 2002; 3 (research0079.1-0079.14)Crossref Google Scholar) and correspond to full-length cDNA clones (48Stapleton M. Liao G. Brokstein P. Hong L. Carninci P. Shiraki T. Hayashizaki Y. Champe M. Pacleb J. Wan K. Yu C. Carlson J. George R. Celniker S. Rubin G.M. Genome Res. 2002; 12: 1294-1300Crossref PubMed Scopus (120) Google Scholar). The first, Sk1 (formerly CG1747, GenBank™ accession number AAF48045), is located on the X chromosome at region 10B13 (Fig. 2B). There are two predicted splice variants (2614 and 2472 bp, respectively) that result in alternative 5′-untranslated sequences. The predicted protein for both transcripts contains 641 amino acids and has a mass of 71 kDa. There are no reported mutant alleles of this gene. The second locus, Sk2 (formerly CG32484, GenBank™ accession number AAF47706), is located on the third chromosome at region 63A3-5 (Fig. 2C) and contains a single intron. The predicted protein product contains 661 amino acids and has a calculated molecular mass of 74 kDa. This gene has one recorded mutant allele, Sk2KG05894, which harbors a P-element insertion in the 5′-untranslated region (49Roseman R.R. Johnson E.A. Rodesch C.K. Bjerke M. Nagoshi R.N. Geyer P.K. Genetics. 1995; 141: 1061-1074Crossref PubMed Google Scholar). SK1 and SK2 proteins are 39% identical and 63% similar to one another. Both proteins contain five domains conserved within the diacylglycerol kinase putative catalytic domain family. Both SK1 and SK2 are more similar to mammalian Sphk2 than to Sphk1, with Drosophila SK1 demonstrating 33% identity and 47% similarity to murine Sphk2 versus 27% identity and 42% similarity to murine Sphk1, and Drosophila SK2 demonstrating 32% identity and 45% similarity to murine Sphk2 versus 27% identity and 39% similarity to murine Sphk1. Similarities among SK1, SK2, and the mammalian Sphk2 proteins are distributed throughout the length of the proteins and include a domain near the N terminus of ∼100 amino acids shared by these three proteins but which is not present in murine or human Sphk1. A conserved SGDGX17–21K sphingosine kinase motif has been proposed in which the second conserved glycine is critical for ATP binding, and the conserved downstream lysine facilitates nucleotide orientation within the binding pocket (21Pitson S.M. Moretti P.A. Zebol J.R. Zareie R. Derian C.K. Darrow A.L. Qi J. D'Andrea R.J. Bagley C.J. Vadas M.A. Wattenberg B.W. J. Biol. Chem. 2002; 277: 49545-49553Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). This motif is present in murine and human Sphk1 proteins and human Sphk2, whereas in murine Sphk2 the lysine has been replaced by an arginine. Similarly, in Drosophila SK1, the SGDG is conserved, and an arginine replaces the lysine. However, in SK2 the motif is altered to GGDG, and an arginine is present in the 12th position, reminiscent of phosphatidylinositol 4-phosphate kinases. Murine Sphk2 was also reported recently (32Liu H. Toman R.E. Goparaju S. Maceyka M. Nava V.E. Sankala H. Payne S.G. Bektas M. Ishii I. Chun J. Milstien S. Spiegel S. J. Biol. Chem. 2003; 278: 40330-40336Abstract Full Text Full Text PDF PubMed Scopus (308) Google" @default.
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