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• W2051917023 abstract "Sphingosine kinases catalyze the production of the bioactive lipid molecule sphingosine 1-phosphate. Mice have two isoforms of sphingosine kinase type 1, SPHK1a and SPHK1b. In addition to the previously reported difference in their enzyme activities, we have found that these isoforms differ in several enzymatic characteristics. First, SPHK1b is unstable, whereas SPHK1a is highly stable. Degradation of SPHK1b occurs at the membrane and is inhibited by a proteasome inhibitor. Second, only SPHK1b exhibits abnormal mobility on SDS-PAGE, probably due to its SDS-resistant structure. Third, SPHK1a and SPHK1b are predominantly detected in the soluble and membrane fractions, respectively, when their degradation is inhibited. Fourth, only SPHK1b is modified with lipid, on its unique Cys residues (Cys-4 and Cys-5). Site-directed mutagenesis at these Cys residues resulted in increased sphingosine kinase activity, suggesting that the modification is inhibitory to the enzyme. Finally, SPHK1b tends to form homo-oligomers, whereas most SPHK1a is presented as monomers. We have also determined that the lipid modification of SPHK1b is involved in its homo-oligomerization. Thus, although these two proteins differ only in a few N-terminal amino acid residues, their enzymatic traits are extremely different. Sphingosine kinases catalyze the production of the bioactive lipid molecule sphingosine 1-phosphate. Mice have two isoforms of sphingosine kinase type 1, SPHK1a and SPHK1b. In addition to the previously reported difference in their enzyme activities, we have found that these isoforms differ in several enzymatic characteristics. First, SPHK1b is unstable, whereas SPHK1a is highly stable. Degradation of SPHK1b occurs at the membrane and is inhibited by a proteasome inhibitor. Second, only SPHK1b exhibits abnormal mobility on SDS-PAGE, probably due to its SDS-resistant structure. Third, SPHK1a and SPHK1b are predominantly detected in the soluble and membrane fractions, respectively, when their degradation is inhibited. Fourth, only SPHK1b is modified with lipid, on its unique Cys residues (Cys-4 and Cys-5). Site-directed mutagenesis at these Cys residues resulted in increased sphingosine kinase activity, suggesting that the modification is inhibitory to the enzyme. Finally, SPHK1b tends to form homo-oligomers, whereas most SPHK1a is presented as monomers. We have also determined that the lipid modification of SPHK1b is involved in its homo-oligomerization. Thus, although these two proteins differ only in a few N-terminal amino acid residues, their enzymatic traits are extremely different. The bioactive lipid molecule sphingosine 1-phosphate (S1P) 2The abbreviations used are: S1P, sphingosine 1-phosphate; HEK, human embryonic kidney; 3×FLAG, triple FLAG; PBS, phosphate-buffered saline. regulates several cellular processes such as cell proliferation, cell migration, and differentiation through binding to its cell surface receptors, which are S1P/Edg family members (1.Pyne S. Pyne N.J. Biochem. J. 2000; 349: 385-402Crossref PubMed Scopus (662) Google Scholar, 2.Spiegel S. Milstien S. Nat. Rev. Mol. Cell Biol. 2003; 4: 397-407Crossref PubMed Scopus (1768) Google Scholar, 3.Taha T.A. Argraves K.M. Obeid L.M. Biochim. Biophys. Acta. 2004; 1682: 48-55Crossref PubMed Scopus (165) Google Scholar). In addition to its extracellular action, S1P is presumed to act intracellularly in Ca2+ mobilization, cell proliferation, and apoptosis inhibition (1.Pyne S. Pyne N.J. Biochem. J. 2000; 349: 385-402Crossref PubMed Scopus (662) Google Scholar, 2.Spiegel S. Milstien S. Nat. Rev. Mol. Cell Biol. 2003; 4: 397-407Crossref PubMed Scopus (1768) Google Scholar). S1P is abundant in blood (4.Yatomi Y. Igarashi Y. Yang L. Hisano N. Qi R. Asazuma N. Satoh K. Ozaki Y. Kume S. J. Biochem. (Tokyo). 1997; 121: 969-973Crossref PubMed Scopus (412) Google Scholar) and is physiologically important, especially in the vascular and immune systems (5.Hla T. Semin. Cell Dev. Biol. 2004; 15: 513-520Crossref PubMed Scopus (350) Google Scholar). Its importance in the vascular system is evident in S1P1/Edg1-null mice, which die in utero with severe hemorrhage resulting from impaired vessel integrity due to a deficiency in smooth muscle cell recruitment (6.Liu 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). Its influence in the immune system has been demonstrated using the synthetic immunosuppressant FTY720, which is phosphorylated in vivo and binds to S1P receptors (7.Brinkmann 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 (1320) Google Scholar, 8.Mandala 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 (1443) Google Scholar). S1P and the S1P1 receptor also have important functions in the egress of lymphocytes from lymphoid organs, and phosphorylated FTY720 induces the down-regulation of S1P1 on lymphocytes and inhibits their recirculation (9.Allende M.L. Dreier J.L. Mandala S. Proia R.L. J. Biol. Chem. 2004; 279: 15396-15401Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 10.Matloubian M. Lo C.G. Cinamon G. Lesneski M.J. Xu Y. Brinkmann V. Allende M.L. Proia R.L. Cyster J.G. Nature. 2004; 427: 355-360Crossref PubMed Scopus (2091) Google Scholar). Sphingosine kinases catalyze the production of S1P from sphingosine and are also responsible for the phosphorylation of FTY720 (11.Billich 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, 12.Kharel Y. Lee S. Snyder A.H. Sheasley-O'neill S L. Morris M.A. Setiady Y. Zhu R. Zigler M.A. Burcin T.L. Ley K. Tung K.S. Engelhard V.H. Macdonald T.L. Pearson-White S. Lynch K.R. J. Biol. Chem. 2005; 280: 36865-36872Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Although sphingosine kinases possess no apparent enzymatic motif or transmembrane domain, there are five regions, termed C1 to C5, that are conserved among sphingosine kinases (13.Kohama 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). The C1 to C3 regions bind to Mg2+-ATP (14.Pitson 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), whereas the C4 is involved in sphingosine binding (15.Yokota S. Taniguchi Y. Kihara A. Mitsutake S. Igarashi Y. FEBS Lett. 2004; 578: 106-110Crossref PubMed Scopus (40) Google Scholar). Two mammalian sphingosine kinases are known, SPHK1, which was identified by purification of the enzyme and subsequent sequence determination (13.Kohama 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), and SPHK2, which was cloned based on its homology to SPHK1 (16.Liu 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). Both sphingosine kinases are expressed ubiquitously among tissues, although their tissue-specific patterns differ (16.Liu 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). The enzymes may share redundant functions, since the single knock out of either kinase confers no apparent phenotype (12.Kharel Y. Lee S. Snyder A.H. Sheasley-O'neill S L. Morris M.A. Setiady Y. Zhu R. Zigler M.A. Burcin T.L. Ley K. Tung K.S. Engelhard V.H. Macdonald T.L. Pearson-White S. Lynch K.R. J. Biol. Chem. 2005; 280: 36865-36872Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 17.Allende M.L. Sasaki T. Kawai H. Olivera A. Mi Y. van Echten-Deckert G. Hajdu R. Rosenbach M. Keohane C.A. Mandala S. Spiegel S. Proia R.L. J. Biol. Chem. 2004; 279: 52487-52492Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar). Intracellular S1P levels are regulated by a balance between synthesis by sphingosine kinase and degradation by either phosphohydrolase or lyase. Sphingosine kinases are activated by stimuli such as treatment with platelet-derived growth factor (18.Olivera A. Spiegel S. Nature. 1993; 365: 557-560Crossref PubMed Scopus (818) Google Scholar), tumor necrosis factor α (19.Xia P. Gamble J.R. Rye K.A. Wang L. Hii C.S. Cockerill P. Khew-Goodall Y. Bert A.G. Barter P.J. Vadas M.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 14196-14201Crossref PubMed Scopus (358) Google Scholar), or phorbol ester (20.Mazurek N. Megidish T. Hakomori S. Igarashi Y. Biochem. Biophys. Res. Commun. 1994; 198: 1-9Crossref PubMed Scopus (89) Google Scholar) as well as the cross-linking of FcγR1 (21.Melendez 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) or FcϵR1 (22.Choi O.H. Kim J.-H. Kinet J.P. Nature. 1996; 380: 634-636Crossref PubMed Scopus (385) Google Scholar). Additionally, SPHK1 is known to be regulated by protein-protein interactions (23.Xia 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, 24.Lacana E. Maceyka M. Milstien S. Spiegel S. J. Biol. Chem. 2002; 277: 32947-32953Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 25.Hayashi S. Okada T. Igarashi N. Fujita T. Jahangeer S. Nakamura S. J. Biol. Chem. 2002; 277: 33319-33324Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 26.Fukuda Y. Aoyama Y. Wada A. Igarashi Y. Biochim. Biophys. Acta. 2004; 1636: 12-21Crossref PubMed Scopus (41) Google Scholar, 27.Maceyka M. Nava V.E. Milstien S. Spiegel S. FEBS Lett. 2004; 568: 30-34Crossref PubMed Scopus (61) Google Scholar), phosphorylation (28.Johnson 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, 29.Pitson S.M. Moretti P.A. Zebol J.R. Lynn H.E. Xia P. Vadas M.A. Wattenberg B.W. EMBO J. 2003; 22: 5491-5500Crossref PubMed Scopus (463) Google Scholar), and translocation to the plasma membrane (28.Johnson 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, 29.Pitson S.M. Moretti P.A. Zebol J.R. Lynn H.E. Xia P. Vadas M.A. Wattenberg B.W. EMBO J. 2003; 22: 5491-5500Crossref PubMed Scopus (463) Google Scholar). Although such regulation is involved in the stimuli-dependent activation of SPHK1, the precise molecular mechanisms that link the stimuli and the activation still remain largely unknown in most cases. Two isoforms for mouse SPHK1 have been reported, SPHK1a and SPHK1b (13.Kohama 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). Interestingly, the activity of SPHK1b is significantly lower (30-200-fold) than that of SPHK1a (13.Kohama 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), although these proteins differ only in a few amino acid residues in their N termini. In the present study we have demonstrated that SPHK1b does not accumulate within the cell due to its low stability. We also found that SPHK1b differs from SPHK1a in several enzymatic characteristics. These include structural resistance toward SDS, membrane localization, protein stability, post-translational modification, and oligomer formation. Thus, the N terminus plays an important role in the determination of the enzymatic properties of SPHK1. Cell Culture and Transfection—Mouse F9 embryonal carcinoma cells and human embryonic kidney (HEK) 293T cells were grown in Dulbecco's modified Eagle's medium (D6429; Sigma) containing 10% fetal calf serum and supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin in 0.1% gelatin- and 0.3% collagen-coated dishes, respectively. Transfections were performed using Lipofectamine™ 2000 reagent (Invitrogen) for F9 cells and Lipofectamine Plus™ reagent (Invitrogen) for HEK 293T cells. Plasmids—The pCE-puro SPHK1a, pCE-puro SPHK1a2, and pCE-puro SPHK1b plasmids are derivatives of the pCE-puro vector (30.Kihara A. Ikeda M. Kariya Y. Lee E.Y. Lee Y.M. Igarashi Y. J. Biol. Chem. 2003; 278: 14578-14585Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) and encode SPHK1a, SPHK1a2, and SPHK1b, respectively. The pCE-puro SPHK1a-3×FLAG, pCE-puro SPHK1b-3×FLAG, pCE-puro SPHK1a-Myc, and pCE-puro SPHK1b-Myc plasmids encode SPHK1 proteins tagged with triple FLAG (3×FLAG) or Myc epitopes at the C termini. The pCE-puro 3×FLAG-SPHK1a-Myc and pCE-puro 3×FLAG-SPHK1b-Myc plasmids encode SPHK1 proteins tagged with both 3×FLAG epitopes at their N termini and Myc epitopes at their C termini. The SPHK1a, SPHK1a2, and SPHK1b cDNAs were amplified by PCR using the pCE-puro HA-SPHK1a plasmid (30.Kihara A. Ikeda M. Kariya Y. Lee E.Y. Lee Y.M. Igarashi Y. J. Biol. Chem. 2003; 278: 14578-14585Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) as a template. The primers used included a common primer, 5′-GGTTTGTCCAAACTCATCAA-3′, a SPHK1a-specific primer, 5′-TGCCACCATGGAACCAGAATGCCCTCG-3′, a SPHK1a2-specific primer, 5′-GCCACCATGGAACCAGTAGAATGCCCTCGAGGACTGCTCCC-3′, and a SPHK1b-specific primer, 5′-CGATTAGCTTGCCACCATGTGGTGGTGTTGTGTTTTGTTTGTAGTAGAATGCCCTCGAGGACTG-3′. Each amplified fragment was first cloned into the pGEM-T Easy vector (Promega, Madison, WI), and the NotI-NotI fragment of the resulting plasmid was then cloned into the NotI site of the pCE-puro vector, generating the pCE-puro SPHK1a, pCE-puro SPHK1a2, or pCE-puro SPHK1b plasmid. The pCE-puro SPHK1b-C1, pCE-puro SPHK1b-C2, and pCE-puro SPHK1b-C1C2 plasmids were constructed by site-directed mutagenesis using a QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) from the pCE-puro SPHK1b plasmid. The primers used are as follows: for pCE-puro SPHK1b-C1, 5′-GCTTGCCACCATGTGGTGGAGTTGTGTTTTGTTTGTAGTAG-3′ and 5′-CTACTACAAACAAAACACAACTCCACCACATGGTGGCAAGC-3′; for pCE-puro SPHK1b-C2, 5′-GCTTGCCACCATGTGGTGGTGTAGTGTTTTGTTTGTAGTAG-3′ and 5′-CTACTACAAACAAAACACTACACCACCACATGGTGGCAAGC-3′; for pCE-puro SPHK1b-C1C2, 5′-GCTTGCCACCATGTGGTGGAGTAGTGTTTTGTTTGTAGTAG-3′ and 5′-CTACTACAAACAAAACACTACTCCACCACATGGTGGCAAGC-3′. The pCE-puro SPHK1b-C1C2-3×FLAG plasmid was constructed by cloning a 0.48-kilobase EcoRI-SgrAI fragment of pCE-puro SPHK1b-C1C2 into the EcoRI-SgrAI site of pCE-puro SPHK1b-3×FLAG. The pCE-puro SPHK1b-C1C2-Myc plasmid was similarly constructed using pCE-puro SPHK1b-C1C2 and pCE-puro SPHK1b-Myc. The pCE-puro 3×FLAG-Ub plasmid, which encodes ubiquitin tagged with a 3×FLAG epitope at the N terminus, was constructed as follows. The ubiquitin cDNA was amplified from 17-day mouse embryo cDNA (BD Biosciences Clontech, Palo Alto, CA) using primers 5′-AGGATCCATGCAGATCTTTGTGAAGACCCTG-3′ and 5′-TTTAGCCACCTCTGAGGCGAAGGACCAGG-3′. The amplified fragment was cloned into the pGEM-T Easy vector, creating the pGEM Ub plasmid. The pCE-puro 3×FLAG-Ub plasmid was constructed by cloning the 0.26-kilobase BamHI-NotI fragment of the pGEM Ub plasmid into the BamHI-NotI site of the pCE-puro 3×FLAG-1 vector, which had been designed to produce an N-terminal 3×FLAG tagged protein. Real-time Quantitative PCR—First-strand cDNAs from various mouse tissues and embryonic development stages were purchased from BD Biosciences Clontech (Mouse Multiple Tissue cDNA (MTC) panel I). Real-time quantitative PCR was performed using the first-strand cDNAs, Platinum Quantitative PCR SuperMix UDG (Invitrogen), and an ABI PRISM™ 7700 sequence detector (Applied Biosystems, Foster City, CA). 6-Carboxyfluorescein (FAM)-labeled LUX™ primers (5′-CGAGGTATGGAACCAGAATGCCCT(FAM)G-3′ for SPHK1a and 5′-CTACAAATGGTGGTGTTGTGTTTTGTTTG(FAM)AG-3′ for SPHK1b) and the common unlabeled primer (5′-GCCTTGCCCTTGCCACCCTGGGG-3′) were used as primers. The expression level of glyceraldehyde-3-phosphate dehydrogenase was examined using Certified LUX™ Primer Set (100 M-01; Invitrogen). Immunoblotting—Immunoblotting was performed as described previously (30.Kihara A. Ikeda M. Kariya Y. Lee E.Y. Lee Y.M. Igarashi Y. J. Biol. Chem. 2003; 278: 14578-14585Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Anti-SPHK1 (1/1000 dilution) (31.Fukuda Y. Kihara A. Igarashi Y. Biochem. Biophys. Res. Commun. 2003; 309: 155-160Crossref PubMed Scopus (107) Google Scholar), anti-FLAG M2 (1 μg/ml; Stratagene), anti-Myc PL14 (1 μg/ml; Medical & Biological Laboratories, Nagoya, Japan), anti-glyceraldehyde-3-phosphate dehydrogenase (1 μg/ml; Ambion, Austin, TX), and anti-calnexin (H-10) antibodies (0.2 μg/ml; Santa Cruz Biotechnology, Santa Cruz, CA) were used as primary antibodies. Horseradish peroxidase-conjugated anti-mouse or anti-rabbit IgG F(ab′)2 fragment (both from Amersham Biosciences and diluted 1:7500) were used as secondary antibodies. When immunoprecipitated proteins were subjected to immunoblotting with the same rabbit antibodies used for the immunoprecipitation, horseradish peroxidase-conjugated rabbit IgG TrueBlot™ (eBioscience, San Diego, CA), which does not react with SDS-denatured rabbit IgGs, was used as the secondary antibody. Labeling was detected using ECL™ Reagents or an ECL Plus system for Western blotting detection (both from Amersham Biosciences). Immunofluorescence Microscopy —Microscopic immunofluorescence analysis was performed as described previously (32.Ogawa C. Kihara A. Gokoh M. Igarashi Y. J. Biol. Chem. 2003; 278: 1268-1272Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar) with affinity-purified anti-SPHK1 antibodies (1:100) and Alexa Fluor 488 goat anti-rabbit IgG (H+L)-conjugated antibody (5 μg/ml, Molecular Probes, Eugene, OR). The stained cells were analyzed by fluorescence microscopy (Axioskop 2 PLUS; Carl Zeiss, Oberkochen, Germany). Pulse-Chase and Immunoprecipitation —HEK 293T cells were transfected with the pCE-puro SPHK1a, pCE-puro SPHK1b, or pCE-puro SPHK1b-C1C2 plasmid. Eighteen h after transfection, the cell layer was disrupted by a trypsin/EDTA solution, and the cell suspension was divided into 4 or 5 aliquots. Each aliquot was added to a new 30-mm culture dish and incubated at 37 °C for 24 h. Culture medium was then changed to 1 ml of Dulbecco's modified Eagle's medium without Met/Cys (D0422, Sigma) and incubated at 37 °C for 1 h. Cells were pulse-labeled with [35S]Met/[35S]Cys (22 μCi/dish EXPRESS™ protein labeling mix; PerkinElmer Life Sciences) for 20 min and chased with unlabeled Met (final concentration 0.5 mg/ml) and Cys (final concentration 0.1 mg/ml) in 1 ml of Dulbecco's modified Eagle's medium (D6429) containing 10% fetal calf serum. At predetermined times cells were washed with phosphate-buffered saline (PBS), treated with 1 ml of radioimmune precipitation assay buffer (50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 1 mm EDTA, 1% Triton X-100, 0.1% SDS, 0.1% sodium deoxycholate, 1 mm dithiothreitol, 1 mm phenylmethylsulfonyl fluoride, and 1× protease inhibitor mixture (Complete™ EDTA free; Roche Diagnostics)), and kept on ice. Cells were disrupted by 5 passages through a 21-gauge needle, and debris was removed by centrifugation at 20,000 × g for 5 min at 4 °C. Cell lysates with equal radioactivity were incubated with affinity-purified anti-SPHK1 antibodies and protein A-Sepharose (Amersham Biosciences) at 4 °C for 14 h. After 2 washes with 1 ml of radioimmune precipitation assay buffer and 1 with 1 ml of 10 mm Tris-HCl (pH 8.0), beads were suspended in 2× SDS sample buffer (125 mm Tris-HCl (pH 6.8), 4% SDS, 20% glycerol, and a trace amount of bromphenol blue) containing 10% 2-mercaptoethanol and boiled for 3 min. The precipitates were then separated by SDS-PAGE. Radioactivities associated with SPHK1 were quantified using a Bio-Imaging Analyzer BAS2500 (Fuji Photo Film, Tokyo, Japan). For detection of SPHK1-ubiquitin conjugations, HEK 293T cells were transfected with pCE-puro 3×FLAG-Ub and either pCE-puro SPHK1a or pCE-puro SPHK1b, then incubated for 40 h at 37 °C. Preparation of cell lysates and immunoprecipitation with anti-SPHK1 antibodies were performed as described above. Precipitates were separated by SDS-PAGE and subjected to immunoblotting using an anti-FLAG M2 antibody. To detect homo-oligomer formation of SPHK1, HEK 293T cells were transfected with two plasmids, one carrying 3×FLAG-tagged SPHK1 genes and the other harboring Myc-tagged SPHK1 genes. Twenty-two hours after transfection, 20 μm MG132 was added to inhibit the proteasome-dependent SPHK1 degradation, and the cells were incubated for 4 h at 37 °C. The cells were washed twice with PBS, suspended in buffer A (PBS, 1 mm dithiothreitol, 1× protease inhibitor mixture, and 1 mm phenylmethylsulfonyl fluoride), and sonicated. After a centrifugation at 300 × g for 3 min at 4 °C, the resulting supernatant was treated with 1% Triton X-100 for 30 min at 4 °C. Samples were centrifuged at 100,000 × g for 30 min at 4 °C, and the supernatant was incubated with an M2 affinity gel (Sigma) for 2 h at 4°C. The gel was washed 3 times with PBS containing 0.1% Triton X-100, suspended in 2× SDS sample buffer, and boiled. The obtained precipitates were treated with 10% 2-mercaptoethanol, boiled for 3 min, separated by SDS-PAGE, and subjected to immunoblotting with an anti-FLAG M2 antibody or an anti-Myc PL14 antibody. In Vivo [3H]Palmitic Acid Labeling— HEK 293T cells grown on a 30-mm dish were transfected with plasmids and incubated for 23 h at 37 °C. Culture medium was then changed to 1.5 ml of serum-free Dulbecco's modified Eagle's medium. After a 30-min incubation at 37 °C, 3 μg/ml cerulenin, which inhibits fatty acid synthesis, and 20 μm MG132 were added to the medium, and the cells were incubated for 30 min at 37 °C. Cells were labeled with 0.2 mCi of [3H]palmitic acid (60 Ci/mmol; American Radiolabeled Chemical, St. Louis, MO) at 37 °C for 3 h. After washing with PBS, cells were treated with 1 ml radioimmune precipitation assay buffer. SPHK1 was immunoprecipitated using anti-SPHK1 antibodies and protein A-Sepharose as described above. Immunoprecipitates suspended in 2× SDS sample buffer containing 10 mm 2-mercaptoethanol were boiled for 3 min, separated by SDS-PAGE, and visualized by autoradiography using the fluorographic reagent EN3HANCE™ (PerkinElmer Life Sciences). Preparation of Soluble and Membrane Fractions—HEK 293T cells transfected with plasmids were washed twice with PBS, suspended in buffer A, and sonicated. After removal of cell debris by centrifugation at 300 × g for 3 min at 4 °C, cell lysates were centrifuged at 100,000 × g for 1 h at 4 °C. The resulting supernatant and pellet were used as soluble and membrane fractions, respectively. Sphingosine Kinase Assay —Sphingosine kinase assays were performed as described elsewhere (33.Olivera A. Kohama T. Tu Z. Milstien S. Spiegel S. J. Biol. Chem. 1998; 273: 12576-12583Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar). Tissue-specific Expression of SPHK1 Isoforms—There are variations in mouse SPHK1 mRNAs due to the differences in transcriptional initiation sites, and as a result, two different polypeptides, SPHK1a and SPHK1b, are produced (34.Imamura T. Miyauchi-Senda N. Tanaka S. Shiota K. J. Vet. Med. Sci. 2004; 66: 1387-1393Crossref PubMed Scopus (18) Google Scholar). At least four types of mRNAs, differing in the first exons, encode the same SPHK1a protein, whereas only one mRNA is known for SPHK1b (34.Imamura T. Miyauchi-Senda N. Tanaka S. Shiota K. J. Vet. Med. Sci. 2004; 66: 1387-1393Crossref PubMed Scopus (18) Google Scholar). Translation of the SPHK1a protein is initiated near the end of the common second exon (Fig. 1). By searching EST clone databases, we found that certain SPHK1a-encoding mRNAs contain a 3-bp insertion in the coding region. This newly identified variation, named here SPHK1a2, is produced by an altered junction between the second and third exons. Consequently, the SPHK1a2 protein contains an extra Val residue. In contrast, transcription of SPHK1b mRNA is initiated within the intron region between the second and third exons of SPHK1a/SPHK1a2, so the 3′-half of the first exon of the SPHK1b gene is common to the third exon of SPHK1a/SPHK1a2 (34.Imamura T. Miyauchi-Senda N. Tanaka S. Shiota K. J. Vet. Med. Sci. 2004; 66: 1387-1393Crossref PubMed Scopus (18) Google Scholar). Translation of the SPHK1b protein is initiated from 28 bp upstream of the 5′-terminal end of the third exon of SPHK1a2 (Fig. 1). Thus, there are three types of SPHK1 proteins, which differ only in their N termini. The N-terminal sequences specific to each are: SPHK1a, Met-Glu-Pro; SPHK1a2, Met-Glu-Pro-Val; SPHK1b, Met-Trp-Trp-Cys-Cys-Val-Leu-Phe-Val-Val. Of the 23 EST clones registered, we found that 8 represented SPHK1a, 12 represented SPHK1a2, and 3 represented SPHK1b, suggesting that SPHK1a2 may be the most abundant isoform, and SPHK1b may be only a minor isoform. We examined tissue- and embryonic development-specific expression patterns of SPHK1a/a2 and SPHK1b, although we were unable to prepare a specific primer that would distinguish SPHK1a mRNA from SPHK1a2 mRNA. Real-time quantitative PCR analysis revealed that SPHK1a/a2 mRNA is ubiquitously expressed, although levels vary among tissues and embryonic stages (Table 1). However, very little expression of SPHK1a/a2 mRNA was detectable in skeletal muscle, consistent with the extremely low sphingosine kinase activity in this tissue (31.Fukuda Y. Kihara A. Igarashi Y. Biochem. Biophys. Res. Commun. 2003; 309: 155-160Crossref PubMed Scopus (107) Google Scholar). The expression of SPHK1b mRNA was more restrictive than that of SPHK1a (Table 1). Little SPHK1b mRNA expression was detected in heart, brain, liver, and skeletal muscle. The expression levels of SPHK1b mRNA were observed to be lower than those of SPHK1a/a2 mRNA regardless of the tissue, which is consistent with the small number of the EST clones carrying SPHK1b. The amount of SPHK1b mRNA was 10-20% that observed for SPHK1a/a2 mRNA in spleen, lung, kidney, and testis. In embryonic stages, days 11-15, the discrepancy was somewhat less, with SPHK1b mRNA levels at 67 and 37% of SPHK1a/a2 mRNA levels. Thus, it would appear that the expression of SPHK1a/a2 mRNA and that of SPHK1b mRNA are regulated differently among tissues and embryonic stages.TABLE 1Expression levels of SPHK1a/a2 and SPHK1b in various tissues and during embryonic development The mRNA expression of SPHK1a/a2 and SPHK1b was investigated in the indicated tissues and embryonic stages by real-time quantitative PCR as detailed under “Materials and Methods.” Values provided are relative to the GAPDH mRNA levels in the respective tissues or embryonic stages and represent the mean ± S.D. from three independent experiments.Tissue or embryonic stageRelative SPHK1 levelSPHK1a/a2SPHK1b×10–4Heart2.9 ± 0.400.96 ± 0.93Brain6.4 ± 1.51.1 ± 0.38Spleen190 ± 8.121 ± 3.2Lung240 ± 8248 ± 4.4Liver2.4 ± 0.500.52 ± 0.45Skeletal muscle0.48 ± 0.0300.062 ± 0.053Kidney19 ± 3.04.3 ± 0.56Testis51 ± 136.5 ± 6.57 Day430 ± 2462 ± 2.411 Day11 ± 1.97.4 ± 2.515 Day15 ± 1.75.5 ± 2.317 Day26 ± 1.43.7 ± 1.0 Open table in a new tab Abnormal Mobility of SPHK1b on SDS-PAGE—SPHK1b reportedly has a much lower activity than SPHK1a (13.Kohama 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), which we also observed (data not shown), yet the reason was unclear. To study this phenomenon, we cloned each of the three isoforms and expressed them in mouse F9 cells. In immunoblots using anti-SPHK1 antibodies, SPHK1a and SPHK1a2 were detected at ∼43 kDa (Fig. 2A), in accordance with their predicted molecular masses (both 42.4 kDa). We found no differences in enzymatic characteristics (activity, gel mobility, membrane localization, stability, and oligomerization) between SPHK1a and SPHK1a2 (Fig. 2A and data not shown), so we described hereafter only SPHK1a. In contrast, the mobility of SPHK1b deviated from its predicted molecular mass (43.3 kDa) and was instead detected at 34 kDa (Fig. 2A). This mobility was not cell-specific, since it was also observed for HEK 293T cells (Figs. 3, 4, 5, 6). An additional band at 68 kDa, which may represent a SDS-resistant dimer (see Fig. 6), was also detected, although its level varied with the experimental conditions. A similar upper band was often observed in SPHK1a blots (Figs. 3B, 4A, and 6A); however, the intensity of the band was always lower than that observed for SPHK1b blots.FIGURE 4Ubiquitination of SPHK1. HEK 293T cells were transfected with a plasmid encoding 3×FLAG-ubiquitin together with one encoding no protein, SPHK1a, or SPHK1b. A, total cell lysates were prepared, separated by SDS-PAGE, and subjected to immunoblotting with anti-SPHK1 antibodies. B, SPHK1 and any associated proteins were immun" @default.
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• W2051917023 title "Mouse Sphingosine Kinase Isoforms SPHK1a and SPHK1b Differ in Enzymatic Traits Including Stability, Localization, Modification, and Oligomerization" @default.
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