AU2001251002A1 - Mammalian sphingosine kinase type 2 isoforms, cloning, expression and methods of use thereof - Google Patents

Description

MAMMALIAN SPHINGOSINE KINASE TYPE 2 ISOFORMS, CLONING, EXPRESSION AND METHODS OP USE THEREOF'

CROSS-REFERENCE TO RELATED APPLICATION

The present application claims the benefit of Provisional Application Serial No. SO/194, 313, filed April 3, 2000, wherein priority under 35 USC 119-(e) is claimed.

GOVERNMENT RIGHTS

This invention was made with United States government support under Grant GM 43880 from the National Institutes of Health and a Postdoctoral Fellowship BC961968 from the United States Army Medical Research and Materiel Command, Prostate Cancer Research Program (VEN) . The United States government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention concerns mammalian (such as mouse and human) sphingosine inase type 2 isoforms, the molecular cloning of such isoforms and methods of use of such isoforms . Sphingosine kinase type 2 has distinct characteristics when compared to sphingosine kinase type 1.

Background Information

Sphingosine-1-phosphate (SPP) is a bioactive sphingolipid metabolite which regulates diverse biological processes acting both inside ceils as a second messenger to regulate proliferation, and survival and outside cells as a ligand for G- protein coupled receptors of the EDG-1 subfamily (Spiegel, S., J. Leukoc. Biol., 65, (1999), 341-344; Goetzl, E.J., An, S. FASSB J. , 12, (1998), 1589-1598). Thus, SPP plays important roles as a second messenger to regulate cell growth and survival (Olivera, A., Spiegel, S., Nature , 363, (1993), 557- 560; Cuvillier, 0., Pirianov, G., Kleuser, B., Vanek, P. G. , Coso, 0. A., Gutkind, S., and Spiegel, S., Nature, 381, (1996), 800-803) . Many external stimuli, particularly growth and survival factors, activate sphingosine kinase ("SPHK") , the enzyme that forms SPP from sphingosine. This rapidly growing list includes platelet-derived growth factor ("PDGF") (Olivera, A., Spiegel, S., Nature, 365, (1993), 557-560; Pyne, S., Chapman, J. Steele, L., and Pyne, N.J., Eur. J. Biochem. , 237, (1996), 819-826; Coroneos, E., Martinez, M. , McKenna, S. and Kester, M., J. Biol. Chem. , 270, (1995) , 23305-23309) , nerve growth factor

("NGF") (Edsall, L. C, Pirianov, G. G. , and Spiegel, S., J. Neurosci . , 17, (1997), 6952-6960; Rius, R.A. , Edsall, L.C., and Spiegel, S., FEBS Lett . , 417, (1997), 173-176), vitamin D3

(Kleuser, B., Cuvillier, 0., and Spiegel, S., Cancer Res . _r 58 ,

(1998) 1817-1824) , muscarinic acetylcholine agonists (Meyer zu Heringdorf, D., Lass, H., Alemany, R. , Laser, K.T., Neumann, E. Zhang, C, Schmidt, M. , Rauen, U. , Jakobs, K.H., and van Koppen, C.J., EMBO J. , 17, (1998), 2830-2837), TNF-a (Xia, P., Gamble, J.R., Rye, K.A. , Wang, L. , Hii, C.S.T., Cockerill, P., Khew-Goodall, Y. , Bert, A.G. , Barter, P.J., and Vadas, M.A. , Proc. Natl. Acad. Sci . USA, 95, (1998) , 14196-14201) , and the cross-linking of the immunoglobulin receptors FceRl (Choi, O. H., Kim, J.-H., and Kinet, J.-P., Nature , 380, (1996), 634-636) and FcgRl (Melendez, A., Floto, R. A., Gillooly, D. J. , Harnett, M. M. , and Allen, J.M., J. Biol. Che . , 273 (1998), 9393-9402) .

Intracellular SPP, in turn, mobilizes calcium from internal stores independently of InsP3 (Meyer zu Heringdorf, D., Lass, H. , Alemany, R. , Laser, K.T., Neumann, E. Zhang, C. , Schmidt, M. , Rauen, U. , Jakobs, K.H. , and van Koppen, C.J., EMBO J . , 17, (1998), 2830-2837; Mattie, M. , Brooker, G, and Spiegel, S., Biol. Chem., 269, (1994), 3181-3188), as well as eliciting diverse signaling pathways leading to proliferation

(Rani, C.S., Berger, A., Wu, J. , Sturgill, T. W. , Beitner- Johnson, D., LeRoith, D. , Varticovski, L. , and Spiegel, S., J. Biol. Chem., 272, (1997), 10777-10783; Van Brooklyn, J. R. , Lee, M. J. , Menzeleev, R, Olivera, A., Edsall, L. , Cuvillier, O., Thomas, D. M. , Coopman, P. J. P., Thangada, S., Hla, T., and Spiegel, S., J. Cell Biol. , 142, (1998), 229-240) and suppression of apoptosis (Cuvillier, O., Pirianov, G., Kleuser, B., Vanek, P. G. , Coso, O. A., Gutkind, S., and Spiegel, S., Nature , 381, (1996), 800-803; Edsall, L. C, Pirianov, G. G. , and Spiegel, S, J. Neurosci. , 17, (1997), 6952-6960; Van Brooklyn, J.R., Lee, M. J., Menzeleev, R. , Olivera, A., Edsall, L., Cuvillier, O., Thomas, D. M. , Coopman, P. J. P., Thangada, S., Hla, T., and Spiegel S., J. Cell Biol. , 142, (1998), 229- 240) .

Moreover, competitive inhibitors of sphingosine kinase block formation of SPP and selectively inhibit calcium mobilization, cellular proliferation and survival induced by these various stimuli (Spiegel, S., J. Leukoc . Biol . , 65, (1999) , 341-344) . Thus, it has been suggested that the dynamic balance between levels of the sphingolipid metabolites, ceramide and SPP, and the consequent regulation of opposing signaling pathways, is an important factor that determines the fate of cells (Cuvillier, O., Rosenthal, D. S., Smulson, M. E., and Spiegel, S., J. Biol. Chem. , 273, (1998), 2910-2916). For example, stress stimuli increase ceramide levels leading to apoptosis, whereas survival factors stimulate SPHK leading to increased SPP levels, which suppress apoptosis (Cuvillier, O., Rosenthal, D. S., Smulson, M. E., and Spiegel, S., J. Biol. Chem. , 273, (1998) , 2910-2916) .

Furthermore, the SPHK pathway, through the generation of SPP, is critically involved in mediating TNF-a-induced endothelial cell activation (Xia, P., Gamble, J.R. , Rye, K.A. , Wang, L. , Hii, C.S.T., Cockerill, P., Khew-Goodall, Y., Bert, A.G., Barter, P.J., and Vadas, M.A. , Proc . Natl . Acad. Sci. USA, 95, (1998) , 14196-14201) and the ability of high density lipoproteins (HDL) to inhibit cytokine-induced adhesion molecule expression has been correlated with its ability to reset this sphingolipid rheostat (Xia, P., Gamble, J.R., Rye, K.A., Wang, L., Hii, C.S.T., Cockerill, P., Khew-Goodall, Y. , Bert, A.G., Barter, P.J., and Vadas, M.A. , Proc . Natl . Acad. Sci. USA, 95, (1998) , 14196-14201) . This has important implications for the protective function of HDL against the development of atherosclerosis and associated coronary heart disease . Recent data has also connected the sphingolipid rheostat to allergic responses (Prieschl, E., E., Csonga, R. ,

Novotny, V., Kikuchi, G. E., and Baumruker, T. , J. Exp. Med. , 190, (1999) , 1-8) .

Interest in SPP has accelerated recently with the discovery that it is a ligand of the G-protein coupled cell surface receptor EDG-1 (Van Brooklyn, J. R. , Lee, M. J. , Menzeleev, R., Olivera, A., Edsall, L., Cuvillier, 0., Thomas, D. M., Coopman, P. J. P., Thangada, S, Hla, T., and Spiegel, S., J. Cell Biol. , 142, (1998), 229-240; Lee, M. J. , Van Brocklyn, J. R. , Thangada, S., Liu, C. H. , Hand, A. R. , Menzeleev, R. , Spiegel, S., and Hla, T., Science, 279, (1998), 1552-1555) . This rapidly led to the identification of several other related receptors, named EDG-3, -5, -6, and -8, which are also specific SPP receptors (Goetzl, E. J. , and An, S., FASEB J. , 12, (1998), 1589-1598; Spiegel, S., and Milstein, S., Biochem.Biophys . Acta . , 1484 (2-3) : 107-16, (2000)). Sphinganine-1-phosphate, which is structurally similar to SPP and lacks only the trans double bond at the 4-position, but not lysophosphatidic acid or sphingosylphosphorylcholine, also binds to these receptors (Van Brocklyn, J. R. , Tu, Z., Edsall, L. C, Schmidt, R. R., and Spiegel, S., J. Biol. Chem. , 274 , (1999) 4626-4632) , demonstrating that EDG-1 belongs to a family of G-protein coupled receptors that bind SPP with high affinity and specificity (Goetzl, E. J. and An, S., FASEB J. , 12 , (1998), 1589-1598; Spiegel, S. and Milstien, S., Biochem. Biophys . Acta . , 1484 (2-3) : 107-16, (2000)).

The EDG-1 family of receptors are differentially expressed, mainly in the cardiovascular and nervous systems, and are coupled to a variety of G-proteins and thus can regulate diverse signal transduction pathways culminating in pleiotropic responses depending on the cell type and relative expression of EDG receptors. Although the biological functions of the EDG-1 family of GPCRs are not completely understood, recent studies suggest that binding of SPP to EDG-1 stimulates migration and chemotaxis (Wang, F., Van Brocklyn, J. R. , Hobson, J. P., Movafagh, S., Zukowska-Grojec, Z., Milstien, S., and Spiegel, S. J. Biol. Chem., 274, (1999), 35343-35350; English, D., Kovala, A. T. , Welch, Z., Harvey, K. A., Siddiqui, R. A., Brindley, D. N. , and Garcia, J. G. , J. Hematother. Stem Cell Res. , 8 , (1999) , 627-634) , and as a consequence, may regulate angiogenesis (Wang, F., Van Brocklyn, J. R., Hobson, J. P., Movafagh, S., Zukowska-Grojec, Z., Milstien, S., and Spiegel, S. J. Biol. Chem. , 274, (1999), 35343-35350; Lee, O. H., Kim, Y. M., Lee, Y. M. , Moon, E. J. , Lee, D. J., Kim, J. H. , Kim, K. W., and Kwon, Y. G. , Biochem. Biophys. Res. Commun. , 264, (1999) 743-750; Lee, M. J. , Thangada, S., Claffey, K. P., Ancellini, N. , Liu, C. H. , Kluk, M. , Volpi, Sha'afi, R. I., and Hla, T. , Cell, 99_, (1999), 301-312). EDG-5 may play a role in cytoskeletal reorganization during neurite retraction, which is important for neuronal differentiation and development (Van Brocklyn, J. R. , Tu, Z., Edsall, L. C, Schmidt, R. R., and Spiegel, S., J. Biol. Chem. , 274, (1999), 4626-4632; MacLennan, A. J. , Marks, L., Gaskin, A. A., and Lee, N., Neuroscience, 79, (1997), 217-224).

Critical evaluation of the role of SPP requires cloning of the enzymes that regulate its metabolism. Recently, rat kidney SPHK has been purified to apparent homogeneity (Olivera, A. , Kohama, T., Tu, Z., Milstien, S., and Spiegel, S., J. Biol. Chem. , 273, (1998) , 12576-12583) and subsequently the first mammalian SPHK, designated mSPHKl (Kohama, T., Olivera, A., Edsall, L., Nagiec, M. M., Dickson, R., and Spiegel, S., J. Biol. Chem., 273, (1998), 23722-23728) was cloned. Independently, two genes, termed LCB4 and LCB5, were also shown to code for SPHKs in Saccharo yces cerevisiae (Nagiec, M. M. ,

Skrzypek, M. , Nagiec, E. E., Lester, R. L., and Dickson, R. C, J. Biol. Chem. , 273, (1998) 19437-19442) . Moreover, databases identify homologues of mSPHKl in numerous widely disparate organisms, including worms, plants and mammals, demonstrating that the enzyme is encoded by a member of a highly conserved gene family (Kohama, T., Olivera, A., Edsall, L., Nagiec, M. M., Dickson, R., and Spiegel, S., J. Biol. Chem. , 273 , (1998), 23722-23728) . Comparison of the predicted a ino acid sequences of the known SPHKls revealed five blocks of highly conserved amino acids (Kohama, T., Olivera, A., Edsall, L., Nagiec, M. M., Dickson, R., and Spiegel, S., J. Biol. Chem. , 273, (1998), 23722-23728) . However, several lines of evidence indicate that there may be multiple mammalian SPHK isoforms.

The finding that SPHK activity in platelets could be chro atographically fractionated into several forms with differing responses to detergents and inhibition by known SPHK inhibitors, indicate the presence of multiple enzyme forms in human platelets (Banno, Y. , Kato, M. , Hara, A. , and Nozawa, Y. , Biochem. J. , 335, (1998), 301-304). Moreover, homology searches against a comprehensive nonredundant database revealed that several of the expressed sequence tags (dbEST) at NCBI had significant homology to conserved domains of mSPHKla (Kohama, T., Olivera, A., Edsall, L., Nagiec, M. M. , Dickson, R. , and Spiegel, S., J. Biol. Chem. , 273, (1998), 23722-23728), yet had substantial sequence differences.

USP 5,374,616 concerns compositions containing sphingosylphosphorylcholine for promoting cellular proliferation of mammalian cells.

WO 99/61581 describes DNA fragments which encoded murine sphingosine SPHKla (381 amino acids) and SPHKlb (388 amino acids) .

SUMMARY OF THE INVENTION

It is an object of the present invention to provide isolated and purified DNAs which encode mammalian (such as a mouse or human) sphingosine kinase type 2 isoforms and peptides encoded therefrom.

It is a further object of the present invention to provide recombinant DNA constructs comprising a vector and the above described DNAs and host cells transformed with such recombinant DNA constructs .

It is a still further object of the present invention to furnish a method for producing mouse and human sphingosine type 2 isoform peptides by culturing such host cells.

It is an additional object of the present invention to provide a method for detecting an agent or a drug which inhibits or promotes sphingosine kinase activity.

It is yet another object of the present invention to provide a method for regulating a biological process; for treating or ameliorating a disease resulting from increased or decreased cell proliferation or increased or decreased cell death; and for treating or ameliorating a disease resulting from abnormal migration or motility of cells such as cancer, restenosis or diabetic neuropathy.

The present invention is also directed to an isolated and purified DNA which encodes a peptide of a sphingosine kinase type 2 isoform, the DNA comprising a sequence selected from the group consisting of the sequence of Genbank Accession No. bankit325787 and the sequence of Genbank Accession No. bankit325752.

The present invention also concerns methods for detecting an agent or a drug which inhibits or promotes sphingosine kinase type 2 activity comprising:

(a) providing a recombinant DNA construct as discussed above, into a cell such that sphingosine kinase type 2 isoform is produced in the cell;

(b) adding at least one drug or agent to the cell, and

(c) detecting whether or not the drug or agent inhibits or promotes sphingosine kinase type 2 activity by measuring sphingosine kinase-dependent phosphorylation of lipids in the cells and comparing the resultant measurement to a control which did not receive the drug or agent, wherein a decrease in the amount of sphingosine kinase-dependent phosphorylation of lipids as compared to the control indicates an inhibitory drug or agent, or an increase in the amount of sphingosine kinase- dependent phosphorylation of lipids in the cell as compared to the control indicates a stimulatory drug or agent.

As described hereinabove, the present invention also relates to methods of regulating a biological process (such as mitogenesis, apoptosis, neuronal development, chemotaxis, angiogenesis and inflammatory responses) in a mammal comprising administering to a mammal (such as a human) in need thereof, a pharmaceutically effective amount of a peptide as described above .

Also as described hereinabove, the present invention is further directed to methods for the treatment or amelioration of a disease resulting from increased cell death or decreased cell proliferation, comprising administering to a mammal (such as a human) in need thereof, a pharmaceutically effective amount of a peptide as described above.

Further as described above, the present invention also relates to methods for the treatment or administration of a disease resulting from decreased cell death or increased cell proliferation comprising administering to a mammal (such as a human) in need thereof, a pharmaceutically effective amount of an antibody to a peptide as described above.

Additionally as described above, the present invention further concerns methods for treatment or amelioration of a disease resulting from abnormal migration or otility of cells selected from the group consisting of cancer, restenosis and diabetic neuropathy, the method comprising administering to a mammal (such as a human) in need thereof, a pharmaceutically effective amount of an antibody to a peptide as described above .

The present invention further relates to compositions for (a) regulating biological processes, (b) treating or ameliorating diseases resulting from increased cell death or decreased cell proliferation, (c) treating or ameliorating diseases resulting from decreased cell death or increased cell proliferation, or (d) treating or ameliorating diseases resulting from abnormal migration or motility of cells (such as cancer, restenosis and diabetic neuropathy) comprising (i) a pharmaceutically effective amount of a peptide as described above or an antibody to such peptide as described above, and (ii) a pharmaceutically acceptable carrier.

The present invention also involves a method for screening agents or drugs which reduce or eliminate sphingosine kinase type 2 activity, the method comprising detecting a decrease in sphingosine kinase type 2 enzyme activity in the presence of the agent or drug.

Furthermore, the present invention is directed to a method for detecting the presence of sphingosine kinase type 2 isoform in a sample comprising:

(i) contacting a sample with antibodies which recognize sphingosine kinase type 2; and

(ii) detecting the presence or absence of a complex formed between sphingosine kinase type 2 and antibodies specific therefor.

The present invention also concerns a method for detecting sphingosine kinase type 2 in a sample comprising subjecting the sample to a poly erase chain reaction and detecting for the presence of sphingosine kinase type 2.

The present invention is additionally directed to a diagnostic kit for detecting sphingosine kinase type 2 RNA/cDNA in a sample comprising primers or oligonucleotides specific for sphingosine kinase type 2 RNA or cDNA suitable for hybridization to sphingosine kinase type 2 RNA or cDNA and/or amplification of sphingosine kinase type 2 sequences and suitable ancillary reagents.

Sphingosine kinase catalyzes the phosphorylation of sphingosine to yield SPP. Based on sequence homology to murine and human sphingosine kinase-1 (SPHK1) , which was recently cloned (Kohama, et al . , J. Biol. Chem. , 273 , 23722-23728, (1998) ) , the present invention is directed to the cloning, functional characterization, and tissue distribution of a second type of mouse and human sphingosine kinase (mSPHK2 and hSPHK2) . mSPHK2 and hSPHK2 of the present invention encode proteins of 617 and 618 amino acids, respectively, both much larger than SPHK1, and both contain the conserved domains previously found in SPHK1, but their sequences diverge considerably in the centers and at the amino termini . Northern blot analysis of multiple human and murine tissues revealed that SPHK2 mRNA expression was strikingly different from that of SPHK1 and was highest in brain, heart, kidney, testes, and liver. Whereas SPHK1 expression is greatest at mouse embryonic day 7, SPHK2 expression is only detectable at embryonic day 11 and increases thereafter.

Human embryonic 293 kidney cells transiently transfected with mSPHK2 or hSPHK2 expression vectors had marked increases in SPHK activity resulting in elevated SPP levels. Notably, SPHK2 had somewhat different substrate specificity than SPHK1. D - erytiirσ-sphinganine (dihydrosphingosine, DHS) was an even better substrate than D-erythro-sphingosine for SPHK2, while

DHS was a potent inhibitor of SPHK1.

SPHK2 also catalyzed the phosphorylation of phytosphingosine and D, L- threo-dihydrosphingosine, albeit to a lesser extent. DMS, a competitive inhibitor of SPHK1, surprisingly was a non-competitive inhibitor of SPHK2. Although increasing ionic strength inhibited SPHK1, KCl and NaCl markedly stimulated SPHK2 activity. Moreover, Triton X-100 and BSA inhibited SPHK2 , in contrast to their effects on SPHK1, whereas phosphatidylserine stimulated both types. The data herein indicate that SPHK2 is a novel member of this growing class of lipid kinases, which is important in the regulation of diverse biological processes, including mitogenesis, apoptosis, neuronal development, chemotaxis, angiogenesis, and inflammatory responses.

BRIEF DESCRIPTION OF THE DRAWINGS

For the purposes of illustrating the invention, features, aspects and advantages are shown in the drawings. It is to be understood, however, that the present invention is not limited to that which is depicted in the drawings

Fig. 1A shows predicted amino acid sequences of murine and human type 2 sphingosine kinase based on non-ClustalW alignment of the predicted amino acid sequences of ("mSPHK2") and human sphingosine kinase 2 ("hSPHK2"). Identical and conserved amino acid substitutions are shaded dark and light gray, respectively. The dashes represent gaps in sequences and numbers on the right refer to the amino acid sequence of mSPHK2. The conserved domains (Cl to C5) are indicated by lines.

Fig. IB is a schematic representation of conserved regions of SPHK1 and SPHK2. The primary sequence of mSPHK2 is compared to that of mSPHKla.

Figs. 2A, 2B and 2C are Northern blots which show the tissue specific expression of type 1 and type 2 sphingosine kinase.

In Fig. 2A, mSPHK2 (upper panel) and mSPHKla (middle panel) probes were end labeled and hybridized to poly (A) + RNA blots from the indicated mouse tissues as described hereinbelow. Lanes: 1, heart; 2, brain; 3, spleen; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; 8, testis. A β-actin probe (lower panel) was used as a loading control.

Fig. 2B shows the tissue specific expression of hSPHK2. Lanes 1, brain; 2, heart; 3, skeletal muscle; 4, colon; 5, thymus; 6, spleen; 7, kidney; 8, liver; 9, small intestine; 10, placenta; 11, lung; 12, leukocyte.

Fig. 2C shows the expression of mSPHKla and mSPHK2 during mouse embryonic development . Poly (A) + RNA blots from days 7 , 11, 15 and 17 mouse embryos were probed as in Fig. 2A.

Figs. 3A and 3B are graphs which show the enzymatic activity of recombinant SPHK2.

In Fig. 3A, HEK 293 cells were transiently transfected with an empty vector or with mSPHK2 or hSPHK2 expression vectors. After 24 hours, SPHK activity was measured in cytosol (open bars) and particulate fractions (filled bars) . The data are means ± S.D. Parental and vector-transfected cells had basal SPHK activities of 26 and 37 pmol/min/mg, respectively.

Fig. 3B shows the changes in mass levels of SPP after transfection with SPHK2. Mass levels of SPP in HEK293 cells transfected with an empty vector (open bars) or with mSPHK2

(filled bars) or with hSPHK2 (hatched bars) were measured as described hereinbelow. The data are expressed as pmol/nmol phospholipid.

Figs . 4A to 4D are graphs which show the substrate specificity of mSPHK2.

Fig. 4A is a graph which shows SPHK-dependent phosphorylation of various sphingosine analogs or other lipids (50 mM) which was measured in cytosol from HEK293 cells transfected with mSPHK2. Lanes: 1, D-erythro-sphingosine ("D-er thro-Sph") ; 2, D-erythro-dihydrosphingosine

( "D- ery thro-DHS" ) ; 3, D, L- threo-DHS; 4, N,N- dimethylsphingosine ("DMS"); 5, C2-ceramide; 6, C16-ceramide; 7, diacylglycerol; 8, phosphatidylinositol; 9, phytosphingosine. Data are expressed as percentage of phosphorylation of D-er thro-Sph.

Figs. 4A to 4D are graphs which show the noncompetitive inhibition of recombinant SPHK2 by N,N-dimethylsphingosine .

Fig. 4B shows the dose-dependent inhibition of mSPHK2 by DMS. SPHK activity in HEK293 cell lysates after transfection as in Fig. 4A was measured with 10 μM D-erythro-sphingosine in the presence of increasing concentrations of DMS . Fig. 4C shows a kinetic analysis of DMS inhibition. SPHK activity was measured with varying concentrations of D-eryfchro-sphingosine in the absence (open circles) or presence of 10 μM (filled squares) or 20 μM DMS (filled triangles) .

Fig. 4D are Lineweaver-Burk plots. The Km for D-erythro-sphingosine was 3.4 μM. The Ki value for DMS was 12 μM.

Figs . 5A to 5E are graphs which show the pH dependence and salt effects on mSPHK2.

Fig. 5A shows cytosolic SPHK2 activity in transfected HEK 293 cells that was measured in a kinase buffer with the pH adjusted using the following buffers: 200 mM sodium acetate (pH 4.5-5.5, open circles); 200 mM MES (pH 6-7, filled circles); 200 mM potassium phosphate (pH 6.5-8, open squares); 200 mM HEPES (pH 7-7.5, filled squares); 200 mM Tris-HCl (pH 7.5-9, open triangles) ; and 200 mM borate (pH 10, filled triangle) .

Figs. 5B to 5E show that salts stimulate SPHK2, but inhibit SPHK1.

In Figs. 5B and 5C, the SPHK activity in HEK293 cell lysates was measured 24 hours after transfection with mSPHKl (Fig. 5B) or mSPHK2 (Fig. 5C) in the absence or presence of increasing concentrations of NaCl (open squares) or KCl (filled circles) .

Fig. 5D shows a kinetic analysis of SPHK2 activation by KCl. mSPHK2 activity was measured with varying concentrations of D-erythro-sphingosine in the absence (open circles) , or presence of 50 mM KCl (open squares) , or 200 mM KCl (filled squares) .

Fig. 5E are Lineweaver-Burk plots of data from Fig 5D. The Km value not affected by the presence of KCl . Vmax values were 0.1, 0.3 and 1 (nmol/min/mg) in the presence of 0, 50, and 200 mM KCl, respectively.

Figs. 6A to 6B are graphs which show that Triton X-100 and bovine serum albumin ("BSA") have differential effects on the activity of SPHK1 and SPHK2. HEK293 cells were transfected with mSPHKla (open circles) or mSPHK2 (filled circles) and the activities of each in cell lysates were measured after 24 hours in the presence of the indicated concentrations of Triton X-100

(Fig. 6A) or BSA (Fig. 6B) .

Fig. 6C is a graph which shows that phosphatidylserine has similar effects on the activity of SPHK1 and SPHK2. HEK293 cells were transfected with mSPHKla (circles) or mSPHK2

(triangles) and the activities of each in cell lysates were measured after 24 hours in the presence of the indicated concentrations of phosphatidylserine (filled symbols) or phosphatidylcholine (open symbols) . Data are expressed as percentage of control activity measured without any additions.

DETAILED DESCRIPTION OF THE INVENTION

In one embodiment, the present invention relates to a DNA or cDNA segment which encodes mammalian (such as mouse and human) sphingosine kinase type 2 isoforms.

In addition, isolated nucleic acid molecules of the invention include DNA molecules which comprise sequences substantially different from those described above but which, due to the degeneracy of the genetic code, still encode mammalian sphingosine kinase type 2 isoforms. Of course, the genetic code and species-specific codon preferences are well known in the art. Thus, it would be routine for one of ordinary skill in the art to generate the degenerate variants described above, for instance, to optimize codon expression for a particular host (e.g., change codons in the human mRNA to those preferred by a bacterial host such as E. coli) .

Nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand, also known as the "sense strand", or it may be the noncoding strand, also referred to as the "antisense strand" .

By "isolated" nucleic acid molecule (s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention. Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution. Isolated RNA molecules include in vivo or in vi tro RNA transcripts of the

DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.

The present invention is further directed to nucleic acid molecules encoding portions or fragments of the nucleotide sequences described herein. Fragments include portions of the nucleotide sequences of Fig. 1A for mSPHK2 and hSPHK2 at least 10 contiguous nucleotides in length selected from any two integers, one of which 5 representing a 5' nucleotide position and a second of which representing a 3 ' nucleotide position, where the first nucleotide for each nucleotide sequence in Fig. 1A is position 1. That is, every combination of a 5 ' and 3' nucleotide position that a fragment at least 10 contiguous nucleotide bases in length or any integer between 10 and the length of an entire nucleotide sequence of mSPHK2 or hSPHK2 of Fig. 1A minus 1.

Further, the present invention includes polynucleotides comprising fragments specified by size, in nucleotides, rather than by nucleotide positions . The present invention includes any fragment size, in contiguous nucleotides, selected from integers between 1 and the entire length of an entire nucleotide sequence minus 1. Preferred sizes include 20 to 50 nucleotides; sizes of 50 to 300 nucleotides are useful as primers and probes . Regions from which typical sequences may be derived include, but are not limited to, for example, regions encoding specific epitopes or domains within said sequence, such as domains C1-C5 shown in Fig. 1A.

In another aspect, the invention provides isolated nucleic acid molecules comprising polynucleotides which hybridize under stringent hybridization conditions to a polynucleotide sequence 30 of the present invention described above, for instance, a nucleic acid sequence shown in Fig. 1A or a specified fragment thereof. By "stringent hybridization conditions" is intended overnight incubation at 42°C in a solution comprising: 50% 35 formamide, 5X SSC (150 mM NaCl, 15 mM trisodium citrate) , 50 MM sodium phosphate (pH 7.6), 5X Denhardt ' s solution, 10% dextran sulfate, and 20 g/ml denatured sheared salmon sperm DNA, followed by washing the filters in 0. IX SSC at about 65°C.

The sequences encoding the polypeptides of the present invention or portions thereof may be fused to other sequences which provide additional functions known in the art such as a marker sequence, or a sequence encoding a peptide which facilitates purification of the fused polypeptide, peptides having antigenic determinants known to provide helper T-cell stimulation, peptides encoding sites for post-translational modifications, or amino acid sequences in which target the fusion protein to a desired location, e.g., a heterologous leader sequence.

The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the sphingosine kinase type 2 isoform polypeptides shown in Fig. 1A. Variants may occur naturally, such as a natural allelic variant. By an "allelic variant" is intended one of several alternate forms of a gene occupying a given locus of a chromosome of an organism. Non-naturally occurring variants may be produced by known mutagenesis techniques. Such variants include those produced by nucleotide substitution, deletion or addition of one or more nucleotides in the coding or noncoding regions or both. Alterations in the coding regions may produce conservative or nonconservative amino acid substitutions, deletions, or additions. Especially preferred among these are silent substitutions, additions, and deletions which do not alter the properties and activities of sphingosine kinase type 2 isoform polypeptides disclosed herein or portions thereof. Also preferred in this regard are conservative substitutions.

Nucleic acid molecules with at least 90-99% identity to a nucleic acid molecule which encodes a sphingosine kinase type 2 isoform shown in Fig. 1A is another aspect of the present invention. These nucleic acids are included irrespective of whether they encode a polypeptide having sphingosine kinase activity. By "a polypeptide having sphingosine kinase type 2 activity" is intended polypeptides exhibiting activity similar, but not identical, to an activity of the sphingosine kinase type 2 isoform of the present invention, as measured in the assays described below. The biological activity or function of the polypeptides of the present invention are expected to be similar or identical to, polypeptides from other organisms that share a high degree of structural identity/similarity.

In another embodiment, the present invention relates to a recombinant DNA molecule that includes a vector and a DNA sequence as described above . The vector can take the form of a plasmid, phage, cosmid, YAC, an eukaryotic expression vector such as a DNA vector, Pichia pastor is , or a virus vector such as for example, baculovirus vectors, retroviral vectors or adenoviral vectors, and others known in the art. The cloned gene may optionally be placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences, or sequences which may be inducible and/or cell type-specific . Suitable promoters are known to a person with ordinary skill in the art. The expression construct will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation. Among the vectors preferred for use include pCMV-SPORT2 (Life Technologies, Inc.), pcDNA3 (Invitrogeni), to name a few.

Introduction of the construct into the host cell can be effected by calcium phosphate transfection, electroporation, infection, and other methods known in the art and described in standard laboratory manuals such as Current Protocols in Molecular Biology, Ausubel, F. M. et al . (Eds), Wiley & Sons, Inc. All documents cited herein supra and infra are hereby incorporated in their entirety by reference thereto.

In a further embodiment, the present invention relates to host cells stably transformed or transfected with the above-described recombinant DNA constructs. The host cell can be prokaryotic (for example, bacterial) , lower eukaryotic (for example, yeast or insect) or higher eukaryotic (for example, all mammals, including, but not limited to, rat and human) .

Both prokaryotic and eukaryotic host cells may be used for expression of desired coding sequences, when appropriate control sequences which are compatible with the designated host are used. Among prokaryotic hosts, E. coli is most frequently used. Expression control sequences for prokaryotes include promoters, optionally containing operator portions, and ribosome binding sites. Transfer vectors compatible with prokaryotic hosts are commonly derived from, for example, pBR322, a plasmid containing operons conferring ampicillin and tetracycline resistance, and the various pUC vectors, which also contain sequences conferring antibiotic resistance markers. These markers may be used to obtain successful transformants by selection. See, for example, Maniatis, Fitsch and Sambrook, Molecular, Cloning: A Laboratory Manual, (1982) or DNA Cloning-, Volumes I and II (D. N. Glover ed. , 1985) for general cloning methods .

A transformant having a plasmid in which a cDNA encoding human SPHK2 is inserted, namely E. coli pCR3. l-hSPHK2 SANK

70200 has been deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology, 1-3, Higashi 1-chome, Tsukuba-shi, Ibaraki-ken 305-8566, Japan, accession number FERM BP-7110, deposited March 29, 2000.

The DNA sequence can be present if the vector operably linked to a sequence encoding an IgG molecule, an adjuvant, a carrier, or an agent for aid in purification of SPHK, such as glutathione S-transferase, or a series of histidine residues also known as a histidine tag. The recombinant molecule can be suitable for transfecting eukaryotic cells, for example, mammalian cells and yeast cells in culture systems. Saccharomyces cerevisiae, Saccharomyces carl sber gens is, and

Pichia pastoris are the most commonly used yeast hosts, and are convenient fungal hosts . Control sequences for yeast vectors are know in the art. Mammalian cell lines are available as hosts for expression are known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC) , such as HEK293 cells, and NIH 3T3 cells, to name a few. Suitable promoters are also known in the art and include viral promoters such as that from SV40, Rous sarcoma virus ("RSV") , adenovirus ("ADV") , bovine papilloma virus ("BPV"), and cytomegalovirus ("CMV").

Mammalian cells may also require terminator sequences and poly A addition sequences; enhancer sequences which increase expression may also be included, and sequences which cause amplification of the gene may also be desirable. These sequences are known in the art .

The transformed or transfected host cells can be used as a source of DNA sequences described above. When the recombinant molecule takes the form of an expression system, the transformed or transfected cells can be used as a source of the protein described below.

In another embodiment, the present invention relates to the employment of nucleotide sequences corresponding to GenBank/EMBL Data Bank accession nos. bankit325787 and bankit325752. A polypeptide or amino acid sequence expressed from the nucleotide sequences discussed above, refers to polypeptide having an amino acid sequence identical to that of a polypeptide encoded from the sequence, or a portion thereof wherein the portion contains at least 2 to 5 amino acids, and more preferably at least 8 to 10 amino acids, and even more preferably at least 15 amino acids, or which is immunologically identifiable with a polypeptide encoded in the sequence.

A recombinant or derived polypeptide is not necessarily translated from a designated nucleic acid sequence; it may be generated in any manner, including, for example, chemical synthesis, or expression of a recombinant expression system. In addition the polypeptide can be fused to other proteins or polypeptides which increase its antigenicity, such as adjuvants, for example.

As noted above, the methods of the present invention are suitable for production of any polypeptide of any length, via insertion of the above-described nucleic acid molecules or vectors into a host cell and expression of the nucleotide sequence encoding the polypeptide of interest by the host cell. Introduction of the nucleic acid molecules or vectors into a host cell to produce a transformed host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as in Davis et al . , Basic Methods In Molecular Biology, (1986) .

Once transformed host cells have been obtained, the cells may be cultivated under any physiologically compatible conditions of pH and temperature, in any suitable nutrient medium containing assimilable sources of carbon, nitrogen and essential minerals that support host cell growth. Recombinant polypeptide-producing cultivation conditions vary according to the type of vector used to transform the host cells. For example, certain expression vectors comprise regulatory regions which require cell growth at certain temperatures, or addition of certain chemicals or inducing agents to the cell growth medium, to initiate the gene expression resulting in the production of the recombinant polypeptide. Thus, the term "recombinant polypeptide-producing conditions," as used herein, is not meant to be limited to any one set of cultivation conditions. Appropriate culture media and conditions for the above-described host cells and vectors are well-known in the art. Following its production in the host cells, the polypeptide of interest may be isolated by several techniques. To liberate the polypeptide of interest from the host cells, the cells are lysed or ruptured. This lysis may be accomplished by contacting the cells with a hypotonic solution, by treatment with a cell wall-disrupting enzyme such as lysozyme, by sonication, by treatment with high pressure, or by a combination of the above methods. Other methods of bacterial cell disruption and lysis that are known to one of ordinary skill may also be used.

Following disruption, the polypeptide may be separated from the cellular debris by any technique suitable for separation of particles in complex mixtures. The polypeptide may then be purified by well-known isolation techniques. Suitable techniques for purification include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, electrophoresis, immunoadsorption, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, immunoaffinity chromatography, size exclusion chromatography, liquid chromatography (LC) , high performance LC (HPLC) , fast performance LC (FPLC) , hydroxylapatite chromatography and lectin chromatography.

The recombinant or fusion protein can be used as a diagnostic tool and in a method for producing sphingosine-1-phosphate, detectably labeled and unlabeled, and in a method for measuring levels of SPP in samples as described below. In addition, the recombinant protein can be used as a therapeutic agent to reduce cell death and/or increase cell proliferation. The transformed host cells can be used to analyze the effectiveness of drugs and agents which inhibit SPHK2 function, such as host prote.ins or chemically derived agents or other proteins which may interact with the cell to down-regulate or alter the expression of SPHK2, or its cofactors.

In another embodiment, the present invention relates to monoclonal or polyclonal antibodies specific for the above-described recombinant proteins (or polypeptides) . For instance, an antibody can be raised against a peptide described above, or against, a portion thereof of at least 10 amino acids, preferably 11 to 15 amino acids. Persons with ordinary skill in the art using standard methodology can raise monoclonal and polyclonal antibodies to the protein (or polypeptide) of the present invention, or a unique portion thereof. Material and methods for producing antibodies are well known in the art (see, for example, Goding in Monoclonal Antibodies: Principles and Practice, Chapter 4, 1986) .

The level of expression of sphingosine kinase type 2 can be detected at several levels. Using standard methodology well known in the art, assays for the detection and quantitation of sphingosine kinase type 2 RNA can be designed and include northern hybridization assays, in si tu hybridization assays, and PCR assays, among others. See, for example, Maniatis, Fitsch and Sambrook, Molecular Cloning, A Laboratory- Manual, (1982) or DNA Cloning, Volumes I and II (D. N. Glover ed. 1985), or Current Protocols in Molecular Biology, Ausubel, F. M. et al . , (Eds), Wiley & Sons, Inc. for a general description of methods for nucleic acid hybridization.

Polynucleotide probes for the detection of sphingosine kinase type 2 RNA can be designed from the sequence available at accession numbers AF068748 and/or AF068749 for the mouse sequence (Kohama, T., et al., J. Biol. Chem. , 273:23722-23728). For example, RNA isolated from samples can be coated onto a surface such as a nitrocellulose membrane and prepared for northern hybridization. In the case of in si tu hybridization of biopsy samples, for example, the tissue sample can be prepared for hybridization by standard methods known in the art and hybridized with polynucleotide sequences which specifically recognize sphingosine kinase type 2 RNA. The presence of a hybrid formed between the sample RNA and the polynucleotide can be detected by any method known in the art such as radiochemistry, or immunochemistry, to name a few.

One of skill in the art may find it desirable to prepare probes that are fairly long and/or encompass regions of the amino acid sequence which would have a high degree of redundancy in the corresponding nucleic acid sequences . In other cases, it may be desirable to use two sets of probes simultaneously, each to a different region of the gene. While the exact length of any probe employed is not critical, typical probe sequences are no greater than 500 nucleotides, even more typically they are no greater than 250 nucleotides; they may be no greater than 100 nucleotides, and also may be no greater than 75 nucleotides in length. Longer probe sequences may be necessary to encompass unique polynucleotide regions with differences sufficient to allow related target sequences to be distinguished. For this reason, probes are preferably from about 10 to about 100 nucleotides in length and more preferably from about 20 to about 50 nucleotides.

The DNA sequence of sphingosine kinase type 2 can be used to design primers for use in the detection of sphingosine kinase type 2 using the polymerase chain reaction (PCR) or reverse transcription PCR (RT-PCR) . The primers can specifically bind to the sphingosine kinase type 2 cDNA produced by reverse transcription of sphingosine kinase type 2 RNA, for the purpose of detecting the presence, absence, or quantifying the amount of sphingosine kinase type 2 by comparison to a standard. The primers can be any length ranging from 7 to 40 nucleotides, preferably 10 to 35 nucleotides, most preferably 18 to 25 nucleotides homologous or complementary to a region of the sphingosine kinase type 2 sequence .

Reagents and controls necessary for PCR or RT-PCR reactions are well-known in the art. The amplified products can then be analyzed for the presence or absence of sphingosine kinase type 2 sequences, for example, by gel fractionation, by radiochemistry, and immunochemical techniques. This method is advantageous, since it requires a small number of cells. Once sphingosine kinase type 2 is detected, a determination of whether the cell is overexpressing or underexpressing sphingosine kinase type 2 can be made by comparison to the results obtained from a normal cell using the same method. Increased sphingosine kinase type 2 RNA levels correlate with increased cell proliferation and reduced cell death.

In another embodiment, the present invention relates to a diagnostic kit for the detection of sphingosine kinase type 2 RNA in cells . The kit comprises a package unit having one or more containers of sphingosine kinase type 2 oligonucleotide primers for detection of sphingosine kinase type 2 by PCR or RT-PCR or sphingosine kinase tpye 2 polynucleotides for the detection of sphingosine kinase type 2 RNA in cells by in si tu hybridization or Northern analysis, and in some kits including containers of various reagents used for the method desired. The kit may also contain one or more of the following items : polymerization enzymes, buffers, instructions, controls, detection labels. Kits may include containers of reagents mixed together in suitable proportions for performing the methods in accordance with the invention. Reagent containers preferably contain reagents in unit quantities that obviate measuring steps when performing the subject methods.

In a further embodiment, the present invention provides a method for identifying and quantifying the level of sphingosine Pi PJ φ P t &

Pi Ω

K_ K tr

O X Φ in PJ 3

Φ 3 H- ø TJ _-^■ so H d

CQ φ 3 φ CQ H-

- 0

0 Φ

CQ Hi CQ rt Ω

PJ CQ Φ

TJ d P

P^J H- rr rr

So (-^■

0 tr PJ

Ω tr

O Φ φ

Ω I-¹

Ω Φ CQ

SD 0 •

I-¹ N

I 0 o d 1 φ i Ω

I _-^■ H

Φ PJ

PJ tr

CQ

Φ I-¹

- CQ i H-

Φ 0

I-" Ω

H

5" d a σi φ

CQ 3 rt PJ

Φ I-¹

H PJ

O rr

H- Φ o,

isomerase, yeast-alcohol dehydrogenase, alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase, peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate hydrogenase, glucoamylase, acetylcholine esterase, etc.

Examples of suitable radioisotopic labels include ³H , ^mIn, ¹²⁵I , ³²P , ³⁵S , ¹⁴C , ⁵⁷To , ⁵⁸Co , ⁵⁹Fe , ⁷⁵Se , ¹⁵²Eu , ⁹⁰Y , ⁶⁷Cu , ²¹Ci , ²¹¹At , ²¹²Pb , ⁴⁷Sc , ¹⁰⁹Pd, ^UC , ¹⁹F and ¹³¹I .

Examples of suitable non-radioactive isotopic labels include ¹⁵Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Tr and ⁴⁶Fe.

Examples of suitable fluorescent labels include a ¹⁵²Eu label, a fluorescein label, an isothiocyanate I label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label and a fluorescamine label .

Examples of chemiluminescent labels include a luminal label, an isoluminal label, an aromatic acridinium ester label, an imidazole label, an acridinium salt label, an oxalate ester label, a luciferin label and a luciferase label.

Those of ordinary ,skill in the art will know of other suitable labels which may be employed in accordance with the present invention. The binding of these labels to ligands and to antibodies or fragments thereof can be accomplished using standard techniques commonly known to those of ordinary skill in the art. Typical techniques are described by Kennedy, J. H., et al., (1976), Clin. Chem. Acta. , 70, 1-31, and Schurs, A. H. W. M., et al., (1977), Clin. Chem. Acta. , 81, 1-40. Coupling techniques mentioned in the latter are the glutaraldehyde method, the periodate method, the dalemide method, and others.

The detection of the antibodies (or fragments of antibodies) of the present invention can be improved by the use of carriers. Well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and n ii K W ii CQ 3 CQ tr i Hi 0 J TJ J μ- μ- 0 d CQ d rt g rt Hi φ 0 Hi g J μ- w

PJ Φ 0 μ- PJ J Φ TJ Φ φ H Hi 0 H ø 3 ii CQ TJ CQ μ- tr O tr X ii tr 0 0 ø 0

■Q Hi μ^j 0 i φ rt φ rt o rt J CQ g Pi μ- tr Φ iQ φ ø φ PJ rt d _-^■ CQ < 3 φ Φ H¹ Ω μ- Ω tr Ω S3 PJ 3 CQ μ- rt d rt d μ- ø μ- P. PJ 0 rt φ Ω CQ Φ CQ Φ φ

CQ H PJ tr 0 rr 0 μ- Φ rt TJ tr Φ ii tr ø ø iQ ø ø d Ω PJ ii ^■ _ - Ω μ- 0 φ ø Φ μ- >i p. Hi Φ Φ J tr 0 Hi Φ 0 PJ iQ rt Pi 3 CQ ii CQ 0 M d tr rt φ r

H 0 Pi 3 3 0 CQ μ- ø i μ- Pi 0 PJ g H PJ 0 0 CQ tr φ o rt d PJ μ- rt μ- X O Ω μ- 3 Q Ω TJ ø μ- K Ω J * ø CQ Φ 0 Ω μ-¹ ø P Φ Φ 0 rt r

<-3 Φ rθ Q d Ω Ω CQ Pi Φ iQ φ Φ ϋ rt *< μ- Λ 0 0 PJ s tr Pi φ ø Φ

1 d rt ø 0 0 rt TJ μ- H 0 CQ PJ rt μ- CQ ø d Hi μ-¹ Hi μ- Q ii μ- CQ ø • to tr tr K φ J 3 t tr CQ ^"CQ CQ ,-_^, Ω T 0 φ PJ μ- PJ d μ- CQ CQ rt rt o Φ H < J 3 φ μ- φ 0 μ- CQ 3 μ- μ- Hi 0 CQ iQ H PJ r-¹ CQ ii Ω d ii σι ii μ- PJ 0 ii ø PJ 0 ø TJ Φ • PJ J H-" ^ rt TJ J 0 0 Hi PJ TJ Ω d H 0 φ CQ μ- CQ O ø Φ CQ CQ φ φ 3 Φ CQ P⁾ μ- μ- tr ø d rt PT PJ PJ TJ PJ Ω tr ii t

0 μ- tr ø CQ Hi 0 φ CQ Ω tr • Pi CQ ø rt μ- Pi rt μ- ø Ω 0 TJ rt φ

Hi ø μ- PJ μ- 0 CQ . d μ- H μ- J μ- <! J PJ ø CQ μ- tr 0 CQ Φ K J d μ- ø S ^ Ω ø H μ- CQ μ- Hi PJ J ^• : ø J CQ rt Q ø 0 s3 ⁴ J rt tr ii CO ø

W iQ 0 0 ø TJ ø μ- ø PJ IQ CQ H φ μ- 0 o Φ J μ^j 3 rt Ω φ ι-3 φ PJ Ω Φ t μ- <! CQ H θ- Φ 0 CQ _-^■ PJ d CQ

0 r

^ g Φ Hi Ω Φ _-^■ TJ o r

Pi CO π φ J 0 tr PJ tr Ω CQ CQ 0 tr φ rt Φ μ- φ PJ rt μ- 0 TJ μ- J 0 Ω ^ ii Φ 0 Φ rt φ Hi -— « 0 CQ Φ Φ rt ø J X ø PJ ø ø 0 Ω 0 d i

0 ø 3 d μ- μ- Φ 0 Φ rt ft ^><; ^

TJ tr 3 PJ tr ø TJ Pi rt H • μ- μ- Ω J 8 -¹ φ 0 J 0 ^• _ 0 Hi Hi 0 ii tr μ- Pi Φ rt Φ ϋ H H rr μ- ø rt H

3 s3 J PJ Φ Φ ii Φ PJ ii ii rr rt CQ Φ 0 3 iQ Ω • ii 0 0 μ- H 0 0 ø- iQ tr Hi φ

3 μ- 0 CQ rt Hi to ii μ- tr μ- £, rr rt P. Φ d μ- μ- 3 d Ω Hi

^ Pi Φ CQ Hi Φ J -. CQ PJ • ø 0 3 μ- tr Φ ii ø IQ J Hi ø tr ω 0 ^•^ Φ Φ tr d CQ α CQ μ- _P PJ Pi φ Φ CQ μ- PJ Pi d rt 0 r φ μ- d CQ rt ct ø tr to μ- J J CQ CQ d 3 d CQ μ- ø μ-¹ K rt l ø rt μ- ii Φ ii t

PJ 0 ø d φ a Ω J - ø J J μ- Ω 3 CQ PJ Φ φ rt t-¹ CQ μ- ø J i φ ^Q Pi Ω Ω Φ <l iQ 0 0 ^ ø tr d d μ-¹ CQ J tr CQ CQ rt μ- 0 Q rt μ- rt

CQ CO . 0 ! tr rt φ μ- PJ 0 μ- ii iQ ø Ω μ- rt rt Φ d rt Φ P. ø μ- PJ tr Ω

CQ PJ • Q μ- Φ O ø 0 CQ rt rt Ω J 0 tr rt 3 μ- μ- ii ii Φ rt 0 φ PJ

PJ ^ < ø Q PJ Pi to Hi iQ Pi μ- H — - J CQ CQ PJ PJ 0 0 J rt μ- 0 g 0 ø ϋ

<! CQ -— - 0 CQ P 0 0 rt CQ i rt K 0 tr PJ TJ J CQ PJ •_^ 3 TJ ii

H tr f 0 tr μ- PJ J Ω φ Ω Hi PJ ii CQ Φ μ- • h-¹ tr • rt d "< CQ PJ d μ

S J o • ; J P. Hi ^■ : P 0 φ 0 tr ø J PJ rt < to Hi Φ μ- ii TJ CQ ^ ii Φ φ ϋ I tr H ii CQ 0 ?r ii φ Pi Pi ^ φ Φ H Φ <! Hi tr tr 0 TJ ii rt φ ∞ 2! o Q, d J rt Φ TJ rt μ- PJ μ- CQ Ω Pi PJ rt m Φ J Φ μ- tr 0 tr — 0 ii Φ o ø tr CQ tr J ø d Φ Ω H 0 rt ^>< μ- IQ O Ω tr Ω ø H" so CQ Ω

0 Pi - ii PJ CQ ii ^> Φ d μ- Ω PJ X 0 Pi μ- W μ- CQ 3 PJ o ^ Φ CQ iQ 0 φ PJ

Pi φ I rt Ω Φ _-^■ ø rt CQ 0 J 0 3 0 0 Pi Ω Φ J Ii CQ TJ 0 0 Φ CQ ø

CQ μ- 0 ii CQ 0 CQ rt iQ μ- φ CQ 3 CQ 3 3 0 ø Φ d 0 CQ ii φ 0 tr CQ CQ

Ω ø H μ- Ω Hi so μ- 0 ø Φ TJ rt Φ d 3 ct CQ rt Ω μ- rr Hi Φ μ- 0 t φ ϋ Ω HI < Ti Φ 3 ø CQ iQ rr ϋ rt ø ø 3 φ CQ Q φ φ 0 tr H ø PJ μ- Hi φ

Pi μ- 0 • rt 0 rt TJ iQ μ- S 3 Φ d tr o PJ Ω Φ ii H Hi φ PJ μ- Φ CQ >i μ- tr ϋ CQ ι-3 μ- rt tr ø μ- φ - Ω 0 PJ rt ^•<. rt Pi 0 rt CQ Ω rt rt φ rt Φ J • φ 0 Φ φ Ω φ rt Φ 3 rt i CQ 0 Hi PJ 0 CQ rr PJ ^ rt d tr μ

Φ 0 Ω 0 PJ 0 tr PJ Φ 0 CQ tr rt PJ μ- Hi H d Φ H μ- tr J φ r

P. K tr 0 PJ § *- 3 t ii Pi H¹ J TJ Φ tr tr J ø O P. ii CQ - ø Φ I-¹ t tr J Φ ø 0 rt ø ø TJ μ- μ- PJ g 0 ^• ; Φ Φ 0 0 ii μ- Hi rr PJ H" TJ Φ tr "< rt rt μ- Hi μ- 0 Pi ø rt o ø μ- tr IQ CQ H J < rt rt ø PJ J CQ Ω ^■^ ii H

Φ Q tr s3 φ J tr H Φ Ω ^ ^< ~ CQ Ω TJ Φ μ- ø- tr PJ Ω rt CQ φ o Φ s! Pi PJ d PJ 0 ø PJ X CQ — ii 0 Ω ϋ tr Φ μ- ϋ Φ d d PJ CQ CQ

W μ- H Φ 8" ø Φ J J 0 Ω s3 0 0 Φ 3 O ø ^■ ; tr μ- rt J ø φ 0 μ- P. tr . Q ^ Pi rt Hi rt 0 Φ ω 3 CQ J Pi Q Pi 3 φ ø 3 H ^> 0 I i Φ ^■ _ Φ μ- 0 rt Q TJ μ- J l-¹ TJ Φ ^ μ- J μ- CQ So Φ rt d ft⁴ J rr tr ii 3 PJ 0 rt rr μ-¹ 0 I-¹ 0 φ g ø ^ ^ PJ φ a P. t tr J ø Φ 0 3 g ii ii μ- Hi μ- Ω tr CQ φ Q μ- 0 h-

PJ rt O Pi Ω i Φ Φ TJ rt PJ ø ^ CQ Φ Φ M tr K tr to Φ

3 ΓT μ- i μ- r iQ ø tr 0 tr μ-¹ Φ φ rt μ- φ to φ 0 μ- 0 φ 0 ^■<_ rt J r tr 0 CQ 0 0 0 P. Hi μ- tr i 0 ø ø ø ø ø φ

and Hunter, E. & S. Livingstone, Edinburgh, 1970.

The diagnostic methods of this invention are predictive of proliferation and metastatic potentials in patients suffering from cancers including carcinomas of the lung, such as small cell carcinoma, large cell carcinoma, squamous carcinoma, and adenocarcinoma, stomach carcinoma, prostatic adenocarcinoma, ovarian carcinoma such as serous cystadenocarcinoma and ucinous cystadenocarcinoma, ovarian germ cell tumors, testicular carcinomas, and germ cell tumors, pancreatic adenocarcinoma, biliary adenocarcinoma, heptacellular carcinoma, renal cell adenocarcinoma, endometrial carcinoma including adenocarcinomas and mixed Mullerian tumors (carcinosarcomas) , carcinomas of the endocervix, ectocervix, and vagina such as adenocarcinoma and squamous carcinoma, basal cell carcinoma, melanoma, and skin appendage tumors, esophageal carcinoma, carcinomas of the nasopharynx and oropharynx including squamous carcinoma and adenocarcinomas, salivary gland carcinomas, brain and central nervous system tumors, including tumors of glial, neuronal, and meningeal origin, tumors of peripheral nerve, soft tissue sarcomas and sarcomas of bone and cartilage. Cells of these tumors which express increased levels of sphingosine kinase type 2, RNA or sphingosine kinase type 2 protein, have increased proliferation and decreased cell death.

The protein can be used to identify inhibitors of sphingosine kinase type 2 activity. Using an enzyme assay, natural and synthetic agents and drugs can be discovered which result in a reduction or elimination of sphingosine kinase type 2 enzymatic activity. Knowledge of the mechanism of action of the inhibitor is not necessary as long as a decrease in the activity of sphingosine kinase type 2 is detected. Inhibitors may include agents or drugs which either bind or sequester the enzyme's substrate(s) or cofactor(s), or inhibit the enzyme itself directly, for example, by irreversible binding of the agent or drug to the enzyme or indirectly, for example, by introducing an agent which binds the sphingosine kinase type 2 substrate. Agents or drugs related to the present invention may result in partial or complete inhibition of sphingosine kinase type 2 activity.

Inhibitors of sphingosine kinase type 2 include DL-threo-dihydrosphingosine (DHS) and the more recently discovered inhibitor N,N-dimethylsphingosine ("DMS") described in Edsall, L. C. et al . , (1998), Biochemistry, 37, 12892-12898. Inhibitors of sphingosine kinase type 2 may be used in the treatment or amelioration of diseases such as cancer, atherosclerosis, neurodegenerative disorders, i.e., stroke and Alzheimer's disease.

Agents which decrease the level of sphingosine kinase type 2 (i.e., in a human or an animal) or reduce or inhibit sphingosine kinase type 2 activity may be used in the therapy of any disease associated with the elevated levels of sphingosine kinase type 2 or diseases associated with increased cell proliferation, such as cancer. An increase in the level of sphingosine kinase tupe 2 is determined when the level of sphingosine kinase type 2 in a tumor cell is about 2 to 3 times the level of sphingosine kinase type 2 in the normal cell, up to about 10 to 100 times the amount of sphingosine kinase type 2 in a normal cell. Agents which decrease sphingosine kinase type 2 RNA include, but are not limited to, one or more ribozymes capable of digesting sphingosine kinase type 2 RNA, or antisense oligonucleotides capable of hybridizing to sphingosine kinase type 2 RNA, such that the translation of sphingosine kinase type 2 is inhibited or reduced resulting in a decrease in the level of sphingosine kinase type 2. These antisense oligonucleotides can be administered as DNA, as DNA entrapped in proteoliposomes containing viral envelope receptor proteins (Kanoda, Y. et al . , (1989), Science, 5, 243, 375) or as part of a vector which can be expressed in the target cell such that the antisense DNA or RNA is made. Vectors which are expressed in particular cell types are known in the art, for example, for the mammary gland. See Furth, J. Mammary Gland Biol. Neopl. , 2 , (1997), 373, for examples of conditional control of gene expression in the mammary gland.

Alternatively, the DNA can be injected along with a carrier. A carrier can be a protein such as a cytokine, for example, interleukin or a polylysine-glycoprotein carrier. Such carrier proteins and vectors and methods of using same are known in the art. In addition, the DNA could be coated onto tiny gold beads and such beads can be introduced into the skin with, for example, a gene gun (Ulmer, T. B. et al . , Science, 259, (1993) , 1745) .

Alternatively, antibodies, or compounds capable of reducing or inhibiting sphingosine kinase type 2, that is reducing or inhibiting either the expression, production or activity of sphingosine kinase type 2, such as antagonists, can be provided as an isolated and substantially purified protein, or as part of an expression vector capable of being expressed in the target cell, such that the sphingosine kinase type 2 reducing or inhibiting agent is produced. In addition, co-factors such as various ions, i.e., Ca²⁺ or factors which affect the stability of the enzyme can be administered to modulate the expression and function of sphingosine kinase type 2. These formulations can be administered by standard routes. In general, the combinations may be administered by the topical, transdermal, intraperitoneal, oral, rectal, or parenteral (e.g., intravenous, subcutaneous, or intramuscular) route. In addition, sphingosine kinase type 2 inhibiting compounds may be incorporated into biodegradable polymers being implanted in the vicinity of where drug delivery is desired, for example, at the site of a tumor so that the sphingosine kinase type 2 inhibiting compound is slowly released systemically. The biodegradable polymers and their use are described, for example, in detail in Brem et al., J. Neurosurg. , 74, (1991), 441-446. These compounds are intended to be provided to recipient subjects in an amount sufficient to effect the inhibition of sphingosine kinase type 2. Similarly, agents which are capable of negatively affecting the expression, production, stability or function of sphingosine kinase type 2 , are intended to be provided to recipient subjects in an amount sufficient to effect the inhibition of sphingosine kinase type 2. An amount is said to be sufficient to "effect" the inhibition or induction of sphingosine kinase type 2 if the dosage, route of administration, etc., of the agent are sufficient to influence such a response.

In line with the function of sphingosine kinase type 2 in cell proliferation, agents which stimulate the level of sphingosine kinase type 2, such as agonists of SPHK2, may be used in the therapy of any disease associated with a decrease of SPHK2, or a decrease in cell proliferation, wherein SPHK2 is capable of increasing such proliferation, e.g., developmental retardation.

In providing a patient with agents which modulate the expression or function of sphingosine kinase type 2 to a recipient patient, the dosage of administered agent will vary depending upon such factors as the patient's age, weight, height, sex, general medical condition, previous medical history, etc. In general, it is desirable to provide the recipient with a dosage of agent which is in the range of from about 1 pg/kg to 10 mg/kg (body weight of patient) , although a lower or higher dosage may be administered.

A composition is said to be "pharmacologically acceptable" if its administration can be tolerated by a recipient patient. Such an agent is said to be administered in a "therapeutically effective amount", if the amount administered is physiologically significant. An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient patient. The compounds of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington' s Pharmaceutical Sciences, 16th Ed., Osol, A. ed. , Mack Easton PA. (1980) . In order to form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the above-described compounds together with a suitable amount of carrier vehicle.

Additional pharmaceutical methods may be employed to control the duration of action. Control release preparations may be achieved through the use of polymers to complex or absorb the compounds. The controlled delivery may be exercised by selecting appropriate macromolecules (for example, polyesters, polyamino acids, polyvinyl, pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the method of incorporation in order to control release. Another possible method to control the duration of action by controlled release preparations is to incorporate the compounds of the present invention into particles of a polymeric material, such as polyesters, polyamino acids, hydrogels, poly (lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, interfacial polymerization, for example, hydroxymethylcellulose for gelatin-microcapsules and poly (methylmethacrylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions . Such techniques are disclosed in Remington's Pharmaceutical Sciences (1980) . The present invention also provides kits for use in the diagnostic or therapeutic methods described above. Kits according to this aspect of the invention may comprise one or more containers, such as vials, tubes, ampules, bottles and the like, which may comprise one or more of the compositions of the invention.

The kits of the present invention may comprise one or more compounds or compositions of the present invention, and one or more excipients, diluents or adjuvants.

Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the present invention.

The following Materials and Methods were used in the Examples described below.

EXAMPLES

Materials

SPP, sphingosine, and N,N-dimethylsphingosine were from Biomol Research Laboratory Inc. (Plymouth Meeting, PA) . All other lipids were purchased from Avanti Polar Lipids

(Birmingham, AL) . [g-32P]ATP (3000 Ci/mmol) was purchased from Amersham (Arlington Heights, IL) . Poly-L-lysine and collagen were obtained from Boehringer Mannheim (Indianapolis, IN) . Restriction enzymes were obtained from New England Biolabs

(Beverly, MA) . Poly (A) + RNA blots of multiple mouse adult tissues were purchased from Clontech (Palo Alto, CA) .

"Lipofectamine PLUS" and "Lipofectamine" were obtained from Life Technologies, Inc. (Gaithersburg, MD) .

Example 1 : cDNA Cloning of Murine Sphingosine Kinase Type 2 (mSPHK2)

BLAST searches of the EST database identified a mouse EST clone (GenBank accession number AA839233) which had significant homology to conserved domains of mSPHKla (Kohama, T., Olivera, A., Edsall, L. , Nagiec, M. M. , Dickson, R. and Spiegel, S., J. Biol. Chem., 273, (1998), 23721-23728), yet had substantial sequence differences. Using this EST, a second isoform of SPHK, denoted mSPHK2, was cloned by two different PCR approaches .

In the first approach, the method PCR cloning from a mouse cDNA library (Stratagene) was used. Approximately 1 x 10⁶ phage were plated on twenty 150 mm plates, plaques were collected, and plasmids were isolated using standard procedures (Ausubel, F. M. , Brent, R. , Kingston, R. E., Moore, D. D., Smith, J. A., Seidman, J. G. , and Struhl, K. , Current Protocols in Molecular Biology, Green Publishing Associates and Wiley- Interscience, New York (1987)) . An initial PCR reaction was carried out with a sequence specific primer (M-3-1, 5' -CCTGGGTGCACCTGCGCCTGTATTGG (SEQ ID NO: 1)) and the M13 reverse primer. The longest PCR products were gel purified and used as the template for a second PCR which contained a sequence specific antisense primer (M-3-2,

5' -CCAGTCTTGGGGCAGTGGAGAGCC-3' (SEQ ID NO: 2)) and the T3 primer. The final PCR products were subcloned using a "TOPO TA" cloning kit (Invitrogen) and then sequenced. Platinum high fidelity DNA poly erase (Life Technologies) was used for the PCR amplifications with the following cycling parameters: 30 cycles of 94°C for 30 seconds, 55°C for 45 seconds, and 70°C for 2 minutes with a final primer extension at 72°C for 5 minutes.

In a second approach, 5 'RACE PCR was performed with the 5 'RACE System for Rapid Amplification of cDNA ends according to the manufacturer's protocol (Life Technologies). Poly (A) + RNA was isolated from Swiss 3T3 fibroblasts using a Quick Prep mRNA Purification kit (Pharmacia) . The first strand cDNA was synthesized at 42 °C for 50 minutes with 5 mg of Swiss 3T3 poly (A) + RNA using a target antisense primer designed from the sequence of AA839233 (m-GSPl, 5 ' -AGGTAGAGGCTTCTGG (SEQ ID NO: 3)) and Superscript II reverse transcriptase (Life Technologies) . Two consecutive PCR reactions using this cDNA as a template and LA Taq polymerase (TaKaRa) were carried out as follows: first PCR, 94 °C for 2 minutes followed by 30 cycles of 94 °C for 1 minute, 55°C for 1 minute, 72°C for 2 minutes, and primer extension at 72°C for 5 minutes with 5 'RACE Abridged Anchor Primer, 5 ' -GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG (SEQ ID NO: 4) and the target specific antisense primer m-GSP2, 5 ^■ -GCGATGGGTGAAAGCTGAGCTG (SEQ ID NO: 5); for the second PCR, the same conditions were employed, except that the annealing temperature was 65°C, with Abridged Universal Amplification Primer (AUAP) , 5 ' -GGCCACGCGTCGACTAGTAC (SEQ ID NO: 6) and m-GSP3, 5 ' -AGTCTCCAGTCAGCTCTGGACC (SEQ ID NO: 7). PCR products were cloned into pCR2.1 and sequenced. The PCR products were subcloned into pCR3.1 and pcDNA 3 expression vectors.

Example 2 : cDNA Cloning of Human Sphingosine Kinase-2 (hSPHK2)„ Poly (A) ⁺ RNA from HEK293 cells was used for a 5 'RACE reaction. Target specific antisense primers (h-GSPl, 5'- CCCACTCACTCAGGCT (SEQ ID NO: 8); h-GSP2, 5'- GAAGGACAGCCCAGCTTCAGAG (SEQ ID NO: 9); and h-GSP3, 5'- ATTGACCAATAGAAGCAACC (SEQ ID NO: 10)) were designed according to the sequence of a human EST clone (accession number AA.295570) . A first strand cDNA was synthesized with 5 μg of HEK293 mRNA and h-GSPl. This cDNA was used as a template in an initial PCR reaction using 5 'RACE Abridged Anchor Primer and h-GSP2. Then, a nested PCR was carried out using the AUAP primer and h-GSP3. The resulting PCR products were cloned and sequenced as described above.

Example 3 : Overexpression and Activity of SPHK2 Human embryonic kidney cells (HEK293, ATCC CRL-1573) and NIH 3T3 fibroblasts (ATCC CRL-1658) were cultured as described in Olivera, A., Kohama, T., Edsall, L. C, Nava, V., Cuvillier, O., Poulton, S., and Spiegel, S., J. Cell Biol. , 147, (1999), 545-558. HEK293 cells were seeded at 6 x 10⁵ per well in poly-L-lysine coated 6 well plates. After 24 hours, cells were transfected with 1 μg of vector alone or with vectors containing sphingosine kinase constructs and 6 μl of "Lipofectamine PLUS" reagent plus 4 μl of "Lipofectamine" reagent per well. One to three days after transfection, cells were harvested and lysed by freeze-thawing as described in Kohama, T., Olivera, A., Edsall, L., Nagiec, M. M. , Dickson, R., and Spiegel, S., J. Biol. Chem. , 273, (1998), 23722-23728. In some experiments, cell lysates were fractionated into cytosol and membrane fractions by centrifugation at 100,000 x g for 60 minutes. SPHK activity was determined in the presence of sphingosine, prepared as a complex with 4 mg/ml BSA, and [g-32P]ATP in kinase buffer (Olivera, A. and Spiegel, S. in Methods in Molecular Biology, (Bird, I.M. ed.), (1998), Vol. 105, 233-242, Humana Pres, Inc., Totowa, N.J.), containing 200 mM KCl, unless indicated otherwise. 32P-SPP was separated by TLC and quantified with a phosphoimager as previously described.

Example 4 : Lipid Extraction and Measurement of SPP Cells were washed with PBS and scraped in 1 ml of methanol containing 2.5 μl concentrated HCl . Lipids were extracted by adding 2 ml chloroform/lM NaCl (1:1, v/v) and 100 μl 3N NaOH and phases separated. The basic aqueous phase containing SPP, and devoid of sphingosine, ceramide, and the majority of phospholipids, was transferred to a siliconized glass tube. The organic phase was re-extracted with 1 ml methanol/lM NaCl (1:1, v/v) plus 50 μl 3N NaOH, and the aqueous fractions were combined. Mass measurement on SPP in the aqueous phase and total phospholipids in the organic phase were measured exactly as described in Edsall, L. C, Pirianov, G. G. , and Spiegel, S., J. Neurosci. , 17, (1997) 6952-6960; Edsall, L. C. , and Spiegel, S., Anal. Biochem. , 272, (1999) 80-86). Example 5 : Northern Blotting Analysis

Poly (A) + RNA blots containing 2 μg of poly (A) + RNA per lane from multiple adult mouse and human tissues and mouse embryos were purchased from Clontech. Blots were hybridized with the 1.2 kb PSTI fragment of mouse EST AA389187 (mSPHKl probe), the 1.5 kb EcoRI fragment of pCR3. l-mSPHK2, or the 0.3 kb PvuII fragment of pCR3.1-hSPHKl, after gel-purification and labeling with [a-32P]dCTP. Hybridization in "ExpressHyb" buffer (Clontech) at 65 'C overnight was carried out according to the manufacturer's protocol. Blots were reprobed with b-actin as a loading control (Clontech) . Bands were quantified using a Molecular Dynamics Phosphoimager.

Results

Cloning of Type 2 Sphingosine Kinase

Blast searches of the EST data base identified several ESTs that displayed significant homology to the recently cloned mSPHKla sequence. Specific primers were designed from the sequences of these ESTs and were used to clone a new type of mouse and human sphingosine kinase (named mSPHK2 and hSPHK2) by the approaches of PCR cloning from a mouse brain cDNA library and 5' -RACE PCR.

ClustalW alignment of the amino acid sequences of mSPHK2 and hSPHK2 is shown in Fig. 1A. The open reading frames of mSPHK2 and hSPHK2 encode polypeptides of 617 and' 618 amino acids, respectively, with 83% identity and 90% similarity. Five highly conserved regions (Cl to C5) , identified previously in SPHKls (Kohama, T. , Olivera, A., Edsall, L. , Nagiec, M. M. , Dickson, R., Spiegel, S., J. Biol. Chem., 273, (1998) 23722- 23728), are also present in both type 2 kinases . Interestingly, the invariant GGKGK positively charged motif in the Cl domain of SPHK1 is modified to GGRGL in SPHK2, suggesting that it may not be part of the ATP binding site as previously proposed (Kohama, T., Olivera, A., Edsall, L., Nagiec, M. M. , Dickson, R. , Spiegel, S., J. Biol. Chem. , 273 , (1998) 23722-23728) . A motif search also revealed that a region beginning just before the conserved Cl domains of mSPHK2 and hSPHK2 (amino acid 147 to 284) also has homology to the diacylglycerol kinase catalytic site.

Compared to SPHK1, both SPHK2s encode much larger proteins containing 236 additional amino acids (Fig IB) . Moreover, their sequences diverge considerably from SPHK1 in the center and at the amino termini. However, after amino acid 140 of mSPHK2 , the sequences of type 1 and type 2 SPHK have a large degree of similarity. These sequences (amino acid 9 to 226 for mSPHKl; 141 to 360 for mSPHK2) , which encompass domains Cl to C4, have 47% identity and 79% similarity (Fig. IB) . In the C terminal portion of the proteins there are also large homologous regions, which include the C5 domain, from amino acid 227 to 381 for mSPHKl and 480 to 617 for mSPHK2, with 43% identity and 78% similarity (Fig. IB) . The divergence in these domains suggests that SPHK2 probably did not arise as a simple gene duplication event.

Tissue Distribution of Sphingosine Kinase Type 2 The tissue distribution of SPHK2 mRNA expression in adult mouse was compared to that of SPHK1 by Northern blotting (Fig. 2A) . In most tissues, including adult liver, heart, kidney, testis and brain, a predominant 3.1 kb SPHK2 mRNA species was detected, indicating ubiquitous expression. However, the level of expression was markedly variable and was highest in adult liver and heart and barely detectable in skeletal muscle and spleen (Fig. 2A) . In contrast, the expression pattern of mSPHKl was quite different, with the highest mRNA expression in adult lung, spleen, and liver, although expression in the liver did not predominate as with mSPHK2. mSPHKl and mSPHK2 were both temporally and differentially expressed during embryonic development. mSPHKl was expressed highly at mouse embryonic day 7 (E7) and decreased dramatically after Ell (Fig. 2B) . In contrast, at E7, mSPHK2 expression was much lower than mSPHKl, and gradually increased up to E17. The hSPHK2 2.8 kb mRNA transcript was mainly expressed in adult kidney, liver and brain, with much lower expression in other tissues (Fig. 2C) . Interestingly, expression of SPHK2 in human kidney was very high and relatively much lower in the mouse, while the opposite pattern held for the liver.

Activity of Recombinant Sphingosine Kinase Type 2 To investigate whether mSPHK2 and hSPHK2 encode bona fide SPHKs, HEK293 cells were transiently transfected with expression vectors containing the corresponding cDNAs . Because previous studies have indicated that SPHK might be present in cells in both soluble and membrane-associated forms (Olivera, A., and Spiegel, S., Nature, 365, (1993) 557-560; Banno, Y. , Kato, M. , Hara, A., and Nozawa, Y. , Biochem. J. , 335 , (1998) 301-304; Buehrer, B. M. , and Bell, R. M. , J. Biol. Chem. , 267, 3154-3159; Oivera, A. Rosenthal, J. , and Spiegel, S., Anal. Biochem. , 223 , (1994) 306-312; Ghosh, T. K. , Bian, J., and Gill, D. L., J. Biol. Chem., 269, (1994), 22628-22635), recombinant SPHK2 activity was measured both in cytosol and in membrane fractions of transfected cells. As previously described in Kohama, T., Olivera, A., Edsall, L., Nagiec, M. M., Dickson, R., and Spiegel, S., J. Biol. Chem. , 273, (1998) 23722-23728, untreated or vector transfected HEK 293 cells have low levels of SPHK activity (Fig. 3A) . Twenty four hours after transfection with mSPHK2 and hSPHK2, in vi tro SPHK activity was increased by 20 and 35 fold, respectively, and then decreased thereafter (Fig.3A). In contrast, SPHK activity from cells transfected with mSPHKl was much higher, 610-fold more than basal levels 24 hours after transfection and remaining at this level for at least 3 more days (data not shown) . As in HEK293 cells, transfection of NIH 3T3 fibroblasts with mSPHKl resulted in much higher SPHK activity than with mSPHK2. It was previously found that, similar to untransfected cells, the majority of SPHK activity in cells transfected with mSPHKl was cytosolic (Kohama, T. , Olivera, A., Edsall, L., Nagiec, M. M. , Dickson, R., and Spiegel, S., J. Biol. Chem.. 273, (1998) 23722-23728) . Similarly, in cells transfected with either mSPHK2 or hSPHK2, 17% and 26%, respectively, of the SPHK activity was membrane-associated (Fig. 3B) , although Kyte-Doolittle hydropathy plots did not suggest the presence of hydrophobic membrane-spanning domains.

Transfection of HEK 293 cells with mSPHK2 and hSPHK2 also resulted in 2.2- and 3.3-fold increases in SPP, the product formed by SPHK, respectively (Fig.3C) , was in agreement with previous studies of sphingolipid metabolite levels after transfections with mSPHKla showing a lack of correlation of fold increases in levels and in vitro enzyme activity (Kohama, T., Olivera, A., Edsall, L., Nagiec, M. M. , Dickson, R. , and Spiegel, S., J. Biol. Chem., 273, (1998) 23722-23728; Olivera, A., Kohama, T., Edsall, L. C, Nava, V., Cuvillier, O. , Poulton, S., and Spiegel, S., J. Cell Biol. , 147, (1999), 545- 558) .

Characteristics of Recombinant mSPHK2 Subs tra te specif i ci tγ

Although SPHK2 is highly homologous to SPHK1, there are substantial sequence differences. Therefore, it was of interest to compare their enzymatic properties. Typical Michaelis-Menten kinetics were observed for recombinant SPHK2 (data not shown) . The Km for D-erythro-sphingosine as substrate is 3.4 μM, almost identical to the Km previously found for SPHK1 (Olivera, A., Kohama, T. , Tu, Z., Milstien, S., and Spiegel, S., J. Biol. Chem. , 273, (1998), 12576-12583). Although the naturally occurring D-eryth-ro-sphingosine isomer was the best substrate for SPHK1 (Kohama, T., Olivera, A., Edsall, L., Nagiec, M. M. , Dickson, R. , and Spiegel, S., J. Biol. Chem., 273, (1998) 23722-23728) , D-eryt ro-dihydrosphingosine was a better substrate for SPHK2 than D-erythro-sphingosine (Fig. 4A) . Moreover, although D,L-threo-dihydrosphingosine and phytosphingosine were not phosphorylated at all by SPHK1, they were significantly phosphorylated by SPHK2, albeit much less efficiently than sphingosine. Like SPHK1, other lipids including N,N-dimethylsphingosine (DMS) , C2- or C16-ceramide, diacylglycerol, and phosphatidylinositol, were not phosphorylated by SPHK2 (Fig. 6A) , suggesting high specificity for the sphingoid base .

DMS and DHS have previously been shown to be a potent competitive inhibitors of SPHK1 (Edsall, L. C. , Van Brocklyn, J. R., Cuvillier, O., Kleuser, B., and Spiegel, S., Biochemistry, 37, (1998), 12892-12898) and have been used to block increases in intracellular SPP levels resulting from various physiological stimuli (Olivera, A., and Spiegel, S., Nature , 365, (1993), 557-560 ;^' Cuvillier, 0., Pirianov, G. , Kleuser, B., Vanek, P. G. , Coo, 0. A., Gutkind, S., and Spiegel, S., Nature , 381, (1996), 800-803; Edsall, L. C, Pirianov, G. G., and Spiegel, S., J. Neurosci, 17, (1997), 6952-6960; Meyer zu Heringdorf, D., Lass, H. , Alemany, R. , Laser, K. T., Neumann, E., Zhang, C, Schmidt, M. , Rauen, U. , Jakobs, K. H. , and van Koppen, C. J. , EMBO J. , 17, 2830-2837; Choi, O, H., Kim, J.-H., and Kinet, J.-P., Nature, 380, (1996), 634-636; Melendez, A., Floto, R. A., Gillooly, D. J. , Harnett, M. M., and Allen, J. M., J. Biol. Chem. , 273, 9393-9402; Machwate, M., Rodan, S. B., Rodan, G. A., and Harada, S. I., Mol . Pharmacol . , 54, (1998) , 70-77) . However, because DHS is a substrate for SPHK2 and the product, dihydro SPP, is as potent as SPP in binding to and activating cell surface SPP EDG-1 family receptors, it cannot be used as a tool to investigate the role of SPHK2. Thus, it was important to characterize the inhibitory potential of the non-substrate DMS on SPHK2. Surprisingly, it was found that although DMS was also a potent inhibitor of SPHK2 (Fig. 4B) , it acted in a non-competitive manner (Fig. 4C and Fig. 4D) . The Ki for DMS with SPHK2 was 12 μM, slightly higher than the Ki of 4 μM with SPHKl, making it a useful tool to inhibit both types of SPHK. mSPHK2 had highest activity in the neutral pH range from 6.5 to 8 with optimal activity at pH 7.5 (Fig. 5A) , a pH dependency similar to that of SPHKl (data not shown) . The activity decreased markedly at pH values below and above this range .

Effects of KCl and NaCl

Most of the SPHK activity in human platelets is membrane-associated and extractable with 1 M NaCl (Banno, Y. , Kato, M. , Hara, A. and Nozawa, Y., Biochem. J. , 335, (1998), 301-304) . Furthermore, the salt extractable SPHK from platelets has different properties than the cytosolic enzyme. It was thus of interest to determine the effect of high salt concentrations on recombinant SPHKl and SPHK2. Interestingly, it was found that high ionic strength had completely opposite effects on their activities. SPHKl was inhibited markedly inhibited by either NaCl and KCl with each causing 50% inhibition at a concentration of 200 mM (Fig. 5B) . In contrast, SPHK2 activity was dramatically stimulated by increasing the salt concentration, with a maximal effect at a concentration of 400 mM, although KCl was much more effective than NaCl. However, above this concentration, SPHK2 activity decreased sharply although remaining elevated even at 1 M salt (Fig. 5C) . Thus, the activities of SPHKl and SPHK2 have completely opposite responses to changes in ionic strength. Kinetic analysis of mSPHK2 in the presence and absence of high concentrations of salt indicated that the Km for sphingosine was unaltered but the V ax was increased (Fig. 5D and Fig. 5E) . The physiological significance of these observations remains to be determined but it might be related to different subcellular localizations . Substrate presentation

Because sphingolipids are highly lipophilic, in in vi tro

SPHK assays, sphingosine is usually presented in micellar form with Triton X-100 or as a complex with BSA (Olivera, A., Rosenthal, J. , and Spiegel, S., J. Cell. Biochem. , 60, (1996), 529-537; Olivera, A., Barlow, K. D., and Spiegel, S., Methods Enzymol , 311, (2000) , 215-223) . Furthermore, detergents such as Triton X-100 have been shown to stimulate the activity of SPHK in rat brain extracts (Buehrer, B. M. , and Bell, R. M. , J. Biol. Chem., 267, (1992) , 3154-3159) and the enzyme from rat kidney (Olivera, A., Kohama, T., Tu, Z., Milstien, and Spiegel, S., J. Biol. Chem. , 273, (1998), 12576-12583), and it was previously found that the stability of rat kidney SPHK was increased in the presence of certain detergents (Olivera, A. , Kohama, T., Tu, Z., Milstien, and Spiegel, S., J. Biol. Chem. , 273 , (1998) , 12576-12583) . However, when the effect of increasing concentrations of Triton X-100 on the activities of SPHKl and SPHK2 were compared, some unexpected results were found. Concentrations of detergent below 0.005% had no effect, but at higher concentrations, SPHK2 activity was inhibited and SPHKl activity was markedly increased (Fig. 6A) . At a concentration of Triton X-100 of 0.5%, SPHKl activity was increased by more than 4 fold while SPHK2 was almost completely inhibited.

Interestingly, increasing the BSA concentration from the usual SPHK assay conditions with sphingosine-BSA complex as a substrate, i.e. 0.2 mg/ml BSA, caused a concentration-dependent inhibition of SPHK2 activity without affecting SPHKl activity (Fig. 6B) . Therefore, when measuring SPHK activity in cell or tissue extracts, the method of substrate preparation, whether in mixed micelles or in BSA complexes, must be carefully optimized because the differential effects of Triton X-100 and BSA on activity could yield different results depending on the relative expression of the two types of SPHK. Effects of phospholipids

Acidic phospholipids, particularly phosphatidylserine, and phosphatidic acid and phosphatidylinositol, and cardiolipin to a lesser extent, induce a dose-dependent increase in SPHK activity Swiss 3T3 fibroblast lysates, whereas neutral phospholipids had no effect (Olivera, A., Rosenthal, J., and Spiegel, S., J. Cell. Biochem., 60 , (1996), 529-537). In agreement, recombinant SPHKl and SPHK2 were stimulated by phosphatidylserine; the activity of both was maximally increased 4-fold at a concentration of 40 μg/ml (Fig. 6C) and inhibited by higher concentrations in a dose-dependent manner. These effects of phosphatidylserine appeared to be specific since other phospholipids, including phosphatidylcholine, had no effect on the enzyme activity. In contrast, the activities of the three major forms of SPHK in human platelets are not affected by phosphatidylserine (Banno, Y. , Kato, M. , Hara, A, and Nozawa, Y. , Biochem. J. , 335, (1998), 301-304).

The mechanism by which phosphatidylserine enhances the enzymatic activity of SPHK is not yet understood. One possibility is that phosphatidylserine possesses unique membrane-structuring properties which better present the substrate, sphingosine. A second possibility is that SPHK contains determinants that specifically recognize the structure of the serine headgroup and that these determinants may only become exposed upon interaction of SPHK with membranes. In this regard, the molecular basis for the remarkable specificity of protein kinase C for phosphatidylserine has been the subject of much debate. However, recent data reveal that lipid structure and not membrane structure is the major determinant in the regulation of protein kinase C by phosphatidylserine (Johnson, J. E., Zimmerman, M. L., Daleke, D. L., and Newton, A. C, Biochemistry, 37, (1998), 12020- 12025) .

The presence of multiple ESTs in the database with significant homologies to SPHKl as well as the identification of several genes in S. cerevisiae encoding different SPHKs

(Nagiec, M. M., Skrzypek, M., Nagiec, E. E., Lester, R. L., and Dickson, R. C, J. Biol. Chem. , 273, (1998), 19437-19442) suggests that there may be a large and important SPHK gene family. Although SPHK2 has a high degree of homology to SPHKl, especially in the previously identified conserved domains identified in type 1 SPHKs (Kohama, T., Olivera, A., Edsall, L., Nagiec, M. M. , Dickson, R. , and Spiegel, S., J. Biol. Chem. , 273, (1998), 23722-23728), it is much larger (65.2 and 65.6 kDa for SPHKl and SPHK2, respectively versus 42.4 kDa for mSPHKla) and contains an additional 236 amino acids. Furthermore, its differential tissue expression, temporal developmental expression, cellular localization, and kinetic properties in response to increasing ionic strength and detergents, are completely different from SPHKl, suggesting that it most likely has a different function and regulates levels of SPP in a different manner than SPHKl which is known to play a prominent role in regulating cell growth and survival. Thus, type 2 SPHK is considered to be involved in regulation of some of the numerous biological responses attributed to SPP, such as angiogenesis and allergic responses.

Sequence for GenBank 1 EMBC Bank Accession No. bankit325787

1 aattcggcac gagggaggac cgagtaaacc gaggcttcca gaaccaaaga gaagtcagcc 61 tgaggaaagg gctgggaccc ggagcctctc tggcctttcc ccgtccctgc tctaacactc 121 tccaggggta aagggaccgg agaatcagag acatgatcgg agcttgctgg acgagtcgcg 181 tggtgactct ctggccgcac gccgaccgct tctcggtggc tcgcggagga cccggtgggc 241 tgtgtgtcgg agcctccgaa gtagctggaa tcaccgtctt tcaacacttg gcctggctct 301 gccatttaaa gttgtgatct tggaggctgg tccaggagct gaccacaagc caagagccta 361 ggagtgcttg ggactgaacc agggtcatgg σcccaccacc actactgcca gtggctgcca 421 gcactccaat cctgcacggc gagtttggtt cctacccggc caacggccca cggtttgccc 481 tcaccctcac aacacaagcc ctacacatac agcgactacg cccaaagcca gaagcccggc 541 cccgagatgg tctagtctct ctggatgagg tctcgggctg tggcaccctg cagagccgta 601 gccccgagga cactgcagcc tacttctgca tctacacct cccacgtggc cgtcgagggg 661 gccggcgcag agctacgcgg accttccggg cggatggggc caccacttat gaggagaatc 721 gtgcagaggc ccagcgctgg gccactgccc tcacgtgtct cctccgagga gtgcctctgt 781 caggggacca ggaaatcacc cctgaattgσ tgccccggaa gcccaggctg ctcatattgg 841 tcaatccctt tggggggcgg ggcctggcct ggcagcgctg tatggaccac gtggtgccaa 901 tgatctctga agctgggctg tccttcaacc tcatacagac agaacgaσag aaccatgccc 961 gtgagctggt gcaggggtta agcctgagtg agtgggaagg cattgtcact gtgtctggag 1021 acgggσtgct ttacgaggtg ctgaatgggc tccttgatcg gccagactgg gaggatgccg 1081 tgσggatgcσ σattggtgtc ctcccctgtg gatcgggcaa tgcgctagct ggggcggtga 1141 gccatcatgg cgggtttgag caggttgtcg gtgttgacct gttgctcaac tgctcgcttc 1201 ttctσtgccg tggtggcagc catcctctgg acttgctctc tgtgacgcta gcctcgggat 1261 cccgctgttt ttccttcctg tcagtggcct ggggattctt gtcagatgtg gacattcaca 1321 gtgagcgctt cagggccctg ggcagcgctc gattcacact gggtgcagtg ctaggcctgg 1381 cctcgttgca tacctaccgt ggacgcctct cctacctccc cgctaccaca gaaccagcct 1441 tgcccatccc aggccacagt ctgcctcgag ccaagtcaga actagtcttg gctccagccc 1501 cagcσcccgc cgccacccac tσgcctctac atcgatctgt gtctgacctg cccctgcccc 1561 ttccccagcc tgccttggtc tcccctggct cccctgagcc cctgcctgac ctgtccctca 1621 atggtggtgg tccagagctg actggagact ggggaggagc tggggatgca cctσtgtccc 1681 cagacccact gctgccttca tcccccaacg ctctcaaaac agctcagctt tcacccatcg 1741 ctgaagggcc cσcagaaatg ccagσatctt cggggttcct gcctcccacc cacagtgccσ 1801 cagaagcctc tacctggggc ccagtggacc acctcctccc tcccctgggc tctccactgσ 1861 cccaagactg ggtgacaata gagggggagt ttgtactcat gttgggcatc ttgacgagcσ 1921 acσtctgcgc agacctgatg gcagccccac atgcacgctt tgatgatggc gttgtgcacc 1981 tgtgttgggt gcggagcggc atctcacggg ctgcacttct acgσattttt ctggccatgg 2041 agcatggaaa ccacttcagc ctgggctgcc cccatctggg ctatgctgca gcacgtgcct 2101 tccgccttga accactcacg σctcgtggσc tgctcactgt agatggggag ttagtggagt

2161 atgggccaat acaggcgcag gtgcacccag gtctcgccac gctgctcact gggcctgcag

2221 gtcaaaagcc acaagcctga acgagcσtaa aagcatggcg agttggtgga accagcgccc

2281 cataggctaa gatctatcat ttacaggtag aagtggggcσ σgcactcaga actgtgagga

2341 gggtggagag tggtcctgac cctcagttcc cagaggacct agaggctcga gggtggggcc

2401 tgcctttctt gatgtccaat gatggggcct ggaatgtatg agctagcaag gcttcttcag

2461 cttattgacc agσcagggtt tcttcttgcc tactccggtg σctctacttg actggccaat

2521 cagcccttga ggggcaggtt cccccaggtg gtccccagat ttgcactaat gttcctcccc

2581 tggccagtta gggatgggat gttctgtgtc ttgtgtgtcc ctctccctag tctaaaaagc

2641 aattgaaaag gtctatgcaa taaaggttgt tgcttccctc taaaaaaaaa aaaaaaaa

Sequence for Gen. Bank 1 EMBC Bank Accession No.bankit325752

1 gccaccatgg ccccgccσcc accgccactg gctgccagca ccccgctcct cσatggcgag 61 tttggctcct acccagcccg aggccσacgc tttgccctca cccttacatc gcaggccctg 121 cacatacagc ggctgcgccc caaacctgaa gccaggcccc ggggtggcσt ggtcccgttg 181 gccgaggtct caggctgctg caccctgcga agccgcagcc σctcagactc agcggcctac 241 ttctgcatct acacctaccc tcggggccgg cgcggggcσc ggcgcagagc σactcgcacc 301 ttccgggcag atggggccgc cacctacgaa gagaaccgtg ccgaggccca gcgctgggcc 361 actgccctca σctgtctgct ccgaggactg cσactgcccg gggatgggga gatcacccct 421 gacctgctac ctcggccgcc ccggttgctt ctattggtca atccctttgg gggtcggggc 481 ctggcctggc agtggtgtaa gaaccacgtg cttcccatga tctctgaagc tgggctgtcc 541 ttcaacctca tccagacaga acgacagaac cacgcσcggg agctggtcca ggggctgagc 601 ctgagtgagt gggatggcat cgtcacggtc tcgggagacg ggctgctcca tgaggtgctg 661 aacgggctcc tagatcgccc tgactgggag gaagctgtga agatgcctgt gggcatcctc 721 ccctgcggct cgggcaacgc gctggccgga gcagtgaacc agcacggggg atttgagcca 781 gccctgggcc tcgacctgtt gctcaactgc tσactgttgc tgtgccgggg tggtggccac 841 ccactggacc tgctctccgt gacgctggcσ tcgggctccc gctgtttctc cttcctgtct 901 gtggcctggg gcttcgtgtc agatgtggat atccagagcg agcgcttcag ggccttgggc 961 agtgcccgct tcacactggg cacggtgctg ggcctcgcca cactgcacac ctaccgcgga 1021 cgcctctcct acctccccgc cactgtggaa cσtgcctcgc ccacccctgc ccatagcctg 1081 cctcgtgcca agtcggagct gaccctaacc ccagacccag ccccgcccat ggcccactca 1141 cccctgcatc gttctgtgtc tgacctgcct σttcccctgc cccagcctgc cctggcctct 1201 cσtggctcgc cagaacccct gcccatcctg tccctcaacg gtgggggccc agagctggct 1261 ggggactggg gtggggctgg ggatgctccg ctgtccccgg acσcactgct gtcttcacct 1321 cctggctctc cσaaggcagc tctacactca cccgtctccg aaggggcccc cgtaattccc 1381 ccatσctctg ggctcccact tcccacσcct gatgcσσggg taggggcctσ cgacctgcggc 1441 ccgccσgacc aσctgσtgcσ tσcgctgggσ accσσgσtgc cσσσagaσtg gtgacgctg 1501 gagggggact ttgtgctcat gttggccatc tcgcccagcc acctaggσgc tgacctggtg 1561 gσagctccgc atgcgσgctt cgacgacggc ctggtgcacc tgtgσtgggt gσgtagcggc 1621 atctcgcggg ctgcgctgct gcgccttttc ttggccatgg agcgtggtag ccacttcagc

1681 ctgggctgtc cgcagctggg ctacgccgcg gcccgtgcct tccgcctaga gcσgσtσaca

1741 cσacgcggcg tgctcacagt ggacggggag caggtggagt atgggccgct acaggcacag

1801 atgcaσcctg gcatcggtac aσtgctcaσt gggcctcσtg gσtgcccggg gcgggagccc

1861 tgaaactaaa σaagcttggt aσσσgccggg ggcggggcct acattσσaat ggggcggagc

1921 ttgagctagg gggtgtggcσ tggσtgctag agttgtggtg gcaggggcσc tggccccgtc

1981 tcaggattgc gctcgctttσ atgggaccag acgtgatgct ggaaggtggg cgtcgtcaσg

2041 gttaaagaga aatgggσtcg tcccgagggt agtgcσtgat σaatgagggc ggggcctggα

2101 gtctgatctg gggccgccct tacggggσag ggctcagtσσ tgaσgσttgc caσctgσtcc

2161 taccσggcca ggatggctga gggσggagtc tattttacgσ gtcgccσaat gaσaggacct

2221 ggaatgtact ggσtggggta ggcctcagtg agtcggσσgg tσagggccσg cagcctcgσσ

2281 σcatccactc cggtgcctcc atttagctgg ccaatσagσσ caggaggggc aggttcccσg

2341 gggσcggcgc taggatttgc aσtaatgttc σtctcσσcgc

SEQ ID NO. 14

Amino aσid sequences of human SPHK2 MAPPPPPLAASTPLLHGEFGSYPARGPRFALTLTSQALHIQRLRPKPEARPRGGLVPLAEVSGCGTLRSR SPSDSAAYFCIYTYPRGRRGARRRATRTFRADGAATYEENRAEAQR ATALTCLLRGLPLPGDGEITPDL LPRPPRLLLLVNPFGGRGLAWQ CK_[fflVLPMISEAGLSFNLIQTERQimARELVQGLSLSEWDGIVTVSG DGLLHEVLNGLLDRPD EEAVKMPVGILPCGSGNALAGAVNQHGGFEPALGLDLLLNCSLLLCRGGGHPL DLLSVTIASGSRCFSFLSVA GFVSDVUIQSERFRALGSARFTLGTVLGLATLHTYRGRLSYLPATVEPA SPTPAHSLPRAKSΞLTLTPDPAPPN-AHSPLHRSVSDLPLPLPQPAI-ASPGSPEPLPILSLNGGGPELAGD WGGAGDAPLSPDPLLSSPPGSPKAALHSPVSEGAPVIPPSSGLPLPTPDARVGASTCGPPDHLLPPLGTP LPPDWVTLEGDFVIiMI_AISPSHLGADLVAAPHARFDDGLVHLCWVRSGlSRAALLRLF--AMERGSHFSLG CPQLGYAAARAFRLEPLTPRGVLTVDGEQVEYGPLQAQMHPGIGTLLTGPPGCPGREP

SEQ ID NO. 12

Amino Acid Sequenσe of mouse SPHK2 MAPPPLLPVAASTPILHGEFGSYPANGPRFALTLTTQALHIQRLRPKPEARPRDGLVSLDEVSGCGTLQS RSPEDTAAYFCIYTYPRGRRGGRRRATRTFRADGATTYEENRAEAQRWATALTCLLRGVPLSGDQEITPE LLPRKPRLLILVNPFGGRGIΛWQRCIΦHVVPMISEAGLSFNLIQTERQIIΗARELVQGLSLSEWEGIVTVS GDGLLYEVLNGLLDRPDWEDAVRMPIGVLPCGSGNALAGAVSHHGGFEQVVGVDLLLNCSLLLCRGGSHP LDLLSVTLASGSRCFSFLSVA GFLSDVDIHSERFRALGSARFTLGAVLGLASLHTYRGRLSYLPATTEP ALPIPGHSLPRAKSELVLAPAPAPAATHSPLHRSVSDLPLPLPQPALVSPGSPEPLPDLSLNGGGPELTG D GGAGDAPLSPDPLLPSSPNALKTAQLSPIAEGPPEMPASSGFLPPTHSAPEAST GPVDHLLPPLGSP LPQDWTIEGEFVL LGILTSHLCADIJVIAAPHARFDDGVVHLCWWSGI^ CPHLGYAAARAFRLEPLTPRGLLTVDGELVEYGPIQAQVHPGLATLLTGPAGQKPQA SEQUENCE LISTING

<110> SANKYO COMPANY. LIMITED GEORGETOWN UNIVERSITY

<120> Mamπalian Sphingosiπa Kinase Type 2 Isoforms. Cloning, Expression and Methods of Use Thereof

<130> 00170PCT HG

<140> <141>

<150> US βO/194, 318 <151> 2000-04-03

<160> 15

<170> PatentIn Ver. 2.0

210> 1

<211> 26

<212> DNA

<2.3> Mus musculus

<400> 1 cctggfftEca cctgcgcctff tattgg 26

<210> 2

<211> 24

<212> DNA

<213> Mus musculus

<400> 2 eeagtcttgg ggcagtggag agec 24 <210> 3

<211> 16

<212> DNA

<213> Mus musculus

<400> 3 aggtagaggc ttctgg IS

<210> 4

<211> 36 212> DNA

<213> Ar ificial Sequence

<220>

<223> Description of Artificial Sequence: 5^* RACE Abridged Anchor Primer

<220>

<221> mo ified_base <222> (24).. (25) <223> i

<220>

<221> modified_basβ <222> (29).. (30) <223> i

<220>

<221> modified_base <222 (34).. (35) <223> i

<400> 4 ggccacgcgt cgactagtac gggπngggπn gggπng 36

<210> 5 <211> 22

<212> DNA

<213> Mus musculus

<400> 5 gcgatgggtg aaagctgaec tg 22

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Description of Artificial Sequence: Abridged Universal Amplifica ion Primer

<400> β ggccacgcgt egaetagtac 20

<210> 7

<211> 22

<212> DNA

<213> Mus musculus

<400> 7 agtctccagt cagctetgga ec 22

<210> 8

<211> 16

<212> DNA

<2.3> Homo sapiens

<400> 8 cccactcact caggct 16 <210> 9

<2.1> 22

<212> DNA

<213> Homo sa iens

<400> 9 gaaggacagc eeagcttcag ag 22

<210> 10

<211> 20

<212> DNA

<213> Homo sapiens

<400> 10 attgaccaat agaageaacc 20

<210> 11

<211> 2698

<212> DNA

<213> Mus musculus

<220>

<221> CDS

<222> (387) .. (2237)

<300>

<302> Molecular cloning and functional characteriza ion of a novel mammalian sphingosine kinase type 2 isoform <303> J. Biol. Chem. <304> 275 <305> 26

<306> 19513-19520 <308> AF245448

<400> 11 aatteggeac gagggaggac cgagtaaacc gaggcttcca gaaccaaaga gaagtcagce 60

tgaggaaagg getgggaece ggagcctctc tggcctttcc ecgtecctgc tctaacactc 120

tecaggggta aagggacegg agaatcagag acatgategg agcttgctgg acgagtcgcg 180

tggtgactct ctggccgcae gccgacegct tctcggtggc tcgcggagga cccggtgggc 240

tgtgtgtegg agccteegaa gtagctggaa tcaccgtctt tcaaeacttg gcctggctct 300

gecatttaaa gttgtgatct tggaggetgg tecaggaget gaccaeaagc caagageeta 360

ggagtget g ggactgaacc agggtc atg gee cca cca cea eta etg eca gtg 413

Met Ala Pro Pro Pro Leu Leu Pro Val 1 5

get gee age act cca ate etg cac ggc gag ttt ggt tee tac ccg gee 461 Ala Ala Ser Thr Pro Me Leu His Gly Glu Phe Sly Ser Tyr Pro Aia 10 15 20 25

aac ggc cea egg ttt gee etc ace etc aea aca caa gcc eta cac ata 509 Asπ Gly Pro Arg Phe Ala Leu Thr Leu Thr Thr Gin Ala Leu His I le 30 35 40

cag cga eta egc cca aag eca gas gcc egg eec cga gat gg eta gtc 557 Gin Arg Leu Arg Pro Lys Pro GJu Ala Arg Pro Arg Asp Gly Leu Val 45 50 55

ct etg gat gag gtc teg ggc tgt ggc ace etg cag age cgt age ece 605 Ser Leu Asp Glu Val Ser Gly Cys Gly Thr Leu Gin Ser Arg Ser Pro 60 65 70

gag gac act gca ee tac ttc tge ate tac ace tac eca cgt ggc cgt 653 Glu Asp Thr Ala Ala Tyr Phe Cys lie Tyr Thr Tyr Pro Arg Gly Arg 75 80 85

cga ggg ggc egg egc aga get aeg egg ace ttc egg gcg gat ggg gcc 701 Arg Gly Gly Arg Arg Arg Ala Thr Arg Thr Phe Arg Ala Asp Gly Aia 90 95 100 105

ace act tat gag gag aat cgt gca gag gee eag egc tgg gcc act gcc 749 Thr Thr Tyr Glu Glu Asn Arg Ala Glu Ala Gin Arg Trp Ala Thr Ala HO 115 120

etc acg tgt etc etc cga gga gtg cct etg tea ggg gac cag gaa ate 797 Leu Thr Cys Leu Leu Arg Gly Val Pro Leu Ser Gly Asp Gin Glu I le 125 130 135

ace cct gaa ttg etg ecc egg aag ecc agg etg etc ata ttg gtc aat 845 Thr Pro iu Leu Leu Pro Arg Lys Pro Arg Leu Lau 1 le Leu Val Asn 140 145 150

ecc ttt ggg ggg egg ggc etg gcc tgg eag egc tgt atg gac cac gtg 893 Pro Phe Gly Gly Arg Gly Leu Ala Trp Gin Arg Cys Met Asp His Val 155 160 185

gtg cca atg ate tet gaa get ggg etg tec ttc aac etc ata cag aca 941 Val Pro Met I le Ser Glu Ala Gly Leu Ser Phe Asn Leu I le Gin Thr 170 175 180 185

gaa cga cag aac eat gcc cgt gag etg gtg cag ggg tta age etg agt 989 Glu Arg Gin Asn His Ala Arg Glu Leu Val Gin Gly Leu Ser Leu Ser 190 195 200

gag tgg gaa ggc att gtc act gtg tet gga gac ggg etg ctt tac gag 1037 Glu Trp Glu Gly lie Val Thr Val Ser Gly Asp Gly Leu Leu Tyr Giu 205 210 215

gtg etg aat ggg etc ctt gat egg cea gac tgg gag gat gcc gtg egg 1085 Val Leu Asn Gly Leu Leu Asp Arg Pro Asp Trp Glu Asp Ala Val Arg 220 225 230

atg eec att ggt gtc etc ecc tgt gga teg ggc aat geg eta get ggg 1133 Met Pro i le Gly Val Leu Pro Cys Gly Ser Gly Aβn Ala Leu Ala Gly 235 240 245 geg gtg age eat cat gge ggg ttt gag eag gtt gte ggt gtt gae etg 1181 Ala Val Ser His His Gly Gly Phe Glu Gin Val Val Gly Val Asp Leu 250 255 260 265

ttg etc aac tge teg eft ctt etc tge cgt ggt ggc age eat cct etg 1229 Leu Leu Asn Cys Ser Leu Leu Leu Cys Arg Gly Gly Ser His Pro Leu 270 275 280

gae ttg cte tet gtg aeg eta gcc teg gga tec egc tgt ttt tee ttc 1277 Asp Leu Leu Ser Val Thr Leu Ala Ser Gly- Ser Arg Cys Phe Ser Phe 285 290 295

etg tea gtg gee tgg gga ttc ttg tea gat gtg gae att cac agt gag 1325 Leu Ser Val Ala Trp Gly Phe Leu Ser Asp Val Asp lie His Ser Glu 300 305 310

egc ttc agg gcc etg ggc age get ega ttc aca etg ggt gca gtg eta 1373 Arg Phe Arg Ala Leu Gly Ser Ala Arg Phe Thr Leu Gly Ala Va l Leu 315 320 325

ggc etg gee teg ttg eat ace tac egt gga egc cte tec tac etc ecc 1 21 Gly Leu Ala Ser Leu His Thr Tyr Arg Gly Arg Leu Ser Tyr Leu Pro 330 335 340 345

get ace aca gaa eca gcc ttg cee ate cca ggc eae agt etg cct ega 1469 Ala Thr Thr Glu Pro Ala Leu Pro He Pro Gly His Ser Leu Pro Arg 350 355 360

gcc aag tea gaa eta gte ttg get cea gcc cca gcc cee gee gcc ace 1517 Ala Lys Ser Glu Leu Val Leu Aia Pro Ala Pro Ala Pro Ala. Ala Thr 365 370 375

cac teg eet eta cat cga tet gtg tet gac etg ecc etg ecc ctt cee 1565 His Ser Pro Leu His Arg Ser Val Ser Asp Leu Pre Leu Pro Leu Pro 380 385 390

cag ect gcc ttg gtc tec cct ggc tec cct gag ecc etg eet gae etg 1613 Gin Pro Ala Leu Val Ser Pro Gly Ser Pro Glu Pro Leu Pro Asp Leu 395 400 405

tec etc aat ggt ggt ggt cea gag etg act gga gac tgg gga gga get 1661 Ser Leu Asn Gly Gly Gly Pro Giu Leu Thr Gly Asp Trp Gly Gly Ala 410 415 420 425

ggg gat gca cct etg tec cca gae eca etg etg cct tea tec ecc aac 1709 Gly Asp Ala Pro Leu Ser Pro Asp Pro Leu Leu Pro Ser Ser Pro Asn 430 435 440

get etc aaa aca get eag ctt tea ecc ate get gaa ggg ecc cca gaa 1757 Ala Leu Lys Thr Ala Gin Leu Ser Pro lie Ala Giu Gly Pro Pro Glu 445 450 455

atg cca gca tet teg ggg ttc etg eet ecc ace eae agt gcc eca gaa 1805 Met Pro Ala Ser Ser Gly Phe Leu Pro Pro Thr His Ser Ala Pro Glu 460 465 470

gcc tet aee tgg ggc cca gtg gac cac etc etc cct ecc etg ggc tet 1853 Ala Ser Thr Trp Gly Pro Val Asp His Leu Leu Pro Pro Leu Gly Ser 475 480 485

eca etg cee eaa gac tgg. gtg aca ata gag ggg gag ttt gta cte atg 1901 Pro Leu Pro Gin Asp Trp Val Thr lie Glu Gly Glu Phe Val Leu Met 490 495 500 505

ttg ggc ate ttg aeg age eae etc tge gca gac etg atg gca gee cea 1949 Leu Gly lie Leu Thr Ser His Leu Cys Ala Asp Leu Met Aia Ala Pro 510 515 520

cat gca egc ttt gat gat ggc gtt gtg cac etg tgt tgg gtg egg age 1997 His Aia Arg Phe Asp Asp Gly Val Val His Leu Cys Trp Val Arg Ser 525 530 535

ggc ate tea egg get gca ctt eta egc att ttt etg gee atg gag cat 2045 Gly I le Ser Arg Aia Ala Leu Leu Arg I le Phe Leu Ala Met Giu His 540 545 550 gga aac cac ttc age etg ggc tge cee eat etg gge tat get gca gca 2093 Gly Asn His Phe Ser Leu Gly Cyβ Pro His Leu Gly Tyr Ala Ala Ala 555 560 565

cgt gee ttc egc ctt gaa cca ete aeg cct cgt gge etg cte act gta 2141 Arg Ala Phe Arg Leu Glu Pro Leu Thr Pro Arg Gly Leu Leu Thr Val 570 575 580 585

gat ggg gag tta gtg gag tat ggg cca ata cag geg cag gtg cac cca 2189 Asp Gly Glu Leu Val Giu Tyr Gly Pro lie Gin Ala Gin Val His Pro 590 595 600

ggt cte gee aeg etg etc act ggg cct gca ggt caa aag eca caa gcc 2237 Gly Leu Ala Thr Leu Leu Thr Gly Pro Ala Gly Gin Lys Pro Gin Ala 605 610 615

tgaacgagce taaaagcatg gegagttggt ggaaceagcg ccceataggc taagatetat 2297

catttacagg tagaagtggg geccgcaetc agaactgtga ggagggtgga gagtggtcct 2357

gaecctcagt teeeagagga eetagagget cgagggtggg gcctgcettt ettgaigtce 2417

aatgatgggg cctggaatgt atgagctagc aaggcttctt cagcttattg accageeagg 2477

gtttcttctt gectactecg gtgcctetae ttgactggcc aateagccct tgaggggcag 2537

gttcccccag gtggtcccea gatttgcact aatgtteetc ccctggecag ttagggatgg 2597

gatgttetgt gtcttgtgtg tceetetccc tagtetaaaa agcaattgaa aaggtctatg 2657

eaataaaggt tgttgcttcc ctctaaaaaa aaaaaaaaaa a 2698

<210> 12 <211> 617 212> PRT <213> Mus musculus <400> 12

Met Ala Pro Pro Pro Leu Leu Pro Val Ala Ala Ser Thr Pro lie Leu 1 5 10 15

His Gly Glu Phe Gly Ser Tyr Pro Ala Asn Gly Pro Arg Phe Ala Leu 20 25 30

Thr Leu Thr Thr Gin Ala Leu His Me Gin Arg Leu Arg Pro Lys Pro 35 40 45

Glu Ala Arg Pro Arg Asp Gly Leu Val Ser Leu Asp Glu Val Ser Gly 50 55 60

Cys Gly Thr Leu Gin Ser Arg Ser Pro Glu Asp Thr Aia Ala Tyr Phe 65 70 75 80

Cys lie Tyr Thr Tyr Pro Arg Gly Arg Arg Gly Gly Arg Arg Arg Ala 85 90 95

Thr Arg Thr Phe Arg Ala Asp Gly Ala Thr Thr Tyr Glu Glu Asn Arg 100 105 110

Ala Glu Ala Gin Arg Trp Ala Thr Ala Leu Thr Cys Leu Leu Arg Gly 115 120 125

Val Pro Leu Ser Gly A3p. Gin Glu I le Thr Pro Glu Leu Leu Pro Arg 130 135 140

Lys Pro Arg Leu Leu I le Leu Val Asn Pro Phe Gly Gly Arg Gfy Leu 145 150 155 160

Ala Trp Gin Arg Cys Met Asp His Val Val Pro Met Me Ser Glu Ala 165 170 175

Gly Leu Ser Phe Asn Leu Me Gin Thr Glu Arg Gin Asn His Ala Arg 180 185 190

Glu Leu Val Gin Gly Leu Ser Leu Ser Glu Trp Glu Gly Me Val Thr 195 200 205

Val Ser Gly Asp Gly Leu Leu Tyr Glu Val Leu Asn Gly Leu Leu Asp 210 215 220

Arg Pro Asp Trp Glu Asp Ala Val Arg Met Pro He Gly Val Leu Pro 225 230 235 240

Cys Gly Ser Gly Asn Ala Leu Ala Gly Ala Val Ser His His Gly Gly 245 250 255

Phe Glu Gin Val Vaf Gly Val Asp Leu Leu Leu Asn Cys Ser Leu Leu 260 265 270

Leu Cys Arg Gly Gly Ser His Pro Leu Asp Leu Leu Ser Val Thr Leu 275 280 285

Ala Ser Gly Ser Arg Cys Phe Ser Phe Leu Ser Val Ala Trp Gly Phe 290 295 300

Leu Ser Asp Val Asp I le His Ser GJ-u Arg Phe Arg Ala Leu Gly Ser 305 310 315 320

Ala Arg Phe Thr Leu Gly Ala Val Leu Gly Leu Ala Ser Leu His Thr 325 330 335

Tyr Arg Gly Arg Leu Ser Tyr Leu Pro Ala Thr Thr Glu Pro Ala Leu 340- 345 350

Pro Me Pro Gly His Ser Leu Pro Arg Ala Lys Ser Glu Leu Val Leu 355 360 365

Ala Pro Ala Pro Ala Pro Ala Ala Thr His Ser Pro Leu His Arg Ser 370 375 380

Val Ser Asp Leu Pro Leu Pro Leu Pro Gin Pro Ala Leu Val Ser Pro 385 390 395 400 Gly Ser Pro Glu Pro Leu Pro Asp Leu Ser Leu Asn Gly Gly Gly Pro 405 410 415

Glu Leu Thr Gly Asp Trp Gly Gly Ala Gly Asp Ala Pro Leu Ser Pro 420 425 430

Asp Pro Leu Leu Pro Ser Ser Pro Asn Ala Leu Lys Thr Ala Gin Leu 435 440 445

Ser Pro Me Ala Glu Gly Pro Pro Giu Met Pro Ala Ser Ser Gly Phe 450 455 460

Leu Pro Pro Thr His Ser AJa Pro Glu Ala Ser Thr Trp Gly Pro Val 465 470 475 480

Asp His Leu Leu Pro Pro Leu Gly Ser Pro Leu Pro Gin Asp Trp Val 485 490 495

Thr Me Glu Gly Glu Phe Val Leu Met Leu Gly Me Leu Thr Ser His 500 505 510

Leu Cys Ala Asp Leu Met Ala Ala Pro His Ala Arg Phe Asp Asp Gly 515 520 525

Val Val His Leu Cys Trp Val Arg Ser Gly Me Ser Arg Ala Ala Leu 530 535 540

Leu Arg lie Phe Leu Ala Met Glu His Giy Asn His Phe Ser Leu Gly 545 550 555 560

Cys Pro His Leu Gly Tyr Ala Ala Ala Arg Ala Phe Arg Leu Glu Pro 565 570 575

Leu Thr Pro Arg Gly Leu Leu Thr Val Asp Gly Glu Leu Val Glu Tyr

580 585 590

Gly Pro Me Gin Ala Gin Val His Pro Gly Leu Ala Thr Leu Leu Thr

595 600 605 Gly Pro Ala Gly Gin Lys Pro Gin Ala 670 615

<210> 13

<211> 2380

<212> DNA

<213> Homo sapiens

<220>

<221> CDS

<222> (7).. (1860)

<300>

<302 Molecular cloning and func ional characte ization of a novel mamnalian sphingosine kinase type 2 isoform <303> J. Biol. Chem. <304> 275 <305> 26

<306> 19513-19520 <308> AF245447

<400> 13 geeaee atg gee ecg ecc cca ccg cca etg get gcc age aee ecg etc 48 Met Ala Pro Pro Pro Pro Pro Leu Ala Ala Ser Thr Pro Leu 1 5 10

etc cat gge gag ttt ggc tee tac eca gcc cga gge eca egc ttt gee 96 Leu His Gly Glu Phe Gly Ser Tyr Pro Ala Arg Gly Pro Arg Phe Ala 15 20 25 30

etc ace ctt aca teg cag gee etg eae ata cag egg etg egc cee aaa 144 Leu Thr Lau Thr Ser Gin Ala Leu His Me Gin Arg Leu Arg Pro Lys 35 40 45

cct gaa gcc agg cee egg ggt .ggc etg gtc ecg ttg gcc gag gtc tea 192 Pro Glu Ala Arg Pro Arg Gly Gly Leu Val Pro Lau Ala Glu Val Ser 50 55 60

ggc tge tge aec etg ega age ege age ecc tea gac tea geg gee tac 240 Gly Cys Cys Thr Leu Arg Ser Arg Ser Pro Ser Asp Ser Ala Ala Tyr 65 70 75

ttc tge ate tac ace tac eet egg ggc egg egc ggg gee egg egc aga 288 Phe Cys I le Tyr Thr Tyr Pro Arg Gly Arg Arg Gly Ala Arg Arg Arg 80 85 90

gcc act ege aec ttc egg gca gat ggg gee gcc aec tae gaa gag aac 336 Ala Thr Arg Thr Phe Arg Ala Asp Gly Ala Ala Thr Tyr Glu Glu ten 95 100 105 110

cgt gee gag gee eag egc tgg gee act gcc etc aec tgt etg etc cga 384 Arg Ala Glu Ala Gin Arg Trp Ala Thr Ala Leu Thr Cys Leu Leu Arg 115 120 125

gga etg cca etg eee ggg gat ggg gag ate ace eet gac etg eta eet 432 Gly Leu Pro Leu Pro Gly Asp Gly Glu I le Thr Pro Asp Leu Leu Pro 130 135 140

egg ecg eee egg ttg ctt eta ttg gtc aat ecc ttt ggg ggt egg ggc 480 Arg Pro Pro Arg Leu Leu Leu Leu Val Asn Pro Phe Gly Gly Arg Gly 145 150 155

etg gee tgg eag tgg tgt aag aae cac gtg ctt eee atg ate tet gaa 528 Leu Ala Trp Gin Trp Cys Lys Asn His Val Leu Pro Met Me Ser Glu 160 165 170

get ggg etg tec ttc aac etc ate cag aca gaa ega cag aae eae gcc 576 Ala Gly Leu Ser Phe Asn Leu 1 le Gin Thr Glu Arg Gin Asn His Ala 175 180 185 190

egg gag etg gte cag ggg etg age etg agt gag tgg gat ggc ate gtc 624 Arg Glu Leu Val Gin Gly Leu Ser Leu Ser Glu Trp Asp Gly I le Val 195 200 205 aeg gtc teg gga gac ggg etg cte eat gag gtg etg aac ggg etc eta 672 Thr Val Ser Gly Asp Gly Leu Leu His Glu Val Leu Asn Gly Leu Leu 210 215 220

gat ege cct gac tgg gag gaa get gtg aag atg cct gtg ggc ate cte 720 Asp Arg Pro Asp Trp Glu Glu Aia Val Lys Met Pro Val Giy I le Leu 225 230 235

ecc tge ggc teg ggc aae geg etg gee gga gca gtg aac cag eae ggg 768 Pro Cys Gly Ser Gly Asn Ala Leu Ala Gly Aia Val Asn Gin His Gly 240 245 250

gga ttt gag cca gee etg gge etc gae etg ttg ete aac tge tea etg 816 ly Phe Glu Pro Ala Leu Gly Leu Asp Leu Leu Leu Asn Cys Ser Leu 255 260 265 270

ttg etg tge 'egg ggt ggt ggc eae cca etg gac etg etc tec gtg aeg 864 Leu Leu Cys Arg Gly Gly Gly His Pro Leu Asp Leu Leu Ser Val Thr 275 280 285

etg gee teg gge tec ege tgt tte tec tte etg tet gtg gcc tgg ggc 91 Leu Ala Ser Gly Ser Arg Cys Phe Ser Phe Leu Ser Val Ala Trp Gly 290 295 300

ttc gtg tea gat gtg gat ate eag age gag egc tte agg gcc ttg ggc 960 Phe Val Ser Asp Val Asp Me Gin Ser Glu Arg Phe Arg Ala Leu Gly 305 310 315

agt gcc egc ttc aca etg ggc aeg gtg etg ggc etc gee aea etg eae 1008 Ser Ala Arg Phe Thr Leu Gly Thr Val Leu Giy. Leu Ala Thr Leu His 320 325 330

aec tac egc gga ege etc tee tae etc eee gcc act gtg gaa eet gee 1056 Thr Tyr Arg Gly Arg Leu Ser Tyr Lau Pro Ala Thr Val Glu Pro Ala 335 340 345 350

teg ecc ace cct gcc cat age etg eet cgt gee aag teg gag etg aec 1104 Ser Pro Thr Pro Ala His Ser Leu Pro Arg Ala Lys Ser Glu Leu Thr 355 360 365

eta ace cca gae eca gee ceg cee atg gee eae tea eee etg cat cgt 152 Leu Thr Pro Asp Pro Ala Pro Pro Met Ala His Ser Pro Leu His Arg 370 375 380

tet gtg tet gae etg cct ett eee etg eee eag cct gee etg gee tet 1200 Ser Val Ser Asp Leu Pro Leu Pro Leu Pro Gin Pro Ala Leu Ala Ser 385 390 395

eet gge teg eca gaa ecc etg ecc ate etg tee cte aac ggt ggg ggc 1248 Pro Gly Ser Pro Glu Pro Leu Pro I le Leu Ser Leu Asn Gly Gly Gly 400 405 410

cea gag etg get ggg gac tgg ggt ggg get ggg gat get ecg etg tee 1296 Pro Glu Leu Ala Gly Asp Trp Gly Gly Ala Gly Asp Aia Pro Leu Ser 415 420 425 430

ecg gae eea etg etg tet tea cct cct ggc tet ecc aag gca get eta 1344 Pro Asp Pro Leu Leu Ser Ser Pro Pro Gly Ser Pro Lys Ala Ala Leu 435 440 445

cac tea ecc gtc tee gaa ggg gee ecc gta att ecc eea tec tet ggg 1392 His Ser Pro Vai Ser Glu Gly Ala Pro Val Me Pro Pro Ser Ser Gly 450 455 460

ete eca ett eee ace cct gat gee egg gta ggg gcc tec ace tge ggc 1440 Leu Pro Leu Pro Thr Pro Asp Ala Arg Val Gly Ala Ser Thr Cys Gly 465 470 . 475

ceg cee gac cac etg etg cct ecg etg ggc ace ceg etg cee eea gac 1488 Pro Pro Asp His Leu Leu Pro Pro Leu Gly Thr Pro Leu Pro Pro Asp 480 485 490

tgg gtg aeg etg gag ggg gac ttt gtg ete atg ttg gee ate teg eee 1536 Trp Val Thr Leu Glu Gly Asp Phe Vai Leu Met Leu Ala Me Ser Pro 495 500 505 510 age cac eta ggc get gac etg gtg gca get ecg eat geg ege ttc gac 1584 Ser His Lau Gly Ala Asp Leu Val Ala Ala Pro His Ala Arg Phe Asp 515 520 525

gac ggc etg gtg cac etg tge tgg gtg cgt age gge ate teg egg get 1632 Asp Gly Leu Vat His Leu Cys Trp Val Arg Ser Gly I le Ser Arg Ala 530 535 540

geg etg etg egc ett ttc ttg gee atg gag cgt ggt age cac tte age 1680 Ala Leu Leu Arg Leu Phe Leu Aia Met Glu Arg Gly Smr His Phe Ser 545 550 555

etg ggc tgt ceg cag etg gge tae gcc geg gee cgt gee ttc egc eta 1728 Leu Gly Cys Pro Gin Leu Gly Tyr Ala Ala Ala Arg Ala Phe Arg Leu 560 565 570

gag ecg etc aea cca egc gge gtg etc aca gtg gac ggg gag eag gtg 1776 Glu Pro Leu Thr Pro Arg Gly Val Leu Thr Vai Asp Gly Glu Gin Val 575 580 585 590

gag tat ggg ecg eta cag gca eag atg cac eet ggc ate ggt aca etg 1824 Glu Tyr Gly Pro Leu Gin Ala Gin Met His Pro Gly Me Gly Thr Leu 595 600 605

etc act ggg eet cct ggc tge ecg ggg egg gag ecc tgaaactaaa 1870 Leu Thr Gly Pro Pro Gly Cys Pro Gly Arg Glu Pro 610 615

caagcttggt aeccgccggg ggeggggcct aeatteeaat ggggcggage ttgagctagg 1930

gggtgtggee tggetgctag agttgtggtg gcaggggece tggccccgte tcaggattgc 1990

getcgetttc atgggaecag acgtgatget ggaaggtggg egtcgtcacg gttaaagaga 2050

aat ggctcg tcccgagggt agtgcctgat eaatgagggc ggggcctggc gtetgatctg 2110

gggccgecet tacggggeag ggctcagtcc tgaegettgc cacctgetec tacccggcca 2170 ggatggctga gggcggagte tattttacge gtcgeceaat gacaggaect ggaatgtact 2230

SSCtggggta ggcctcagtg agtcggccgg tcagggeecg eageetegec eeatecacte 2290

eggtgcctcc atttagctgg ccaatcagec eaggagggge aggttecccg gggecggege 2350

taggatttge aetaatgtte etctcceege 2380

<210> 14

<211> 618

<212> PRT

<213> Homo sapiens

<400> 14

Met Aia Pro Pro Pro Pro Pro Leu Ala Ala Ser Thr Pro Leu Leu His 1 5 10 15

Gly Glu Phe Gly Ser Tyr Pro Ala Arg Gly Pro Arg Phe Ala Leu Thr 20 25 30

Leu Thr Ser Gin Ala Leu His I le Gin Arg Leu Arg Pro Lys Pro Glu 35 40 45

Ala Arg Pro Arg Gly Gly Leu Val Pro Leu Ala Glu Val Ser Gly Cys 50 55 60

Cys Thr Leu Arg Ser Arg Ser Pro Ser Asp Ser Ala Ala Tyr Phe Cys 85 70 75 80

I iβ Tyr Thr Tyr Pro Arg Gly Arg Arg Gly Ala Arg Arg Arg Ala Thr 85 90 85

Arg Thr Phe Arg Ala Asp Gly Aia Ala Thr Tyr Glu Glu Asn Arg Ala 100 105 110

Glu Ala Gin Arg Trp Ala Thr Aia Leu Thr Cys Leu Leu Arg Gly Leu 115 120 125 Pro Lsu Pro Gly Asp Gly Glu Me Thr Pro Asp Leu Leu Pro Arg Pro 130 135 140

Pro Arg Leu Leu Leu Leu Val Asn Pro Phe Gly Gly Arg Gly Leu Ala 145 150 155 160

Trp Gin Trp Cys Lys Asn His Val Leu Pro Met Me Ser Glu Aia Gly 165 170 175

Leu Ser Phe Asn Leu 11« Gin Thr Glu Arg Gin Asn His Ala Arg Glu 180 185 190

Leu Val Gin Gly Leu Ser Leu Ser Glu Trp Asp Gly Me Val Thr Val 195 200 205

Ser Gly Asp Gly Leu Leu His Glu Val Leu Asn Gly Leu Leu Asp Arg 210 215 220

Pro Asp Trp Glu Glu Ala Val Lys Met Pro Val Gly lie Leu Pro Cys 225 230 235 240

Gly Ser Gly Asn Ala Leu Aia Gly Ala Val Asn Gin His Gly Gly Phe 245 250 255

Glu Pro Al Leu Gly Leu Asp Leu Leu Leu Asn Cys Ser Leu Leu Leu 260 265 270

Cys Arg Gly Gly Gfy His Pro Leu Asp Leu Leu Ser Val Thr Lau Ala 275 280 285

Ser Gly Ser Arg Cys Phe Ser Phe Leu Ser Val Ala Trp Gly Phe Val 290 295 300

Ser Asp Val Asp Me Gin Ser Glu Arg Phe Arg Ala Leu Gly Ser Ala 305 310 315 320

Arg Phe Thr Leu Gly Thr Val Leu Gly Leu Ala Thr Lau His Thr Tyr 325 330 335

Arg Gly Arg Leu Ser Tyr Leu Pro Ala Thr Val Glu Pro Ala Ser Pro 340 345 350

Thr Pro Ala His Ser Leu Pro Arg Ala Lys Ser Glu Leu Thr Leu Thr 355 360 365

Pro Asp Pro A/a Pro Pro Met Ala His Ser Pro Leu His Arg Ser Val 370 375 380

Ser Asp Leu Pro Leu Pro Leu Pro Gin Pro Aia Leu Aia Ser Pro Gly 385 390 395 400

Ser Pro Glu Pro Leu Pro I le Leu Ser Leu Asn Gly Gly Gly Pro Glu 405 410 415

Leu Ala Gly Asp Trp Gly Gly Ala Giy Asp Ala Pro Leu Ser Pro Asp 420 425 430

Pro Leu Leu Ser Ser Pro Pro Gly Ser Pro Lys Ala Ala Leu His Ser 435 440 445

Pro Val Ser Glu Giy Ala Pro Val Me Pro Pro Ser Ser Gly Leu Pro 450 455 460

Leu Pro Thr Pro Asp Ala Arg Val Gly Ala Ser Thr Cys Gly Pro Pro 465 470 475 480

Asp His Leu Leu Pro Pro Leu Gly Thr Pro Leu Pro Pro Asp Trp Val 485 490 . 495

Thr Leu Glu Gly Asp Phe Val Leu Met Leu Ala I le Ser Pro Ser His 500 505 510

Leu Giy Ala Asp Leu Val Ala Aia Pro His Aia Arg Phe Asp Asp Gly 515 520 525 Leu Val His Leu Cys Trp Val Arg Ser Gly t le Ser Arg Ala Ala Leu 530 535 540

Leu Arg Leu Phe Leu Ala Met Glu Arg Gly Ser His Phe Ser Leu Gly 545 550 555 560

Cys Pro Gin Leu Gly Tyr Aia Aia Ala Arg Ala Phe Arg Leu Glu Pro 565 570 575

Leu Thr Pro Arg Gly Vai Leu Thr Val Asp Gly Glu Gin Val Glu Tyr 580 585 590

Gly Pro Leu Gin Ala Gin Met His Pro Gly Me Gly Thr Leu Leu Thr

595 600 605

Gly Pro Pro Gly Cys Pro Gly Arg Glu Pro 610 615

<210> 15

<211> 388

<212> PRT

<213> Mus musculus

<300>

<302> Molecular cloning and functional characterization of uri e sphingosine kinase <303> J. Biol. Chem. <304> 273 <305> 37

<306> 23722-23728 <308> AAC81693

<400> 15

Met Trp Trp Cys Cys Val Leu Phe Val Val Glu Cys Pro Arg Gly Leu 1 5 10 15

Leu Pro Arg Pro Cys Arg Val Leu Val Leu Leu Asn Pro Gin Gly Gly 20 25 30

Lys Gly Lys Ala Leu Gin Leu Phe Gin Ser Arg Val Gin Pro Phe Leu 35 40 45

Giu Glu Ala Glu Me Thr Phe Lys Lau Me Leu Thr Glu Arg Lya Asn 50 55 60

His Ala Arg Glu Leu Val Cys Ala Glu Glu Leu Gly His Trp Asp Ala 65 70 75 80

Leu Ala Vai Met Ser Gly Asp Gly Leu Met His Glu Val Vai Aβn Gly 85 90 95

Leu Met Glu Arg Pro Asp Trp Glu Thr Ala I le Gin Lys Pro Leu Cys 100 105 110

Ser Leu Pro Gly Gly Ser Gly Asn Ala Leu Ala Aia Ser Val Asn His 115 120 125

Tyr Ala Gly Tyr Glu Gin Vai Thr Asn Glu Asp Leu Leu Me Asn Cys 130 135 140

Thr Leu Leu Leu Cys Arg Arg Arg Leu Ser Pro Met Asn Leu Leu Ser 145 150 155- 160

Leu Hie Thr Ala Ser Gly Lau Arg Leu Tyr Ser Val Leu Ser Leu Ser 165 170 175

Trp Gly Phe Val Ala Asp Val Asp Leu Glu Ser Glu Lys Tyr Arg Arg 180 135 190

Leu Gly Glu I le Arg Phe Thr Val Gly Thr Phe Phe Arg Leu Ala Ser 195 200 205

Leu Arg Me Tyr Gin Gly Gin Leu Ala Tyr Leu Pro Val Gly Thr Val 210 215 . 220 Aia Ser Lys Arg Pro Aia Ser Thr Leu Val Gin Lys Giy Pro Vai Asp 225 230 235 240

Thr His Leu Val Pro Leu Glu Glu Pro Vai Pro Ser His Trp Thr Val 245 250 255

Vai Pro Glu Gin Asp Phe Vai Leu Val Leu Val Leu Leu His Thr His 260 265. 270

Leu Ser Ser Glu Leu Phe Ala Aia Pro Met Gly Arg Cys Glu Ala Giy 275 280 . 285

Val Met His Leu Phe Tyr Val Arg Ala Gly Val Ser Arg Ala Aia Leu 290 295 300

Leu Arg Leu Phe Leu Ala Met Gin Lys Gly Lys His Met Glu Leu Asp 305 310 315 320

Cys Pro Tyr Leu Val His Vai Pro Val Val Ala Phe Arg Leu Glu Pro 325 330 335

Arg Ser Gin Arg Gly Val Phe Ser Val Asp Gly Glu Leu Met Val Cys 340 345 350

Glu Ala Val Gin Gly Gin Val His Pro Asn Tyr Leu Trp Mat Val Cys 355 . 360 365

Giy Ser Arg Asp Ala Pro Ser Gly Arg Asp Ser Arg Arg Gly Pro Pro 370 375 380

Pro Glu Glu Pro 385 It will be appreciated that the instant specification is set forth by way of illustration and not limitation, and that various modifications and changes may be made without departing from the spirit and scope of the present invention.

INDICATIONS RELATING TO DEPOSITED MICROORGANISM OR OTHER BIOLOGICAL MATERIAL

(PCT Rule 13- .s)

The indications made below relate to the deposited microorganism or other biological material referred to in the description on page 1 8 , line 27 to 33

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | [

Name of depositary institution

National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology

Address of depositary institution (including postal code and country)

1-3, Higas i 1 -chome Tsukuba-shi, Ibaraki-ken 305-8566

Date of deposit Accession Number

March 29 , 2000 FERM BP- 71 1 0

C. ADDITIONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet Q

D. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

E. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable)

The indications listed below will be submitted to the International Bureau later (specify the general nature of the indications ag., "Accession Number of Deposit")

For receiving Office use only For International Bureau use only

This sheet was received with the international application | | This sheet was received by the International Bureau on:

Authorized officer Authorized officer Λ r t

Claims (50)

WHAT IS CLAIMED IS:

1. An isolated and purified DNA which encodes a mammalian sphingosine kinase type 2 isoform.

2. The isolated and purified DNA of claim 1, which encodes a mouse sphingosine kinase type 2 isoform DNA.

3. The isolated and purified DNA of claim 1, which encodes a human sphingosine kinase type 2 isoform DNA.

4. The isolated and purified DNA of claim 2 , which encodes a peptide of 617 amino acids.

5. The isolated and purified DNA of claim 3 , which encodes a peptide of 618 amino acids.

6. A peptide encoded by the DNA of claim 4.

7. A peptide encoded by the DNA of claim 5.

8. An amino acid sequence of human SPHK2 consisting essentially of SEQ ID NO: 14.

9. An amino acid sequence of urine SPH 2 consisting essentially of SEQ ID NO: 12.

10. An isolated and purified DNA which encodes a peptide of a sphingosine kinase type 2 isoform, said DNA comprising a sequence selected from the group consisting of the sequence of Genbank Accession No. nkit325787 and the sequence of Genbank Accession No. bankit325752.

11. A recombinant DNA construct comprising:

(a) a vector and

(b) the DNA of claim 2.

12. A recombinant DNA construct comprising:

(a) a vector and

(b) the DNA of claim 3.

13. The recombinant DNA construct according to claim 11, wherein said vector is an expression vector.

14. The recombinant DNA construct according to claim 11, wherein said vector is a prokaryotic vector.

15. The recombinant DNA construct according to claim 11, wherein said vector is a eukaryotic vector.

16. The recombinant DNA construct according to claim 12 , wherein said vector is an expression vector.

17. The recombinant DNA construct according to claim 12, wherein said vector is a prokaryotic vector.

18. The recombinant DNA construct according to claim 12, wherein said vector is a eukaryotic vector.

19. A host cell transformed with the recombinant DNA construct according to claim 11.

20. The host cell according to claim 19, wherein said cell is prokaryotic.

21. The host cell according to claim 19, wherein said cell is eukaryotic .

22. A host cell transformed with the recombinant DNA construct according to claim 12.

23. The host cell according to claim 22, wherein said cell is prokaryotic .

24. The host cell according to claim 22, wherein said cell is eukaryotic.

25. A method for producing a mouse sphingosine kinase type 2 isoform peptide which comprises culturing the host cell according to claim 19, under conditions such that an isolated mouse sphingosine kinase type 2 isoform DNA is expressed and said mouse sphingosine kinase type 2 isoform peptide is thereby produced.

26. A method for producing a human sphingosine kinase type 2 isoform peptide which comprises culturing the host cell according to claim 22, under conditions such that an isolated human sphingosine kinase type 2 isoform DNA is expressed and said human sphingosine kinase type 2 isoform peptide is thereby produced.

27. A method for detecting an agent or a drug which inhibits or promotes sphingosine kinase type 2 activity comprising:

(a) providing a recombinant DNA construct according to claim 11, into a cell such that sphingosine kinase type 2 isoform is produced in said cell;

(b) adding at least one drug or agent to said cell, and

(c) detecting whether or not said drug or agent inhibits or promotes sphingosine kinase type 2 activity by measuring sphingosine kinase-dependent phosphorylation of lipids in said cells and comparing the resultant measurement to a control which did not receive said drug or agent, wherein a decrease in the amount of sphingosine kinase-dependent phosphorylation of lipids as compared to the control indicates an inhibitory drug or agent, or an increase in the amount of sphingosine kinase-dependent phosphorylation of lipids in said cell as compared to the control indicates a stimulatory drug or agen .

28. A method for detecting an agent or a drug which inhibits or promotes sphingosine kinase type 2 activity comprising:

(a) providing a recombinant DNA construct according to claim 12, into a cell such that sphingosine kinase type 2 isoform is produced in said cell;

(b) adding at least one drug or agent to said cell, and

(c) detecting whether or not said drug or agent inhibits or promotes sphingosine kinase type 2 activity by measuring sphingosine kinase-dependent phosphorylation of lipids in said cells and comparing the resultant measurement to a control which did not receive said drug or agent, wherein a decrease in the amount of sphingosine kinase-dependent phosphorylation of lipids as compared to the control indicates an inhibitory drug or agent, or an increase in the amount of sphingosine kinase-dependent phosphorylation of lipids in said cell as compared to the control indicates a stimulatory drug or agent .

29. An agent or drug detected by the method of claim 27.

30. An agent or drug detected by the method of claim 28.

31. A method of regulating a biological process in a mammal comprising administering to a mammal in need thereof a pharmaceutically effective amount of the peptide according to claim 6.

32. A method of regulating a biological process in a mammal comprising administering to a mammal in need thereof a pharmaceutically effective amount of the peptide according to claim 7.

33. The method of claim 31, wherein the biological process is selected from the group consisting of mitogenesis, apoptosis, neuronal development, chemotaxis, angiogenesis and inflammatory responses .

34. The method of claim 31, wherein the biological process is angiogenesis .

35. The method of claim 32, wherein the biological process is selected from the group consisting of mitogenesis, apoptosis, neuronal development, chemotaxis, angiogenesis and in lammatory responses .

36. The method of claim 32, wherein the biological process is angiogenesis .

37. A method for the treatment or amelioration of a disease resulting from increased cell death or decreased cell proliferation, comprising administering to a mammal in need thereof a pharmaceutically effective amount of a peptide according to claim 6.

38. A method for the treatment or amelioration of a disease resulting from increased cell death or decreased cell proliferation, comprising administering to a mammal in need thereof a pharmaceutically effective amount of a peptide according to claim 7.

39. A method for the treatment or administration of a disease resulting from decreased cell death or increased cell proliferation comprising administering to a mammal in need thereof a pharmaceutically effective amount of an antibody to a peptide according to claim 6.

40. A method for the treatment or administration of a disease resulting from decreased cell death or increased cell proliferation comprising administering to a mammal in need thereof a pharmaceutically effective amount of an antibody to a peptide according to claim 7.

41. A method for the treatment or amelioration of a disease resulting from abnormal migration or motility of cells selected from the group consisting of cancer, restenosis and diabetic neuropathy, the method comprising administering to a mammal in need thereof, a pharmaceutically effective amount of an antibody to a peptide according to claim 6.

42. A method for the treatment or amelioration of a disease resulting from abnormal migration or motility of cells selected from the group consisting of cancer, restenosis and diabetic neuropathy, the method comprising administering to a mammal in need thereof, a pharmaceutically effective amount of an antibody to a peptide according to claim 7.

43. The method of claim 41, wherein the disease is cancer.

44. The method of claim 42, wherein the disease is cancer.

45. A composition for treating or ameliorating a disease resulting from increased cell death or decreased cell proliferation comprising a pharmaceutically effective amount of a peptide according to claim 6, and a pharmaceutically acceptable carrier.

46. A composition for treating or ameliorating a disease resulting from increased cell death or decreased cell proliferation comprising a pharmaceutically effective amount of a peptide according to claim 7, and a pharmaceutically acceptable carrier.

47. A method for screening agents or drugs which reduce or eliminate sphingosine kinase type 2 activity, the method comprising detecting a decrease in sphingosine kinase type 2 enzyme activity in the presence of said agent or drug.

48. A method for detecting the presence of a sphingosine kinase type 2 isoform in a sample comprising

(i) contacting a sample with antibodies which recognize sphingosine kinase type 2; and

(ii) detecting the presence or absence of a complex formed between sphingosine kinase type 2 and antibodies specific therefor.

49. A method for detecting sphingosine kinase type 2 in a sample comprising subjecting the sample to a polymerase chain reaction and detecting for the presence of sphinosine kinase type 2.

50. A diagnostic kit for detecting sphingosine kinase type 2 RNA/cDNA in a sample comprising primers or oligonucleotides specific for sphingosine kinase type 2 RNA or cDNA suitable for hybridization to sphingosine kinase type 2 RNA or cDNA and/or amplification of sphingosine kinase type 2 sequences and suitable ancillary reagents.