WO2003082322A1

WO2003082322A1 - A method of modulating cellular activity

Info

Publication number: WO2003082322A1
Application number: PCT/AU2003/000388
Authority: WO
Inventors: Stuart M. Pitson; Pu Xia; Paul A. Moretti; Julia R. Verwey; Mathew A. Vadas; Brian W. Wattenberg
Original assignee: Medvet Science Pty. Ltd.
Priority date: 2002-03-28
Filing date: 2003-03-28
Publication date: 2003-10-09
Also published as: EP1499343A1; CA2480661A1; AU2009201639A1; EP1499343A4; JP2005526809A; AU2003215426A1

Abstract

The present invention relates generally to a method of modulating cellular activity and to agents for use therein. More particularly, the present invention provides a method of modulating cellular activity by modulating phosphorylation of sphingosine kinase and, thereby, its activation. In a related aspect, the present invention provides a method of modulating sphingosine kinase functional activity via modulation of its phosphorylation and agents for use therein. The present invention still further extends to sphingosine kinase variants and to functional derivatives, homologues or analogues, chemical equivalents and mimetics thereof exhibiting reduced and/or ablated capacity to undergo phosphorylation. The method and molecules of the present invention are useful, inter alia, in the treatment and/or prophylaxis of conditions characterised by aberrant, unwanted or otherwise inappropriate cellular and/or sphingosine kinase functional activity. The present invention is further directed to methods for identifying and/or designing agents capable of modulating sphingosine kinase phosphorylation.

Description

A METHOD OF MODULATING CELLULAR ACTIVITY

FIELD OF THE INVENTION

The present invention relates generally to a method of modulating cellular activity and to agents for use therein. More particularly, the present invention provides a method of modulating cellular activity by modulating phosphorylation of sphingosine kinase and, thereby, its activation. In a related aspect, the present invention provides a method of modulating sphingosine kinase functional activity via modulation of its phosphorylation and agents for use therein. The present invention still further extends to sphingosine kinase variants and to functional derivatives, homologues and analogues, thereof exhibiting reduced and/or ablated capacity to undergo phosphorylation. The method and molecules of the present invention are useful, mter alia, in the treatment and/or prophylaxis of conditions characterised by aberrant, unwanted or otherwise inappropriate cellular and/or sphingosine kinase functional activity. The present invention is further directed to methods for identifying and/or designing agents capable of modulating sphingosine kinase phosphorylation.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

Sphingosine kinase catalyses the formation of sphingosine 1-phosphate (SIP), a lipid messenger that plays important roles in a wide variety of mammalian cellular processes (Pyne et al. 2000; Spiegel 1999). SIP is mitogenic in various cell types and triggers a diverse range of important regulatory pathways including; mobilisation of intracellular calcium by an inositol triphosphate independent pathway (Mattie et al. 1994), activation of phospholipase D (Desai et al. 1992), inhibition of c-Jun N-terminal kinase (JNK) (Cuvilliver et al. 1998), inhibition of caspases (Cuvilliver et al. 1998), adhesion molecule expression (Xia et al. 1998), and stimulation of DNA binding activity of NF- B (Xia et al. 2002) and transcription factor activator protein-1 (AP-1) (Su et al. 1994).

In addition to its role in cellular proliferation and survival, SIP appears to have other functions in the cell. For example, recent studies have shown that SIP is an obligatory signalling intermediate in adhesion molecule expression of vascular endothelial cells (Xia et al 1998), suggesting a likely role in inflammation and atherosclerosis.

Cellular levels of SIP are largely mediated by the activity of sphingosine kinase, and to a lesser extent by its degradation by SIP lyase (Van Neldhoven et al. 2000) and SIP phosphatase (Mandala et al. 2000) activities. Basal levels of SIP in the cell are generally low (Spiegel et al. 1998), but can increase rapidly and transiently when cells are exposed to various mitogenic agents. This response is a direct consequence of an increase in sphingosine kinase activity in the cytosol and can be prevented by the addition of sphingosine kinase inhibitors. This places sphingosine kinase, and its activation, in a central and obligatory role in mediating the^'bbserved effects attributed to SIP in the cell. However, at present almost nothing is known of the mechanism(s) leading to sphingosine kinase activation.

Sphingosine kinase can be very rapidly activated by wide variety of cell agonists. While the response differs between cell types, these stimuli include TΝF (Xia et al. 1998); Pitson et al. 2000) (Fig 1), platelet-derived growth factor (Olivera et al, 1993), epidermal growth factor (Meyer zu Heringdorf et al. 1999), nerve growth factor (Rius et al, 1997), vitamin D3 (Kleuser et al. 1998), phorbol esters (Pitson et al. 2000; Buehrer et al. 1996), acetylcholine (muscarinic agonists) (Meyer zu Heringdorf et al. 1998), and crosslinking of the immunoglobulin receptors FcεRl (Choi et al. 1996) and FcγRl (Melendez et al 1998). In all cases this sphingosine kinase activation increases the V_max of the reaction while leaving the substrate affinities (K_m) unaltered. The molecular mechanisms that couple these agonists to activation of sphingosine kinase activity remain largely unknown. Accordingly, there is a need to elucidate these mechanisms and to develop methods of regulating cellular activities via regulation of the sphingosine kinase signalling pathways.

In work leading up to the present invention, the inventors have determined that phosphorylation of sphingosine kinase is essential for its activation. Further the inventors have identified the phosphorylation sites on the sphingosine kinase molecule. Further, it has been determined that phosphorylation of sphingosine kinase is performed by a proline- directed protein kinase, more particularly ERK2.

SUMMARY OF THE INVENTION

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

The subject specification contains nucleotide sequence information prepared using the programme Patentln Version 3.1, presented herein after the bibliography. Each nucleotide sequence is identified in the sequence listing by the numeric indicator <201> followed by the sequence identifier (eg. <210>1, <210>2, etc). The length, type of sequence (DNA, etc) and source organism for each nucleotide sequence is indicated by information provided in the numeric indicator fields <211>, <212> and <213>, respectively. Nucleotide sequences referred to in the specification are identified by the indicator SEQ ID NO: followed by the sequence identifier (eg. SEQ ID NO: 1 , SEQ ID NO:2, etc.). The sequence identifier referred to in the specification correlates to the information provided in numeric indicator field <400> in the sequence listing, which is followed by the sequence identifier (eg. <400>1, <400>2, etc). That is SEQ ID NO:l as detailed in the specification correlates to the sequence indicated as <400>1 in the sequence listing.

Specific mutations in amino acid sequence are represented herein as "XaaιnXaa₂" where

Xaai is the original amino acid residue before mutation, n is the residue number and Xaa is the mutant amino acid. The abbreviation "Xaa" may be the three letter or single letter amino acid code. A mutation in single letter code is represented, for example, by XιnX₂ where Xi and X₂ are the same as Xaai and Xaa₂ respectively. In terms of both the mutation and the human sphingosine kinase protein sequence in general, the amino acid residues for human sphingosine kinase are numbered with the residue serine(S) in the motif KTPASPV Q SEQ ID NO:l being numbered 225.

One aspect of the present invention provides a method of modulating sphingosine kinase functional activity, said method comprising contacting said sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of said sphingosine kinase wherein inducing or otherwise agonising said phosphorylation up-regulates said sphingosine kinase activity and inhibiting or otherwise antagonising said phosphorylation down-regulates sphingosine kinase activity.

Another aspect of the present invention provides a method of modulating human sphingosine kinase functional activity, said method comprising contacting said human sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of said human sphingosine kinase wherein inducing or otherwise agonising said phosphorylation up-regulates said human sphingosine kinase activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said human sphingosine kinase activity.

Yet another aspect of the present invention provides a method of modulating sphingosine kinase functional activity, said method comprising contacting said sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate the phosphorylation of said sphingosine kinase at S²²⁵, wherein inducing or otherwise agonising said phosphorylation up-regulates said sphingosine kinase activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said sphingosine kinase activity.

Still another aspect of the present invention is directed to a method of modulating sphingosine kinase functional activity, said method comprising contacting said sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate proline-directed protein kinase catalysed sphingosine kinase phosphorylation wherein inducing or otherwise agonising said phosphorylation up-regulates said sphingosine kinase activity and inhibiting or otherwise antagonising said phosphorylation down-regulates sphingosine kinase activity.

In a related aspect the present invention provides a method of modulating sphingosine kinase functional activity, said method comprising contacting said sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of said sphingosine kinase, which agent binds, links or otherwise associates with serine , wherein inducing or otherwise agonising said phosphorylation up-regulates said sphingosine kinase activity and inhibiting or otherwise antagonising said phosphorylation down-regulates sphingosine kinase activity.

Yet another aspect of the present invention is directed to a method of modulating cellular activity, said method comprising contacting said cell with an effective amount of an agent for a time and under conditions sufficient to modulate the phosphorylation of sphingosine kinase wherein inducing or otherwise agonising said phosphorylation up-regulates said cellular activity and inhibiting or otherwise antagonising said phosphorylation down- regulates said cellular activity.

A further aspect of the present invention is directed to a method of modulating cellular activity, said method comprising contacting said cell with an effective amount of an agent for a time and under conditions sufficient to modulate the phosphorylation of human sphingosine kinase wherein inducing or otherwise agonising said phosphorylation up- regulates said cellular activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said cellular activity.

In another further aspect, the present invention is directed to a method of modulating human cellular activity, said method comprising contacting said cell with an effective amount of an agent for a time and under conditions sufficient to modulate the phosphorylation of sphingosine kinase at S wherein inducing or otherwise agonising said phosphorylation up-regulates said human cellular activity and inl ibiting or otherwise antagonising said phosphorylation down-regulates said human cellular activity.

Yet another aspect of the present invention is directed to a method for the treatment and/or prophylaxis of a condition in a mammal, which condition is characterised by aberrant, unwanted or otherwise inappropriate cellular activity, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of sphingosine kinase wherein inducing or otherwise agonising said phosphorylation up-regulates said cellular activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said cellular activity.

In still another aspect, the present invention is directed to a method for the treatment and/or prophylaxis of a condition in a human, which condition is characterised by aberrant, unwanted or otherwise inappropriate cellular activity, said method comprising administering to said human an effective amount of an agent for a time and under conditions sufficient to modulate the phosphorylation of sphingosine kinase at S²²⁵ wherein inducing or otherwise agonising said phosphorylation up-regulates said cellular activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said cellular activity.

Another aspect of the present invention contemplates the use of an agent, as hereinbefore defined, in the manufacture of medicament for the treatment of a condition in a mammal, which condition is characterised by aberrant, unwanted or otherwise inappropriate cellular activity, wherein said agent modulates the phosphorylation of sphingosine kinase and wherein inducing or otherwise agonising said phosphorylation up-regulates said cellular activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said cellular activity.

In yet another further aspect, the present invention contemplates a pharmaceutical composition comprising the modulatory agent as hereinbefore defined together with one or more pharmaceutically acceptable carriers and/or diluents. These agents are referred to as the active ingredients.

Yet another aspect of the present invention relates to the agent as hereinbefore defined, when used in the method of the present invention. A further aspect of the present invention provides a method for detecting an agent capable of modulating the phosphorylation of sphingosine kinase or its functional equivalent or derivative thereof said method comprising contacting a cell or extract thereof containing said sphingosine kinase and phosphorylation catalyst or its functional equivalent or derivative with a putative agent and detecting an altered expression phenotype associated with said phosphorylation.

Still another aspect of the present invention is directed to agents identified in accordance with the screening method defined herein and to said agents for use in the methods of the present invention. Said agent should be understood to extend to monoclonal antibodies which bind to the phosphorylation sites of sphingosine kinase, and in particular, amino acids S²²⁵.

Still a further aspect of the present invention is directed to sphingosine kinase variants comprising a mutation in a region of said sphingosine kinase which region comprises a phosphorylation site, wherein said variant exhibits ablated or reduced phosphorylation capacity relative to wild type sphingosine kinase or a derivative, homologue, analogue, chemical equivalent or pneumatic of said sphingosine kinase variant.

In another aspect there is provided a human sphingosine kinase variant comprising an amino acid sequence with a single or multiple amino acid substitution and/or deletion of amino acids S¹⁴⁸, S¹⁸¹, Y¹⁸⁴, S²²⁵ and/or T²⁵⁰ wherein said variant exhibits ablated or reduced phosphorylation capacity relative to wild-type sphingosine kinase or a derivative, homologue, analogue, chemical equivalent or mimetic of said sphingosine kinase variant.

In yet another aspect, the present invention extends to genetically modified animals, which animals have been modified to express a sphingosine kinase variant as hereinbefore defined. Single and three letter abbreviations used throughout the specification are defined in Table 1.

TABLE 1 Single and three letter amino acid abbreviations Amino Acid Three-letter One-letter Abbreviation Symbol

Alanine Ala ~

Arginine Arg R

Asparagine Asn Ν Aspartic acid Asp D Cysteine Cys C Glutamine Gin Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine He I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine The T Tryptophan Trp w Tyrosme Tyr Y Valine Nal V Any residue Xaa X BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graphical representation of the phosphorylation of hSKl paralleling its activation. TNFα simulation of hSKl -transfected HEK293T cells resulted in an in vivo phosphorylation of hSKl which was paralleled by an increase in sphingosine kinase activity in these cells.

Figure 2 is an image of the inactivation and lack of phosphorylation, in response to TNFo of a mutant which is unable to bind TRAF2. Unlike wild-type hSKl, hSK^TB2, a hSKl mutant defective in its ability to bind TRAF2 (Xia et al. 2002) is neither activated nor phosphorylated in response to TNFα treatment of HEK293T cells. In contrast, PMA treatment of these cells results in both phosphorylation and activation of wild-type hSKl and hSK^TB2.

Figure 3 is a schematic representation of the prediction of phosphorylated amino acids in hSKl using NetPhos. Circled residues represent those amino acids predicted to be phosphorylated in hSKl (and its mouse and monkey homologues) by the NetPhos program.

Figure 4 is an image of the ablation of phosphorylation of hSKl expressing an Ser²²⁵ -» Ala mutation. Metabolic labelling of HEK293T cells transfected with wild-type hSKl, hSK^sl48A, hSK^sl81A, hSK^Y184A, hSK^S225A, hSK^T250A, or empty vector and either untreated or treated with PMA showed that only the Ser²²⁵ — » Ala mutation ablated phosphorylation of hSKl.

Figure 5 is an image of the phosphorylation of hSKl, expressing an Ser²²⁵ - Ala mutation, at adjacent sites. Metabolic labelling of HEK293T cells transfected with wild- type hSKl , hSK^S220A, hSK^S225A, or hSK^T222A and either untreated or treated with PMA again showed that only the Ser²²⁵ - Ala mutation ablated phosphorylation of hSKl. This indicated that the Ser²²⁵ -» Ala mutation directly effects phosphorylation of hSKl at Ser²²⁵ rather than indirectly effecting phosphorylation at adjacent sites (i.e. Ser²²⁰ and Thr²²²) Figure 6 is a graphical representation of the inactivation of hSK^S255A by TNFα or PMA. Unlike wild-type hSKl -transfected HEK293T cells, hSK^S255A-transfected HEK293T cells show no increase in sphingosine kinase activity following treatment with TNFα or PMA.

Figure 7 is an image of the in vitro phosphorylation of hSKl. In vitro phosphorylation of purified recombinant hSKl was examined with ERK1, ERK2 and CDK2. Quantitation of the specific activity of phosphorylation showed ERK2 phosphorylated purified recombinant hSKl with greatest efficiency.

Figure 8 is an image of the in vitro phosphorylation of hSKl by ERK2 occurring only at Ser²²⁵. In vitro phosphorylation of purified recombinant wild-type hSKl, hSK^S225A, hSK^sl48A and hSK^T222A was examined with ERK2. Only hSK^S225A was not phosphorylated by ERK2 indicating that ERK2 specifically phosphorylates hSKl at Ser²²⁵.

Figure 9 is an image of ERKl/2 phosphorylation and activation of hSKl in vivo, a and b, Agonist-induced phosphorylation and activation of hSKl in HEK293T cells is blocked by ERKl/2 pathway inhibitors. Phosphorylation and activation of hSKl in transiently transfected HEK293T cells (a) was followed by Western blot using the phospho-hSKl specific polyclonal antibodies (phospho-hSKl) and sphingosine kinase enzyme assays. Total hSKl levels were determined via the FLAG epitope, while ERKl/2 activation were followed by phospho- ERK1/2 and ERKl/2 antibodies. Phosphorylation of hSKl in untransfected HEK293T cells (b) showed similar results, c, Immunoprecipitation of ERKl/2 and hSKl. HEK293T cells overexpressing either wild type hSKl or hSKl^TB2 were treated with TNFc or PMA for 30 min. hSKl (via their FLAG epitope) or ERKl/2 from clarified cell lysates were immunoprecipitated, subjected to SDS-PAGE, and the protem complexes probed by Western blot for ERKl/2, FLAG or directly for hSKl. d, The time course of sphingosine kinase and ERKl/2 activation by TNFc. (•) and PMA (o) correlate in both empty vector and hSKl- transfected cells.

Figure 10A is a graphical representation which indicates that hSKl is directly activated by in vitro phosphorylation of Ser²²⁵ with ERK2. Data show sphingosine kinase activity (k_cat) 99-r of hSKl prior to and following in vitro phosphorylation at Ser by ERK2. Values are corrected for the proportion of hSKl phosphorylated in the assay mix.

Figure 1 OB is a graphical representation of the substrate kinetics with ATP of in vitro

99 • phosphorylated hSKl . The data indicate that phosphorylation of hSKl at Ser results m a 13.6-fold increase in k_cai (93 s^"1 to 1265 s^"1). R alues for ATP were 81 ± 12 μM and 56 ± 8 μM for non-phosphorylated and phosphorylated hSKl, respectively.

Figure IOC is a graphical representation of the substrate kinetics with sphingosine of in vitro phosphorylated hSKl . The data indicate that phosphorylation of hSKl at Ser²²⁵ results in a 13.6-fold increase in k_c__t (93 s^"1 to 1265 s^"1). Ku values for sphingosine were 15 ± 4 μM and 13 + 3 μM for non-phosphorylated and phosphorylated hSKl, respectively.

Figure 11 is a graphical representation demonstrating that a Ser²²⁵ -» Glu mutation in hSKl does not create a constitutively activated hSKl . hSK^WT and hSK^S225E were expressed in HEK293T cells and the resultant sphingosine kinase activity in cell lysates quantitated with respect to protein expression levels.

Figure 12 is a schematic representation of the targets for sphingosine kinase antagonists.

Figure 13 is a graphical representation of an ELISA of phospho-hSK antiserum. Crude open symbols) and affinity purified (closed symbols) antiserum from rabbits injected with a KLH-conjugated phosphopeptide designed around Ser²²⁵ of hSKl was analysed by ELISA with the phosphopeptide (circles) and corresponding non-phosphorylated peptide (squares).

Figure 14 is an image illustrating that the phospho-hSKl antiserum specifically reacts with phosphorylated hSKl in Western blot analysis. Western blot analysis of hSKl with the affinity purified phospho-hSKl antiserum. Lane 1, recombinant hSKl ; Lane 2, recombinant hSKl phosphorylated in vitro by ERK2; Lane 3, recombinant hSKl phosphorylated in vitro by ERK2 and subsequently dephosphorylated by alkaline phosphatase.

Figure 15 is an image illustrating that the phosph-hSKl antiserum allows phosphorylation of in vivo hSKl to be followed. HEK293T cells were transfected with wild-type hSKl or hSK^S225A and treated with TNFα or PMA, harvested and analysed by Western blot with the phospho-hSKl antiserum and anti-FLAG antibody.

Figure 16 is a graphical representation of the proliferation of HEK293T cells in response to overexpression of wild type hSKl and hSKl^S225A. Cells transfected with wild type hSKl (triangles), hSKl^S225A (diamonds), or empty vector (circles) were grown in DMEM media containing either 5 % foetal calf serum (FCS), 1 % FCS or no serum (0.1 % BSA). Cell numbers were determined using the MTT assay.

Figure 17 is a graphical representation of the transformation of NIH3T3 cells in response to overexpression of wild type hSKl and hSKl . (A) Panels on the left show colony formation in soft agar of NIH3T3 cells transfected with either empty vector, wild type hSKl or hSKl^S225A. Panels on the right show colony formation in soft agar of NIH3T3 cells co-transfected with constitutively active V12-RAs and either empty vector, wild type hSKl or hSKl^S225A. (B) Quantitation of colonies formed.

Figure 18 shows that phosphorylation of hSKl leads to its translocation to membranes and increased intracellular and extracellular sphingosine 1 -phosphate levels, a and b, Translocation of hSKl from the cytosol to the plasma membrane is dependent on its phosphorylation. Cell lysates were fractionated into cytosol to membranes, and probed via Western blot for total hSKl (with anti-FLAG) and phospho-hSKl to show phosphorylation- dependent translocation to the membrane fraction (a). Immunoblots for E-cadherin, an integral plasma membrane protein, were used to show equal membrane loading, b, Confocal microscopy of HEK293T cells transfected with either wildtype hSKl-GFP or hSKl^S225A-GFP with or without 1 μM PMA for 30 min showed phosphorylation-dependent translocation of hSKl from the cytosol to the plasma membrane. Confocal images are representative of >50% of cells observed in three independent experiments, c, Intracellular and extracellular SIP levels were determined in HEK293T cells transiently transfected with empty vector, or plasmids encoding for wild type hSKl and hSKl^S225A following TNFα and PMA treatment, and in the presence or absence of the ERKl/2 pathway inhibitor U0126. Data are mean (± SD) from three experiments.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated, in part, on the determination that sphingosine kinase activation is mediated by a phosphorylation event and, further, the identification of the sphingosine kinase phosphorylation site itself. The inventors have still further determined that sphingosine kinase phosphorylation can be mediated by members of the proline- directed protein kinase family and, most preferably, ERK2. These determinations now permit the rational design of therapeutic and/or prophylactic methods for treating conditions, such as those characterised by aberrant or unwanted cellular activity and/or sphingosine kinase functional activity. Further, there is facilitated the identification and/or design of agents which specifically modulate sphingosine kinase phosphorylation.

Accordingly, one aspect of the present invention provides a method of modulating sphingosine kinase functional activity, said method comprising contacting said sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of said sphingosine kinase wherein inducing or otherwise agonising said phosphorylation up-regulates said sphingosine kinase activity and inhibiting or otherwise antagonising said phosphorylation down-regulates sphingosine kinase activity.

Reference to "sphingosine kinase functional activity" should be understood as a reference to any one or more of the activities which sphingosine kinase can perform, for example, its activity as a key regulatory enzyme in the functioning of the sphingosine kinase signalling pathway. In this regard, it is thought that sphingosine kinase is central to the generation of sphingosine- 1 -phosphate during activation of this pathway. It should be understood that modulation of sphingosine kinase functional activity encompasses both up and down- regulation of any one or more of the functional activities attributable to sphingosine kinase, such as the induction or cessation of a given activity or a change to the level or degree of any given activity. Without limiting the invention to any one theory or mode of action, sphingosine kinase is thought to exhibit two levels of catalytic activity. At the first level, sphingosine kinase exhibits baseline catalytic activity. At the second level, sphingosine kinase exhibiting baseline activity can be activated such that the Vmax of the enzyme is increased. In the context of the preferred embodiment of the present invention, the ablation or reduction of sphingosine kinase catalytic activity will be achieved where the baseline activity and or the activation of sphingosine kinase beyond that of baseline activity is ablated or reduced. Preferably, activation is ablated or reduced and even more preferably activation is ablated.

In a preferred embodiment, antagonising said phosphorylation prevents activation of sphingosine kinase and agonising said phosphorylation results in activation of said sphingosine kinase.

Reference to "sphingosine kinase" should be understood to include reference to all forms of sphingosine kinase protein or derivatives, mutants, homologues or analogues thereof. In this regard, "sphingosine kinase" should be understood as being a molecule which is, mter alia, involved in the generation of sphingosine- 1 -phosphate during activation of the sphingosine kinase signalling pathway. This includes, for example, all protein forms of sphingosine kinase or its functional derivatives, mutants, homologues or analogues thereof, including, for example, any isofonns which arise from alternative splicing of sphingosine kinase mRNA or allelic or polymorphic variants of sphingosine kinase. Reference to a "functional" derivative, mutant, homologue or analogue thereof should be understood as a reference to a molecule which exhibits any one or more of the functional activities of sphingosine kinase. Preferably, said sphingosine kinase is human sphingosine kinase.

Accordingly, the present invention provides a method of modulating human sphingosine kinase functional activity, said method comprising contacting said human sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of said human sphingosine kinase wherein inducing or otherwise agonising said phosphorylation up-regulates said human sphingosine kinase activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said human sphingosine kinase activity. Reference to "phosphorylation" should be understood as a reference to the addition of a phosphate group to the hydroxyl groups on proteins, in particular to the side chains of the amino acids serine, threonine or tyrosine. Without limiting the present invention to any one theory or mode of action, this process of phosphorylating proteins is normally catalysed by a protein kinase (such molecules herein being referred to as "phosphorylation catalysts"), often a specific protein kinase, with ATP acting as the phosphate donor. The phosphorylation of proteins is generally found to regulate the activity of the subject protein.

As detailed hereinbefore, the phosphorylation of a protein is an event which occurs in the context of specific amino acid residues of a subject protein. In this regard, the inventors have identified S¹⁴⁸, S¹⁸¹, Y¹⁸⁴, S²²⁵ and T²⁵⁰ as being the amino acid residues which are relevant to phosphorylation of the sphingosine kinase molecule, in general. However, in terms of the human sphingosine kinase molecule, the inventors have still further identified S²²⁵ as the primary physiological phosphorylation site of this molecule.

Accordingly, in a preferred embodiment, the present invention provides a method of modulating sphingosine kinase functional activity, said method comprising contacting said sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate the phosphorylation of said sphingosine kinase at one or more of S¹⁴⁸, S¹⁸¹, Y¹⁸⁴, S²²⁵ or T²⁵⁰, wherein inducing or otherwise agonising said phosphorylation up-regulates said sphingosine kinase activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said sphingosine kinase activity.

In a most preferred embodiment, said sphingosine kinase is human sphingosine kinase and said phosphorylation modulation occurs at the amino acid residue S 22S

Reference to "modulating" either sphingosine kinase functional activity or a phosphorylation event should be understood as a reference to up-regulating or down- regulating the subject functional activity or phosphorylation event. Reference to up- regulating and down-regulating in this regard should be understood to include both increasing or decreasing the level, degree or rate at which the functional activity or phosphorylation event occurs, in addition to including reference to inducing or ablating the subject functional activity or phosphorylation event. Accordingly, the agent which is utilised in accordance with the method of the present invention may be an agent which induces the activity/event, agonises an activity or event which has already undergone onset, antagonises a pre-existing activity or event or entirely ablates such an activity or event.

Accordingly, reference to "inducing or otherwise agonising" phosphorylation should be understood as a reference to:

(i) inducing the phosphorylation of sphingosine kinase, for example, inducing the interaction of sphingosine kinase with a proline-directed protein kinase, such as ERK2, which effects sphingosine kinase phosphorylation; or

(ii) up-regulating, enhancing or otherwise agonising an existing phosphorylation event, for example, increasing the affinity of or otherwise stabilising the interaction of sphingosine kinase with a phosphorylating molecule.

Conversely, "inhibiting or otherwise antagonising" phosphorylation should be understood as a reference to:

(i) preventing the interaction of sphingosine kinase with a phosphorylating molecule; or

(ii) antagonising an existing interaction between sphingosine kinase and a phosphorylating molecule such that the phosphorylation of sphingosine kinase is rendered ineffective or less effective.

It should be understood that modulation (either in the sense of up-regulation or down- regulation) of the phosphorylation of sphingosine kinase may be partial or complete. Partial modulation occurs where only some of the sphingosine kinase phosphorylation events which would normally occur in a given cell or on a given molecule are affected by the method of the present invention while complete modulation occurs where all sphingosine kinase phosphorylation events are modulated.

Modulation of the phosphorylation of sphingosine kinase may be achieved by any one of a number of techniques including, but not limited to:

(i) introducing into a cell a proteinaceous or non-proteinaceous agent which antagonises the interaction between sphingosine kinase and a phosphorylation catalyst;

(ii) introducing into a cell a proteinaceous or non-proteinaceous agent which agonises the interaction between sphingosine kinase and a phosphorylation catalyst;

(iii) introducing into a cell a proteinaceous or non-proteinaceous agent which acts as a phosphorylation catalyst;

(iv) introducing into a cell a nucleic acid molecule which encodes an agent which acts as a phosphorylation catalyst.

Preferably, said phosphorylation occurs at Ser²²⁵ of sphingosine kinase, and even more preferably, at Ser²²⁵ of human sphingosine kinase.

Reference to "agent" should be understood as a reference to any proteinaceous or non- proteinaceous molecule which modulates (i.e. up-regulates or down-regulates) the interaction of sphingosine kinase with a phosphorylation catalyst, for example, the molecules detailed in points (i) - (ii), above. "Agent" should also be understood to extend to molecules which, of themselves, function as a phosphorylation catalyst, for example, the molecules detailed in (iii) - (iv), above. The subject agent may be linked, bound or otherwise associated with any proteinaceous or non-proteinaceous molecule. For example, it may be associated with a molecule which permits targeting to a localised region.

Said proteinaceous molecule may be derived from natural, recombinant or synthetic sources including fusion proteins or following, for example, natural product screening. Said non-proteinaceous molecule may be derived from natural sources, such as for example natural product screening or may be chemically synthesised. For example, the present invention contemplates chemical analogues of sphingosine kinase or phosphorylation catalyst, such as a kinase molecule (for example, a proline-directed protein kinase such as ERK2), capable of acting as agonists or antagonists of sphingosine kinase phosphorylation. Chemical agonists may not necessarily be derived from sphingosine kinase or the phosphorylation catalyst but may share certain conformational similarities. Alternatively, chemical agonists may be specifically designed to mimic certain physiochemical properties of sphingosine kinase or phosphorylation catalyst. Antagonists may be any compound capable of blocking, inhibiting or otherwise preventing sphingosine kinase phosphorylation. Antagonists include antibodies (such as monoclonal and polyclonal antibodies) specific for sphingosine kinase or the phosphorylation catalyst, or parts of said sphingosine kinase, and antisense nucleic acids which prevent transcription or translation of genes or mRNA in the subject cells. Modulation of expression may also be achieved utilising antigens, RNA, ribosomes, DNAzymes, RNA aptamers, antibodies or molecules suitable for use in co-suppression. Screening methods suitable for use in identifying such molecules are described in more detail hereinafter.

Said proteinaceous or non-proteinaceous molecule may act either directly or indirectly to modulate the interaction of sphingosine kinase with a phosphorylating molecule (such as a phosphorylating kinase). Said molecule acts directly if it associates with the sphingosine kinase or the phosphorylation catalyst. Said molecule acts indirectly if it associates with a molecule other than the sphingosine kinase or phosphorylation catalyst, which other molecule either directly or indirectly modulates the interaction of sphingosine kinase with the phosphorylation catalyst. Accordingly, the method of the present invention encompasses regulation of sphingosine kinase phosphorylation via the induction of a cascade of regulatory steps.

It should be understood that reference to "phosphorylation catalyst" is intended to encompass both molecules which catalyse the transfer of phosphate from a molecule such as ATP to sphingosine kinase. However, it should also be understood to encompass reference to molecules which otherwise achieve the phosphorylation of sphingosine kinase, such as molecules which are able to transfer phosphate groups from molecules other than ATP or which can directly transfer a phosphate group from the phosphorylation catalyst itself to the sphingosine kinase molecule.

"Derivatives" include fragments, parts, portions and variants from natural, synthetic or recombinant sources including fusion proteins. Parts or fragments include, for example, active regions of sphingosine kinase or the phosphorylation catalyst. Derivatives may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. An example of substitutional amino acid variants are conservative amino acid substitutions. Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins.

A "variant" or "mutant", in this context, should be understood to mean a variant or mutant form of sphingosine kinase which exhibits at least some of the functional activity of sphingosine kinase. The variation or mutation may take any form and may be naturally or non-naturally occurring.

Reference to "homologues" should be understood as a reference to nucleic acid molecules or proteins derived from alternative species.

Equivalents of nucleic acid or protein molecules should be understood as molecules exhibiting any one or more of the functional activities of these molecules and may be derived from any source such as being chemically synthesized or identified via screening processes such as natural product screening.

The derivatives include fragments having particular epitopes or parts of the entire protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules.

Analogues contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose confoπnational constraints on the proteinaceous molecules or their analogues.

Derivatives of nucleic acid sequences may similarly be derived from single or multiple nucleotide substitutions, deletions and/or additions including fusion with other nucleic acid molecules. The derivatives of the nucleic acid molecules of the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in cosuppression and fusion of nucleic acid molecules. Derivatives of nucleic acid sequences also include degenerate variants.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH₄; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5 -phosphate followed by reduction with NaBH₄.

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4- chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2- chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N- bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3- hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenyl glycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acids contemplated herein is shown in Table 2. TABLE 2

Non-conventional Code Non-conventional Code amino acid amino acid

α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine Dgln L-N-methylnorvaline Nmnva

D-glutamic acid Dglu L-N-methylomithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine Dile L-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn •-L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine Me D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

D-valine Dval c-methyl- -aminobutyrate Mgabu

D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen

D-cc-methylasparagine Dmasn α-methyl-c.-napthylalanine Manap

D-α-methylaspartate Dmasp c-methylpenicillamine Mpen

D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu

D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn

D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu

D-α-methylleucine Dmleu c-napthylalanine Anap

D-c-methyllysine Dmlys N-benzylglycine Nphe

D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln D-c-methylornithine Dmorn N-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu

D-c--methylproline Dmpro ^"N-(carboxymethyl)glycine Nasp

D-α-methylserine Dmser N-cyclobutylglycine Ncbut

D-o!-methylthreonine Dmthr N-cycloheptylglycine Nchep D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D-c-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-c-methylvaline Dmval N-cylcododecylglycine Ncdod

D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D-N-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycineNbhe

D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-(l-hydroxyethyl) glycine Nthr D-N-methylhistidine Dn his N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dn ile N-(imidazolylethyl))glycine Nhis

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nht

D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnmmet

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine Nala D-N-methylphenylalanine Dnmphe

N-methylaminoisobutyrate N aib D-N-methylproline Dnmpro N-(l-methylpropyl)glycine Nile D-N-methylserine Dnmser

N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr

D-N-methyltryptophan Dnmtrp N-( 1 -methylethyl) glycine Nval

D-N-methyltyiOsine Dnmtyr N-methyla-napthylalanine Nmanap

D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr

L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys

L-ethylglycine Etg penicillamine Pen

L-homophenylalanine Hphe L-C--rnethylalanine Mala

L-o:-methylarginine Marg L-α-methylasparagine Masn L-α-methylaspartate Masp L-θ!-methyl-t-butylglycine Mtbug

L-α-methylcysteine Mcys L-methylethylglycine Metg

L-α-methylglutamine Mgln L-α-methylglutamate Mglu

L-α-methylhistidine Mhis L-α-methylhomophenylalanineMhphe

L-c-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet L-α-methylleucine Mleu L-α-methyllysine Mlys

L-α-methylmethionine Mmet L-c-methylnorleucine Mnle

L-α-methylnorvaline Mnva L-α-methylornithine Morn

L-c-methylphenylalanine Mphe L-Oi-methylproline Mpro

L-α-methylserine Mser L-α-methylthreonine Mthr L-c-methyltryptophan Mtrp L-α-methyltyrosine Mtyr

L-α-methylvaline Mval L-N-methylhomophenylalanin Nmhphe

N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine 1 -carboxy- 1 -(2,2-diphenyl-Nmbc ethylamino)cyclopropane Crosslinkers can be used, for example, to stabilise 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH₂)_n spacer groups with n=l to n=6, glutaraldehyde, N-hydroxysuccinimide esters .and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety.

Without limiting the present invention to any one theory or mode of action, the inventors have utilised alanine mutagenesis to determine that serine ²²⁵ is the primary physiologic phosphorylation site on human sphingosine kinase 1. Further, it has been determined that the kinases of the pro line-directed protein kinase family are able to phosphorylate human sphingosine kinase. Specifically, ERK1, ERK2 and CDK2 phosphorylate human sphingosine kinase 1 to varying degrees of efficiency. ERK2, however, has been demonstrated to show the greatest efficiency in human sphingosine kinase phosphorylation.

Accordingly, in a most preferred embodiment the present invention is directed to a method of modulating sphingosine kinase functional activity, said method comprising contacting said sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate proline-directed protein kinase catalysed sphingosine kinase phosphorylation wherein inducing or otherwise agonising said phosphorylation up- regulates said sphingosine kinase activity and inhibiting or otherwise antagonising said phosphorylation down-regulates sphingosine kinase activity.

Most preferably, said proline directed protein kinase is ERK1, ERK2 or CDK2 .and, even more particularly, ERK2.

Most preferably, said phosphorylation is modulated at S²²⁵.

Reference to proline-directed protein kinase molecules, such as ERK1, ERK2 and CDK2, and any other phosphorylation catalyst should be understood to encompass reference to functional derivatives, mutants, homologues, analogues, equivalents and mimetics of these molecules.

h a related aspect the present invention provides a method of modulating sphingosine kinase functional activity, said method comprising contacting said sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of said sphingosine kinase, which agent binds, links or otherwise associates with serine ²²⁵, wherein inducing or otherwise agonising said phosphorylation up-regulates said sphingosine kinase activity and inhibiting or otherwise antagonising said phosphorylation down-regulates sphingosine kinase activity.

In a most preferred embodiment, said modulation of sphingosine kinase activity is down- regulation.

Since sphingosine kinase is a molecule which is central to the functioning of an intracellular signalling pathway, the method of the present invention provides a means of modulating cellular activity which is regulated or controlled by sphingosine kinase signalling. For example, the sphingosine kinase signalling pathway is known to regulate cellular activities, such as those which lead to inflammation, cellular transformation, apoptosis, cell proliferation, up-regulation of the production of inflammatory mediators such as cytokines, chemokines, eNOS and up-regulation of adhesion molecule expression. Said up-regulation may be induced by a number of stimuli including, for example, inflammatory cytokines such as tumour necrosis factor and interleukin 1, endotoxin, oxidised or modified lipids, radiation or tissue injury. In this regard, reference to "modulating cellular activity" is a reference to up-regulating, down-regulating or otherwise altering any one or more of the activities which a cell is capable of performing pursuant to sphingosine kinase signalling such as, but not limited, one or more of chemokine production, cytokine production, nitric oxide synthesis, adhesion molecule expression and production of other inflammatory modulators. Although the preferred method is to down- regulate sphingosine kinase activity, thereby down-regulating unwanted cellular activity, the present invention should nevertheless be understood to encompass up-regulating of cellular activity, which may be desirable in certain circumstances.

Accordingly, yet another aspect of the present invention is directed to a method of modulating cellular activity, said method comprising contacting said cell with an effective amount of an agent for a time and under conditions sufficient to modulate the phosphorylation of sphingosine kinase wherein inducing or otherwise agonising said phosphorylation up-regulates said cellular activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said cellular activity.

Preferably, the present invention is directed to a method of modulating cellular activity, said method comprising contacting said cell with an effective amount of an agent for a time and under conditions sufficient to modulate the phosphorylation of human sphingosine kinase wherein inducing or otherwise agonising said phosphorylation up- regulates said cellular activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said cellular activity.

In a most preferred embodiment the present invention is directed to a method of modulating human cellular activity, said method comprising contacting said cell with an effective amount of an agent for a time and under conditions sufficient to modulate the phosphorylation of sphingosine kinase at S²²⁵ wherein inducing or otherwise agonising said phosphorylation up-regulates said human cellular activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said human cellular activity.

Most preferably, said modulation is down-regulation.

A further aspect of the present invention relates to the use of the invention in relation to the treatment and/or prophylaxis of disease conditions. Without limiting the present invention to any one theory or mode of action, the broad range of cellular functional activities which are regulated via the sphingosine kinase signalling pathway renders the regulation of sphingosine kinase functioning an integral component of every aspect of both healthy and disease state physiological processes. Accordingly, the method of the present invention provides a valuable tool for modulating aberrant or otherwise unwanted cellular functional activity which is regulated via the sphingosine kinase signalling pathway.

Accordingly, yet another aspect of the present invention is directed to a method for the treatment and/or prophylaxis of a condition in a mammal, which condition is characterised by aberrant, unwanted or otherwise inappropriate cellular activity, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of sphingosine kinase wherein inducing or otherwise agonising said phosphorylation up-regulates said cellular activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said cellular activity.

Still another aspect of the present invention is directed to a method for the treatment and/or prophylaxis of a condition in a mammal, which condition is characterised by aberrant, unwanted or otherwise inappropriate sphingosine kinase functional activity, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of sphingosine kinase wherein inducing or otherwise agonising said phosphorylation up-regulates said sphingosine kinase activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said sphingosine kinase activity.

Preferably, said phosphorylation event is phosphorylation of sphingosine kinase at S²²⁵.

In a most preferred embodiment, the present invention is directed to a method for the treatment and/or prophylaxis of a condition in a human, which condition is characterised by aberrant, unwanted or otherwise inappropriate cellular activity, said method comprising administering to said human an effective amount of an agent for a time and under conditions sufficient to modulate the phosphorylation of sphingosine kinase at S²²⁵, wherein inducing or otherwise agonising said phosphorylation up-regulates said cellular activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said cellular activity.

hi another most preferred embodiment, the present invention is directed to a method for the treatment and/or prophylaxis of a condition in a human, which condition is characterised by aberrant, unwanted or otherwise inappropriate sphingosine kinase functional activity, said method comprising administering to said human an effective amount of an agent for a time and under conditions sufficient to modulate the phosphorylation of sphingosine kinase at S²²⁵ wherein inducing or otherwise agonising said phosphorylation up-regulates said sphingosine kinase functional activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said sphingosine kinase functional activity.

Most preferably, said modulation is down-regulation.

Reference to "aberrant, unwanted or otherwise inappropriate" cellular activity should be understood to be understood as a reference to overactive cellular activity, to physiological normal cellular activity which is inappropriate in that it is unwanted or to insufficient cellular activity. This definition applies in an analogous manner in relation to "aberrant, unwanted or otherwise, inappropriate" sphingosine kinase activity. For example, TNF production during tumour cell growth has been shown to support cellular proliferation and to provide anti-apoptotic characteristics to the neoplastic cells. Accordingly, to the extent that a cell is neoplastic, it is desirable that the promotion of cellular proliferation and anti- apoptotic characteristics be down-regulated. Similarly, diseases which are characterised by inflammation, such as rheumatoid arthritis, atherosclerosis, asthma, autoimmune disease and inflammatory bowel disease, are known to involve cellular activation by cytokines such as TNF, leading to the synthesis and secretion of inflammatory mediators, such as adhesion molecules. In such situations, it is also desirable to down-regulate such activity. In other situations, it may be desirable to agonise or otherwise induce sphingosine kinase phosphorylation in order to stimulate cellular proliferation. Preferably, said condition is an inflammatory condition and said cellular activity is down- regulation of inflammatory mediator production, in particular adhesion molecule expression. Most preferably, said condition is rheumatoid arthritis, atherosclerosis, asthma, autoimmune disease or inflammatory bowel disease.

In another preferred embodiment said condition is a neoplasm and said cellular activity is down regulation of cellular proliferation and/or antiapoptotic characteristics.

In accordance with the previous aspects of the present invention, the subject agent is preferably PD98059 or U0126 or functional derivative, mutant, analogue, equivalent or mimetic thereof.

The term "mammal" as used herein includes humans, primates, livestock animals (eg. sheep, pigs, cattle, horses, donkeys), laboratory test animals (eg. mice, rabbits, rats, guinea pigs), companion animals (eg. dogs, cats) and captive wild animals (eg. foxes, kangaroos, deer). Preferably, the mammal is human or a laboratory test animal Even more preferably, the mammal is a human.

An "effective amount" means an amount necessary at least partly to attain the desired response, or to delay the onset or inhibit progression or halt altogether, the onset or progression of a particular condition being treated. The amount varies depending upon the health and physical condition of the individual to be treated, the taxonomic group of individual to be treated, the degree of protection desired, the formulation of the composition, the assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that can be determined through routine trials.

Reference herein to "treatment" and "prophylaxis" is to be considered in its broadest context. The term "treatment" does not necessarily imply that a subject is treated until total recovery. Similarly, "prophylaxis" does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylaxis" may be considered as reducing the severity or onset of a particular condition. "Treatment" may also reduce the severity of an existing condition.

The present invention further contemplates a combination of therapies, such as the administration of the agent together with subjection of the mammal to other agents, drugs or treatments which may be useful in relation to the treatment of the subject condition such as cytotoxic agents or radiotherapy in the treatment of cancer.

Administration of the modulatory agent, in the form of a pharmaceutical composition, may be performed by any convenient means. The modulatory agent of the pharmaceutical composition is contemplated to exhibit therapeutic activity when admimstered in an amount which depends on the particular case. The variation depends, for example, on the human or animal and the modulatory agent chosen. A broad range of doses may be applicable. Considering a patient, for example, from about 0.1 mg to about 1 mg of modulatory agent may be administered per kilogram of body weight per day. Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation.

The modulatory agent may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intraperitoneal, intramuscular, subcutaneous, intradermal or suppository routes or implanting (e.g. using slow release molecules). The modulatory agent may be administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g. with zinc, iron or the like (which are considered as salts for purposes of this application). Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If the active ingredient is to be administered in tablet form, the tablet may contain a binder such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate.

Routes of administration include, but are not limited to, respiratorally, intratracheally, nasopharyngeally, intravenously, intraperitoneally, subcutaneously, intracranially, intradermally, intramuscularly, intraoccularly, intrathecally, intracereberally, intranasally, infusion, orally, rectally, via TV drip patch and implant.

In accordance with these methods, the agent defined in accordance with the present invention may be coadministered with one or more other compounds or molecules. By "coadministered" is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. For example, the subject agent may be administered together with an agonistic agent in order to enhance its effects. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules. These molecules may be administered in any order.

Still another aspect of the present invention contemplates the use of an agent, as hereinbefore defined, in the manufacture of medicament for the treatment of a condition in a mammal, which condition is characterised by aberrant, unwanted or otherwise inappropriate sphingosine kinase functional activity, wherein said agent modulates the phosphorylation of sphingosine kinase and wherein inducing or otherwise agonising said phosphorylation up-regulates said sphingosine kinase functional activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said sphingosine kinase functional activity.

Preferably, said phosphorylation is phosphorylation of sphingosine kinase at S²²⁵.

Even more preferably, said mammal is a human, and said modulation is down-regulation.

As detailed hereinbefore, and without limiting the present invention to any one theory or mode of action, it is thought that phosphorylation of sphingosine kinase activates sphingosine kinase exhibiting baseline activity such that the Vmax of the enzyme is increased. Accordingly, screening individuals for the presence, absence or degree of sphingosine kinase phosphorylation provides a means for diagnosing, monitoring or , screening patients for conditions characterised by aberrantly activated sphingosine kinase activity and/or cellular activity. The present invention should therefore be understood to extend to such diagnostic assays.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion or may be in the form of a cream or other form suitable for topical application. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation. Generally, dispersions are prepared by incorporating the various sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile inj ectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 μg and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cheny flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical fonn of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as-cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release preparations and formulations.

The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule encoding a modulatory agent. The vector may, for example, be a viral vector.

Yet another aspect of the present invention relates to the agent as hereinbefore defined, when used in the method of the present invention.

The present invention should also be understood to encompass a method for screening for agents which modulate the phosphorylation of sphingosine kinase, particularly at Ser²²⁵, such as agents which agonise or antagonise the interaction of sphingosine kinase with a phosphorylation catalyst (such as ERK2) or which themselves phosphorylate sphingosine kinase, for example by functioning as a phosphorylation catalyst. Screening for the modulatory agents hereinbefore defined can be achieved by any one of several suitable methods including, but in no way limited to, contacting a cell comprising sphingosine kinase and a phosphorylation catalyst (such as ERK2) with an agent and screening for the modulation of sphingosine kinase phosphorylation or modulation of the activity or expression of a downstream sphingosine kinase cellular target such as NF-κB. Detecting such modulation can be achieved utilising techniques such as Western blotting, electrophoretic mobility shift assays and/or the readout of reporters of sphingosine kinase activity such as luciferases, CAT and the like.

It should be understood that the sphingosine.kinase or phosphorylation catalyst may be naturally occuning in the cell which is the subject of testing or the genes encoding them may have been transfected into a host cell for the purpose of testing. Further, the naturally occurring or transfected gene may be constitutively expressed - thereby providing a model useful for, inter alia, screening for agents which down-regulate sphingosine kinase phosphorylation or the gene may require activation - thereby providing a model useful for, inter alia, screening for agents which modulate sphingosine kinase phosphorylation under certain stimulatory conditions. Further, to the extent that a sphingosine kinase nucleic acid molecule is transfected into a cell, that molecule may comprise the entire sphingosine kinase gene or it may merely comprise a portion of the gene such as a portion comprising Ser²²⁵.

In another example, the subject of detection could be a downstream sphingosine kinase regulatory target, rather than sphingosine kinase itself, such as NF-/cB. Yet another example includes sphingosine kinase binding sites ligated to a minimal reporter. For example, modulation of sphingosine kinase phosphorylation can be detected by screening for the modulation of the downstream signalling components of a TNF stimulated cell. Where the cell which is the subject of the screening system is a neoplastic cell, for example, modulation of sphingosine kinase phosphorylation could be detected by screening for the induction of apoptosis of that cell. Suitable agents may also be identified and/or designed utilising well known methods such as combinatorial chemistry or high throughput screening of recombinant libraries or following natural product screening.

For example, libraries containing small organic molecules may be screened, wherein organic molecules having a large number of specific parent group substitutions are used. A general synthetic scheme may follow published methods (eg., Bunin BA, et al. (1994) Proc. Natl. Acad. Sci. USA, 91.4708-4712; DeWitt SH, et al. (1993) Proc. Natl. Acad. Sci. USA, 90:6909-6913). Briefly, at each successive synthetic step, one of a plurality of different selected substituents is added to each of a selected subset of tubes in an anay, with the selection of tube subsets being such as to generate all possible permutation of the different substituents employed in producing the library. One suitable permutation strategy is outlined in US. Patent No. 5,763,263.

There is cunently widespread interest in using combinational libraries of random organic molecules to search for biologically active compounds (see for example U.S. Patent No. 5,763,263). Ligands discovered by screening libraries of this type may be useful in mimicking or blocking natural ligands or interfering with the naturally occurring ligands of a biological target. In the present context, for example, they may be used as a starting point for developing sphingosine kinase phosphorylation agonists or antagonists which exhibit properties such as more potent pharmacological effects. Sphingosine kinase or a functional part thereof and/or phosphorylation catalyst may according to the present invention be used in combination libraries formed by various solid-phase or solution-phase synthetic methods (see for example U.S. Patent No. 5,763,263 and references cited therein). By use of techniques, such as that disclosed in U.S. Patent No. 5,753,187, millions of new chemical and/or biological compounds may be routinely screened in less than a few weeks. Of the large number of compounds identified, only those exhibiting appropriate biological activity are further analysed.

With respect to high throughput library screening methods, oligomeric or small-molecule library compounds capable of interacting specifically with a selected biological agent, such as a biomolecule, a macromolecule complex, or cell, are screened utilising a combinational library device which is easily chosen by the person of skill in the art from the range of well-known methods, such as those described above. In such a method, each member of the library is screened for its ability to interact specifically with the selected agent. In practising the method, a biological agent is drawn into compound-containing tubes and allowed to interact with the individual library compound in each tube. The interaction is designed to produce a detectable signal that can be used to monitor the presence of the desired interaction. Preferably, the biological agent is present in an aqueous solution and further conditions are adapted depending on the desired interaction. Detection may be performed for example by any well-known functional or non-functional based method for the detection of substances.

Accordingly, another aspect of the present invention provides a method for detecting an agent capable of modulating the phosphorylation of sphingosine kinase or its functional equivalent or derivative thereof said method comprising contacting a cell or extract thereof containing said sphingosine kinase and phosphorylation catalyst or its functional equivalent or derivative with a putative agent and detecting an altered expression phenotype associated with said phosphorylation.

Reference to "sphingosine kinase" and "phosphorylation catalyst" should be understood as a reference to either the sphingosine kinase or phosphorylation catalyst expression product or to a portion or fragment of the sphingosine kinase or phosphorylation catalyst, such as the Ser²²⁵ sphingosine kinase phosphorylation site. In this regard, the sphingosine kinase or phosphorylation catalyst expression product is expressed in a cell. The cell may be a host cell which has been transfected with the sphingosine kinase or phosphorylation catalyst nucleic acid molecule or it may be a_cell which naturally contains the sphingosine kinase gene. Reference to "extract thereof should be understood as a reference to a cell free transcription system.

Reference to detecting an "altered expression phenotype associated with said phosphorylation" should be understood as the detection of cellular changes associated with modulation of the phosphorylation of sphingosine kinase. These may be detectable, for example, as intracellular changes or changes observable extracellularly. For example, this includes, but is not limited to, detecting changes in downstream product levels or activities (e.g. MF-KB).

Still another aspect of the present invention is directed to agents identified in accordance with the screening method defined herein and to said agents for use in the methods of the present invention. Said agent should be understood to extend to monoclonal antibodies which bind to the phosphorylation sites of sphingosine kinase, and in particular, amino acid S²²⁵.

Still a further aspect of the present invention is directed to sphingosine kinase variants comprising a mutation in a region of said sphingosine kinase which region comprises a phosphorylation site, wherein said variant exhibits ablated or reduced phosphorylation capacity relative to wild type sphingosine kinase or a functional derivative, homologue or analogue of said sphingosine kinase variant.

The present invention also extends to variants which exhibit enhanced or up-regulated activity due to the nature of the mutation of an existing phosphorylation site or the incorporation of additional phosphorylation sites.

Reference to "mutation" should be understood as a reference to any change, alteration or other modification, whether occurring naturally or non-naturally, which modulates the capacity of said sphingosine kinase to undergo phosphorylation. Said modulation may be up-regulation or down-regulation. Although the present invention is preferably directed to variants which exhibit ablated activation capacity, it should be understood that the present invention extends to the generation of variants which exhibit additional phosphorylation sites or an improved level of phosphorylation capacity of existing sites. Reference to a "functional" derivative, homologue, or analogue in this context should be understood as a reference to the subject molecule exhibiting the defined modulated phosphorylation capacity. The change, alteration or other modification may take any form including, but not limited to, a structural modification (such an alteration in the secondary, tertiary or quaternary structure of the sphingosine kinase molecule), a molecular modification (such as an addition, substitution or deletion of one or more amino acids from the sphingosine kinase protein) or a chemical modification. The subject modification should also be understood to extend to the fusion, linking or binding of a proteinaceous or non-proteinaceous molecule to the sphingosine kinase protein or to the nucleic acid molecule encoding a sphingosine kinase protein. It should also be understood that although it is necessary that the subject mutation is expressed by the sphingosine kinase expression product, the creation of the mutation may be achieved by any suitable means including either mutating a wild-type sphingosine kinase protein, synthesising a sphingosine kinase variant or modifying a nucleic acid molecule encoding a wild-type sphingosine kinase protein such that the expression product of said mutated nucleic acid molecule is a sphingosine kinase protein variant. Preferably, said mutation is a single or multiple amino acid sequence substitution, addition and/or deletion.

In accordance with this prefened embodiment there is provided a human sphingosine kinase variant comprising an amino acid sequence with a single or multiple amino acid substitution and/or deletion of amino acids S¹⁴⁸, S¹⁸¹, Y¹⁸⁴, S²²⁵ and or T²⁵⁰ wherein said variant exhibits ablated or reduced phosphorylation capacity relative to wild-type sphingosine kinase or a functional derivative, homologue or analogue of said sphingosine kinase variant.

• • • 99 Preferably, amino acid is ammo acid S and even more preferably said substitution is a Ser²²⁵ Ala substitution.

In terms of the present invention, reference to "wild-type" sphingosine kinase is a reference to the forms of sphingosine kinase expressed by most individuals in a given population. There may be greater than one wild-type form of sphingosine kinase (for example due to allelic or isoform variation) and the level of phosphorylation exhibited by said wild-type sphingosine kinase molecules may fall within a range of levels. However, it should be understood that "wild-type" does not include reference to a naturally occurring form of sphingosine kinase which cannot be phosphorylated. Such a variant form of sphingosine kinase may, in fact, constitute a naturally occurring mutant form of sphingosine kinase within the context of the present invention. ^"

Reference to "agent" as hereinbefore defined should be understood to include reference to the sphingosine kinase variants herein defined.

In yet another aspect, the present invention extends to genetically modified animals, which animals have been modified to express a sphingosine kinase variant as hereinbefore defined.

The present invention is further described by reference to the following non-limiting description.

EXAMPLE 1 Methods

Generation of phospho-hSKl -specific polyclonal antibodies

Polyclonal antibodies were raised in rabbits against a phosphopeptide (CGSKTPApSPVVVQQ) designed from the hSKl sequence around Ser²²⁵. Prior to injection into rabbits, the phosphopeptide was conjugated to maleimide activated keyhole limpet hemocyanin (Pierce) via its iV-terminal cysteine. Antibodies active against the non- phosphorylated peptide were removed from the antiserum using SulfoLink™ beads (Pierce) to which the non-phosphorylated peptide (CGSKTPASPVVVQQ SEQ ID NO:2)was conjugated using the manufactures inst xctions.

Construction and expression ofhSK mutants

Wild-type human SKI (hSKWT) cDNA (Pitson et al 2000) (Genbank accession number AF200328) was FLAG epitope tagged at the 3' end and subcloned into pALTER (Promega Inc., Madison, WI) site directed mutagenesis vector, as previously described (Pitson et al. 2000). Single-stranded DNA was prepared and used as template for oligonucleotide directed mutagenesis as detailed in the manufacturer's protocol. The mutagenic oligonucleotides shown in Table 1 were designed to generate the required mutants which were subsequently sequenced to verify incorporation of the desired modification. The mutant cDNA were then subcloned into pcDNA3 (Invitrogen Corp., San Diego, CA) for transient transfection into HEK293T cells of pGEX-4T2 for expression as glutathione--?- transferase (GST)-fusion proteins in E. coli. hSK^TB2 was generated as previously described (Xia et al. 2002).

Cell Culture, transfection and cell fractionation

Human embryonic kidney cells (HEK293T, ATCC CRL- 1573) were cultured on

Dulbecco's modified Eagle's medium (CSL Biosciences, Parkville, Australia) containing 10% fetal calf serum, 2 mM glutamine, 0.2% (w/v) sodium bicarbonate, penicillin (1.2 mg/ml), and gentamycin (1.6 mg/ml) i Transfections were performed using the calcium phosphate precipitation method. Cells were harvested 24 h after transfection and lysed by sonication (2 watts for 30 s at 4 °C) in lysis buffer A containing 50 mM Tris/HCl (pH 7.4), 10% (w/v) glycerol, 0.05% (w/v) Triton X-100, 150 mM NaCl, 1 mM dithiothreitol, 2 mM Na3VO4, 10 mM NaF, 1 mM EDTA and protease inhibitors (Complete™; Boehringer Mannheim). Activation of hSKl was assessed by treatment of cells with either phorbol 12-myristate 13-acetate (PMA; Sigma) for 30 min, or tumor necrosis factor-α (TNFα; R&D Systems Inc., Minneapolis, MN) for 10 min. For cell fractionation, HEK293T cells were scraped into lysis buffer lacking Triton X-100, sonicated, and centrifuged at 1,000 x g for 10 min. The supernatants were then centrifuged at 100,000 x g for 60 min at 4 °C to yield membrane and cytosolic fractions. The membrane fraction was then resuspended in lysis buffer containing 0.8% Triton X-100, sonicated, left on ice for 30 min, and centrifuged at 10,000 x g for 10 min at 4 °C to remove Triton-insoluble material. Protein concentrations in cell homogenates were determined with Coomassie Brilliant Blue (Sigma) reagent using BSA as standard.

Generation of recombinant proteins in E. coli

Recombinant proteins were generated of in E. coli and purified as previously described (Pitson et al. 2000).

Metabolic labelling of cells with P

HEK293T cells were cultured and transfected as described above. The cultures, in 10 cm dishes, were incubated for 30 min with phosphate-free, serum- free Dulbecco's modified Eagle's medium (DMEM). This medium was then replaced with fresh phosphate-free, serum-free DMEM containing 500 μCi [³²P]orthophosphate and the cultures incubated for 3 hr. Cultures were then stimulated with 1 ng/ml TNF or 100 ng/ml PMA for 30 min. Cells were then washed 3 times with cold PBS and scaped into 1.2 ml of lysis buffer B containing 50 mM Tris/HCl (pH 7.4), 1 % (w/v) Trition X-100, 1 % (w/v) deoxycholate, 0.1% SDS, 150 mM NaCl, 50 mM NaF, 10 mM sodium orthovanadate, 1 mM EDTA, 1 mM DTT, and protease inhibitors (Complete™). Cells were subjected to three freeze-thaw cycles to ensure complete disruption. The cell debris was then removed by centrifugation (13,000 g for 20 min at 4°C) and the supernatant incubated with 12 μg of M2 anti-FLAG antibody (Sigma) overnight with mixing at 4°C. The immunocomplexes were then collected with Protein A-Separose and washed four times with lysis buffer B. The immunoprecipitates were then subjected to SDS-PAGE on 12 % acrylamide gels, the gels dried and the phosphorylated hSK quantitated by Phosphorimager (Molecular Dynamics).

In vitro phosphorylation ofhSKl

Purified recombinant hSKl (1.5 μg) was incubated with recombinant ERK2 (0.1 μg; Upstate Biotechnology), 125 μM ATP and 5 μCi [γ³²P]ATP in 20 mM MOPS (pH 7.2) containing 20 mM MgCl₂, 25 mM β-glycerophosphate, 5 mM EGTA, 1 mM sodium orthovanadate, and 1 mM DTT. The reaction was stopped after 20 min at 37°C by the addition of SDS-PAGE sample buffer, the mixture boiled for 5 min, subjected to SDS- PAGE on 12 % acrylamide gels, the gels dried, and the phosphorylated hSKl quantitated by Phosphorimager.

Sphingosine kinase assays

SK activity was routinely determined using D-erythro-sphingosine (Biomol, Plymouth Meeting, PA) and [γ32P]ATP (Geneworks, Adelaide, South Australia) as substrates, as described previously (Pitson et al. 2000). A unit (U) of SK activity is defined as the amount of enzyme required to produce 1 pmol SlP/min.

SIP levels

To determine both intracellular and extracellular SIP levels HEK293T cells were metabolically labeled with [³²P]orthophosphate as described above. Cells were then transferred to fresh phosphate-free DMEM and treated with 1 ng ml^"1 TNFα or 100 ng ml^"1

PMA for 30 min. To determine extracellular SIP release following stimulation, the media was then removed, centrifuged at 1,000 x g, and 2.5 ml of the supernatant added to 2.5 ml chlorofonn, 2.5 ml methanol and 20 μl cone. HCl. The organic phase was then dried under vacuum, resuspended in chloroform and SIP resolved by TLC on silica gel 60 with 1- butanol/ethanol/acetic acid water (8:2:1:2, w/v). Intracellular SIP levels were determined by harvesting the cells into 400 μl methanol containing 25 μl cone. HCl. Lipids were then extracted under alkaline conditions by the addition of 400 μl chloroform, 400 μl KC1 and 40 μl 3 M NaCl. The aqueous phase, containing SIP under these conditions, was then acidified through the addition of 50 μl cone. HCl and re-extracted with 400 μl chloroform. The organic phase organic phase was then dried under vacuum, resuspended in chloroform and SIP resolved by TLC as described above.

Confocal microscopy

For fluorescence microscopy wild type hSKl and hSKl^S225A were tagged at the N-terminus with GFP. Briefly, EGFP was PCR amplified from pEGFP-1 (Clontech) with the following primers 5*-TAAAGCTTGCCACCATGGTGAGCAAG-3' (SEQ ID NO: 13) and 5'-ATGGATCCATCTTGTACAGCTCGTCCATG-3' (SEQ ID NO: 14), and the resultant product digested with Hindlll and BamHI and cloned into pcDNA3-hSKl-FLAG¹⁰ and pcDNA3-hSKl^S225A-FLAG. HEK293T cells were transfected with these plasmids, as described above, cultured for 24 h, and then plated onto eight well glass chamber slides (Nalge Nunc international) at 1 x 10⁴ cells / well. After 24 h cells were treated for 30min with 1 μg/ml PMA or DMSO control, fixed with 4% paraformaldehyde, and viewed using a 60X oil- immersion objective on an Olympus 1X70 inverted microscope linked to a BioRad Radiance 2100 confocal microscope after excitation at 488 nm. EXAMPLE 2 Results

hSKl is phosphorylated in HEK293 cells in response to cell agonists

The in vivo phosphorylation of hSKl in HEK293 cells in response to TNFα and phorbol esters (PMA) was examined since phosphorylation is a common mechanism of regulating the catalytic activity of many eukaryotic. Metabolic labelling, with [ Pjorthophosphate, of HEK293 cells stably over-expressing hSKl 1000-fold revealed that hSKl is, indeed, phosphorylated, and that this phosphorylation increases rapidly in response to cell exposure to TNFα (Fig 1) and PMA. Furthermore, this increase in hSKl phosphorylation is highly conelated with the observed increase in sphingosine kinase activity in these cells in response to TNFα (Fig 1) and PMA. These observations suggested that phosphorylation plays a role in hSKl activation. Further evidence for this was also obtained using a hSKl mutant defective in its ability to bind TRAF2. This hSKl mutant was is neither activated (Xia et al. 2002) or phosphorylated in response to TNFα (Fig 2). PMA, however, induces both activation (Xia et al. 2002)and phosphorylation of this mutant (Fig 2).

hSKl is phosphorylated at Ser

Analysis of the hSKl sequence with the NetPhos phosphorylation site prediction program (Blom et al. 1999) allowed the identification of several possible phosphorylation sites in hSKl that were also conserved in the mouse and monkey SKI isoforms (Fig 3). Alanine mutagenesis of these possible sites revealed that the hSKl mutant containing the Ser²²⁵ - Ala mutation was not phosphorylated (Fig 4). This suggested Ser²²⁵ as the sole physiologic phosphorylation site in hSKl . Further mutagenesis to alanine of Ser and Thr , two potentially phosphorylatable amino acids in the region around Ser²²⁵ had no effect on hSKl phosphorylation (Fig 5). This indicated that the Ser²²⁵ - Ala mutation directly blocks phosphorylation at Ser²²⁵ rather than having a secondary effect on phosphorylation at an adjacent site (i.e. Ser²²⁰ or Thx²²²). Activation ofhSKl requires phosphorylation at Ser^'"

The hSK^S225A mutant, when expressed in HEK293 cells, cannot be activated by cell treatment with TNFα or PMA (Fig 6). This provides strong evidence that phosphorylation

Ser²²⁵ is essential for hSKl activation.

ERK1, ERK2, and CDK2 specifically phosphorylate hSKl at Ser²²⁵ in vitro

The amino acid sequence around Ser²²⁵ is SKTPAS²²⁵PVVVQ (SEQ ID NO:3). The presence of a proline immediately C-terminal to Ser²²⁵ suggests a member of the proline- directed protein kinase family is responsible for hSKl phosphorylation. In particular, the PASP sequence of this region is reminiscent of the ERKl/2 substrate recognition motif, PxS/TP, where x represents a small, neutral amino acid (Chen et al. 2001). Experiments examining in vitro phosphorylation of purified recombinant hSKl with ERK1, ERK2 and cyclin-dependent kinase 2 (CDK2) showed that all three kinases can phosphorylate hSKl (Fig 7). Of these kinases, however, ERK2 showed the greatest efficiency of hSKl phosphorylation. Further in vitro phosphorylation analysis with purified recombinant hSK^S225A mutant showed that ERK2 could not phosphorylate this protein (Fig 8). ERK2 was, however, still able to phosphorylate recombinant hSKl mutants containing alaine mutations the only other potential proline-dependent kinase phosphorylation sites in hSKl (Ser¹⁴⁸ and Thr²²²). This indicated that ERK2 specifically phosphorylates hSKl at Ser²²⁵.

Increases in hSKl phosphoiylation in cells are blocked by a ERKl/2 pathway inhibitor, but not a CDK inhibitor

The fact that ERK2 efficiently phosphorylates hSKl in vitro, and does so at the conect site suggested this enzyme may the physiologically relevant protein kinase that phosphorylates hSKl in vivo. To test the involvement of ERKl/2 in intact cells, the effect of the well characterised ERKl/2 pathway inhibitors, PD98059 and U0126, on hSKl phosphorylation and activation was examined. These both act specifically on MEKl/2 to block ERKl/2 activation (Davies et al., 2000). Both chemical inhibitors potently blocked TNFα and PMA-induced increases in hSKl phosphorylation and catalytic activity (Fig. 9a) in cells overexpressing hSKl. Consistent with this, the U0126 inhibitor also blocked increases in phosphorylation (Fig 9b) and catalytic activity of the endogenous enzyme in non- overexpressing cells. This is strong evidence for an in vivo role for ERKl/2 in the activation of hSKl. To establish that there is a direct interaction between ERKl/2 and hSKl in vivo, as suggested by the in vitro experiments, co-immunoprecipitation of the two proteins from cell extracts (Fig. 9c) was examined. An interaction between hSKl and ERKl/2 using antibodies against either component for immunoprecipitation was detected. Importantly, an interaction between endogenous ERKl/2 and endogenous hSKl was detected, confirming that this was not an interaction that was forced by overexpression. If ERKl/2 is the directly activating kinase for hSKl, the time-course of activation of these kinases should parallel the activation of sphingosine kinase activity. This was found to be the case for both TNFα or PMA activation (Fig. 9d). Notably, the activation of hSKl by TNFα is sustained in cells overexpressing hSKl . This is only partially explained by the more sustained activation of ERKl/2 in the cells, which is consistent with previous studies (Lacana et al., 2002), and may also indicate that overexpression of hSKl saturates the hSKl dephosphorylation mechanism. The combined results of the in vitro and in vivo experiments demonstrate activation and association of hSKl with ERKl/2, .and is a strong indication that hSKl is a bonafide direct effector of ERKl/2.

Interaction of hSKl with TRAF2 is essential for TNFα-induced hSKl activation (Xia et al., 2002). Consistent with this, it has been found that TNFα-induced hSKl phosphorylation is also dependent on the interaction of hSKl with TRAF2. Data indicate TRAF2 interacts with the pre- formed hSKl -ERKl/2 complex. This may facilitate the recruitment of upstream components of the MAP kinase pathway to this complex following engagement of TNFα, allowing for localised activation of ERKl/2 and subsequent phosphorylation of the associated hSKl. Such an adaptor molecule role for TRAF2 would allow for enhanced hSKl activation and explain how TNFα, a relatively weak ERKl/2 activator, results in similar hSKl activation as PMA. The role of phosphorylation in the mechanism ofhSKl activation

The-ability to specifically phosphorylate recombinant hSKL at Ser²²⁵ in vitro with ERK2 afforded the opportunity to examine the direct effect of this phosphorylation on the catalytic activity of hSKl . It has been found that phosphorylation of hSKl resulted in a dramatic increase in its sphingosine kinase activity. Quantitation of ³²P incorporation into the recombinant hSKl following this in vitro phosphorylation showed 42 % of the hSKl protein became phosphorylated, signifying that phosphorylation of hSKl at Ser²²⁵ results in an approximate 14-fold increase in its catalytic activity (Fig. 10A). Substrate kinetic analysis demonstrated that phosphorylation essentially increased the turnover number (/ _cat) of hSKl (Table 4). In contrast, the phosphorylated hSKl had only a slightly lower K value for ATP, and an unaltered Ku value for sphingosine, compared to the non- phosphorylated enzyme (Fig. 10B, IOC and Table 4). Thus, although it has previously been shown that hSKl possesses considerable intrinsic catalytic activity in the absence of post-translational modifications, phosphorylation of this enzyme at Ser²²⁵ directly results in a marked, 14-fold increase in its catalytic efficiency.

Mutagenesis of Ser —> Glu does not create a constitutively activated hSKl

By virtue of their negative charge the acidic amino acids glutamate and aspartate can sometimes mimic phosphorylated amino acids and create a protein conformation resembling the activated state. However, a hSKl mutant containing the Ser²²⁵ - Glu mutation had similar activity to the wild-type hSKl (Fig 11). Therefore, unlike the case in some other enzymes, this mutation does not create a constitutively activated hSKl .

Phospho-hSKl -specific polyclonal antibodies

The crude polyclonal antisera raised in rabbits against the phosphopeptide centred around Ser²²⁵ of hSKl was highly reactive in ELISA towards the phosphopeptide, but also showed similar reactivity to the non-phosphorylated peptide (Fig 13). However, following affinity chromatography using the non-phosphorylated peptide to remove reactivity to this peptide, the antiserum showed high specificity in ELISA to the phosphopeptide (Fig 13). Furthermore, high specificity was also shown for the phosphorylated hSKl protein in Western blots (Eig 1_4). In these Western blots, the antiserum showed reactivity towards ERK2 phosphorylated hSKl, but showed little reactivity towards the non-phosphorylated wildtype hSKl (Fig 14) or the hSK^S225A mutant (Fig 15). Thus, we have used this purified antiserum to show TNFα and PMA-dependent increases in hSKl phosphorylation (Fig 15).

Phosphoiylation/ activation ofhSKl is responsible for its effects on proliferation and transformation

It has previously been shown that overexpression of wild type hSKl significantly enhanced cell proliferation and resulted in cell transformation (Xia et al., 2000). These effects were shown to be dependent on the catalytic activity of hSKl . However, since this enzyme has considerable intrinsic catalytic activity (Pitson et al., 2000a) it was not clear from these studies whether hSKl activation played a role in these effects. Certainly, it appeared that activation of hSKl may be important in Ras-mediated transformation (Xia et al., 2000), making it tempting to speculate that the activated form of hSKl may also be the key factor in tr.ansformation resulting from hSKl overexpression. Elucidation of the activation mechanism of hSKl afforded the ability to examine this further through the use of the non-activatable Ser²²⁵ — » Ala mutant of hSKl. Consistent with previous findings (Xia et al., 2000), HEK293T cells overexpressing wild type hSKl displayed enhanced proliferation and serum-independent growth (Fig. 16). In contrast, cells overexpressing the non-phosphorylatable hSKl mutant, hSKl displayed no difference in proliferative rate or serum-dependence to the control cells transfected with empty vector (Fig. 16). These marked differences in the observed proliferative effects of wild type hSKl and hSKl^S225Λ were seen even though the expression levels of both proteins were similar in these cells, as were the cellular sphingosine kinase activities.

Similar results were also observed in cellular transformation assays performed by colony formation in soft agar. As previously reported NIH3T3 cells transfected with wild type hSKl displayed considerable transforming activity over that seen with control cells (Xia et al., 2000). In contrast, however, cells transfected with hSKl had remarkably less transforming activity (Fig. 17)_ This is despite the cells expressing similar levels of the.. . transfected proteins and possessing similar overall sphingosine kinase activities. This data provides compelling evidence that it is the phosphorylated, activated form of hSKl that is responsible for the transforming capacity of this enzyme. Interestingly, it was also found that hSKl blocked Ras-induced cell transformation Fig. 17), providing further evidence for the involvement of hSKl activation in this process. Since the hSKl- transfected cells already possess a high level of sphingosine kinase activity, the effect of activation on cell transformation may not just be a consequence of the resultant increases in catalytic activity of this enzyme. Instead, it may also be related to an activation-induced change in subcellular localisation of the activated hSKl.

Translocation of hSKl to membranes and increased intracellular and extracellular sphingosine 1 -phosphate levels depends on hSKl phosphorylation.

Recent reports suggest that activation of hSKl by PMA or PDGF is accompanied by translocation of the enzyme from the cytosol to membrane fractions. To test whether this translocation depends on agonist-dependent phosphorylation of hSKl the levels of both total and phosphorylated hSKl in cytosolic and membrane fractions with and without stimulation by TNFα or PMA of HEK293T cells overexpressing either wild type hSKl or the phosphorylation deficient hSKl^S225A mutant (Fig. 18a) were measured. Stimulation with PMA, or TNFα consistently induced an increase in membrane-associated wild type hSKl . In contrast the Ser²²⁵ - Ala mutation completely blocked any increase in membrane-associated hSKl, confirming that phosphorylation at that site is required for the translocation event. Further confocal microscopy of HEK293T cells expressing hSKl-green fluorescent protein (GFP) fusion proteins also confirmed phosphorylation dependent translocation of hSKl since PMA induced cytosol to plasma membrane translocation of wild type hSKl, but not the phosphorylation deficient hSKl^S225A mutant (Fig. 18b).

Phosphorylation is not only required for agonist-stimulated increases in enzyme activity, as demonstrated above, but is also essential for the activation-dependent elevation in SIP, the second messenger produced by hSKl (Fig. 18b). Overexpression of wild type hSKl resulted in large increases in both intracellular and extracellular SIP over that of vector-transfected cells, even in the absence of stimulus. Stimulation of these cells with either TNFα or PMA, however, resulted in further increases in both intracellular and extracellular SIP. This mirrored well the changes in SIP, at lower overall levels, in empty vector transfected cells. In contrast, mutation of the Ser²²⁵ phosphorylation site completely eliminated any agonist- dependent changes in either intracellular or extracellular SIP. In addition, compared with wild type hSKl transfected cells, reduced basal SIP levels were observed in cells overexpressing the hSKl^S225A mutant. This was most marked for extracellular SIP, and was despite the wild type hSKl and hSKl^S225A-overexpressing cells possessing similar basal sphingosine kinase activities. Consistent with ERKl/2 being the activator(s) of endogenous as well as overexpressed hSKl, inhibition with the ERKl/2 pathway inhibitor U0126 blocked TNFα- and PMA-induced increases in intracellular and extracellular SIP in both vector transfected and wild type hSKl transfected cells. The inability of this inhibitor to reduce SIP levels to tthhaatt oofftthhee hhSSKKll^5S225A mutant is explained by an incomplete block of basal hSKl phosphorylation.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds refened to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Table 3 Mutagenic oligonucleotides used for site-directed mutagenesis of hSKl

Mutation Sequence

S148A CGGCTGCTGGCGCCCATGAAC - SEQ LD NO:4

S181A TGTGGACCTCGAGGCTGAGAAGTA - SEQ ID NO:5

Yl 84A AGTGAGAAGGCTCGGCGCCTGGGGGAG - SEQ ID NO:6

S220A AAGAGTGGGCGCCAAGACAC - SEQ ID NO:7

T222A AAGAGTGGGATCCAAGGCGCCTGCCTCC - SEQ ID NO: 8

S225A AAGACACCTGCGGCGCCCGTTGTG - SEQ ID NO:9

S225E ACACCTGCCGAACCGGTTGTGGTC - SEQ ID NO:10

T250A TCTCACTGGGCAGTGGTGC - SEQ ID NO: 11

Table 4. Substrate kinetics of in vitro phosphorylated hSKl

k_cai (s 9^" ^ ) (ATP) (μM) u (Sphingosine) (μM)

hSKl 93 + 15 81 + 12 15 + 4

phospho-hSKl 1265 + 86 56 + 8 13 + 3

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Claims

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A method of modulating sphingosine kinase functional activity, said method comprising contacting said sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of said sphingosine kinase wherein inducing or otherwise agonising said phosphorylation up-regulates said sphingosine kinase activity and inhibiting or otherwise antagonising said phosphorylation down-regulates sphingosine kinase activity.

2. A method of modulating cellular activity, said method comprising contacting said cell with an effective amount of an agent for a time and under conditions sufficient to modulate the phosphorylation of sphingosine kinase wherein inducing or otherwise agonising said phosphorylation up-regulates said cellular activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said cellular activity.

3. The method according to claim 1 or 2 wherein said sphingosine kinase is human sphingosine kinase.

4. The method according to any one of claims 1-3 wherein said phosphorylation is modulated at S²²⁵.

5. The method according to claim 4 wherein said agent binds, links or otherwise associates with S²²⁵.

6. The method according to any one of claims 1-5 wherein modulation of said phosphorylation is modulation of proline-directed protein kinase catalysed phosphorylation.

7. The method according to claim 6 wherein said proline directed ldnase is ERK1, ERK2 or CDK2.

8. The method according to claim 7 wherein said proline directed kinase is ERK2.

9. . „ The method according to any one of claims 1-8 wherein said modulation is down- regulation.

10. The method according to claim 9 wherein said agent is U0126.

11. The method according to claim 9 wherein said agent is PD98059.

12. A method for the treatment and/or prophylaxis of a condition in a mammal, which condition is characterised by abenant, unwanted or otherwise inappropriate cellular activity, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of sphingosine kinase wherein inducing or otherwise agonising said phosphorylation up- regulates said cellular activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said cellular activity.

13. A method for the treatment and/or prophylaxis of a condition in a mammal, which condition is characterised by abenant, unwanted or otherwise inappropriate sphingosine kinase functional activity, said method comprising administering to said mammal an effective amount of an agent for a time and under conditions sufficient to modulate phosphorylation of sphingosine kinase wherein inducing or otherwise agonising said phosphorylation up-regulates said sphingosine kinase functional activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said sphingosine kinase functional activity.

14. The method according to claim 12 or 13 wherein said sphingosine kinase is human sphingosine kinase.

15. The method according to any one of claims 12-14 wherein said phosphorylation is modulated at S²²⁵.

16. The method according to claim 15 wherein said agent binds, links or otherwise

99^ associates with S ...

17. The method according to any one of claims 12-16 wherein modulation of said phosphorylation is modulation of proline-directed protein kinase catalysed phosphorylation.

18. The method according to claim 17 wherein said proline directed kinase is ERKl , ERK2 or CDK2.

19. The method according to claim 18 wherein said proline directed kinase is ERK2.

20. The method according to any one of claims 12-19 wherein said modulation is down-regulation.

21. The method according to claim 20 wherein said cellular activity is induced by TNF.

22. The method according to claim 21 wherein said condition is a neoplastic condition and said cellular activity is TNF-induced cellular proliferation and or anti-apoptotic characteristic.

23. The method according to claim 21 wherein said condition is an inflammatory condition and said cellular activity is the production of inflammatory mediators.

24. The method according to claim 23 wherein said inflammatory mediator is adhesion molecular expression.

25. The method according to claim 23 or 24 wherein said inflammatory condition is rheumatoid arthritis, atherosclerosis, asthma, autoimmune disease or inflammatory bowel disease.

26. The method according to any one of claims 20-25 wherein said agent is U0126.

27. The method according to any one of claims 20-25 wherein said agent is PD98059.

28. Use of an agent in the manufacture of a medicament for the treatment of a condition in a mammal, which condition is characterised by abenant, unwanted or otherwise inappropriate cellular activity, wherein said agent modulates the phosphorylation of sphingosine kinase and wherein inducing or otherwise agonising said phosphorylation up-regulates said cellular activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said cellular activity.

29. Use of an agent in the manufacture of a medicament for the treatment of a condition in a mammal, which condition is characterised by abenant, unwanted or otherwise inappropriate sphingosine kinase activity, wherein said agent modulates the phosphorylation of sphingosine kinase and herein inducing or otherwise agonising said phosphorylation up-regulates said sphingosine kinase activity and inhibiting or otherwise antagonising said phosphorylation down-regulates said sphingosine kinase activity.

30. Use according to claim 28 or 29 wherein said sphingosine kinase is human sphingosine kinase.

31. Use according to any one of claims 28-30 wherein said phosphorylation is modulated at S²²⁵.

32. Use according to claim 31 wherein said agent binds, links or otherwise associates with S²²⁵.

33. Use according to any one of claims 28-32 wherein modulation of said phosphorylation is modulation of proline-directed protein kinase catalysed phosphorylation.

34. Use according to claim 33 wherein said proline directed kinase is ERK1 , ERK2 or CDK2.

35. Use according to claim 34 wherein said proline directed kinase is ERK2.

36. Use according to any one of claims 28-35 wherein said modulation is down- regulation.

37. Use according to claim 36 wherein said cellular activity is induced by TNF.

38. Use according to claim 37 wherein said condition is a neoplastic condition and said cellular activity is TNF-induced cellular proliferation and/or anti-apoptotic characteristic.

39. Use according to claim 37 wherein said condition is an inflammatory condition and said cellular activity is the production of inflammatory mediators.

40. Use according to claim 39 wherein said inflammatory mediator is adhesion molecular expression.

41. Use according to claim 39 or 40 wherein said inflammatory condition is rheumatoid arthritis, atherosclerosis, asthma, autoimmune disease or inflammatory bowel disease.

42. Use according to any one of claims 36-41 wherein said agent is U0126.

43. Use according to any one of claims 36-41 wherein said agent is PD98059.

44. A pharmaceutical composition comprising an agent, which agent modulates phosphorylation of sphingosine kinase, together with one or more pharmaceutically acceptable caniers and/or diluents when used in accordance with the method of any one of claims 1-27.

45. An agent, which agent modulates phosphorylation of sphingosine kinase, when used in accordance with the method of any one of claims 1-27.

46. An isolated sphingosine kinase variant comprising a mutation in a region of said sphingosine kinase which region comprising a phosphorylation site, wherein said variant exhibits ablated or reduced phosphorylation capacity relative to wild-type sphingosine kinase or a functional derivative, homologue or analogue thereof.

47. An isolated sphingosine kinase variant comprising a mutation in a region of said sphingosine kinase which region comprising a phosphorylation site, wherein said variant exhibits enhanced or up-regulated phosphorylation capacity relative to wild-type sphingosine kinase or a functional derivative, homologue or analogue thereof.

48. The isolated variant of claim 46 wherein said variant comprises an amino acid sequence with a single or multiple amino acid substitution and/or deletion of amino acid 225

49. The isolated variant of claim 48 wherein said substitution is a Ser²²⁵ Ala substitution.