WO2006135969A1 - Modulation of sphingosine kinase signalling - Google Patents
Modulation of sphingosine kinase signalling Download PDFInfo
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- WO2006135969A1 WO2006135969A1 PCT/AU2006/000867 AU2006000867W WO2006135969A1 WO 2006135969 A1 WO2006135969 A1 WO 2006135969A1 AU 2006000867 W AU2006000867 W AU 2006000867W WO 2006135969 A1 WO2006135969 A1 WO 2006135969A1
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- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
- C12Y207/01091—Sphinganine kinase (2.7.1.91)
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Definitions
- 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 the level of sphingosine kinase functional activity. In a related aspect, the present invention provides a method of modulating sphingosine kinase mediated signalling via modulation of its intracellular level of activity. The present invention still further extends to novel molecules which exhibit the capacity to induce sphingosine kinase activity.
- the methods 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 functional activity and/or aberrant, unwanted or otherwise inappropriate sphingosine kinase mediated signalling.
- the present invention is further directed to methods for identifying and/or designing agents capable of modulating the level of sphingosine kinase activity.
- Sphingosine kinases are enzymes that catalyse the phosphorylation of sphingosine to generate the bio-active phospholipid, sphingosine 1 -phosphate (SlP) (Pyne & Pyne, 2002, Biochim. Biophys. Acta 1582:121-131; Spiegel & Milstien, 2003, Nat. Rev MoI Cell Biol. 4:397-407).
- SlP can affect many biological processes, including calcium mobilization, mitogenesis, apoptosis, atherosclerosis, inflammatory responses and cytoskeleton rearrangement (Spiegel & Milstien, 2000, FEBS Lett. 476:55-57).
- SK is the main regulator of SlP levels in mammalian cells (Spiegel & Milstien, 2003, supra), the activity and activation of this enzyme plays a central and crucial role in controlling the observed effects attributed to SlP in the cell.
- SKl and SlP appear to be involved in other aspects of cellular regulation. It has been shown that SlP may be involved in inflammation and atherosclerosis through the induction of adhesion molecule expression on vascular endothelial cells (Xia et al, 1999, J. Biol Chem. 274:34499- 34505). Other studies suggest its involvement in hypertension since high levels of SKl can enhance blood vessel constriction (BoIz et al, 2003, Circulation 108, 342-347; Coussin et al, 2002, Circ. Res. 91:151-157).
- hSKl has intrinsic catalytic activity that is independent of post-translational modifications of the protein (Pitson et al. 2000a, Biochem. J. 350:429-441).
- ERK1/2 phosphorylates hSKl in vivo (Pitson et al, 2003, EMBOJ. 22:1-10) This phosphorylation of hSKl results directly in a 14-fold increase in the k C ⁇ t of the enzyme, while having no significant effect on KM values for either sphingosine or ATP.
- hSKl activity correlates with its translocation from the cytosol to the plasma membrane (Rosenfeldt et al, 2001, FASEB J. 15:2649-2659; Young et al, 2003, Cell calcium 33:119-128).
- the phosphorylation at Ser225 not only directly increases the catalytic activity of hSKl, but it was also shown to be necessary for agonist-induced translocation of the protein to the plasma membrane (Pitson et al, 2003, supra).
- Translocation of SKl may be important in localizing it to its potential substrate and hence generate localized signalling of SlP or its secretion to engage cell-surface SlP receptors (Pitson et al, 2003, supra).
- sphingosine kinase isoforms Two human sphingosine kinase isoforms exist (1 and 2), which differ in their tissue distribution, developmental expression, catalytic properties, and somewhat in their substrate specificity (Pitson et al, 2000, supra; Liu et al, 2000, J. Biol. Chem. 275:19513- 19520).
- a number of studies have shown the effects of sphingosine kinase 1 in enhancing cell proliferation and suppressing apoptosis (Olivera et al, 1999, J. Cell Biol 147:545- 558; Xia et al, 2000, Curr. Biol. 10:1527-1530; Edsall et al, 2001, J. Neurochem. 76:1573-1584).
- SphK2 In addition to SphKl, another isoform SphK2 has been cloned and characterized (Liu et ah, 2000, J Biol Chem, 275:19513-19520). Although both SphKl and S ⁇ hK2 contribute to total cellular SphK activity, these two isoforms demonstrate distinct enzyme kinetics and expression patterns (Kohama et ah, 1998, J Biol Chem, 273:23722-23728; Liu et ah, 2000, supra).
- SphKl is described as a cytoplasmic enzyme
- SphK2 was found to localize in the nuclei through its nuclear localization signal sequence (Igarashi et ah, 2003, J Biol Chem, 278:46832-46839). Additional functional distinctions between Sphkl and Sphk2 are also evident. For example, Sphkl enhances cell survival and proliferation, whereas Sphk2 inhibits DNA synthesis (Liu et ah, 2000, supra) and induces apoptosis potentially through a BH3 domain and Bcl-xL interactions (Liu et ah, 2003, J Biol Chem, 278:40330-40336).
- SphK2 is the more primitive of the two mammalian enzymes involved in sphingolipid catabolism (Herr et ah, 2004, J Biol Chem, 279:12685-12694).
- the ability of SphKl to be regulated at both transcriptional and post- transcriptional levels in response to a variety of physiological stimuli may be a relatively recent evolutionary step, facilitating a signaling role in mammalian cells.
- nucleotide and amino acid sequence information prepared using the programme Patentln Version 3.1, presented herein after the bibliography.
- Each nucleotide or amino acid sequence is identified in the sequence listing by the numeric indicator ⁇ 201> followed by the sequence identifier (eg. ⁇ 210>l, ⁇ 210>2, etc).
- the length, type of sequence (DNA, protein, etc) and source organism for each nucleotide or amino acid sequence is indicated by information provided in the numeric indicator fields ⁇ 211>, ⁇ 212> and ⁇ 213>, respectively.
- Nucleotide and amino acid 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.).
- 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>l, ⁇ 400>2, etc). That is SEQ ID NO:1 as detailed in the specification correlates to the sequence indicated as ⁇ 400>l in the sequence listing.
- One aspect of the present invention provides a method of modulating sphingosine kinase mediated signalling, said method comprising contacting sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with SKAMl or functional derivative, variant, homologue or mimetic thereof wherein inducing or otherwise agonising said association upregulates sphingosine kinase catalytic activity and inhibiting or otherwise antagonising said association downregulates said catalytic activity.
- Another aspect of the present invention provides a method of modulating sphingosine kinase 1 mediated signalling, said method comprising contacting sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase 1 with SKAMl or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association upregulates sphingosine kinase 1 catalytic activity and inhibiting or otherwise antagonising said association downregulates said catalytic activity.
- Still another aspect of the present invention provides a method of modulating sphingosine kinase mediated signalling, said method comprising contacting sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with the amino acid region corresponding to residues 51-132 of SEQ ID NO:1 wherein inducing or otherwise agonising said association upregulates sphingosine kinase catalytic activity and inhibiting or otherwise antagonising said association downregulates said catalytic activity.
- Yet another aspect of the present invention provides a method of upregulating sphingosine kinase mediated signalling, said method comprising contacting sphingosine kinase with an effective amount of SKAMl or functional derivative, homologue or mimetic thereof for a time and under conditions sufficient to induce the interaction of sphingosine kinase with SKAMl and thereby upregulate sphingosine kinase catalytic 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 interaction of sphingosine kinase with SKAMl or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association up-regulates said cellular activity and inhibiting or otherwise antagonising said association 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 interaction of sphingosine kinase 1 with SKAMl or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association up-regulates said cellular activity and inhibiting or otherwise antagonising said association down-regulates said cellular activity.
- Another further aspect of 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 interaction of sphingosine kinase with the amino acid region corresponding to residues 51-132 of SEQ ID NO:1 wherein inducing or otherwise agonising said association up-regulates said human cellular activity and inhibiting or otherwise antagonising said association down- regulates said human cellular activity.
- Yet another further aspect of the present invention relates to the use of the invention in relation to the treatment and/or prophylaxis of disease conditions.
- 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.
- 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.
- Still yet another further 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 the interaction of sphingosine kinase with SKAMl or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association up-regulates said cellular activity and inhibiting or otherwise antagonising said association down-regulates said cellular activity.
- Yet 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 interaction of sphingosine kinase with SKAMl or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association up-regulates said sphingosine kinase activity and inhibiting or otherwise antagonising said association down-regulates said sphingosine kinase activity.
- Another aspect of 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 interaction of sphingosine kinase with the amino acid region corresponding to residues 51-132 of SEQ ID NO:1, wherein inducing or otherwise agonising said interaction up-regulates said cellular activity and inhibiting or otherwise antagonising said interaction down-regulates said cellular activity.
- in yet another aspect of 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 interaction of sphingosine kinase with the amino acid region corresponding to residues 51-132 of SEQ ID NO:1 wherein inducing or otherwise agonising said interaction up-regulates said sphingosine kinase functional activity and inhibiting or otherwise antagonising said interaction down-regulates said sphingosine kinase functional activity.
- 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 cellular activity, wherein said agent modulates the interaction of sphingosine kinase and wherein inducing or otherwise agonising said interaction up-regulates said cellular activity and inhibiting or otherwise antagonising said interaction down-regulates said cellular activity.
- Still yet 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 interaction of sphingosine kinase with SKAMl or functional derivative, homologue or mimetic thereof and wherein inducing or otherwise agonising said interaction upregulates said sphingosine kinase functional activity and inhibiting or otherwise antagonising said interaction downregulates said sphingosine kinase functional activity.
- the present invention contemplates a pharmaceutical composition
- a pharmaceutical composition comprising the modulatory agent as hereinbefore defined together with one or more pharmaceutically acceptable carriers and/or diluents.
- Yet another aspect of the present invention relates to the agent as hereinbefore defined, when used in the method of the present invention.
- Still a further aspect of the present invention is directed to a SKAMl mimetic, said mimetic comprising a region corresponding to residues 51-132 of SEQ ID NO:1 or exhibiting at least 40% similarity thereto, wherein said mimetic is capable of interacting with sphingosine kinase and upregulating the intrinsic catalytic activity of said sphingosine kinase.
- Figure 1 is a schematic representation of the sequence alignment and expression of SKAMl isoforms.
- A Alignment of the predicted amino acid sequences of the naturally occurring SKAMl (A-SEQ ID NO:1, B-SEQ ID NO:2, C-SEQ ID NO:3 and D-SEQ ID NO:4) isoforms.
- Y2H (SEQ ID NO: 5) was the original protein isolated that interacted with SKl in a yeast two-hybrid screen.
- SKAMlY SEQ ID NO:6 represents the minimal SKAMl region that is predicted to interact with SKl. Identical and conserved amino acids substitutions are shaded.
- B Schematic representation of conserved regions of SKAMl isoforms.
- C Expression of HA-epitope tagged SKAMl isoforms in HEK293T.
- SKAMlA 28 kDa
- SKAMlB 24 kDa
- SKAMlC (19 kDa)
- SKAMlD 15 kDa
- SKAMlY 10 kDa
- Figure 2 is an image of the interaction between SKAMl isoforms and hSKl by immunoprecipitation with anti-HA antibodies.
- the interactions of SKAMlA, B, C, D and Y with hSKl were assessed by co-immunoprecipitation.
- the co-transfected HA- SKAMlA, B, C, D and Y with hSKl(FLAG) cell lysates were immunoprecipitated with anti-HA antibodies and then analysed by immunoblotting with both chicken anti-SKl antibody and anti-HA monoclonal antibodies.
- Figure 3 is an image of the interaction between the SKAMl isoforms and hSKl by immunoprecipitation with anti-FLAG antibodies.
- the interactions of SKAMlA, B, C, D and Y with hSKl were assessed by co-immunoprecipitation.
- Lysates from cells co- transfected HA-SKAMlA, B, C, D and Y with hSKl (FLAG) cell lysates were immunoprecipitated with anti-FLAG antibodies and then analysed by immunoblotting with anti-SKAMl antibodies or anti-HA monoclonal antibodies as well as anti-FLAG antibodies.
- Proteins of 28 kDa, 24 kDa, 19 kDa, and 15 kDa, representing the SKAMl isoforms were detected indicating that SKAMlA 5 B, C, and D interact with hSKl.
- Figure 4 is an image of the interaction between endogenous SKAMl and overexpressed hSKl. The interactions of overexpressed hSKl and endogenous SKAMl were assessed by immunoprecipitation analyses. Lysates from cells transfected with either empty vector, or plasmids encoding SKAMlA, SKAMlD or hSKl(FLAG) were immunoprecipitated with anti-SKAMl antibodies and then immunoblotted with chicken anti-SKl antibodies.
- Figure 5 is an image of the interaction of SKAMlD with hSKl S225A .
- the interaction of SKAMlD with hSKl was assessed by co-immunoprecipitation. Lysates from cells co- transfected with HA-SKAMlD and hSKl S225 ⁇ were immunoprecipitated with anti-HA antibodies. Immunoblotting with both anti-HA and anti-FLAG antibodies showed expression of the transgenes. The immunoprecipitated complexes were then immunoblotted with chicken anti-SKl antibody and anti-HA monoclonal antibodies.
- Figure 6 is a graphical representation of the effect of SKAMl isoforms on endogenous SK activity.
- HEK-293T cells were transfected with SKAMlA, B, C, D and Y isoforms or empty pCMV(HA) expression vectors (control) and 24 hr after transfection whole cell lysates were prepared and SK activity measured. Data are the mean ( ⁇ S. D.) of duplicate determinations of five individual experiments.
- Figure 7 is an image of the effect of overexpression of the catalytically inactive hSKl (hSKl G82D ) on SKAMl -mediated activation of endogenous SK activity.
- HEK293T cells were transfected with empty pCMV(HA) expression vector (control), SKAMlA and D isoforms alone or in combination with hSKl G82D .
- IgG is the light chain of the anti-HA antibody used in the immunoprecipitations.
- B SK activity was measured in the cytosolic fractions. Data are the mean ( ⁇ standard error) of two individual experiments and each experiment was done in duplicate.
- Figure 8 is an image of the generation of GST-SKAMl proteins. A coomassie stained gel showing the expression of 54 kDa, 50 kDa, 45 kDa, 4IkDa, 35 kDa corresponding to GST- SKAMlA 5 B, C 5 D 5 Y and GST.
- Figure 9 is a graphical representation of the effect of SKAMl in in vitro stability of hSKl.
- GST-SKAMlD or GST were incubated with rec-hSKl at 45 0 C for 4 hr.
- A At various indicated times SK activity assays were performed.
- B The half-life of hSKl alone and in combination with GST-SKAMlD was determined by plotting the data on log scale.
- Figure 10 is a graphical representation of the effect of GST-SKAMlD on rec-hSKl activity.
- GST-SKAMlD or GST was incubated with rec-hSKl and then subjected to in vitro SK activity assays.
- Figure 11 is a graphical representation of the stability of hSKl in the SK assay. Recombinant hSKl was subjected to in vitro SK activity assays and aliquots removed over time and SlP determined.
- Figure 12 is a graphical representation of the titration of GST-SKAMlD on rec-hSKl activity.
- GST-SKAMlD or GST were incubated at increasing concentrations with rec- hSKl and subjected to in vitro SK activity assays.
- the graph shows that SKl activity increased in a dose responsive manner with maximum activity at 20 molar excess of GST- SKAMlD.
- Figure 13 is a graphical representation of the enzyme kinetics of ATP concentration on hSKl .
- GST-SKAMlD and GST at a concentration of 20 molar ratio of hSKl/GST- SKAMl were added to rec-hSKl and were assayed for SK activity with increasing concentrations of ATP.
- A Substrate kinetics of rec-hSKl with ATP over the concentration range of 0 to 500 ⁇ M
- B Lineweaver-Burk plot of substrate kinetics.
- Figure 14 is a graphical representation of the enzyme kinetics of sphingosine concentration on hSKl.
- GST-SKAMlD and GST at a concentration of 20 molar ratio of hSKl/GST-SKAMl were added to rec-hSKl and were assayed for SK activity with increasing concentrations of sphingosine.
- A Substrate kinetics of rec-hSKl with sphingosine over the concentration 0 to 50 ⁇ M
- B Lineweaver-Burk plot of substrate kinetics.
- Figure 15 is a graphical representation of the effect of GST-SKAMl isoforms on hSKl activity.
- GST-SKAMlA, B, C, D 5 and Y at a concentration of 20 molar ratio of hSKl/GST-SKAMl were incubated with rec-hSKl and then subjected to in vitro SK activity assays.
- the graph shows that SKl activity increased approximately four-fold with GST-SKAM 1 A or B and about two-fold with GST-SKAM 1 C, D or Y compared to control GST.
- Figure 16 is a graphical representation of the effect of overexpression of SKAMl on hSKl activation.
- HEK-293T cells were transfected with empty vector or plasmid encoding SKAMl A and D. 24 hr after transfection, cells were serum starved for 5 hr and then treated with TNF ⁇ (l ⁇ g/ml) for 10 min, whole cell lysates prepared, and SK activity measured. Data are the mean ( ⁇ SEM) of duplicate determinations of two individual experiments.
- Figure 17 is an image of the interaction between SKAMl and hSK2.
- the interactions of SKAMl isoforms A and D with hSK2 were assessed by co-immunoprecipitations.
- the co- transfected HA-SKAMlA and D with hSK2(FLAG) cell lysates were immunoprecipitated with anti-HA antibodies.
- the immunoprecipitated complexes were immunoblotted with both anti-HA monoclonal antibodies and anti-FLAG antibodies.
- IgG-H and IgG-L are the heavy and light chains of the anti-HA antibody, respectively.
- Figure 18 is a graphical representation of the effect of GST-SKAMl isoforms on rec- hSK2 activity.
- GST-SKAMlA 5 B 5 C, D 5 and Y at a concentration of 15 molar ratio of hSK2/GST-SKAMl were incubated with rec-hSK2 and then subjected to in vitro SK activity assays.
- Figure 19 is an image of the characterisation of anti-SKAM antibodies.
- A Titration of anti-SKAMl anti-serum with HEK293T cells overexpressing SKAMlD.
- B Detection of HA-SKAMl isoforms with anti-SKAMl antibodies. Representative western blots showing the expression of HA-SKAMl isoforms including A 5 B, C, D and Y at expected molecular masses of 28 kDa, 24 kDa, 19 kDa, 15 kDa and 10 kDa respectively with both anti- SKAMl and anti-HA antibodies.
- C Immunoprecipitation of HA-SKAMl isoforms with anti-SKAMl antibodies. Lysates from cells transfected with SKAMlA, B, C, D and Y cell were immunoprecipitated with anti-SKAMl antibodies and then immunoblotted with anti- SKAMl antibodies.
- Figure 20 is an image of the detection of endogenous SKAM with anti-SKAMl antibodies.
- A Lysates from various human and mouse cell lines were immunoblotted with anti-SKAMl antibodies. Proteins of about 27 kDa and 23 kDa were detected in TMNC, human lymphocytes, MCF-7, HEK-293T & NIH 3T3 cell lysates that were predicted to be endogenous SKAMlA and B. SKAMIs positive control lane represent where all lysates from overexpressed HA-SKAMlA, B, C & D isoforms were pooled. HUVEC is human umbilical vein endothelial cell.
- B Immunodepletion of endogenous SKAMl .
- a representative blot showing the intensity of the proteins representing endogenous SKAMl were greatly decreased after the blot was incubated with anti- SKAMl antibodies which have been immunodepleted with GST-SKAMlD bound to glutathione-Sepharose but not the control where anti-SKAMl antibodies were immunodepleted with just GST bound to glutathione-Sepharose.
- SKAMIs positive control lane represent where all lysates from overexpressed HA-SKAMlA, B, C & D isoforms were pooled together.
- C Immunoprecipitation of endogenous SKAMl with anti-SKAMl antibodies.
- Lysates from TMNC, HEK-293T and NIH 3T3 were immunoprecipitated and immunoblotted with anti-SKAMl antibodies gave two proteins of approximately 27 kDa and 23 kDa corresponding to endogenous SKAMlA and B.
- SKAMIs positive control lane represent where all lysates of overexpressed HA-SKAMlA, B, C & D isoforms were pooled.
- IP control lanes represent where lysates were incubated in the same conditions, but without anti-SKAMl antibody.
- Figure 21 is an image of the immunofluorescence of HA-SKAMl isoforms.
- NIH 3T3 cells were transfected with HA-SKAMlA, B, C and D, fixed and stained with both anti SKAMl and anti-HA antibodies.
- Figure 22 is an image of the immunofluorescence of highly expressed HA-SKAMl isoforms with anti-SKAMl antibodies.
- NIH 3T3 cells were overexpressed with HA- SKAMlA, B 5 C & D, fixed and stained with the anti SKAMl antibodies.
- Figure 23 is an image of the co-localisation of hSKl with SKAMl isoforms.
- GFP-hSKl was transiently expressed alone or in combination with HA-tagged SKAMl isoforms in NIH 3T3 cells. Cells were plated on slide, fixed, permeabilised, and then immunostained with anti-HA antibodies, followed by Alexa594 labelled secondary antibodies.
- the present invention is predicated, in part, on both the determination that SKAMl molecules upregulate the intrinsic catalytic activity of sphingosine kinase and the identification of the SKAMl binding motif by which its interaction with sphingosine kinase occurs. These determinations now permit the rational design of therapeutic and/or prophylactic methods for treating conditions 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 the interaction of SKAMl with sphingosine kinase.
- one aspect of the present invention provides a method of modulating sphingosine kinase mediated signalling, said method comprising contacting sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with SKAMl or functional derivative, variant, homologue or mimetic thereof wherein inducing or otherwise agonising said association upregulates sphingosine kinase catalytic activity and inhibiting or otherwise antagonising said association downregulates said catalytic activity.
- sphingosine kinase mediated signalling should be understood as a reference to a signalling pathway in which the sphingosine kinase molecule forms a functional component. 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 mediated signalling encompasses both up and downregulation of the signalling events, for example the induction or cessation of a given signalling event or a change to the level or degree of any given signalling event.
- antagonising the interaction of sphingosine kinase with SKAMl prevents the completion of a sphingosine kinase mediated signalling event while agonising or otherwise inducing the interaction of sphingosine kinase with SKAMl promotes sphingosine kinase mediated signalling.
- the degree or level of a sphingosine kinase mediated signalling event can be modulated by increasing or decreasing the concentration of interacting molecules. Accordingly, the modulation of signalling need not necessarily equate to the onset or inhibition of signalling but may be designed to regulate the level of sphingosine kinase mediated signalling which occurs.
- sphingosine kinase should be understood to include reference to all forms of sphingosine kinase protein and derivatives, variants, homologues or mimetics thereof.
- sphingosine kinase should be understood as being a molecule which is, inter alia, involved in the generation of sphingosine- 1 -phosphate during activation of the sphingosine kinase signalling pathway.
- sphingosine kinase isoforms exist (1 and 2), which differ in their tissue distribution, developmental expression, catalytic properties, and somewhat in their substrate specificity (Pitson et al, 2000, supra; Liu et at, 2000, supra).
- a number of studies have shown the effects of sphingosine kinase 1 in enhancing cell proliferation and suppressing apoptosis (Olivera et al, 1999, supra; Xia et al, 2000, supra; Edsall et al, 2001, supra).
- hSKl human sphingosine kinase 1
- references to a "functional" derivative, variant, homologue or mimetic thereof should be understood as a reference to a molecule which exhibits any one or more of the functional activities of sphingosine kinase or SKAM 1.
- the present invention provides a method of modulating sphingosine kinase 1 mediated signalling, said method comprising contacting sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase 1 with SKAMl or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association upregulates sphingosine kinase 1 catalytic activity and inhibiting or otherwise antagonising said association downregulates said catalytic activity.
- SKAMl should be understood as a reference to all forms of this protein. This includes, for example, any isoforms which arise from alternative splicing of SKAMl mRNA or functional mutants or polymorphic variants of this molecule.
- SKAMl is a protein present in many organisms, ranging from humans to flat worms. At present, the structure and function of this protein has yet to be elucidated. Furthermore, no recognizable domains can be found in SKAMl by comparison to other known proteins. Results from initial domain searches using the SMART database (Schultz et ah, 1998) reveal that SKAMlA may possess a AAA-domain.
- each of the four known SKAMl isoforms being SKAMlA (SEQ ID NO:1), SKAMlB (SEQ ID NO:2), SKAMlC (SEQ ID NO:3) and SKAMlD (SEQ ID NO:4), can upregulate the catalytic activity of sphingosine kinase.
- SKAMl functional derivatives such as the truncated SKAMlY (SEQ ID NO:6) similarly upregulate sphingosine kinase catalytic activity and therefore fall within the scope of the definition of "SKAMl".
- SKAMl enhances the rate of the enzyme reaction but does not alter the binding affinity of hSKl for its substrates.
- SKAMl also interacts with hSK2 and is the first protein reported to show a regulatory effect on this enzyme.
- SKAMl is also the first protein shown to bind to both hSKl and hSK2.
- the levels of activation of hSK2 by the different SKAMl isoforms are similar to hSKl, suggesting that SKAMl acts on SK2 in the same manner as it does with hSKl.
- SK2 also appears to exhibit a different cellular function to SKl since SKl has pro-proliferative and survival roles (Payne et ⁇ l., 2002; Olivera et ⁇ l., 1999; Edsall et ⁇ l., 2001; Osawa et ⁇ l., 2001, J. Immunol 167:173-180), whereas, SK2 has been reported to have a pro-apoptotic role (Liu et ⁇ l., 2003).
- the present invention therefore more particularly provides a method of modulating sphingosine kinase mediated signalling, said method comprising contacting sphingosine kinase with an effective amount of an agent for a time and under conditions sufficient to modulate the interaction of sphingosine kinase with the amino acid region corresponding to residues 51-132 of SEQ ID NO:1 wherein inducing or otherwise agonising said association upregulates sphingosine kinase catalytic activity and inhibiting or otherwise antagonising said association downregulates said catalytic activity.
- said sphingosine kinase is sphingosine kinase 1 or 2 and most preferably sphingosine kinase 1.
- said sphingosine kinase mediated signalling is upregulated by inducing or agonising the interaction of sphingosine kinase with the amino acid region corresponding to residues 51-132 of SEQ ID NO: 1.
- SKAMl as an upregulator of the catalytic activity of sphingosine kinase and the site of interaction between these two molecules now provides a means for modulating sphingosine kinase mediated cellular activity.
- modulated is meant upregulated or downregulated.
- inducing or otherwise agonising the interaction of sphingosine kinase with SKlAMl provides a means of increasing the level, degree or rate at which the signalling event occurs, in addition to including reference to inducing the subject signalling event thereby effectively inducing, upregulating or sustaining the subject cellular activity.
- the agent which is utilised in accordance with the method of the present invention may be an agent which induces the subject event, agonises an event which has already undergone onset, antagonises a pre-existing event or entirely prevents the onset of such an event.
- modulation of the interaction between sphingosine kinase and SKAMl may be partial or complete. Partial modulation occurs where only some of the sphingosine kinase/SKAMl interactions which may normally occur in a given cell are affected by the method of the present invention (for example, the agent which is contacted with the subject cell is provided in a concentration insufficient to saturate the intracellular sphingosine kinase/SKAMl interactions) while complete modulation occurs where all sphingosine kinase/SKAMl interactions are modulated.
- Modulation of the interaction between sphingosine kinase and SKAMl may be achieved by any one of a number of techniques including, but not limited to:
- agent should be understood as a reference to any proteinaceous or non- proteinaceous molecule which modulates the interaction of sphingosine kinase with SKAMl and includes, for example, the molecules detailed in points (i) - (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 its targeting to a localised region.
- the subject agent is SKAMl itself, or functional derivative, homologue or mimetic thereof, which is introduced to upregulate sphingosine kinase activation.
- a method of upregulating sphingosine kinase mediated signalling comprising contacting sphingosine kinase with an effective amount of SKAMl or functional derivative, homologue or mimetic thereof for a time and under conditions sufficient to induce the interaction of sphingosine kinase with SKAMl and thereby upregulate sphingosine kinase catalytic activity.
- 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.
- the present invention contemplates chemical analogues of SKAMl capable of acting as agonists or antagonists of the sphingosine kinase interaction.
- Chemical agonists may not necessarily be derived from sphingosine kinase or SKAMl but may share certain conformational similarities.
- chemical agonists may be specifically designed to mimic or upregulate certain physiochemical properties of sphingosine kinase or
- 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 SKAMl with an agent and screening for the modulation of sphingosine kinase/SKAMl functional activity (such as a specific cellular activity) or modulation of the activity or expression of a downstream sphingosine kinase or SKAMl cellular target.
- 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 or SKAMl activity such as luciferases, CAT and the like.
- the sphingosine kinase or SKAMl protein may be naturally occurring 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/SKAMl interactivity or the gene may require activation - thereby providing a model useful for, inter alia, screening for agents which modulate sphingosine kinase/SKAMl interactivity under certain stimulatory conditions, such as phage-display and yeast two- or multi-hybrid screening.
- a sphingosine kinase or SKAMl nucleic acid molecule may comprise the entire sphingosine kinase or SKAMl gene or it may merely comprise a portion of the gene such as the SKAMl region which binds to sphingosine kinase.
- the agents which are utilised in accordance with the method of the present invention may take any suitable form.
- proteinaceous agents may be glycosylated or unglycosylated, phosphorylated or dephosphorylated to various degrees and/or may contain a range of other molecules fused, linked, bound or otherwise associated with the proteins such as amino acids, lipids, carbohydrates or other peptides, polypeptides or proteins.
- the subject non-proteinaceous molecules may also take any suitable form. Both the proteinaceous and non-proteinaceous agents herein described may be linked, bound otherwise associated with any other proteinaceous or non-proteinaceous molecules.
- said agent is associated with a molecule which permits its targeting to a localised region, such as a specific tissue.
- expression refers to the transcription and translation of a nucleic acid molecule.
- Reference to “expression product” is a reference to the product produced from the transcription and translation of a nucleic acid molecule.
- Reference to “modulation” should be understood as a reference to upregulation or downregulation.
- “Derivatives” of the molecules herein described include fragments, parts, portions or variants from either natural or non-natural sources.
- Non-natural sources include, for example, recombinant or synthetic sources.
- recombinant sources is meant that the cellular source from which the subject molecule is harvested has been genetically altered. This may occur, for example, in order to increase or otherwise enhance the rate and volume of production by that particular cellular source.
- Parts or fragments include, for example, active regions of the molecule.
- 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 characterised 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 a sequence has been removed and a different residue inserted in its place. Additions to amino acid sequences include fusions with other peptides, polypeptides or proteins, as detailed above.
- Derivatives also include fragments having particular epitopes or parts of the entire protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules.
- sphingosine kinase, SKAMl or derivative thereof may be fused to a molecule in order to facilitate cell membrane localisation.
- Analogs of the molecules 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 conformational constraints on the proteinaceous molecules or their analogs.
- nucleic acid sequences which may be utilised in accordance with the method of the present invention 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 utilised in 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.
- a "variant" of sphingosine kinase or SKAMl should be understood to mean a molecule which exhibits at least some of the functional activity of the form of sphingosine kinase or SKAMl of which it is a variant.
- a variation may take any form and may be naturally or non-naturally occurring.
- a mutant molecule is one which exhibits modified functional activity.
- homologue is meant that the molecule is derived from a species other than that which is being treated in accordance with the method of the present invention.
- “Mimetics” should be understood as molecules exhibiting any one or more of the functional activities of the subject molecule, which functional equivalents may be derived from any source such as being chemically synthesised or identified via screening processes such as natural product screening.
- functional equivalents can be designed and/or identified utilising well known methods such as combinatorial chemistry or high throughput screening of recombinant libraries or following natural product screening. These methods may also be utilised to screen for any of the modulatory agents which are useful in the method of the present invention.
- 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 et a (1994) Proc. Natl. Acad. ScL USA, 91 :4708-4712; DeWitt et a (1993) Proc. Natl. Acad. ScL 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 array, 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.
- 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.
- they may be used as a starting point for developing sphingosine kinase SKAMl agonists or antagonists.
- Sphingosine kinase and/or SKAMl or a relevant part thereof 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).
- 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.
- a selected biological agent such as a biomolecule, a macromolecule complex, or cell
- each member of the library is screened for its ability to interact specifically with the selected agent.
- 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.
- 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.
- “Analogues" of sphingosine kinase, SKAMl or agonistic or antagonistic agents contemplated herein include, but are not limited to, modifications to side chains, incorporating unnatural amino acids and/or derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the analogues.
- the specific form which such modifications can take will depend on whether the subject molecule is proteinaceous or non- proteinaceous. The nature and/or suitability of a particular modification can be routinely determined by the person of skill in the art.
- 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 NaBH 4 .
- 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);
- 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 derivatisation, 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.
- Tyrosine residues 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, phenylglycine, 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 1.
- D-N-methylcysteine Dnmcys N-(3 , 3 -diphenylpropyl)glycine Nbhe D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg
- D-N-methyltryptophan Dnmtrp N-(l-methylethyl)glycine Nval
- D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap
- the method of the present invention may be performed in the context of a cellular source which is located either in vitro or in vivo.
- the method of the present invention provides a means of modulating cellular activity which is regulated or controlled by sphingosine kinase signalling.
- 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.
- inflammatory cytokines such as tumour necrosis factor ⁇ and interleukin 1
- endotoxin oxidised or modified lipids
- radiation or tissue injury e.g., endotoxin, oxidised or modified lipids, radiation or tissue injury.
- 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.
- the preferred method is to down- regulate sphingosine
- 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 interaction of sphingosine kinase with SKAMl or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association up-regulates said cellular activity and inhibiting or otherwise antagonising said association down-regulates said cellular activity.
- 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 interaction of sphingosine kinase 1 with SKAMl or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association up-regulates said cellular activity and inhibiting or otherwise antagonising said association down-regulates said cellular activity.
- 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 interaction of sphingosine kinase with the amino acid region corresponding to residues 51- 132 of SEQ ID NO: 1 wherein inducing or otherwise agonising said association up- regulates said human cellular activity and inhibiting or otherwise antagonising said association down-regulates said human cellular activity.
- said modulation is upregulation of cellular activity which is achieved by inducing or agonising the interaction of sphingosine kinase with the amino acid region corresponding to residues 51-132 of SEQ ID NO:1.
- said agent is SKAMl or a peptide comprising the sequence of SEQ ID NO: 6 itself.
- 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.
- 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.
- 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.
- 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 the interaction of sphingosine kinase with SKAMl or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association up-regulates said cellular activity and inhibiting or otherwise antagonising said association 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 interaction of sphingosine kinase with SKAMl or functional derivative, homologue or mimetic thereof wherein inducing or otherwise agonising said association up-regulates said sphingosine kinase activity and inhibiting or otherwise antagonising said association down-regulates said sphingosine kinase activity.
- said interaction occurs with the amino acid region corresponding to residues 51-132 of SEQ ID NO:1.
- 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 interaction of sphingosine kinase with the amino acid region corresponding to residues 51-132 of SEQ ID NO:1, wherein inducing or otherwise agonising said interaction up-regulates said cellular activity and inhibiting or otherwise antagonising said interaction down-regulates said cellular activity.
- 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 interaction of sphingosine kinase with the amino acid region corresponding to residues 51-132 of SEQ ID NO:1 wherein inducing or otherwise agonising said interaction up-regulates said sphingosine kinase functional activity and inhibiting or otherwise antagonising said interaction down-regulates said sphingosine kinase functional activity.
- said modulation is upregulation and said agent is SKAMl or a peptide comprising the sequence of SEQ ID NO: 6 or functional derivative, homologue or mimetic thereof.
- references to "aberrant, unwanted or otherwise inappropriate” cellular activity should be understood as a reference to overactive cellular activity, to physiologically 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.
- a cell is neoplastic, it is desirable that the promotion of cellular proliferation and anti- apoptotic characteristics be down-regulated.
- diseases which are characterised by inflammation such as rheumatoid arthritis, atherosclerosis, asthma, autoimmune disease and inflammatory bowel disease, are known to involve cellular activation 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 activation in order to stimulate cellular proliferation, for example in order to promote angiogenesis.
- mamal as used herein includes humans, primates, livestock animals (eg. sheep, pigs, cattle, horses, donkeys), laboratory test animals (eg.
- 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.
- treatment does not necessarily imply that a subject is treated until total recovery.
- 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.
- 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.
- the modulatory agent of the pharmaceutical composition is contemplated to exhibit therapeutic activity when administered 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).
- acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like.
- 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.
- a binder such as tragacanth, corn starch or gelatin
- a disintegrating agent such as alginic acid
- a lubricant such as magnesium stearate.
- Routes of administration include, but are not limited to, respiratorally, intratracheally, nasopharyngeal ⁇ , intravenously, intraperitoneally, subcutaneously, intracranially, intradermally, intramuscularly, intraoccularly, intrathecally, intracereberally, intranasally, infusion, orally, rectally, via IV drip patch and implant.
- the agent defined in accordance with the present invention may be coadministered with one or more other compounds or molecules.
- 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.
- the subject agent may be administered together with an agonistic agent in order to enhance its effects.
- 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.
- 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 interaction of sphingosine kinase and wherein inducing or otherwise agonising said interaction up-regulates said cellular activity and inhibiting or otherwise antagonising said interaction down-regulates said cellular activity.
- 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 interaction of sphingosine kinase with SKAMl or functional derivative, homologue or mimetic thereof and wherein inducing or otherwise agonising said interaction upregulates said sphingosine kinase functional activity and inhibiting or otherwise antagonising said interaction downregulates said sphingosine kinase functional activity.
- said interaction is interaction with the amino acid region corresponding to residues 51-132 of SEQ ID NO:1.
- said mammal is a human, and said modulation is upregulation.
- the present invention contemplates a pharmaceutical composition
- 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.
- 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.
- 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.
- 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.
- 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.
- the active ingredients 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.
- the active compound 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 ⁇ g and
- 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 cherry flavouring.
- 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
- a sweetening agent such as sucrose, lactose or saccharin
- a flavouring agent such as peppermint, oil of wintergreen, or
- 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.
- any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
- 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.
- said percent similarity is 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher.
- the present invention should be understood to extend to nucleic acid molecules encoding said mimetics.
- Glutathione and agarose powder were from Sigma Chemical Co (St. Louis, MO, USA).
- Dulbecco's modified eagle's medium (DMEM), HEPES buffer solution, foetal calf serum, penicillin and streptomycin were purchased from CSL Biosciences (Parkville, Australia).
- Protease inhibitors (CompleteTM) were purchased from Roche Diagnostics GMbH (Mannheim, Germany), and BenchmarkTM Pre-stained protein ladder was purchased from Invitrogen (San Diego, CA), nitrocellulose membranes from Schleicher and Schuell (Keene, USA), and isopropyl ⁇ -D- thiogalactoside (IPTG) from Promega (USA).
- FITC-conjugated sheep anti-rabbit and anti-mouse IgG were from AMRAD (Melbourne, Australia).
- Alexa (594)- conjugated anti-rabbit and anti-mouse IgG were from Molecular Probes (Eugene, Oregon USA).
- Fluorescent mounting medium was from DAKO Corporation (CA, USA)
- protein assay dye reagent was purchased from Bio-Rad Laboratories (CA, USA).
- O-erythro- Sphingosine from Biomol Research Laboratories Inc (Plymouth, PA) and [y- 32 P] ATP from Perkin Elmer (Melbourne, Australia).
- Yeast two-hybrid screening was performed using the Matchmaker Gal4 Two-Hybrid System 3 (Clontech) according to the manufacturer's instructions.
- Full-length hSKl cDNA (Genbank accession number AF200328) was cloned into pGBKT7 (Clontech) in-frame with the Gal4 DNA-binding domain.
- This bait construct was then transformed into the yeast strain AHl 07 together with a human leukocyte cDNA library in pACT2 (Clontech). A total of 1 x 10 6 independent clones were screened, yielding ten confirmed positive clones.
- SKAMlA and B were PCR amplified from placenta cDNA, and SKAMlD from total mononuclear cell cDNA.
- the primers were: SKAMlA, 5'-TAGAATTCCAATGAGTTGCACAATTGAGAAG-S' (SEQ ID NO:7) and 5'-TAGAATTCAGCTCTTCCTCAGACTC-S' (SEQ ID NO:8); SKAMlB, 5'- TAGAATTCCAATGAGTTGCACAATTGAGAAG-S' (SEQ ID N0:9) and 5'- TAGAATTC AGCTCTTCCTC AGACTC-3' (SEQ ID NO: 10), and; SKAMlD, 5'- TAGAATTCCAATGAGTTGCACAATTGAGAAG-S' (SEQ ID NO: 11) and 5'- TAGAATTCAGGTCACATTTATAGAATGG-S' (SEQ ID NO: 12).
- PCR products were cut with EcoRl and cloned in frame into pCMV(HA) mammalian expression vector (Clontech).
- SKAMlC and Y were then generated by PCR from ⁇ CMV(HA)-SKAMlA using the primers: SKAMlC, 5'-TAGAATTCCAATGAGTTGCACAATTGAGAAG-S' (SEQ ID NO: 13) and 5'-TAGAATTCTTAGTGTACATTCTGATTTGCTTCCAAGTGC- 3' (SEQ ID NO:14), and: SKAMlY, 5'- TAGAATTCCACAAGAACTTAATGAAGTCGCG-S' (SEQ ID NO: 15) and 5'-
- HEK-293T Human embryonic kidney cells
- DMEM fetal calf serum
- FCS calcium phosphate precipitation method
- HEK-293T transfected cells were harvested the next day by scraping into 10 ml cold PBS and then were centrifuged at 1500 rpm at 4 0 C for 5 min.
- SKl(FLAG) (Pitson et al, 2000a, supra) were lysed by sonication (2 watts for 30 sec at 4 0 C) in extraction buffer containing 50 mM Tris/HCl (pH 7.4), 10 % glycerol, 0.05 % Triton X- 100, 150 mM NaCl, 1 mM dithiothreitol, 2 mM Na 3 VO 4 , 10 mM NaF, 1 mM EDTA and CompleteTM protease inhibitors. Cells transfected with pcDNA3. SK2(FLAG) (Roberts et al., 2004, Anal. Biochem.
- Lysates expressing the hSKl(FLAG) or hSK2(FLAG) alone and/or in combination with HA-SKAMl isoforms were centrifuged at 13,000 g for 10 min at 4 0 C to remove insoluble material.
- Antibodies were added to the lysates and incubated at 4 0 C for 3 hr with constant agitation.
- the immune complexes were then captured by incubation with 50 ⁇ l of a 50 % slurry of protein A Sepharose (Amersham Pharmacia Biotech) for 3 hr at 4 0 C, washed with cold extraction buffer, subjected to SDS-PAGE and the proteins transferred to nitrocellulose membranes.
- hSKl was quantitated with either the monoclonal M2 anti- FLAG antibody (Sigma), or a polyclonal chicken or rabbit anti-hSKl antibodies raised against recombinant hSKl generated in E. coli (Pitson et al, 2000a, supra).
- SKAMl was determined with either anti-HA antibodies (12CA5; Sigma) or anti-SKAMl antibodies.
- the immunocomplexes were detected with HRP-conjugated anti-mouse (Pierce), anti- rabbit (Pierce) or anti-chicken IgG (IMVS, Sydney, Australia) using an enhanced chemiluminescence kit (ECL, Amersham Pharmacia Biotech). Generation of GST-SKAMl isoforms
- pCMV(HA)-SKAMlA, B 3 C, D, and Y were digested with EcoR I then cloned in frame into the pGEX4T2 vector and transformed into E. coli JM 109 by electroporation. Colonies were picked and grown overnight in either 30 ml L-broth or Super broth (containing 35 g/L tryptone, 20 g/Lyeast extract, 5 g/L NaCl, 20 g/L glucose and 100 ⁇ g/ml ampicillin, pH 7.5). The overnight cultures were diluted 1:30 into 300 ml of either L-broth or Super broth and incubated at 37 0 C with shaking until the OD 600 reached about 0.8.
- GST-SKAMl isoforms were isolated from the clarified lysates through the addition of 200 ⁇ l of a 50 % slurry of glutathione Sepharose (Amersham Pharmacia Biotech) and incubation for 3 hr at 4 0 C with constant mixing.
- the glutathione Sepharose with bound GST-SKAMl was harvested by centrifugation (3000 rpm, 4 0 C 5 min) and washed 3 times with cold PBS.
- the GST-SKAMl proteins were then eluted by the addition of 250 ⁇ l of 20 mM glutathione with mixing at 4 0 C for 30 min.
- Sphingosine kinase activity was determined using D-e/yt/jr ⁇ -sphingosine and [y- 52 P] ATP as substrates as described previously (Pitson et ah, 2000a, supra).
- Assays for hSKl assays were performed using sphingosine-solubilised with Triton X-100, while assays for hSK2 (Roberts et ah, 2004, supra) were performed in the same manner, except that sphingosine was supplied as a complex with BSA.
- One unit (U) of activity is defined as 1 pmol of SlP formed per minute per mg of protein.
- SK substrate kinetic analyses were preformed by varying sphingosine concentrations (1 to 50 ⁇ M) with constant ImM ATP, and varying ATP concentrations (5 to 500 ⁇ M) with constant 100 ⁇ M sphingosine (Sph).
- the kinetic data were analysed using Michaelis-Menten kinetics with the non-linear regression program Hyper 1.1s.
- Lysates from total mononuclear cells (TMNC), HEK293-T cells and NIH 3T3s were generated, centrifuged at 13,000 rpm for 15 min at 4 0 C to remove the insoluble material and subjected to SDS-PAGE using 12 % polyacylamide gels.
- the membranes were blocked in PBS containing 5 % SMP and 0.1 % Tween-20 for 30 min at RT on a rocking platform. While the membranes were blocking, anti-SKAMl antibodies at 1:10000 dilution were incubated with either GST-SKAMlD bound to glutathione-Sepharose or with GST bound to glutathione-Sepharose for 1 hr at RT on a rocking platform.
- the glutathione-Sepharose beads were centrifuged at 2000 rpm for 5 min at 4 0 C.
- the supernatants, representing the control and immunodepleted anti-sera SKAMIs were then used for the Western blo
- Triton- insoluble material was then separated by centrifugation at 13,000 rpm at 4 0 C for 10 min. Triton-soluble membrane fractions were collected and pellets, representing the Triton- insoluble cytoskeletal fraction were again resuspended in extraction buffer containing 0.8 % Triton X-IOO, and sonicated at 3 Hz for 30 sec. All fractions were subjected to 15 % and 12 % polyacylamide gels followed by Western blotting using anti HA and anti-FLAG antibodies as in as described earlier.
- Murine embryonic fibroblasts (NIH 3T3) and breast cancer, cell lines (MCF -7) were plated into DMEM in 6 cm culture dishes. After 24 hr, the NIH 3T3 cells were transfected with pCMV(HA)-SKAMl A, IB, 1C and ID either alone or in combination with pcDNA3 vector encoding GFP-SKl (Pitson et ah, 2003, supra) using Lipofectamine 2000 according to the manufacturer's instructions. The next day Labtek slides were coated with 200 ⁇ l of 50ng/ml fibronectin and incubated at 37 0 C for 30 min.
- the transfected NIH 3T3 and untransfected MCF-7 cells were washed in PBS, resuspended in 5 ml DMEM, counted and then diluted in an appropriate volume of DMEM.
- Fibronectin from 8 well labtek slides were removed, 12,500 cells were seeded per well and incubated overnight at 37 0 C.
- the medium was removed and the cells washed with 200 ⁇ l PBS per well, and fixed with 200 ⁇ l 4 % paraformaldehyde for 10 min. Cells were washed twice with 200 ⁇ l of PBS containing 0.1 % Triton X-100 (PBST).
- PBST was removed and incubated with 100 ⁇ l of anti-HA at 1:4000 and/or anti-SKAMl antibodies at either 1 : 1000 or 1 :4000 dilution in 3 % BSA in PBST for 1 hr at RT.
- the antibodies were removed and cells washed 3 x with 200 ⁇ l PBST.
- Cells were incubated with 100 ⁇ l of anti- mouse Alexa 594 dilution 1:500 and anti-rabbit FITC at 1:1000 dilution in 3 % BSA in PBST for 1 hr at RT in the dark.
- Secondary antibodies were removed and washed cells 3 x with 200 ⁇ l PBST before washing in 200 ⁇ l PBS. The slide was drip-dried, and fluorescent mounting medium (Dako) and coverslip applied.
- a yeast two-hybrid screen was performed to identify proteins that interact with hSKl using a human leukocyte cDNA library with full-length hSKl as the bait. Ten putative hSKl- interacting proteins were identified by this screen, including one designated Sphingosine Kinase Activating Molecule 1 (SKAMl). Database searches revealed four different transcripts, that appear to be produced from alternative splicing, which encode for four different naturally occurring SKAMl protein isoforms (A, B, C & D) ( Figure 1).
- the partial SKAMl cDNA isolated via the yeast two-hybrid screen encoded a protein that was identical to the c-terminal region of the SKAMlD isoform ( Figure IA) since it possesses 8 amino acids at the extreme C-terminus that are unique to this isoform.
- SKAMl is a protein present in many organisms, ranging from humans to flat worms. At present, the structure and function of this protein has yet to be elucidated. Furthermore, no recognizable domains can be found in SKAMl by comparison to other known proteins. Results from initial domain searches using the SMART database (Schultz et al, 1998, . Proc. Natl. Acad. Sci. USA 95:5857-5864) revealed that SKAMlA may possess a AAA-domain. AAA (ATPases associated with diverse cellular activities) proteins are involved in ATP-dependent protein folding, assembly of protein complexes, protein transport through membrane fusion and protein degradation (Ogura & Wilkinson, 2001, Genes Cells 6:575-597).
- AAA ATPases associated with diverse cellular activities
- SKAMlA showed some sequence similarity to the AAA domain it does not possess the Walker A (GXXXXGK(T/S)) and Walker B ((DfE)XK) motifs that are essential for ATP binding and classification into the AAA domain family. Furthermore, SKAMlA also does not have other highly conserved amino acid sequences, such as the second region of homology (SRH) or sensor- 1 motif and proline-rich regions, characteristic of the AAA family (Karata et at, 1999, J. Biol. Chem. 274:26225-26232). Cloning and expression of SKAMl isoforms
- SKAMl Y an artificial truncated version of SKAMl was also generated (SKAMl Y) based on the partial SKAMl cDNA isolated by the Y2H screen for proteins that interact with hSKl . This was performed by PCR amplification of the SKAMl isoforms from TMNC and placenta cDNAs and cloning the resultant products into the vector pCMV(HA) for expression in mammalian cells with an N-terminal haemaglutinin (HA) epitope tag.
- SKAMl isoforms A to D
- SKAMlY The four naturally occurring SKAMl isoforms (A to D) and the artificially truncated SKAMlY were then overexpressed in mammalian cells.
- HEK-293T cells were transiently transfected with pCMV(HA)-SKAMl A, B 5 C, D and Y, and 24 hr after transfection the cells were harvested, lysed and the cell lysates immunoblotted for the presence of the overexpressed proteins via their HA-epitope tags.
- SKAMlA 28 kDa
- SKAMlB 24 kDa
- SKAMlC 19 kDa
- SKAMlD 15 kDa
- SKAMlY 10 kDa
- HEK-293T cells were transfected with the pCMV(HA)-SKAMl A, B, C, D, & Y constructs. After 24 hr, the cells were harvested and whole cell lysates analysed for SK enzyme activity.
- the results ( Figure 6) demonstrate that overexpression of all the naturally occurring SKAMl isoforms resulted in increased basal SK activity by approximately two to three-fold. Similarly, expression of the artificially truncated SKAMlY isoform also enhanced cellular SK activity two-fold. Direct interaction between human SKl and SKAMl in cells mediates increased cellular SK activity.
- SKAMl isoforms were generated as purified recombinant GST fusion proteins.
- SKAMlA, B, C, D and Y DNAs were cloned into the pGEX4T2 vector and the recombinant GST-SKAMl proteins were produced in E. coli. The proteins produced were then analysed by SDS-PAGE with Coomassie staining.
- Proteins of molecular masses 54 kDa (GST-SKAMlA), 50 kDa (GST-SKAMlB), 45 kDa (GST- SKAMlC), 41 kDa (GST-SKAMlD) and 35 kDa (GST-SKAMlY) 5 26 kDa (GST) were visualized ( Figure 8).
- the other proteins present in the GST-SKAMlA, D and Y extracts were likely to be degradation products.
- the yield of GST-SKAMl proteins from the preparations were 364 ⁇ g (SKAMlA), 850 ⁇ g (SKAMlB), 870 ⁇ g (SKAMlC), 500 ⁇ g (SKAMlD) and 1500 ⁇ g (SKAMlY) per 300 ml of bacterial culture. Effect of SKAMl on hSKl in vitro
- GST-SKAMl proteins were incubated at 20-fold molar excess with rec-hSKl and the mixtures subjected to in vitro SK activity assays. In the presence of these proteins rec- hSKl activity was increased approximately four-fold with GST-SKAMlA and B and two to three-fold with GST-SKAMlC, D and Y ( Figure 15). Furthermore, the effect of GST- SKAMl isoforms on hSKl were very similar to the effects observed from overexpression of SKAMl isoforms in HEK-293T cells on endogenous hSKl activity ( Figure 6).
- SKAMl enhanced cellular SK activity, its effect on SK activation in cells by TNF ⁇ , which is mediated by phosphorylation of hSKl at Ser225 by ERK1/2 (Pitson et at, 2003, supra), was examined.
- HEK293T cells overexpressing SKAMlA and SKAMlD (the longest and shortest naturally occurring SKAM isoforms), were treated with TNF ⁇ for 10 minutes, harvested, and the cellular SK activity measured.
- SKAMl overexpression increased basal SK activity by approximately two-fold compared with control cells ( Figure 16).
- TNF ⁇ treatment of these cells overexpressing SKAMlA or D resulted in a further increase in SK activity in a similar manner to that observed in control cells, indicating that SKAMl does not effect phosphorylation-mediated activation of SK.
- SKAMl Since SKAMl interacted with hSKl and had a direct effect on its activity, its potential effect on the other human SK isoform, hSK2, was examined.
- the interaction of hSK2 with SKAMlA and D (the longest and smallest naturally occurring SKAMl isoforms) were assessed by co-immunoprecipitation. Lysates from HEK-293T cells co-expressing FLAG- tagged hSK2 with either HA-SKAMlA or D were immunoprecipitated with anti-FLAG antibodies. The presence of SKAMl proteins in the immuno-complexes were then analysed by immunoblots with anti-HA antibodies.
- Proteins of 28 kDa (SKAMlA) and 15 kDa (SKAMlD) were detected in co-immunoprecipitated lysates ( Figure 17), indicating that there was an interaction between hSK2 and SKAMl A and D.
- SKAMlD was chosen as the antigen since it is the smallest naturally occurring SKAMl isoform and almost its complete polypeptide sequence is present in all the other SKAMl isoforms. Thus, it was considered the most likely SKAMl isoform to produce antibodies that would detect all the SKAMl proteins.
- the avidity and specificity of the anti-SKAMl antibody was tested using the final crude anti-serum to detect overexpressed SKAMlD in HEK293T cells.
- TMNC, HEK-293T, and 3T3 cell lysates were immunoprecipitated with anti-SKAMl antibodies and then immunoblotted with anti-SKAMl antibodies.
- Two proteins with molecular masses of approximately 27 kDa and 23 kDa immunoprecipitated from these three cell lysates probably representing endogenous SKAMlA and B ( Figure 20C).
- EDG-I links the PDGF receptor to Src and focal adhesion kinase activation leading to lamellipodia formation and cell migration.
Abstract
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