human embryonic kidney ( hek293 t ) cells and nih3t3 fibroblasts were cultured and harvested as described previously ( 14 ) . stable and transient transfections were performed using the calcium phosphate precipitation method for hek293 t cells and lipofectamine 2000 ( invitrogen ) for nih3t3 cells . stable transfectants were selected for g418 resistance and pooled to avoid the phenotypic artifacts that may arise from the selection and propagation of individual clones from single transfected cells . for subcellular fractionation , postnuclear supernatants of cell lysates were separated into cytosol and membrane fractions as described previously ( 17 ) . h - ras polyclonal antibody was from santa cruz biotechnology , and hrp - conjugated anti  mouse and anti s1p levels were determined following metabolic labeling of cells with [ p]orthophosphate as outlined previously ( 17 ) . a plasma membrane targeted hsk1 construct ( mp - hsk1 ) containing the myristoylation / palmitylation motif of the lck tyrosine kinase ( mgcgcsshpe ) ( 22 ) was generated by pcr with oligonucleotide primers 5-tagaattcgccaccatgggctgtggctgcagctcacacccggaagatccagcgggcggcc-3 and sp6 using pcdna3-hsk1 ( 4 ) as template dna . the resultant product was then cloned into pcdna3 ( invitrogen ) by digestion with ecori . the orientation was determined by restriction analysis , and sequencing verified the integrity of the flag - tagged mp - hsk1 cdna sequence . to generate flag - tagged mp - hsk1 , the kpni pmli fragment representing the wild - type 5 end of pcdna3-hsk1 ( 17 ) was replaced with the 150-bp kpni cells were plated onto fibronectin - coated 8-well glass chamber slides at 10 cells / well and incubated for 24 h. the cells were fixed with 4% paraformaldehyde in pbs for 10 min , permeabilized with 0.1% triton x-100 in pbs , and incubated with m2 anti - flag antibody in pbs containing 3% bsa and 0.1% triton x-100 for 1 h. the immunocomplexes were then detected with fitc - conjugated anti fluorescence microscopy was performed on an olympus bx-51 microscope equipped with a fluorescein excitation filter ( 494 nm ) acquired to a cool snap fx charge - coupled device camera ( photometrics ) . assays for cell growth were performed by incubating cells in 48-well plates ( 2,500 cells per well ) in medium containing 5 or 1% fcs , or serum free medium ( containing 0.1% bsa ) as described previously ( 8) . cell numbers were determined at the indicated times using the mts assay ( promega ) . cells were plated onto 8-well glass chamber slides ( nalge nunc ) coated with fibronectin at 10 cells / well and grown for 24 h in dmem with 2% fcs . cells were then incubated with 10 m brdu for 3 h , and then fixed and stained for its incorporation using an anti  brdu - fluos antibody ( roche ) following the manufacturer 's protocol . cells positive for brdu incorporation were visualized with an olympus bx-51 fluorescence microscope with at least 300 cells scored per point . apoptosis was assessed by staining cells with 1 g / ml dapi in methanol for 15 min at room temperature . apoptotic cells were identified by condensation and fragmentation of nuclei using fluorescence microscopy and expressed as a percentage of the total cells counted . colony formation in soft agar and tumorigenesis assays in mice were performed as detailed previously ( 8) with approval from the institute of medical and veterinary science animal ethics committee . human embryonic kidney ( hek293 t ) cells and nih3t3 fibroblasts were cultured and harvested as described previously ( 14 ) . stable and transient transfections were performed using the calcium phosphate precipitation method for hek293 t cells and lipofectamine 2000 ( invitrogen ) for nih3t3 cells . stable transfectants were selected for g418 resistance and pooled to avoid the phenotypic artifacts that may arise from the selection and propagation of individual clones from single transfected cells . for subcellular fractionation , postnuclear supernatants of cell lysates were separated into cytosol and membrane fractions as described previously ( 17 ) . h - ras polyclonal antibody was from santa cruz biotechnology , and hrp - conjugated anti  mouse and anti s1p levels were determined following metabolic labeling of cells with [ p]orthophosphate as outlined previously ( 17 ) . a plasma membrane  targeted hsk1 construct ( mp - hsk1 ) containing the myristoylation / palmitylation motif of the lck tyrosine kinase ( mgcgcsshpe ) ( 22 ) was generated by pcr with oligonucleotide primers 5-tagaattcgccaccatgggctgtggctgcagctcacacccggaagatccagcgggcggcc-3 and sp6 using pcdna3-hsk1 ( 4 ) as template dna . the resultant product was then cloned into pcdna3 ( invitrogen ) by digestion with ecori . the orientation was determined by restriction analysis , and sequencing verified the integrity of the flag - tagged mp - hsk1 cdna sequence . to generate flag - tagged mp - hsk1 , the kpni pmli fragment representing the wild - type 5 end of pcdna3-hsk1 ( 17 ) was replaced with the 150-bp kpni cells were plated onto fibronectin - coated 8-well glass chamber slides at 10 cells / well and incubated for 24 h. the cells were fixed with 4% paraformaldehyde in pbs for 10 min , permeabilized with 0.1% triton x-100 in pbs , and incubated with m2 anti - flag antibody in pbs containing 3% bsa and 0.1% triton x-100 for 1 h. the immunocomplexes were then detected with fitc - conjugated anti fluorescence microscopy was performed on an olympus bx-51 microscope equipped with a fluorescein excitation filter ( 494 nm ) acquired to a cool snap fx charge - coupled device camera ( photometrics ) . assays for cell growth were performed by incubating cells in 48-well plates ( 2,500 cells per well ) in medium containing 5 or 1% fcs , or serum free medium ( containing 0.1% bsa ) as described previously ( 8) . cell numbers were determined at the indicated times using the mts assay ( promega ) . cells were plated onto 8-well glass chamber slides ( nalge nunc ) coated with fibronectin at 10 cells / well and grown for 24 h in dmem with 2% fcs . cells were then incubated with 10 m brdu for 3 h , and then fixed and stained for its incorporation using an anti  brdu - fluos antibody ( roche ) following the manufacturer 's protocol . cells positive for brdu incorporation were visualized with an olympus bx-51 fluorescence microscope with at least 300 cells scored per point . apoptosis was assessed by staining cells with 1 g / ml dapi in methanol for 15 min at room temperature . apoptotic cells were identified by condensation and fragmentation of nuclei using fluorescence microscopy and expressed as a percentage of the total cells counted . colony formation in soft agar and tumorigenesis assays in mice were performed as detailed previously ( 8) with approval from the institute of medical and veterinary science animal ethics committee .
sphingosine kinase ( sk ) 1 catalyzes the formation of the bioactive lipid sphingosine 1-phosphate , and has been implicated in several biological processes in mammalian cells , including enhanced proliferation , inhibition of apoptosis , and oncogenesis . 
 human sk ( hsk ) 1 possesses high instrinsic catalytic activity which can be further increased by a diverse array of cellular agonists . 
 we have shown previously that this activation occurs as a direct consequence of extracellular signal  regulated kinase 1/2mediated phosphorylation at ser225 , which not only increases catalytic activity , but is also necessary for agonist - induced translocation of hsk1 to the plasma membrane . in this study , we report that the oncogenic effects of overexpressed hsk1 are blocked by mutation of the phosphorylation site despite the phosphorylation - deficient form of the enzyme retaining full instrinsic catalytic activity . 
 this indicates that oncogenic signaling by hsk1 relies on a phosphorylation - dependent function beyond increasing enzyme activity . 
 we demonstrate , through constitutive localization of the phosphorylation - deficient form of hsk1 to the plasma membrane , that hsk1 translocation is the key effect of phosphorylation in oncogenic signaling by this enzyme . 
 thus , phosphorylation of hsk1 is essential for oncogenic signaling , and is brought about through phosphorylation - induced translocation of hsk1 to the plasma membrane , rather than from enhanced catalytic activity of this enzyme .