Determination of Sphingosine Kinase Activity for Cellular Signaling Studies

The sphingolipids sphingosine and sphingosine-1-phosphate (S1P) play crucial roles as signal transduction molecules involved in cell survival and migration.1–6 These second messengers along with the sphingolipid metabolite ceramide are interconvertible, and their dynamic equilibrium is believed to be a determining factor in whether cells will live or die.7 In addition to its role as an intracellular second messenger, S1P also acts as an extracellular ligand making it a pleiotropic signaling molecule with wide-ranging function from calcium homeostasis to chemotaxis.4,7–9 S1P is produced by phosphorylation of sphingosine by sphingosine kinase 1 and 2 (SK1 and SK2) and is reversibly dephosphorylated by two known mammalian phosphatases SPP1 and SPP2.6,9–11 In addition, S1P can be irreversibly degraded by a pyridoxylphosphate-dependent S1P lyase to hexadecenal and phosphoethanolamine.6,12 The balance and interplay of these metabolic pathways remain to be fully elucidated. SK1 is thought to be oncogenic, and inhibitors of SK1 appear to act as effective chemotherapeutic agents in animal studies.7,10,13,14 SK2 is involved in the immune response, and compounds directed at extracellular S1P signaling are showing great promise in clinical trials for autoimmune diseases.10,15–18 Thus, sphingosine signaling is proving to be extremely important in clinical medicine.17–21 Although S1P plays a major role as an extracellular signaling molecule, it is predominately synthesized by SK within cells.6 The precise mechanism and control of S1P cellular export is not clearly understood, although sphingosine and sphingosine derivatives do appear to readily enter and exit living cells.22,23 Recently, the primary reservoir for S1P acting to influence the immune system has been shown to reside in erythrocytes (red blood cells). It remains unclear whether the intracellular pool of S1P is from endogenous kinase activity or from uptake and storage of exogenous S1P.23,24 These studies have been hampered by the inability to quantitatively measure SK activity in cells. The traditional assay for S1P requires a two-step CHCl3 extraction followed by 9-fluorenylmethyl chloroformiate derivatization of the extracts and analysis by HPLC.23 However, these steps reduce the accuracy of quantitative measurements such that only large changes in S1P concentration in the biological sample can be identified.25 Another common method for the determination of SK activity is by radiometric assay.26–28 There are two widely used protocols. The first protocol involves incubation of the purified enzyme or cell lysate with sphingosine and [γ−32P]ATP. Organic solvents are used to extract the sphingoid base 1-phosphates under acidic conditions with recoveries of at best 50%.25,28 The products are separated by thin-layer chromatography (TLC), and [32P]S1P is identified by autoradiography or phosphorimaging.28 An alternative, albeit even less sensitive, assay uses 3H-labeled sphingosine followed by extraction in CHCl3 under basic conditions.26,27 The reaction product [3H]S1P is recovered in an aqueous phase and separated by TLC or directly quantified using liquid-scintillation counting. Major drawbacks of radiometric assays for SK are their very limited sensitivity, semiquantitative results, use of organic solvent extraction, poor resolution of structurally similar radiolabeled species, and intense investment of time and labor.25 Other techniques developed for determination of SK activity include HPLC and mass spectrometry (MS).29–32 To detect endogenous SK activity in cell and tissue extracts, these techniques still require nonquantitative extraction techniques to remove interference from cellular proteins, and their sensitivity remains limited.29 In addition, MS is an expensive technique which may not be readily available to many biology laboratories. Newly developed fluorescence-based assays for the determination of SK activity appear promising.33–35 These nonradioactive assays are simpler, faster, and more amenable to high-throughput analyses. For biological samples, the assays still require organic solvent extractions and are more suited for quantifying the activity of enzymes in purified state or in bulk cell lysates. All of the current platforms lack the sensitivity for analysis of cellular samples on the order of tens to hundreds of cells. A direct approach for measurement of the interconversion of sphingosine and S1P in cells would be of benefit, particularly if a highly sensitive means with the potential for single-cell analysis could be achieved.36 Chemical cytometry, the application of high-sensitivity chemical separations to characterize single or small numbers of cells, is emerging as an important approach for biochemical studies.37 A number of techniques including MS, electrochemistry, and capillary electrophoresis (CE) are the fundamental analytical tools for such studies. CE using laser-induced fluorescence detection (LIF) is the most sensitive of these techniques, and numerous groups have used this separation technique for a variety of biochemical studies in single cells, including assays of enzymatic activity.37 The objective of the current work was to develop a sensitive, fluorescent CE-based assay for SK and sphingoid phosphatase activity in small numbers of cells. To achieve this goal, a CE-based separation of fluorescein-labeled sphingosine (SphFl) and the phosphorylated product fluorescein-labeled sphingosine-1-phosphate (SphFl-1-P) was used to measure the activity of purified enzyme. These results demonstrated the utility of this technique for in vitro biochemical determination of kinase and phosphatase activity. Furthermore, it was demonstrated that SphFl was readily taken up by mammalian cells without the need for rigorous loading protocols. The technique was then applied to study endogenous SK activity in erythrocytes using both bulk cell lysates and very small numbers of whole cells. The highly sensitive and quantitative method was fast, simple, and robust for both in vitro and cell-based assays. The methodology described should prove to be a valuable analytical tool for quantitative cellular studies of the sphingosine kinases and phosphatases, enzymes of intense interest in both cell biology and clinical medicine.