Sphingosine kinase 1 is an agonist-activated signalling enzyme that catalyses the formation of sphingosine 1-phosphate, a lipid second messenger that has been implicated in a number of agonist-driven cellular responses, including stimulation of cell proliferation, inhibition of apoptosis and expression of in¯ammatory molecules. Although agonist-induced stimulation of sphingosine kinase activity is critical in a number of signalling pathways, nothing has been known of the molecular mechanism of this activation. Here we show that this activation results directly from phosphorylation of sphingosine kinase 1 at Ser225, and present several lines of evidence to show compellingly that the activating kinase is ERK1/2 or a close relative. Furthermore, we show that phosphorylation of sphingosine kinase 1 at Ser225 results not only in an increase in enzyme activity, but is also necessary for translocation of the enzyme from the cytosol to the plasma membrane. Thus, these studies have elucidated the mechanism of agonist-mediated sphingosine kinase activation, and represent a key ®nding in understanding the regulation of sphingosine kinase/sphingosine 1-phosphate-controlled signalling pathways.

Sphingosine kinase (SphK) is a highly conserved lipid kinase that phosphorylates sphingosine to form sphingosine-1-phosphate (S1P). S1P/SphK has been implicated as a signalling pathway to regulate diverse cellular functions [1-3], including cell growth, proliferation and survival [4-8]. We report that cells overexpressing SphK have increased enzymatic activity and acquire the transformed phenotype, as determined by focus formation, colony growth in soft agar and the ability to form tumours in NOD/SCID mice. This is the first demonstration that a wild-type lipid kinase gene acts as an oncogene. Using a chemical inhibitor of SphK, or an SphK mutant that inhibits enzyme activation, we found that SphK activity is involved in oncogenic H-Ras-mediated transformation, suggesting a novel signalling pathway for Ras activation. The findings not only point to a new signalling pathway in transformation but also to the potential of SphK inhibitors in cancer therapy.

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 1/2–mediated 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.

Sphingosine kinase (SK) catalyzes the formation of sphingosine 1-phosphate (S1P), a lipid messenger that plays an important role in a variety of mammalian cell processes, including inhibition of apoptosis and stimulation of cell proliferation. Basal levels of S1P in cells are generally low but can increase rapidly when cells are exposed to various agonists through rapid and transient activation of SK activity. To date, elucidation of the exact signaling pathways affected by these elevated S1P levels has relied on the use of SK inhibitors that are known to have direct effects on other enzymes in the cell. Furthermore, these inhibitors block basal SK activity, which is thought to have a housekeeping function in the cell. To produce a specific inhibitor of SK activation we sought to generate a catalytically inactive, dominant-negative SK. This was accomplished by site-directed mutagenesis of Gly 82 to Asp of the human SK, a residue identified through sequence similarity to the putative catalytic domain of diacylglycerol kinase. This mutant had no detectable SK activity when expressed at high levels in HEK293T cells. Activation of endogenous SK activity by tumor necrosis factor-␣ (TNF␣), interleukin-1␤, and phorbol esters in HEK293T cells was blocked by expression of this inactive sphingosine kinase (hSK G82D ). Basal SK activity was unaffected by expression of hSK G82D . Expression of hSK G82D had no effect on TNF␣-induced activation of protein kinase C and sphingomyelinase activities. Thus, hSK G82D acts as a specific dominant-negative SK to block SK activation. This discovery provides a powerful tool for the elucidation of the exact signaling pathways affected by elevated S1P levels following SK activation. To this end we have employed the dominant-negative SK to demonstrate that TNF␣ activation of extracellular signal-regulated kinases 1 and 2 (ERK1,2) is dependent on SK activation.

SK1 (sphingosine kinase 1) plays an important role in many aspects of cellular regulation. Most notably, elevated cellular SK1 activity leads to increased cell proliferation, protection from apoptosis, and induction of neoplastic transformation. We have previously shown that translocation of SK1 from the cytoplasm to the plasma membrane is integral for oncogenesis mediated by this enzyme. The molecular mechanism mediating this translocation of SK1 has remained undefined. Here, we demonstrate a direct role for CIB1 (calcium and integrin-binding protein 1) in this process. We show that CIB1 interacts with SK1 in a Ca 2؉ -dependent manner at the previously identified "calmodulin-binding site" of SK1. We also demonstrate that CIB1 functions as a Ca 2؉ -myristoyl switch, providing a mechanism whereby it translocates SK1 to the plasma membrane. Both small interfering RNA knockdown of CIB1 and the use of a dominant-negative CIB1 we have generated prevent the agonist-dependent translocation of SK1. Furthermore, we demonstrate the requirement of CIB1-mediated translocation of SK1 in controlling cellular sphingosine 1-phosphate generation and associated anti-apoptotic signaling. SK1 (sphingosine kinase 1) catalyzes the formation of sphingosine 1-phosphate (S1P), 2 a bioactive phospholipid that mediates a wide variety of cellular processes. Elevated cellular S1P has been shown to be pro-proliferative and anti-apoptotic (1), and considerable evidence now exists implicating SK1 in cancer. In particular, overexpression of SK1 in NIH3T3 fibroblasts leads to a transformed phenotype and the ability to form tumors in mice, with SK1 activity also involved in oncogenic H-Ras-mediated transformation (2). Furthermore, suppression of cellular SK1 activity by genetic or pharmacologic approaches has been shown to significantly reduce tumor growth in vivo in mice (3-5) and also sensitize tumor cells to other chemotherapeutics (6).We have previously shown that although SK1 has intrinsic catalytic activity (7), its further activation is required for oncogenic signaling (8). This activation, brought about through phosphorylation at Ser-225 by ERK1/2, not only increases the catalytic activity of SK1 but also results in its translocation from the cytoplasm to the plasma membrane (9), which is essential for the oncogenic signaling by this enzyme (8, 10).Although critical in understanding SK1-induced oncogenesis, the mechanisms regulating agonist-induced translocation of SK1 to the plasma membrane are poorly understood. Studies have suggested that SK1 associates with phosphatidylserine in a phosphorylation-dependent manner, providing a possible mechanism for retention of SK1 at the plasma membrane (11). Although this may facilitate retention of SK1 at the plasma membrane, the molecular mechanism mediating the initial rapid agonist-induced translocation of SK1 has not yet been established. Calmodulin (CaM) has been indirectly implicated in this process because W7, a CaM inhibitor, blocked SK1 translocation (12), as did mutation of the CaM-bindin...

Sphingosine 1-phosphate (S1P) is a novel lipid messenger that has important roles in a wide variety of mammalian cellular processes including growth, differentiation and death. Basal levels of S1P in mammalian cells are generally low, but can increase rapidly and transiently when cells are exposed to mitogenic agents and other stimuli. This increase is largely due to increased activity of sphingosine kinase (SK), the enzyme that catalyses its formation. In the current study we have purified, cloned and characterized the first human SK to obtain a better understanding of its biochemical activity and possible activation mechanisms. The enzyme was purified to homogeneity from human placenta using ammonium sulphate precipitation, anion-exchange chromatography, calmodulin-affinity chromatography and gel-filtration chromatography. This resulted in a purification of over 10(6)-fold from the original placenta extract. The enzyme was cloned and expressed in active form in both HEK-293T cells and Escherichia coli, and the recombinant E. coli-derived SK purified to homogeneity. To establish whether post-translational modifications lead to activation of human SK activity we characterized both the purified placental enzyme and the purified recombinant SK produced in E. coli, where such modifications would not occur. The premise for this study was that post-translational modifications are likely to cause conformational changes in the structure of SK, which may result in detectable changes in the physico-chemical or catalytic properties of the enzyme. Thus the enzymes were characterized with respect to substrate specificity and kinetics, inhibition kinetics and various other physico-chemical properties. In all cases, both the native and recombinant SKs displayed remarkably similar properties, indicating that post-translational modifications are not required for basal activity of human SK.

Tumor necrosis factor-␣ (TNF) receptor-associated factor 2 (TRAF2) is one of the major mediators of TNF receptor superfamily transducing TNF signaling to various functional targets, including activation of NF-B, JNK, and antiapoptosis. We investigated how TRAF2 mediates differentially the distinct downstream signals. We now report a novel mechanism of TRAF2-mediated signal transduction revealed by an association of TRAF2 with sphingosine kinase (SphK), a lipid kinase that is responsible for the production of sphingosine 1-phosphate. We identified a TRAF2-binding motif of SphK that mediated the interaction between TRAF2 and SphK resulting in the activation of the enzyme, which in turn is required for TRAF2-mediated activation of NF-B but not JNK. In addition, by using a kinase inactive dominant-negative SphK and a mutant SphK that lacks TRAF2-binding motif we show that the interaction of TRAF2 with SphK and subsequent activation of SphK are critical for prevention of apoptosis during TNF stimulation. These findings show a role for SphK in the signal transduction by TRAF2 specifically leading to activation of NF-B and antiapoptosis. Tumor necrosis factor-␣ (TNF)1 is a pleiotropic cytokine that elicits a wide spectrum of physiologic and pathogenic events such as cell activation, proliferation, cell death, and inflammation. The different cellular responses to TNF are signaled through cell surface receptors (p55, TNFR1 and p75, TNFR2), and their adaptor proteins, initiating different signaling pathways. These distinct signals can lead to opposing cellular effects as best exemplified by TNF's proapoptotic and antiapoptotic role (1). TNF-induced apoptosis primarily depends on the recruitment of a complex of adaptor proteins, including TRADD and FADD/MORT1 leading to the further recruitment and activation of various caspases and, subsequently, to programmed cell death (2, 3). On the other hand, the cell activation, inflammatory reaction, and antiapoptotic function of the TNF receptor superfamily are predominantly mediated by another class of adaptor proteins, TNF receptor-associated factors (TRAF) (1,4,5). To date, six members of TRAF proteins have been identified in mammals from TRAF1 to TRAF6. TRAF2 is the prototypical member of TRAF family. It can interact directly or indirectly with various members of TNF receptor superfamily to mediate the signal transduction of these receptors. TRAF2 can also interact with numerous intracellular proteins, such as I-TRAF/TANK, RIP, MAPK kinase kinase, NIK, and the caspase inhibitors cIAPs, and thereby transduces signals required for the activation of the transcription factor NF-B, the stress-activated protein kinase (SAPK or JNK) and antiapoptosis (6 -9). While structural studies have revealed the complexity and flexibility of TRAF2 (10) as a signal junction to transduce various signal pathways, it is still not clear how TRAF2 can differentially activate its distinct downstream signals such as NF-B and JNK, leading to different biological functions.Sphingolipids have recently emerg...

Sphingosine kinase catalyzes the formation of sphingosine 1-phosphate, a lipid second messenger that has been implicated in a number of agonist-driven cellular responses including mitogenesis, anti-apoptosis, and expression of inflammatory molecules. Despite the importance of sphingosine kinase, very little is known regarding its structure or mechanism of catalysis. Moreover, sphingosine kinase does not contain recognizable catalytic or substrate-binding sites, based on sequence motifs found in other kinases. Here we have elucidated the nucleotide-binding site of human sphingosine kinase 1 (hSK1) through a combination of site-directed mutagenesis and affinity labeling with the ATP analogue, FSBA. We have shown that Gly 82 of hSK1 is involved in ATP binding since mutation of this residue to alanine resulted in an enzyme with an ϳ45-fold higher K m(ATP) . We have also shown that Lys 103 is important in catalysis since an alanine substitution of this residue ablates catalytic activity. Furthermore, we have shown that this residue is covalently modified by FSBA. Our data, combined with amino acid sequence comparison, suggest a motif of SGDGX 17-21 K is involved in nucleotide binding in the sphingosine kinases. This motif differs in primary sequence from all previously identified nucleotide-binding sites. It does, however, share some sequence and likely structural similarity with the highly conserved glycine-rich loop, which is known to be involved in anchoring and positioning the nucleotide in the catalytic site of many protein kinases.