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.

The vascular complications of diabetes mellitus have been correlated with enhanced activation of protein kinase C (PKC). LY333531, a specific inhibitor of the beta isoform of PKC, was synthesized and was shown to be a competitive reversible inhibitor of PKC beta 1 and beta 2, with a half-maximal inhibitory constant of approximately 5 nM; this value was one-fiftieth of that for other PKC isoenzymes and one-thousandth of that for non-PKC kinases. When administered orally, LY333531 ameliorated the glomerular filtration rate, albumin excretion rate, and retinal circulation in diabetic rats in a dose-responsive manner, in parallel with its inhibition of PKC activities.

The signaling pathways that couple tumor necrosis factor-␣ (TNF␣) receptors to functional, especially inf lammatory, responses have remained elusive. We report here that TNF␣ induces endothelial cell activation, as measured by the expression of adhesion protein E-selectin and vascular adhesion molecule-1, through the sphingosine kinase (SKase) signaling pathway. Treatment of human umbilical vein endothelial cells with TNF␣ resulted in a rapid SKase activation and sphingosine 1-phosphate (S1P) generation. S1P, but not ceramide or sphingosine, was a potent dosedependent stimulator of adhesion protein expression. S1P was able to mimic the effect of TNF␣ on endothelial cells leading to extracellular signal-regulated kinases and NF-B activation, whereas ceramide or sphingosine was not. Furthermore, N,N-dimethylsphingosine, an inhibitor of SKase, profoundly inhibited TNF␣-induced extracellular signal-regulated kinases and NF-B activation and adhesion protein expression. Thus we demonstrate that the SKase pathway through the generation of S1P is critically involved in mediating TNF␣-induced endothelial cell activation.Tumor necrosis factor-␣ (TNF␣) was originally described for its antitumor activity, but is now recognized to be one of the most pleiotropic cytokines in mediating systemic inflammatory and immune responses (1, 2). A major site for these TNF␣ actions is the vascular endothelium, where TNF␣ triggers endothelial cells to secrete various cytokines and induces or enhances the expression of adhesion molecules, such as vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 and E-selectin (3). The regulated expression of these adhesion molecules is essential for the recruitment of circulating blood cells to the endothelium during the inflammatory and immune responses (3-5).TNF␣ activity is exerted through binding two distinct membrane receptors, p55 (TNF␣-R1) and p75 (TNF␣-R2). Engagement of the TNF␣ receptors results in recruitment of two distinct classes of receptor-associated proteins, one the TRADD, FADD͞MORT1 and RIP family, and the other, the TRAF family (6-8). Both of these appear to couple TNF␣ receptors to downstream signaling cascades such as cysteine proteases and NF-B activation to regulate cell proliferation, differentiation, and programmed cell death (6). Recently, the lipid second messenger, ceramide, has also received attention in TNF␣ signaling (6, 9). TNF␣ stimulates the activation of sphingomyelinase, yielding ceramide that, in turn, can induce apoptosis and may play a role in apoptotic signaling in various cell types (6, 9). In addition, ceramide can be subsequently metabolized to sphingosine and sphingosine 1-phosphate (S1P), via ceramidase and sphingosine kinase (SKase) activation, respectively (10). These sphingomyelin metabolites were also proposed to play a variety of roles in regulation of cellular activities such as calcium mobilization, cell motility, and mitogenesis (9, 10). In this study, we demonstrate that TNF␣ promoted generation of ceramide that was...