Sphingosine kinase-mediated calcium signaling by muscarinic acetylcholine receptors

Based on the finding that G protein-coupled receptors (GPCRs) can induce Ca2+ mobilization, apparently independent of the phospholipase C (PLC)/inositol-1,4,5-trisphosphate (IP3) pathway, we investigated whether sphingosine kinase, which generates sphingosine-1-phosphate (SPP), is involved in calcium signaling by mAChR and other GPCRs. Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,/N-dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by M2 and M3 mAChRs in HEK-293 cells without affecting PLC activation. Activation of M2 and M3 mAChR rapidly and transiently stimulated production of SPP. Furthermore, microinjection of SPP into HEK-293 cells induced rapid and transient Ca2+ mobilization. Pretreatment of HEK-293 cells with the calcium chelator BAPTA/AM fully blocked mAChR-induced SPP production. On the other hand, incubation of HEK-293 cells with calcium ionophores activated SPP production. Similar findings were obtained for formyl peptide and P2Y2 purinergic receptors in HL-60 cells. On the basis of these studies we propose, that following initial IP3 production by receptor-mediated PLC activation, a local discrete increase in [Ca2+]i induces sphingosine kinase stimulation, which ultimately leads to full calcium mobilization. Thus, sphingosine kinase activation most likely represents an amplification system for calcium signaling by mAChRs and other GPCRs.     

Photolysis of intracellular caged sphingosine-1-phosphate causes Ca2+ mobilization independently of G-protein-coupled receptors

Sphingosine-1-phosphate (S1P), the product of sphingosine kinase, activates several widely expressed G-protein-coupled receptors (GPCR). S1P might also play a role as second messenger, but this hypothesis has been challenged by recent findings. Here we demonstrate that intracellular S1P can mobilize Ca(2+) in intact cells independently of S1P-GPCR. Within seconds, S1P generated by the photolysis of caged S1P raised the intracellular free Ca(2+) concentration in HEK-293, SKNMC and HepG2 cells, in which the response to extracellularly applied S1P was either blocked or absent. Ca(2+) transients induced by photolysis of caged S1P were caused by Ca(2+) mobilization from thapsigargin-sensitive stores. These results provide direct evidence for a true intracellular action of S1P.     

Protease-activated receptor-1-induced calcium signaling in gingival fibroblasts is mediated by sarcoplasmic reticulum calcium release and extracellular calcium influx

Thrombin is a serine protease activated during injury and inflammation. Thrombin and other proteases generated by periodontal pathogens affect the behavior of periodontal cells via activation of protease-activated receptors (PARs). We noted that thrombin and PAR-1 agonist peptide stimulated intracellular calcium levels ([Ca2+]i) of gingival fibroblasts (GF). This increase of [Ca2+]i was inhibited by EGTA and verapamil. U73122 and neomycin inhibited thrombin- and PAR-1-induced [Ca2+]i. Furthermore, 2-APB (75-100 microM, inositol triphosphate [IP3] receptor antagonist), thapsigargin (1 microM), SKF-96365 (200 microM) and W7 (50 and 100 microM) also suppressed the PAR-1- and thrombin-induced [Ca2+]i. However, H7 (100, 200 microM) and ryanodine showed little effects. Blocking Ca2+ efflux from mitochondria by CGP37157 (50, 100 microM) inhibited both thrombin- and PAR-1-induced [Ca2+]i. Thrombin induced the IP3 production of GF within 30-seconds of exposure, which was inhibited by U73122. These results indicate that mitochondrial calcium efflux and calcium-calmodulin pathways are related to thrombin and PAR-1 induced [Ca2+]i in GF. Thrombin-induced [Ca2+]i of GF is mainly due to PAR-1 activation, extracellular calcium influx via L-type calcium channel, PLC activation, then IP3 binding to IP3 receptor in sarcoplasmic reticulum, which leads to intracellular calcium release and subsequently alters cell membrane capacitative calcium entry.     

Antigen-induced Ca2+ mobilization in RBL-2H3 cells: role of I(1,4,5)P3 and S1P and necessity of I(1,4,5)P3 production

Inositol 1,4,5-trisphosphate (IP3) has long been recognized as a second messenger for intracellular Ca2+ mobilization. Recently, sphingosine 1-phosphate (S1P) has been shown to be involved in Ca2+ release from the endoplasmic reticulum (ER). Here, we investigated the role of S1P and IP3 in antigen (Ag)-induced intracellular Ca2+ mobilization in RBL-2H3 mast cells. Antigen-induced intracellular Ca2+ mobilization was only partially inhibited by the sphingosine kinase inhibitor dl-threo-dihydrosphingosine (DHS) or the IP3 receptor inhibitor 2-aminoethoxydiphenyl borate (2-APB), whereas preincubation with both inhibitors led to complete inhibition. In contrast, stimulation of A3 adenosine receptors with N5-ethylcarboxamidoadenosine (NECA) caused intracellular Ca2+ mobilization that was completely abolished by 2-APB but not by DHS, suggesting that NECA required only the IP3 pathway, while antigen used both the IP3 and S1P pathways. Interestingly, however, inhibition of IP3 production with the phospholipase C inhibitor U73122 completely abolished Ca2+ release from the ER induced by either stimulant. This suggested that S1P alone, without concomitant production of IP3, would not cause intracellular Ca2+ mobilization. This was further demonstrated in some clones of RBL-2H3 cells excessively overexpressing a beta isoform of Class II phosphatidylinositol 3-kinase (PI3KC2beta). In such clones including clone 5A4C, PI3KC2beta was overexpressed throughout the cell, although endogenous PI3KC2beta was normally expressed only in the ER. Overexpression of PI3KC2beta in the cytosol and the PM led to depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), resulting in a marked reduction in IP3 production. This could explain the abolishment of intracellular Ca2+ mobilization in clone 5A4C. Supporting this hypothesis, the Ca2+ mobilization was reconstituted by the addition of exogenous PI(4,5)P2 in these cells. Our results suggest that both IP3 and S1P contribute to FcvarepsilonRI-induced Ca2+ release from the ER and production of IP3 is necessary for S1P to cause Ca2+ mobilization from the ER.     

Antigen-induced Ca mobilization in RBL-2H3 cells: Role of I(1,4,5)P3 and S1P and necessity of I(1,4,5)P3 production

Inositol 1,4,5-trisphosphate (IP3) has long been recognized as a second messenger for intracellular Ca2+ mobilization. Recently, sphingosine 1-phosphate (S1P) has been shown to be involved in Ca2+ release from the endoplasmic reticulum (ER). Here, we investigated the role of S1P and IP3 in antigen (Ag)-induced intracellular Ca2+ mobilization in RBL-2H3 mast cells. Antigen-induced intracellular Ca2+ mobilization was only partially inhibited by the sphingosine kinase inhibitor dl-threo-dihydrosphingosine (DHS) or the IP3 receptor inhibitor 2-aminoethoxydiphenyl borate (2-APB), whereas preincubation with both inhibitors led to complete inhibition. In contrast, stimulation of A3 adenosine receptors with N5-ethylcarboxamidoadenosine (NECA) caused intracellular Ca2+ mobilization that was completely abolished by 2-APB but not by DHS, suggesting that NECA required only the IP3 pathway, while antigen used both the IP3 and S1P pathways. Interestingly, however, inhibition of IP3 production with the phospholipase C inhibitor U73122 completely abolished Ca2+ release from the ER induced by either stimulant. This suggested that S1P alone, without concomitant production of IP3, would not cause intracellular Ca2+ mobilization. This was further demonstrated in some clones of RBL-2H3 cells excessively overexpressing a beta isoform of Class II phosphatidylinositol 3-kinase (PI3KC2beta). In such clones including clone 5A4C, PI3KC2beta was overexpressed throughout the cell, although endogenous PI3KC2beta was normally expressed only in the ER. Overexpression of PI3KC2beta in the cytosol and the PM led to depletion of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), resulting in a marked reduction in IP3 production. This could explain the abolishment of intracellular Ca2+ mobilization in clone 5A4C. Supporting this hypothesis, the Ca2+ mobilization was reconstituted by the addition of exogenous PI(4,5)P2 in these cells. Our results suggest that both IP3 and S1P contribute to FcvarepsilonRI-induced Ca2+ release from the ER and production of IP3 is necessary for S1P to cause Ca2+ mobilization from the ER.     

Sphingosine kinase-mediated Ca2+ signalling by G-protein-coupled receptors.

Formation of inositol 1,4,5-trisphosphate (IP3) by phospholipase C (PLC) with subsequent release of Ca2+ from intracellular stores, is one of the major Ca2+ signalling pathways triggered by G-protein-coupled receptors (GPCRs). However, in a large number of cellular systems, Ca2+ mobilization by GPCRs apparently occurs independently of the PLC-IP3 pathway, mediated by an as yet unknown mechanism. The present study investigated whether sphingosine kinase activation, leading to production of sphingosine-1-phosphate (SPP), is involved in GPCR-mediated Ca2+ signalling as proposed for platelet-derived growth factor and FcepsilonRI antigen receptors. Inhibition of sphingosine kinase by DL-threo-dihydrosphingosine and N,N-dimethylsphingosine markedly inhibited [Ca2+]i increases elicited by m2 and m3 muscarinic acetylcholine receptors (mAChRs) expressed in HEK-293 cells without affecting mAChR-induced PLC stimulation. Activation of mAChRs rapidly and transiently stimulated production of SPP in HEK-293 cells. Finally, intracellular injection of SPP induced a rapid and transient Ca2+ mobilization in HEK-293 cells which was not antagonized by heparin. We conclude that mAChRs utilize the sphingosine kinase-SPP pathway in addition to PLC-IP3 to mediate Ca2+ mobilization. As Ca2+ signalling by various, but not all, GPCRs in different cell types was likewise attenuated by the sphingosine kinase inhibitors, we suggest a general role for sphingosine kinase, besides PLC, in mediation of GPCR-induced Ca2+ signalling.     

Sphingosine kinase expression increases intracellular sphingosine-1-phosphate and promotes cell growth and survival.

Sphingosine-1-phosphate (SPP) is a bioactive lipid that has recently been identified as the ligand for the EDG family of G protein-coupled cell surface receptors. However, the mitogenic and survival effects of exogenous SPP may not correlate with binding to cell-surface receptors (Van Brocklyn, J.R., M.J. Lee, R. Menzeleev, A. Olivera, L. Edsall, O. Cuvillier, D.M. Thomas, P.J.P. Coopman, S. Thangada, T. Hla, and S. Spiegel. 1998. J. Cell Biol. 142:229-240). The recent cloning of sphingosine kinase, a unique lipid kinase responsible for the formation of SPP, has provided a new tool to investigate the role of intracellular SPP. Expression of sphingosine kinase markedly increased SPP levels in NIH 3T3 fibroblasts and HEK293 cells, but no detectable secretion of SPP into the medium was observed. The increased sphingosine kinase activity in NIH 3T3 fibroblasts was sufficient to promote growth in low- serum media, expedite the G(1)/S transition, and increase DNA synthesis and the proportion of cells in the S phase of the cell cycle with a concomitant increase in cell numbers. Transient or stable overexpression of sphingosine kinase in NIH 3T3 fibroblasts or HEK293 cells protected against apoptosis induced by serum deprivation or ceramide elevation. N,N-Dimethylsphingosine, a competitive inhibitor of sphingosine kinase, blocked the effects of sphingosine kinase overexpression on cell proliferation and suppression of apoptosis. In contrast, pertussis toxin did not abrogate these biological responses. In Jurkat T cells, overexpression of sphingosine kinase also suppressed serum deprivation- and ceramide-induced apoptosis and, to a lesser extent, Fas-induced apoptosis, which correlated with inhibition of DEVDase activity, as well as inhibition of the executionary caspase-3. Taken together with ample evidence showing that growth and survival factors activate sphingosine kinase, our results indicate that SPP functions as a second messenger important for growth and survival of cells. Hence, SPP belongs to a novel class of lipid mediators that can function inside and outside cells.     

Extracellular release of newly synthesized sphingosine-1-phosphate by cerebellar granule cells and astrocytes

Sphingosine-1-phosphate (S1P) is a potent biomediator that can act as either an intracellular or an intercellular messenger. In the nervous system it exerts a wide range of actions, and specific membrane receptors for it have been identified in various regions. However, the physiological origin of extracellular S1P in the nervous system is largely unknown. We investigated cerebellar granule cells at different stages of differentiation and astrocytes in primary cultures as possible origins of extracellular S1P. Although these cells show marked differences in S1P metabolism, we found that they can all release S1P and express mRNAs for S1P specific receptors. Extracellular S1P derives from the export of newly synthesized intracellular S1P, and not from the action of a released sphingosine kinase. S1P release is rapid, efficient, and can be regulated by exogenous stimuli. Phorbol ester treatment resulted in an increase in sphingosine kinase 1 activity in the membranes, accompanied by a significant increase in extracellular S1P. S1P release in cells from the cerebellum emerges as a regulated mechanism, possibly related to a specific pool of newly synthesized S1P. To our knowledge, this is the first evidence of the extracellular release of S1P by primary cells from the CNS, which supports a role of S1P as autocrine/paracrine physiological messenger in the cerebellum.     

Sphingosine 1-phosphate stimulates tyrosine phosphorylation of focal adhesion kinase and chemotactic motility of endothelial cells via the G(i) protein-linked phospholipase C pathway

We have previously shown that sphingosine 1-phosphate (S1P) stimulates motility of human umbilical vein endothelial cells (HUVECs) (O.-H. Lee et al., Biochem. Biophys. Res. Commun. 264, 743-750, 1999). To investigate the molecular mechanisms by which S1P stimulates HUVEC motility, we examined tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)) which is important for cell migration. S1P induces a rapid increase in tyrosine phosphorylation of p125(FAK). Compared with other structurally related lipid metabolites such as sphingosine, C2-ceramide, and lysophosphatidic acid, S1P uniquely stimulated p125(FAK) tyrosine phosphorylation and migration of HUVECs. The effect of S1P on p125(FAK) tyrosine phosphorylation was markedly reduced by treatment with pertussis toxin or U73122, a phospholipase C (PLC) inhibitor. As a downstream signal of PLC, p125(FAK) tyrosine phosphorylation in response to S1P was totally blocked by depletion of the intracellular calcium pool. However, protein kinase C (PKC) inhibitor had no effect on the response to S1P. Finally, chemotaxis assays revealed that inhibition of PLC but not PKC significantly abrogated S1P-stimulated HUVEC migration. These results suggest that the G(i)-coupled receptor-mediated PLC-Ca(2+) signaling pathway may be importantly involved in S1P-stimulated focal adhesion formation and migration of endothelial cells.     

Role of sphingosine kinase and sphingosine-1-phosphate in inflammatory arthritis

The importance of sphingosine kinase (SphK) and sphingosine-1-phosphate (S1P) in inflammation has been extensively demonstrated. As an intracellular second messenger, S1P plays an important role in calcium signaling and mobilization, and cell proliferation and survival. Activation of various plasma membrane receptors, such as the formyl methionyl leucyl phenylalanine receptor, C5a receptor, and tumor necrosis factor α receptor, leads to a rapid increase in intracellular S1P level via SphK stimulation. SphK and S1P are implicated in various chronic autoimmune conditions such as rheumatoid arthritis, primary Sjögren’s syndrome, and inflammatory bowel disease. Recent studies have demonstrated the important role of SphK and S1P in the development of arthritis by regulating the pro-inflammatory responses. These novel pathways represent exciting potential therapeutic targets.     

The outs and the ins of sphingosine-1-phosphate in immunity

The potent lipid mediator sphingosine-1-phosphate (S1P) is produced inside cells by two closely related kinases, sphingosine kinase 1 (SPHK1) and SPHK2, and has emerged as a crucial regulator of immunity. Many of the actions of S1P in innate and adaptive immunity are mediated by its binding to five G protein-coupled receptors, designated S1PR1-5, but recent findings have also identified important roles for S1P as a second messenger during inflammation. In this Review, we discuss recent advances in our understanding of the roles of S1P receptors and describe the newly identified intracellular targets of S1P that are crucial for immune responses. Finally, we discuss the therapeutic potential of new drugs that target S1P signalling and functions.     

Identification and characterization of a novel human sphingosine-1-phosphate phosphohydrolase,hSPP2

Sphingosine 1-phosphate (S1P) is a bioactive lipid molecule that acts as both an extracellular signaling mediator and an intracellular second messenger. S1P is synthesized from sphingosine by sphingosine kinase and is degraded either by S1P lyase or by S1P phosphohydrolase. Recently, mammalian S1P phosphohydrolase (SPP1) was identified and shown to constitute a novel lipid phosphohydrolase family, the SPP family. In this study we have identified a second human S1P phosphohydrolase, SPP2, based on sequence homology to human SPP1. SPP2 exhibited high phosphohydrolase activity against S1P and dihydrosphingosine 1-phosphate. The dihydrosphingosine-1-phosphate phosphohydrolase activity was efficiently inhibited by excess S1P but not by lysophosphatidic acid, phosphatidic acid, or glycerol 3-phosphate, indicating that SPP2 is highly specific to sphingoid base 1-phosphates. Immunofluorescence microscopic analysis demonstrated that SPP2 is localized to the endoplasmic reticulum. Although the enzymatic properties and localization of SPP2 were similar to those of SPP1, the tissue-specific expression pattern of SPP2 was different from that of SPP1. Thus, SPP2 is another member of the SPP family that may play a role in attenuating intracellular S1P signaling.     

Release of calcium from inositol 1,4,5-trisphosphate receptor-regulated stores by HIV-1 Tat regulates TNF-alpha production in human macrophages

HIV-1 protein Tat is neurotoxic and increases macrophage and microglia production of TNF-alpha, a cytopathic cytokine linked to the neuropathogenesis of HIV dementia. Others have shown that intracellular calcium regulates TNF-alpha production in macrophages, and we have shown that Tat releases calcium from inositol 1,4, 5-trisphosphate (IP3) receptor-regulated stores in neurons and astrocytes. Accordingly, we tested the hypothesis that Tat-induced TNF-alpha production was dependent on the release of intracellular calcium from IP3-regulated calcium stores in primary macrophages. We found that Tat transiently and dose-dependently increased levels of intracellular calcium and that this increase was blocked by xestospongin C, pertussis toxin, and by phospholipase C and type 1 protein kinase C inhibitors but not by protein kinase A or phospholipase A2 inhibitors. Xestospongin C, BAPTA-AM, U73122, and bisindolylmalemide significantly inhibited Tat-induced TNF-alpha production. These results demonstrate that in macrophages, Tat-induced release of calcium from IP3-sensitive intracellular stores and activation of nonconventional PKC isoforms play an important role in Tat-induced TNF-alpha production.     

HDL3, but not HDL2, stimulates plasminogen activator inhibitor-1 release from adipocytes: the role of sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) is a bioactive lysophospholipid that regulates numerous key cardiovascular functions. High-density lipoproteins (HDLs) are the major plasma lipoprotein carriers of S1P. Fibrinolysis is a physiological process that allows fibrin clot dissolution, and decreased fibrinolytic capacity may result from increased circulating levels of plasminogen activator inhibitor-1 (PAI-1). We examined the effect of S1P associated with HDL subfractions on PAI-1 secretion from 3T3 adipocytes. S1P concentration in HDL3 averaged twice that in HDL2. Incubation of adipocytes with increasing concentrations of S1P in HDL3, but not HDL2, or with S1P complexed to albumin stimulated PAI-I secretion in a concentration-dependent manner. Quantitative RT-PCR revealed that S1P_1–3 are expressed in 3T3 adipocytes, with S1P₂ expressed in the greatest amount. Treatment of adipocytes with the S1P₁ and S1P₃ antagonist VPC23019 did not block PAI-1 secretion. Inhibiting S1P₂ with JTE-013 or reducing the expression of the gene coding for S1P₂ using silencing RNA (siRNA) technology blocked PAI-1 secretion, suggesting that the S1P₂ receptor mediates PAI-1 secretion from adipocytes exposed to HDL3 or S1P. Treatment with the phospholipase C (PLC) inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, the protein kinase C (PKC) inhibitor RO-318425, or the Rho-associated protein kinase (ROCK) inhibitor Y27632 all significantly inhibited HDL3- and S1P-mediated PAI-1 release, suggesting that HDL3- and/or S1P-stimulated PAI-1 secretion from 3T3 cells is mediated by activation of multiple, downstream signaling pathways of S1P₂.     

Calcium influx and signaling in yeast stimulated by intracellular sphingosine 1-phosphate accumulation

In mammalian cells, intracellular sphingosine 1-phosphate (S1P) can stimulate calcium release from intracellular organelles, resulting in the activation of downstream signaling pathways. The budding yeast Saccharomyces cerevisiae expresses enzymes that can synthesize and degrade S1P and related molecules, but their possible role in calcium signaling has not yet been tested. Here we examine the effects of S1P accumulation on calcium signaling using a variety of yeast mutants. Treatment of yeast cells with exogenous sphingosine stimulated Ca(2+) accumulation through two distinct pathways. The first pathway required the Cch1p and Mid1p subunits of a Ca(2+) influx channel, depended upon the function of sphingosine kinases (Lcb4p and Lcb5p), and was inhibited by the functions of S1P lyase (Dpl1p) and the S1P phosphatase (Lcb3p). The biologically inactive stereoisomer of sphingosine did not activate this Ca(2+) influx pathway, suggesting that the active S1P isomer specifically stimulates a calcium-signaling mechanism in yeast. The second Ca(2+) influx pathway stimulated by the addition of sphingosine was not stereospecific, was not dependent on the sphingosine kinases, occurred only at higher doses of added sphingosine, and therefore was likely to be nonspecific. Mutants lacking both S1P lyase and phosphatase (dpl1 lcb3 double mutants) exhibited constitutively high Ca(2+) accumulation and signaling in the absence of added sphingosine, and these effects were dependent on the sphingosine kinases. These results show that endogenous S1P-related molecules can also trigger Ca(2+) accumulation and signaling. Several stimuli previously shown to evoke calcium signaling in wild-type cells were examined in lcb4 lcb5 double mutants. All of the stimuli produced calcium signals independent of sphingosine kinase activity, suggesting that phosphorylated sphingoid bases might serve as messengers of calcium signaling in yeast during an unknown cellular response.     

Generation and metabolism of bioactive sphingosine-1-phosphate

Sphingosine-1-phosphate (S1P) is a bioactive lysosphingophospholipid that has been implicated in the regulation of vital biological processes. Abundant evidence indicates that S1P acts as both an intracellular messenger and an extracellular ligand for a family of five specific G protein-coupled S1P receptors (S1PRs). Cellular levels of S1P are tightly regulated in a spatio-temporal manner through its synthesis catalyzed by sphingosine kinases (SphKs) and degradation by S1P lyase (SPL) and specific S1P phosphohydrolases. Over the past decade, the identification and cloning of genes encoding S1P metabolizing enzymes has increased rapidly. Overexpression and deletion of these enzymes has provided important insights into the intracellular and the "inside-out" functions of S1P. The purpose of this review is to summarize the current knowledge of S1P metabolizing enzymes, their enzymatic properties, and their roles in the control of cellular functions by S1P.     

Phospholipase C and protein kinase C-β 2 mediate insulin-like growth factor II-dependent sphingosine kinase 1 activation

We recently reported that IGF-II binding to the IGF-II/mannose-6-phosphate (M6P) receptor activates the ERK1/2 cascade by triggering sphingosine kinase 1 (SK1)-dependent transactivation of G protein-coupled sphingosine 1-phosphate (S1P) receptors. Here, we investigated the mechanism of IGF-II/M6P receptor-dependent sphingosine kinase 1 (SK1) activation in human embryonic kidney 293 cells. Pretreating cells with protein kinase C (PKC) inhibitor, bisindolylmaleimide-I, abolished IGF-II-stimulated translocation of green fluorescent protein (GFP)-tagged SK1 to the plasma membrane and activation of endogenous SK1, implicating PKC as an upstream regulator of SK1. Using confocal microscopy to examine membrane translocation of GFP-tagged PKCα, β1, β2, δ, and ζ, we found that IGF-II induced rapid, transient, and isoform-specific translocation of GFP-PKCβ2 to the plasma membrane. Immunoblotting of endogenous PKC phosphorylation confirmed PKCβ2 activation in response to IGF-II. Similarly, IGF-II stimulation caused persistent membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, which does not dissociate from the membrane after translocation. IGF-II stimulation increased diacylglycerol (DAG) levels, the established activator of classical PKC. Interestingly, the polyunsaturated fraction of DAG was increased, indicating involvement of phosphatidyl inositol/phospholipase C (PLC). Pretreating cells with the PLC inhibitor, U73122, attenuated IGF-II-dependent DAG production and PKCβ2 phosphorylation, blocked membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, and reduced sphingosine 1-phosphate production, suggesting that PLC/PKCβ2 are upstream regulators of SK1 in the pathway. Taken together, these data provide evidence that activation of PLC and PKCβ2 by the IGF-II/M6P receptor are required for the activation of SK1.     

Sphingosine-1-phosphate: dual messenger functions

The sphingolipid metabolite sphingosine-1-phosphate (S1P) is a serum-borne lipid that regulates many vital cellular processes. S1P is the ligand of a family of five specific G protein-coupled receptors that are differentially expressed in different tissues and regulate diverse cellular actions. Much less is known of the intracellular actions of S1P. It has been suggested that S1P may also function as an intracellular second messenger to regulate calcium mobilization, cell growth and suppression of apoptosis in response to a variety of extracellular stimuli. Dissecting the dual actions and identification of intracellular targets of S1P has been challenging, but there is ample evidence to suggest that the balance between S1P and ceramide and/or sphingosine levels in cells is an important determinant of cell fate.