Sphingosine kinase: a mediator of vital cellular functions

Ample evidence indicates that sphingosine-1-phosphate (SPP) can serve as an intracellular second messenger regulating calcium mobilization, and cell growth and survival. Moreover, the dynamic balance between levels of the sphingolipids metabolites, ceramide and SPP, and consequent regulation of opposing signaling pathways, is an important factor that determines whether a cell survives or dies. SPP has also recently been shown to be the ligand for the EDG-1 family of G protein-coupled receptors, which now includes EDG-1, -3, -5, -6, and -8. SPP is thus a lipid mediator that has novel dual actions signaling inside and outside of the cell. This review is focussed on sphingosine kinase, the enzyme that regulates levels of SPP and thus plays a critical role in diverse biological processes.     

Sphingosine stimulates cellular proliferation via a protein kinase C-independent pathway

Sphingosine, a metabolite of membrane sphingolipids, is generally considered to be cytotoxic for a variety of cell types. However, we have found that sphingosine at low concentrations stimulates DNA synthesis and acts synergistically with known growth factors to induce proliferation of quiescent Swiss 3T3 fibroblasts. Structurally related analogs of sphingosine, such as N-stearoylsphingosine and other long chain aliphatic amines, had no mitogenic effects, suggesting that sphingosine did not induce nonspecific membrane perturbations. Sphingosine, which has been proposed to be a physiological inhibitor of protein kinase C, also markedly potentiates the mitogenic effect of the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Sphingosine still stimulates DNA synthesis in cells made protein kinase C deficient by prolonged treatment with phorbol ester. At mitogenic concentrations, sphingosine does not bind to protein kinase C as shown by its lack of effect on phorbol dibutyrate binding. Only at higher concentrations, in the cytotoxic range, was there a displacement of phorbol dibutyrate from its cellular-binding sites. In contrast to sphingosine, H-7, a known inhibitor of protein kinase C, inhibited the mitogenic response to TPA and the TPA-induced phosphorylation of the 80 kDa cellular substrate of protein kinase C. Our results suggest that sphingosine may play an important role as a positive regulator of cell growth acting in a fundamentally different, protein kinase C-independent pathway.     

Sphingosine kinase: biochemical and cellular regulation and role in disease

Sphingolipids have emerged as molecules whose metabolism is regulated leading to generation of bioactive products including ceramide, sphingosine, and sphingosine-1-phosphate. The balance between cellular levels of these bioactive products is increasingly recognized to be critical to cell regulation; whereby, ceramide and sphingosine cause apoptosis and growth arrest phenotypes, and sphingosine-1-phosphate mediates proliferative and angiogenic responses. Sphingosine kinase is a key enzyme in modulating the levels of these lipids and is emerging as an important and regulated enzyme. This review is geared at mechanisms of regulation of sphingosine kinase and the coming to light of its role in disease.     

Focal adhesion kinase: protein interactions and cellular functions

Integrin-mediated cell adhesion to extracellular matrix (ECM) plays important roles in a variety of biological processes. Recent studies suggested that integrins mediate signal transduction across the plasma membrane via activating several intracellular signaling pathways. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that has been shown to be a major mediator of integrin signal transduction pathways. Upon activation by integrins, FAK undergoes autophosphorylation as well as associations with several other intracellular signaling molecules. These interactions in the signaling pathways have been shown to regulation a variety of cellular functions such as cell spreading, migration, cell proliferation, apoptosis and cell survival. Recent progress in the understanding of FAK interactions with other proteins in the regulation of these cellular functions will be discussed in this review.     

Sphingosine 1-phosphate: a Ca2+ release mediator in the balance

Sphingosine 1-phosphate (S1P) is a lipid signalling molecule with Ca(2+) mobilising properties. Importantly for a role as a Ca(2+) release messenger, intracellular levels of S1P can be regulated by a variety of extracellular stimuli, via the enzyme sphingosine kinase. However, neither the mechanism underlying S1P generation, nor its actions at the endoplasmic reticulum are clear. Thus, the role of S1P as an intracellular mediator of Ca(2+) release remains in the balance.     

Sphingosine 1-phosphate, a key mediator of the cytokine network: juxtacrine signaling

Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite, which has emerged as an important signaling mediator participating in the regulation of multiple cellular processes. The discovery of a family of S1P receptors, together with the more recently identified intracellular targets, has provided fundamental understanding of the multi-faceted actions of S1P. Evidence from both in vitro and in vivo studies has implicated the S1P signaling system in the control of immunity, inflammation and many associated diseases. Enigmatically, S1P appears to have both pro- and anti-inflammatory effects depending on the cell context. Here, we review this emerging area and argue for a pivotal role for S1P, as a key mediator of the cytokine network, acting through juxtacrine signaling in the immune system.     

Cell surface phosphorylation by a novel ecto-protein kinase: a key regulator of cellular functions in spermatozoa

Since 1976 many studies have been reported on the occurrence and functional significance of ecto-protein kinases in a variety of cell types although their precise biochemical identity is largely unknown. This study reports for the first time purification to apparent homogeneity of an ecto-protein kinase (ecto-CIK) and some of its characteristics using caprine sperm as the cell model. The ecto-CIK is a unique membrane-specific serine/threonine protein kinase. It is a strongly basic 115 kDa protein made up of two subunits: 63 and 55 kDa. The ecto-kinase undergoes a remarkable lateral movement on the outer cell surface culminating in capping on the sperm acrosomal tip. MPS, its major protein substrate is also located on the acrosomal tip. Both ecto CIK and MPS serve as potential regulators of flagellar motility. This novel enzyme appears to be major kinase responsible for the reported regulation of mammalian cellular functions by modulating phosphorylation of the membrane-bound proteins.     

Sphingosine is a novel activator of 3-phosphoinositide-dependent kinase 1

3-Phosphoinositide-dependent kinase 1 (PDK1) has previously been shown to phosphorylate the activation loop of several AGC kinase family members. In this study, we show that p21-activated kinase 1, the activity of which is regulated by the GTP-bound form of Cdc42 and Rac and by sphingosine, is phosphorylated by PDK1. Phosphorylation of p21-activated kinase 1 by PDK1 occurred only in the presence of sphingosine, which increased PDK1 autophosphorylation 25-fold. Sphingosine increased PDK1 autophosphorylation in a concentration-dependent manner and significantly increased phosphate incorporation into known PDK1 substrates. Studies on the lipid requirement for PDK1 activation found that both sphingosine isoforms and stearylamine also increased PDK1 autophosphorylation. However, C(10)-sphingosine, octylamine, and stearic acid were unable to increase PDK1 autophosphorylation, indicating that both a positive charge and a lipid tail containing at least a C(10)-carbon backbone were required for PDK1 activation. Three PDK1 autophosphorylation sites were identified after stimulation with sphingosine in a serine-rich region located between the kinase domain and the pleckstrin homology domain using two-dimensional phosphopeptide maps and matrix assisted laser desorption/ionization mass spectroscopy. Increased phosphorylation of endogenous Akt at threonine 308 was observed in COS-7 cells expressing wild type PDK1, but not catalytically inactive PDK1, when cellular sphingosine levels were elevated by treatment with sphingomyelinase. Sphingosine thus appears to be a true PDK1 activator.     

Cell surface phosphorylation by a novel ecto-protein kinase: A key regulator of cellular functions in spermatozoa

Since 1976 many studies have been reported on the occurrence and functional significance of ecto-protein kinases in a variety of cell types although their precise biochemical identity is largely unknown. This study reports for the first time purification to apparent homogeneity of an ecto-protein kinase (ecto-CIK) and some of its characteristics using caprine sperm as the cell model. The ecto-CIK is a unique membrane-specific serine/threonine protein kinase. It is a strongly basic 115 kDa protein made up of two subunits: 63 and 55 kDa. The ecto-kinase undergoes a remarkable lateral movement on the outer cell surface culminating in capping on the sperm acrosomal tip. MPS, its major protein substrate is also located on the acrosomal tip. Both ecto CIK and MPS serve as potential regulators of flagellar motility. This novel enzyme appears to be major kinase responsible for the reported regulation of mammalian cellular functions by modulating phosphorylation of the membrane-bound proteins.     

Sphingosine kinase 2 is a negative regulator of inflammatory macrophage activation

Sphingosine kinases (SPHK) generate the sphingolipid sphingosine-1-phosphate, which, among other functions, is a potent regulator of inflammation. While SPHK1 produces S1P to promote inflammatory signaling, the role of SPHK2 is unclear due to divergent findings in studies utilizing gene depletion versus inhibition of catalytic activity. We sought to clarify how SPHK2 affects inflammatory signaling in human macrophages, which are main regulators of inflammation. SPHK2 expression and activity were rapidly decreased within 6 h upon stimulating primary human macrophages with lipopolysaccharide (LPS), but was upregulated after 24 h. At 24 h following LPS stimulation, targeting SPHK2 with the inhibitor ABC294640, a specific siRNA or by using Sphk2^−/− mouse peritoneal macrophages increased inflammatory cytokine production. Downregulation of SPHK2 in primary human macrophages within 6 h of LPS treatment was blocked by inhibiting autophagy. SPHK2 overexpression or inhibiting autophagy 6 h after human macrophage activation with LPS suppressed inflammatory cytokine release. Mechanistically, SPHK2 suppressed LPS-triggered NF-κB activation independent of its catalytic activity and prevented increased mitochondrial ROS formation downstream of LPS. In conclusion, SPHK2 is an anti-inflammatory protein in human macrophages that is inversely coupled to inflammatory cytokine production. This needs consideration when targeting SPHK2 with specific inhibitors.     

Sphingosine kinase 1 deficiency exacerbates LPS-induced neuroinflammation

The pathogenesis of inflammation in the central nervous system (CNS), which contributes to numerous neurodegenerative diseases and results in encephalopathy and neuroinflammation, is poorly understood. Sphingolipid metabolism plays a crucial role in maintaining cellular processes in the CNS, and thus mediates the various pathological consequences of inflammation. For a better understanding of the role of sphingosine kinase activation during neuroinflammation, we developed a bacterial lipopolysaccharide (LPS)-induced brain injury model. The onset of the inflammatory response was observed beginning 4 hours after intracerebral injection of LPS into the lateral ventricles of the brain. A comparison of established neuroinflammatory parameters such as white matter rarefactions, development of cytotoxic edema, astrogliosis, loss of oligodendrocytes, and major cytokines levels in wild type and knockout mice suggested that the neuroinflammatory response in SphK1-/- mice was significantly upregulated. At 6 hours after intracerebroventricular injection of LPS in SphK1-/- mice, the immunoreactivity of the microglia markers and astrocyte marker glial fibrillary acidic protein (GFAP) were significantly increased, while the oligodendrocyte marker O4 was decreased compared to WT mice. Furthermore, western blotting data showed increased levels of GFAP. These results suggest that SphK1 activation is involved in the regulation of LPS induced brain injury. RESEARCH HIGHLIGHTS: ? Lipopolysaccharide (LPS) intracerebral injection induces severe neuroinflammation. ? Sphingosine kinase 1 deletion worsens the effect of the LPS. ? Overexpression of SphK1 might be a potential new treatment approach to neuroinflammation.     

Sphingosine kinase protects lipopolysaccharide-activated macrophages from apoptosis

Lipopolysaccharide (LPS) signaling is critical for the innate immune response to gram-negative bacteria. Here, evidence is presented for LPS stimulation of sphingosine kinase (SPK) in the RAW 264.7 murine macrophage cell line and rat primary hepatic macrophages (HMs). LPS treatment of RAW 264.7 cells resulted in a time- and dose-dependent activation of SPK and membrane translocation of SPK1. Further, LPS-induced SPK activation was blocked by SPK1-specific small interfering RNA (siRNA). Overexpression of Toll-like receptor 4 and MD2, the receptor and coreceptor of LPS, in HEK 293 cells activated SPK activity in the absence of LPS treatment. Inhibition of SPK by the pharmacological inhibitor N,N-dimethylsphingosine (DMS) or SPK1-specific siRNA blocked LPS stimulation of extracellular signal-regulated kinase 1/2 and p38 but enhanced LPS-induced c-Jun N-terminal kinase activation. The SPK inhibitor DMS and dominant-negative SPK1 also blocked LPS activation of Elk-1 and NF-kappaB reporters in RAW 264.7 cells. Inhibition of SPK sensitized RAW 264.7 cells and HMs to LPS-induced apoptosis. These data demonstrate the critical role of SPK1 in LPS signaling in macrophages and suggest that SPK1 is a potential therapeutic target to block hyperimmune responses induced by gram-negative bacteria.     

Sphingosine kinase 2 is a nuclear protein and inhibits DNA synthesis

Sphingosine kinase-1 (SPHK1) is a key enzyme catalyzing the formation of an important bioactive lipid messenger, sphingosine 1-phosphate, and is implicated in the regulation of cell proliferation and antiapoptotic processes. Biological features of another isozyme SPHK2, however, remain unclear. The present studies were undertaken to characterize SPHK2 by comparison with SPHK1. When SPHK2 was transiently expressed in various cell lines, it was localized in the nuclei as well as in the cytosol, whereas SPHK1 was distributed in the cytosol but not in the nucleus. We have mapped a functional nuclear localization signal (NLS) to the N-terminal region of SPHK2. We have observed that the expression of SPHK2 in various cell types causes inhibition of DNA synthesis, resulting in the cell cycle arrest at G1/S phase. We have also demonstrated that an NLS mutant of SPHK2, SPHK2R93E/R94E, failed to enter the nucleus and to inhibit DNA synthesis. Moreover, a fusion protein, NLS-SPHK1, where SPHK1 was fused to the NLS sequence of SPHK2 acquired the ability to enter nuclei and inhibited DNA synthesis. These results indicate that SPHK2 localizes in the nuclei and causes inhibition of DNA synthesis, and this may affect subsequent cellular events.     

Sphingosine kinase: a point of convergence in the action of diverse neutrophil priming agents

Neutrophils are a vital component of the early acute inflammatory response, but can cause profound tissue damage when activated to excess or prevented from undergoing apoptosis. However, much remains unknown about the intracellular signaling pathways regulating neutrophil activity. The structurally diverse neutrophil-priming agents platelet-activating factor, TNF-alpha, and the substance P analog [D-Arg(6), D-Trp(7,9),N(me)Phe(8)]-substance P(6-11) (SP-G) stimulated a rapid increase in sphingosine kinase activity in freshly isolated human neutrophils. This activity was blocked by preincubation with the sphingosine kinase inhibitor N,N-dimethylsphingosine (DMS). DMS also inhibited the increase in intracellular calcium concentration stimulated by platelet-activating factor, fMLP, and SP-G. This suggests that the increase in intracellular calcium concentration by these agents is dependent on sphingosine kinase activation and the generation of sphingosine-1-phosphate. Changes in cell polarization and the augmentation of the fMLP-induced superoxide anion generation, by all priming agents were also inhibited by DMS, while only the superoxide anion release was blocked by the phosphatidylinositol 3-kinase inhibitor LY294002. Moreover, SP-G and GM-CSF inhibited constitutive neutrophil apoptosis which was completely blocked by DMS. These results suggest a novel role for sphingosine kinase in the regulation of neutrophil priming.     

Sphingosine kinase type 2 activation by ERK-mediated phosphorylation

Sphingosine 1-phosphate (S1P), a potent lipid mediator, is a ligand for a family of five G protein-coupled receptors (S1P(1-5)) that have been shown to regulate a variety of biological responses important for cancer progression. The cellular level of S1P is low and tightly regulated in a spatio-temporal manner through its synthesis catalyzed by two sphingosine kinases, denoted SphK1 and SphK2. Many stimuli activate and translocate SphK1 to the plasma membrane by mechanisms that are dependent on its phosphorylation. Much less is known about activation of SphK2. Here we demonstrate that epidermal growth factor (EGF) as well as the protein kinase C activator, phorbol ester, induce rapid phosphorylation of hSphK2 which was markedly reduced by inhibition of MEK1/ERK pathway. Down-regulation of ERK1 blocked EGF-induced phosphorylation of SphK2. Recombinant ERK1 phosphorylated hSphK2 in vitro and increased its enzymatic activity. ERK1 also was found to be in a complex with hSphK2 in vivo. Site-directed mutagenesis indicated that hSphK2 is phosphorylated on Ser-351 and Thr-578 by ERK1 and that phosphorylation of these residues is important for EGF-stimulated migration of MDA-MB-453 cells. These studies provide the first clues to the mechanism of agonist-mediated SphK2 activation and enhance understanding of the regulation of SphK2 activity by phosphorylation and its role in movement of human breast cancer cells toward EGF.     

Sphingosine kinase and sphingosine 1-phosphate in asthma

Sphingolipids are amphiphatic molecules ubiquitously expressed in all eukaryotic cell membranes. Initially characterized as structural components of cell membranes, sphingolipids have emerged as sources of important signalling molecules over the past decade. Sphingolipid metabolites, such as ceramide and S1P (sphingosine 1-phosphate), have been demonstrated to have roles as potent bioactive messengers involved in cell differentiation, proliferation, apoptosis, migration and angiogenesis. The importance of SphK (sphingosine kinase) and S1P in inflammation has been demonstrated extensively. The prevalence of asthma is increasing in many developed nations. Consequently, there is an urgent need for the development of new agents for the treatment of asthma, especially for patients who respond poorly to conventional therapy. Recent studies have demonstrated the important role of SphK and S1P in the development of asthma by regulating pro-inflammatory responses. These novel pathways represent exciting potential therapeutic targets in the treatment of asthma and are described in the present review.