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Sheldon Milstien

Publications and source records attributed to Sheldon Milstien.

45 records · Page 3Linked to original sources
PubMed2002

Cloning and characterization of a protein kinase A anchoring protein (AKAP)-related protein that interacts with and regulates sphingosine kinase 1 activity.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that has novel dual actions. S1P is the ligand for a family of G protein-coupled receptors known as S1PRs that mediate various physiological functions. Growth factors rapidly activate sphingosine kinase type 1 (SPHK1) resulting in phosphorylation of sphingosine to form S1P, which plays important roles in cell growth regulation and protection from apoptosis. However, little is known of the mechanism(s) by which SPHK activity is regulated. Using a yeast two-hybrid screening approach, we cloned a 3-kb cDNA encoding a SPHK1-interacting protein (SKIP). BLAST analysis revealed that SKIP corresponded to the C-terminal region of a larger ( approximately 7 kb) cDNA that encoded a protein with a high degree of similarity to a family of protein kinase A anchor proteins (AKAP). In confirmation of the yeast two-hybrid assay, glutathione S-transferase (GST)-SPHK1 specifically pulled down SKIP, whereas GST did not. Moreover, immunoprecipitation of in vitro translated SPHK1 and SKIP revealed that SKIP and SPHK1 are tightly associated. Furthermore, SKIP overexpression in NIH 3T3 fibroblasts reduced SPHK1 activity and interfered with its biological functions. The apoptotic-sparing effect of SPHK1 against serum deprivation was reduced when co-transfected with SKIP. In addition, SPHK1-enhanced cell proliferation was also abolished by SKIP, with a corresponding decrease in activation of ERK. Taken together, these results indicate that SKIP is a novel protein likely to play a regulatory role in the modulation of SPHK1 activity.

3T3 Cells↗
PubMed2002

Sphingosine-1-phosphate and lipid phosphohydrolases.

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid that acts as both an extracellular ligand for the endothelial differentiation gene-1 (EDG-1) G-protein coupled receptor (GPCR) family and as an intracellular messenger. Cellular levels of S1P are low and tightly regulated in a spatial-temporal manner not only by sphingosine kinase (SPHK) but also by degradation catalyzed by S1P lyase, specific S1P phosphohydrolases, and by general lipid phosphate phosphohydrolases (LPPs). LPPs are characterized as magnesium-independent, insensitive to inhibition by N-ethylmaleimide (NEM) and possessing broad substrate specificity with a variety of phosphorylated lipids, including S1P, phosphatidic acid (PA), and lysophosphatidic acid (LPA). LPPs contain three highly conserved domains that define a phosphohydrolase superfamily. Recently, several specific S1P phosphohydrolases have been identified in yeast and mammalian cells. Phylogenetic and biochemical analyses indicate that these enzymes constitute a new subset of the LPP family. As further evidence, S1P phosphohydrolases exhibit high specificity for phosphorylated sphingoid bases. Enforced expression of S1P phosphohydrolase alters the cellular levels of sphingolipid metabolites in yeast and mammalian cells, increasing sphingosine and ceramide, bioactive sphingolipids that often have opposing biological actions to S1P. By regulating the cellular ratio between ceramide/sphingosine and S1P, S1P phosphohydrolase is poised to be a critical factor in cell survival/cell death decisions. Indeed, expression of S1P phosphohydrolase in mammalian cells increases apoptosis, whereas deletion of S1P phosphohydrolases in yeast correlates with resistance to heat stress. In this review, we discuss the role of phosphohydrolases in the metabolism of S1P and how turnover of S1P can regulate sphingolipid metabolites signaling.

Animals↗
PubMed2002

Ceramide-induced cell death in primary neuronal cultures: upregulation of ceramide levels during neuronal apoptosis.

Ceramide is a sphingolipid that has been implicated both in apoptosis and protection from cell death. We show that in both rat cerebellar granule cells and cortical neuronal cultures application of C(2)-ceramide causes cell death in a dose- and time-dependent manner. Similar effects were observed with the exogenous application of bacterial sphingomyelinase, which hydrolyzes sphingomyelin located on the outer leaflet of the plasma membrane and leads to endogenous ceramide accumulation. Furthermore, endogenous ceramide levels were increased during apoptosis induced by nutrient deprivation or etoposide treatment. These findings suggest that upregulation of ceramide levels, which may be generated through activation of sphingomyelinase, contributes to neuronal apoptosis.

Animals↗
PubMed2002

Involvement of sphingosine kinase in TNF-alpha-stimulated tetrahydrobiopterin biosynthesis in C6 glioma cells.

In C6 glioma cells, the sphingolipid second messenger ceramide potentiates expression of inducible nitric-oxide synthase (iNOS) induced by tumor necrosis factor alpha (TNF-alpha) without affecting GTP cyclohydrolase I (GTPCH), the rate-limiting enzyme in the biosynthesis of 6(R)-5,6,7,8-tetrahydrobiopterin (BH(4)), a cofactor required for iNOS activity. TNF-alpha also stimulates sphingosine kinase, the enzyme that phosphorylates sphingosine to form sphingosine-1-phosphate (SPP), a further metabolite of ceramide. Several clones of C6 cells, expressing widely varying levels of sphingosine kinase, were used to examine the role of SPP in regulation of GTPCH and BH(4) biosynthesis. Overexpression of sphingosine kinase, with concomitant increased endogenous SPP levels, potentiated the effect of TNF-alpha on GTPCH expression and activity and BH(4) biosynthesis. In contrast, enforced expression of sphingosine kinase had no effect on iNOS expression or NO formation. Furthermore, N,N-dimethylsphingosine, a potent sphingosine kinase inhibitor, completely eliminated the increased GTPCH activity and expression induced by TNF-alpha. Surprisingly, we found that, although C6 cells can secrete SPP, which is enhanced by TNF-alpha, treatment of C6 cells with exogenous SPP or dihydro-SPP had no affect on BH(4) biosynthesis. However, both SPP and dihydro-SPP markedly stimulated ERK 1/2 in C6 cells, which express cell surface SPP receptors. Interestingly, although this ERK activation was blocked by PD98059, which also reduced cellular proliferation induced by enforced expression of sphingosine kinase, PD98059 had no effect on GTPCH activity. Collectively, these results suggest that only intracellularly generated SPP plays a role in regulation of GTPCH and BH(4) levels.

Animals↗
PubMed2002

Sphingosine kinases: a novel family of lipid kinases.

Sphingosine kinase (SPHK) catalyzes the formation of sphingosine-1-phosphate (S11). S1P plays an important role in regulation of a variety of biological processes through intracellular and extracellular actions. S1P has recently been shown to be the ligand for the EDG-1 family of G-protein-coupled receptors. To date, seven cloned SPHKs have been reported with confirmed SPHK activity, including human, mouse, yeast, and plant. A computer search of various databases suggests that a new SPHK family is emerging. The cloning and manipulation of SPHK genes will no doubt provide us with important information about the functions of S1P in a wide range of organisms.

Amino Acid Sequence↗
PubMed2002

The roles of sphingosine-1-phosphate in asthma.

Asthma is a complex condition in which exposure to environmental antigens induces inflammatory reactions in the airway characterized by activation of mast cells and eosinophils. Mast cells are known to be the main effector cells in eliciting IgE-mediated allergic response. These cells secrete various substances that perpetuate inflammation and provoke airway smooth muscle (ASM) contraction. A newly recognized addition to the repertoire of FcepsilonRI-mediated signaling events is the activation of sphingosine kinase leading to the generation of the potent sphingolipid mediator, sphingosine-1-phosphate (S1P) from sphingosine. S1P secretion by the lung significantly increases after challenge with an allergen, adding this sphingolipid metabolite to the variety of mediators that are released during an allergic reaction [FASEB J. 15 (2001) 1212]. Indeed, similar to previous reports, we found that FcepsilonRI cross-linking not only increased cellular levels of S1P, it also markedly enhanced its secretion from rat basophilic leukemia RBL-2H3 cells. Moreover, S1P induced degranulation of RBL and bone marrow derived mast cells (BMMCs) cells as determined by hexosaminidase release. Treatment of BMMCs with the sphingosine kinase inhibitors, DL-threo-dihydrosphingosine and dimethylsphingosine, reduced IgE/Ag stimulated histamine release. RT-PCR analysis demonstrated that these mast cells express S1P receptors EDG-1 and EDG-5 but not EDG-3, EDG-6 or EDG-8 transcripts. Further studies are needed to determine whether IgE triggering results in transactivation of EDG-1 or EDG-5 present on mast cells and whether this is a critical event for mast cell activation.

Airway Obstruction↗
PubMed2002

Sphingosine 1-phosphate signaling: providing cells with a sense of direction.

Sphingosine 1-phosphate (S1P) is a sphingolipid metabolite that regulates diverse biological functions. S1P has been identified as a high-affinity ligand for a family of five G-protein-coupled receptors, known as the S1P receptors. The physiological role of the S1P receptor S1P(1) in vascular maturation was recently revealed by gene disruption in mice. In addition to other cellular processes, the binding of S1P to its receptors regulates motility and directional migration of a variety of cell types, including endothelial cells and vascular smooth muscle cells. This review focuses on the important role of S1P and its receptors in cell migration and describes a new paradigm for receptor cross-communication in which transactivation of S1P(1) by a receptor tyrosine kinase (PDGFR) is crucial for cell motility.

Animals↗
PubMed2001

Characterization of murine sphingosine-1-phosphate phosphohydrolase.

In the present study we have characterized mammalian sphingosine-1-phosphate phosphohydrolase (SPP1), an enzyme that specifically dephosphorylates sphingosine 1-phosphate (S1P) and which differs from previously described lipid phosphate phosphohydrolases. Based on sequence homology to murine SPP1, we cloned the human homolog. Transfection of human embryonic kidney 293 and Chinese hamster ovary cells with murine or human SPP1 resulted in marked increases in SPP1 activity in membrane fractions that were used to examine its enzymological properties. Unlike other known type 2 lipid phosphate phosphohydrolases (LPPs), but similar to the yeast orthologs, mammalian SPP1s are highly specific toward long chain sphingoid base phosphates and degrade S1P, dihydro-S1P, and phyto-S1P. SPP1 exhibited apparent Michaelis-Menten kinetics with S1P as substrate with an apparent K(m) of 38.5 microm and optimum activity at pH 7.5. Similar to other LPPs, SPP1 activity was also independent of any cation requirements, including Mg(2+), and was not inhibited by EDTA but was markedly inhibited by NaF and Zn(2+). However, SPP1 has some significantly different enzymological properties than the LPPs: the aliphatic cation propanolol, which is an effective inhibitor of type 1 phosphatidate phosphohydrolase activities and is only modestly effective as an inhibitor of LPPs, is a potent inhibitor of SPP1; the activity was partially sensitive to N-ethylmaleimide but not to the thioreactive compound iodoacetamide; and importantly, low concentrations of Triton X-100 and other non-ionic detergents were strongly inhibitory. Thus, in agreement with Cluster analysis which shows that outside of the consensus motif there is very little homology between SPP1s and the other type 2 lipid phosphohydrolases, SPP1s are significantly different and divergent from the mammalian LPPs.

Amino Acid Sequence↗