Fluorescence-labeled sphingosines as substrates of sphingosine kinases 1 and 2

Ettmayer, Peter; Billich, Andreas; Baumruker, Thomas; Mechtcheriakova, Diana; Schmid, Heide; Nußbaumer, Peter

doi:10.1016/j.bmcl.2003.12.099

Cited by 35 publications

(23 citation statements)

References 8 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…However, the inability of a recently identified CerK-like protein to phosphorylate ceramide (27) has renewed interest in finding alternate pathways of C1P formation; in particular, via acylation of the lysosphingolipid sphingosine 1-phosphate. To investigate this potential pathway, we applied DMB (dimethyl-BODIPY)-labeled sphingosine to WT and Cerk Ϫ/Ϫ BMDM; DMB-sphingosine is a fluorescent sphingosine analog, which has been validated as a substrate for intracellular sphingosine kinases (28). Although we detected DMBsphingosine 1-phosphate, we found no evidence of DMB-C1P under these conditions (Fig.…”

Section: Unique Role For Cerk In the Production Of C1p From Cer-wecontrasting

confidence: 43%

Regulation and Traffic of Ceramide 1-Phosphate Produced by Ceramide Kinase

Boath¹,

Gräf

Lidome³

et al. 2008

Journal of Biological Chemistry

Self Cite

107

View full text Add to dashboard Cite

Ceramide 1-phosphate (C1P) has been characterized as a sphingolipid that participates in cell signaling. Although C1P synthesis is thought to occur via phosphorylation of ceramide by ceramide kinase (CerK), the processes that regulate C1P formation and fate remain largely unknown. In this study we analyzed bone marrow-derived macrophages (BMDM) from CerK-null mice (Cerk ؊/؊ ) and found significant levels of C1P, suggesting that previously unrecognized pathways may also lead to C1P formation. After these experiments we used an overexpression system, BMDM from Cerk ؊/؊ mice, and short-chain fluorescent ceramides to trace CerK-dependent formation of C1P. Because the ceramide analogs can also be converted to glucosylceramide (GlcCer) and sphingomyelin (SM), they allowed us to directly compare all three metabolites. We found that C1P produced by CerK is turned over rapidly when serum is removed or upon calcium chelation, whereas GlcCer and SM are stable under these conditions. We further demonstrated that ceramide must be transported to the Golgi complex to be phosphorylated by CerK. Inhibition of the ceramide transfer protein slowed down SM formation without decreasing C1P, suggesting an alternate route of ceramide transport. Other experiments indicated that, like GlcCer and SM, C1P traffics along the secretory pathway to reach the plasma membrane. Furthermore, in BMDM C1P was secreted more readily than was GlcCer or SM. Altogether, our results indicate that CerK is essential to C1P formation via phosphorylation of Cer, providing the first insights into mechanisms underlying ceramide access to CerK and C1P trafficking as well as clarifying C1P as a signaling entity.

show abstract

Section: Unique Role For Cerk In the Production Of C1p From Cer-wecontrasting

confidence: 43%

Regulation and Traffic of Ceramide 1-Phosphate Produced by Ceramide Kinase

Boath¹,

Gräf

Lidome³

et al. 2008

Journal of Biological Chemistry

Self Cite

107

View full text Add to dashboard Cite

show abstract

“…Although NBD-Sph was readily converted to NBD-S1P following the incorporation of NBD-Sph into erythrocytes (Fig. 1B), the rate of NBD-Sph phosphorylation is estimated to be 20% of that of sphingosine (24,26,27). Consistent with this estimate, the addition of sphingosine to the extracellular buffer with NBD-Sph decreased the useful for assaying many samples and, thus, likely has applications in high-throughput screening.…”

Section: Discussionmentioning

confidence: 63%

Fluorescence-based rapid measurement of sphingosine-1-phosphate transport activity in erythrocytes

Kobayashi

Otsuka

Yamaguchi

et al. 2016

Journal of Lipid Research

View full text Add to dashboard Cite

This article is available online at http://www.jlr.org S1P recruits lymphocytes from the thymus and secondary lymphoid organs to the circulatory system by activating S1P receptors on lymphocytes (5). Many components of blood, such as erythrocytes, platelets, and neutrophils, express sphingosine kinases and produce S1P (6). Platelets contain large amounts of S1P and release it only when activated by stimuli, such as thrombin, whereas erythrocytes constitutively release S1P in the absence of stimuli. Because erythrocytes constitute a large proportion of the blood cells in the body and because they lack S1P-degrading activity catalyzed by S1P lyase (SGPL1), S1P phosphatases (SGPP1 and SGPP2), and phospholipid phosphatases (PLPP1, PLPP2, and PLPP3) (7), they contribute substantially to plasma S1P. In addition to blood cells, endothelial cells constitutively release S1P into the blood without stimuli (8). Thus, plasma S1P is derived mainly from erythrocytes and endothelial cells (9). When the S1P supply from erythrocytes has been disrupted, the plasma S1P level and the number of the lymphocytes in the blood decrease substantially (10). Therefore, plasma S1P supplied by erythrocytes is essential for the correct distribution of lymphocytes in the blood.Erythrocytes transport sphingosine, which is supplied by other cells, to the intracellular side of the membrane and produce S1P by phosphorylation (6,7,11). In addition to the extracellular source of sphingosine, intracellular alkaline ceramidase also produces sphingosine used for S1P synthesis in human erythrocytes (12). Previously, we reported that erythrocytes export intracellularly synthesized S1P into the extracellular space via an ATP-dependent transporter in a time-dependent manner without any Abstract Sphingosine-1-phosphate (S1P) is present in the blood plasma and acts as a pivotal intercellular signal transmitter in the immune system by recruiting lymphocytes from the thymus and secondary lymphoid tissues. The plasma S1P concentration is maintained by the supply of S1P from erythrocytes. Previously, we showed that S1P release from erythrocytes is mediated by an ATP-dependent transporter. In this study, we attempted to establish a rapid and reliable method for measuring the S1P transport activity in erythrocytes by using a fluorescent S1P analog, 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD)-labeled S1P. NBD-S1P was released from erythrocytes in a time-dependent manner. The NBD-S1P release was reduced after exposure to glyburide, which is an inhibitor of the S1P transporter in erythrocytes. Moreover, the release of NBD-S1P and S1P from erythrocytes was competitively inhibited by intracellular S1P and NBD-S1P, respectively. These results showed that the erythrocyte S1P transporter exports NBD-S1P. We optimized the sample-preparation conditions and lipid extraction to increase the sensitivity of the assay. Furthermore, we successfully measured NBD-S1P release without lipid extraction by decreasing the concentration of BSA in the assay buffer to 0.1%. This method will be ...

show abstract

“…BODIPY 540 sphingosine enabled imaging unacylated sphingolipids, which cannot be accomplished with the existing sphingolipid analogs that contain a fl uorescent fatty acid side chain. Furthermore, unlike previously reported fl uorescent sphingosine analogs (7)(8)(9), BODIPY 540 sphingosine can be visualized in parallel with GFP without the use of UV excitation. By exploiting these capabilities, we verifi ed that mammalian cells rapidly internalized BODIPY 540 sphingosine, transported it to the secretory pathway where it was metabolized to more complex fl uorescent sphingolipids, and eventually catabolized these fl uorescent sphingolipids.…”

Section: Discussionmentioning

confidence: 89%

A new, long-wavelength borondipyrromethene sphingosine for studying sphingolipid dynamics in live cells

Kim¹,

Lou²,

Kraft

2013

Journal of Lipid Research

View full text Add to dashboard Cite

apoptosis, and other critical cellular processes during normal cell function and disease ( 1-4 ). Insight into sphingolipid biosynthesis, transport, and subcellular distribution has been acquired by observing fl uorescent sphingolipid analogs within living cells ( 5 ). Sphingolipid derivatives that contain a fl uorophore-labeled N -acyl fatty acid are often used to investigate dynamic processes that involve acylated sphingolipids ( 5 ). Fatty acids that contain a polyene fl uorophore are especially attractive for this purpose because the structure and behavior of polyene-containing lipids are very similar to the native lipid ( 6 ). However, to study the bioactive, unacylated sphingolipids, sphingosine and sphingosine-1-phosphate, the fl uorophore must be incorporated into the sphingosine backbone. Studies have confi rmed that such fl uorescent sphingosine analogs can be metabolized to more complex fl uorescently labeled sphingolipids in living cells ( 7-10 ). Despite their potential utility, only a limited number of fl uorophores, such as pyrene, borondipyrromethene (BODIPY), and nitrobenzo-2-oxa-1,3-diazole, have been incorporated into the sphingosine backbone ( 7-9 ).To increase the utility of fl uorescent sphingosine analogs for investigating sphingolipid dynamics in living cells, derivatives with a wider range of fl uorescence properties must be developed. Lacking in particular is a bioactive fl uorescent sphingosine that has neither an excitation maximum that is in the UV range nor emission that interferes with detecting green fl uorescent protein (GFP), the most common genetically encoded fl uorescent protein label ( 11 ). A probe with these properties is expected to Abbreviations: GFP, green fl uorescent protein; BODIPY, borondipyrromethene; rt, room temperature; ER, endoplasmic reticulum. 1 R. Kim and K. Lou contributed equally to this work.

show abstract

Fluorescence-labeled sphingosines as substrates of sphingosine kinases 1 and 2

Cited by 35 publications

References 8 publications

Regulation and Traffic of Ceramide 1-Phosphate Produced by Ceramide Kinase

Regulation and Traffic of Ceramide 1-Phosphate Produced by Ceramide Kinase

Fluorescence-based rapid measurement of sphingosine-1-phosphate transport activity in erythrocytes

A new, long-wavelength borondipyrromethene sphingosine for studying sphingolipid dynamics in live cells

Contact Info

Product

Resources

About