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A novel approach to the study of sphingolipid metabolism mediated by sphingosine kinase, an oncogenic signaling enzyme
Author(s) -
Siow Deanna,
Wattenberg Binks
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a485-c
Subject(s) - sphingolipid , sphingosine , sphingosine kinase , enzyme , sphingosine kinase 1 , kinase , microbiology and biotechnology , metabolism , chemistry , signal transduction , biochemistry , biology , sphingosine 1 phosphate , receptor
Sphingosine kinase (SK) is an agonist‐stimulated signaling enzyme that promotes carcinogenesis through formation of the anti‐apoptotic and pro‐proliferative lipid, sphingosine‐1‐phosphate (S1P). Yet SK also serves a vital metabolic role in controlling the steady‐state levels of other sphingolipids with signaling functions, including ceramide and sphingosine. To ascertain the mechanism(s) underlying the regulation of these dual roles, we have devised a novel in situ system to study sphingolipid metabolism mediated by SK. This unique in situ system utilizes adherent cells pulsed with radiolabeled ATP, allowing us to monitor the production and immediate metabolism of S1P. Using this system, we report that production of S1P proceeds in a linear fashion for at least 2 hours following a brief, initial lag. Importantly, despite the 10‐ to 20‐ fold increase in S1P production seen with exogenously added sphingosine, reaction kinetics are identical to those for endogenous sphingosine. In addition, we show that similar results are seen for both endogenous SK and overexpressed SK. Furthermore, using digitonin to selectively permeabilize the cells prior to our in situ enzyme assay, we can directly measure the mobility of SK. These results offer a dynamic picture that includes the rapid redistribution of sphingosine to the sites of SK action, and the freely diffusible nature of SK within the cell. We are extending this work to investigate the roles played by the degradative enzymes, S1P lyase and S1P phosphatases, in the downstream metabolism of S1P. Research supported by IPIBS Fellowship and NIH RO1 CA111987‐01.