Intracellular localization of sphingosine kinase 1 alters access to substrate pools but does not affect the degradative fate of sphingosine-1-phosphate

Abstract

Sphingosine-1-phosphate (S1P) has emerged as a potent bioactive lipid with established roles in essential cellular processes such as vascular maturity, cell proliferation, traffi cking of immune cells, cytoskeletal rearrangement, invasion, and angiogenesis ( 1-3 ). Key to such diversity of functions lies in the ability of S1P to act as both an intracellular effector molecule and an extracellular ligand for a family of fi ve G protein-coupled receptors, S1PR 1-5 ( 4-7 ). S1P made within the cell can be secreted from the cell where it binds S1PRs in an autocrine or paracrine fashion, triggering a wide range of cellular responses, including proliferation, enhanced extracellular matrix assembly, stimulation of adherens junctions, formation of stress fibers, and inhibition of apoptosis induced by either ceramide or growth factor withdrawal ( 8-11 ).Like many signaling molecules, basal levels of S1P are tightly controlled by balancing synthesis with degradation. S1P is generated by the ATP-dependent phosphorylation of sphingosine catalyzed by sphingosine kinase (SK), of which there are two mammalian isoforms: SK1 and SK2. After being produced by SK, there are several possible Abstract Sphingosine kinase 1 (SK1) produces sphingosine-1-phosphate (S1P), a potent signaling lipid. The subcellular localization of SK1 can dictate its signaling function. Here, we use artifi cial targeting of SK1 to either the plasma membrane (PM) or the endoplasmic reticulum (ER) to test the effects of compartmentalization of SK1 on substrate utilization and downstream metabolism of S1P. Expression of untargeted or ER-targeted SK1, but surprisingly not PMtargeted SK1, results in a dramatic increase in the phosphorylation of dihydrosphingosine, a metabolic precursor in de novo ceramide synthesis. Conversely, knockdown of endogenous SK1 diminishes both dihydrosphingosine-1-phosphate and S1P levels. We tested the effects of SK1 localization on degradation of S1P by depletion of the ER-localized S1P phosphatases and lyase. Remarkably, S1P produced at the PM was degraded to the same extent as that produced in the ER. This indicates that there is an effi cient mechanism for the transport of S1P from the PM to the ER. In acute labeling experiments, we fi nd that S1P degradation is primarily driven by lyase cleavage of S1P. Counterintuitively, when S1P-specifi c phosphatases are depleted, acute labeling of S1P is signifi cantly reduced, indicative of a phosphatasedependent recycling process. We conclude that the localization of SK1 infl uences the substrate pools that it has access to and that S1P can rapidly translocate from the site where it is synthesized to other intracellular sites. 11 April 2010. Published, JLR Papers in Press, April 11, 2010 DOI 10.1194 Abbreviations: CIB1, calcium and integrin binding protein 1; ER, endoplasmic reticulum; Hek293, human embryonic kidney cell; HeLa, human cervical carcinoma cell; LDH, lactate dehydrogenase; PM, plasma membrane; PMA, phorbol ester; siRNA, small interfering RNA; SK, sphingosine kinase; S1P, ...

An intrinsic lipid-binding interface controls sphingosine kinase 1 function

“…However, it is also likely that CIB1 can "override" the intrinsic mechanism of SK1 membrane binding and force it to the membrane as a function of the myristoyl-switch. It is also possible that interaction with CIB1 is dependent on SK1 membrane interaction and mutation of the hydrophobic patch disrupts SK1 membrane Activity of membrane-associated SK1 has been documented in several studies [17,40,41].…”

Mammalian ORMDL Proteins Mediate the Feedback Response in Ceramide Biosynthesis

“…The metabolic action of sphingosine kinase is one possible regulatory process. Our group and others have suggested that sphingosine kinase, by consuming dihydrosphingosine, a precursor in the ceramide biosynthetic pathway, can regulate ceramide biosynthesis (13,18,19). Alternatively, regulatory subunits of SPT (3-5) may be involved in the serum-dependent regulation of ceramide biosynthesis.…”

“…H Incorporated into Ceramides-At the completion of the labeling protocol, cells were harvested directly on the plates with 2 ϫ 900 l of acidic methanol (150:1, v/v) and scraping and then transferred to 13 ϫ 100-mm glass tubes and sonicated in a bath sonicator to break up cell clumps. Total cellular lipids were extracted using a modified version of the method of Bligh and Dyer (27) as reported previously (13). The bottom organic phase was collected and dried under nitrogen and then spotted onto thin layer chromatography plates Partisil K6 (Silica Gel 60 Å, Whatman).…”

ORMDL/serine palmitoyltransferase stoichiometry determines effects of ORMDL3 expression on sphingolipid biosynthesis

“…One explanation for the lack of suppression of sphingolipid biosynthesis by ORMDL3 overexpression is that described ( 12 ). Prior to extraction, a mixture of C17 sphingolipids was added to each sample (125 pmol/sample, Avanti sphingolipid mix #1) as internal standards.…”

“…Sphingolipids were quantitated by HPLC electrospray ionization tandem mass spectrometry using selected ion monitoring on an ABSciex 4000 Q-Trap instrument as described previously (13)(14)(15). Total phospholipids for each sample were measured using a modifi ed Ames and Dubin assay ( 16 ), as previously described ( 12 ).…”