Abstract
Sphingosine 1-phosphate is a bioactive sphingolipid that regulates cell growth and suppresses programmed cell death. The biosynthesis of sphingosine 1-phosphate is catalyzed by sphingosine kinase (SK) but the mechanism by which the subcellular localization and activity of SK is regulated in response to various stimuli is not fully understood. To elucidate the origin and structural determinant of the specific subcellular localization of SK, we performed biophysical and cell studies of human SK1 (hSK1) and selected mutants. In vitro measurements showed that hSK1 selectively bound phosphatidylserine over other anionic phospholipids and strongly preferred the plasma membrane-mimicking membrane to other cellular membrane mimetics. Mutational analysis indicates that conserved Thr54 and Asn89 in the putative membrane-binding surface are essential for lipid selectivity and membrane targeting both in vitro and in the cell. Also, phosphorylation of Ser225 enhances the membrane affinity and plasma membrane selectivity of hSK1, presumably by modulating the interaction of Thr54 and Asn89 with the membrane. Collectively, these studies suggest that the specific plasma membrane localization and activation of SK1 is mediated largely by specific lipid-protein interactions.
Highlights
Dation maintains the low basal levels of Sphingosine 1-phosphate (S1P)
A recent study suggested that a phorbol ester, phorbol 12-myristate 13-acetate (PMA), induces the protein kinase C (PKC)-mediated phosphorylation and the localization of SK1 to the plasma membrane in human embryonic kidney (HEK) 293 cells [17], which leads to enhanced release of S1P to the media
An earlier report showed that phosphatidylserine (PS) is the most effective activator of sphingosine kinase (SK) [26], whereas a recent report suggested that SK1 has the high affinity for phosphatidic acid (PA) [27]
Summary
Materials—1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid (POPA), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol (POPI), 1-palmitoyl-2oleoyl-sn-glycero-3-phosphoserine (POPS), and D-erythro-SPH (C20) were purchased from Avanti Polar Lipids, Inc. (Alabaster, AL) and used without further purification. Surface Plasmon Resonance Analysis—The kinetic SPR measurements were performed at 23 °C in 20 mM HEPES, pH 7.4, containing 0.16 M KCl using a lipid-coated L1 chip in the BIACORE X system as described previously [21]. For the assays without lipid vesicles, the recombinant hSK1 was added to 20 mM Tris buffer (pH 7.4) containing 0.16 M KCl, 5 M D-erythro-SPH-bovine serum albumin adduct (99.7:0.3 in mole ratio), 1 mM [␥-32P]ATP (Amersham Biosciences; 1 Ci), and 500 M MgCl2. Activity assays in the presence of lipid vesicles were performed in 20 mM Tris buffer (pH 7.4) containing 0.16 M KCl, 5 M D-erythro-SPH incorporated into phospholipid vesicles of various compositions (5 M total concentration), 1 mM [␥-32P]ATP (Amersham Biosciences; 1 Ci), and 500 M MgCl2. Tandem mass spectra for phosphorylated peptides were generated using Xcalibur version 3.1 and manual assignment of fragment ions was performed to confirm the search results
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