The Mechanism of Membrane Targeting of Human Sphingosine Kinase 1

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 Thr 54 and Asn 89 in the putative membrane-binding surface are essential for lipid selectivity and membrane targeting both in vitro and in the cell. Also, phosphorylation of Ser 225 enhances the membrane affinity and plasma membrane selectivity of hSK1, presumably by modulating the interaction of Thr 54 and Asn 89 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.Sphingosine 1-phosphate (S1P) 3 is a recently identified bioactive lipid that can act both extracellularly and intracellularly as a first and a second messenger, respectively (1). It has been shown that S1P is excreted into serum from platelets and binds members of the endothelial differentiation gene receptor family (S1P1-5) to activate cellular processes such as differentiation, migration, and mitogenesis (2). Intracellularly, S1P has been implicated in signaling cascades that lead to cytoskeletal changes, motility, release of intracellular Ca 2ϩ stores, and protection from apoptosis (3-6). S1P is formed from sphingosine (SPH) by sphingosine kinase (SK) and is degraded by S1P lyase and S1P phosphatases (1). In the resting state of cells, the balance between S1P formation and degradation maintains the low basal levels of S1P. However, cellular S1P levels have been shown to increase rapidly and transiently by agonists that activate SK, such as tumor necrosis factor-␣ (7, 8), platelet-derived growth factor (9), nerve growth factor (10, 11), muscarinic acetylcholine agonists (12), or phorbol esters (13,14).Two types of mammalian SKs (SK1 and SK2) have been characterized so far (15, 16), both of which are primarily cytosolic proteins. 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. Subsequently, it was reported that PMA and tumor necrosis factor-␣ induced the phosphorylation of Ser 225 of SK1 through the activation of mitogen-activated protein kinase, extracellular signal-regulated kinase 1/2, which resulted in pla...

Membrane-permeable Calmodulin Inhibitors (e.g. W-7/W-13) Bind to Membranes, Changing the Electrostatic Surface Potential

“…To cite just one example of a peripheral protein, Young et al (68) showed that 50 M W-7 prevents the binding of sphingosine kinase to the plasma membrane and reasonably attributed this effect to W-7 inhibiting Ca 2ϩ /CaM; recent mutation experiments appear to support this interpretation (69). Our results, however, suggest that this effect could equally well be due to W-7 reducing the surface potential of the plasma membrane, particularly because acidic lipids appear to mediate the membrane association of sphingosine kinase (70). An example of an integral membrane protein is the polymeric immunoglobulin receptor, pIgR, which has a 17-residue membrane-proximal cytoplasmic segment, residues 653-670, that binds Ca 2ϩ /CaM with high affinity (71).…”

Insulin-like Growth Factors Mediate Heterotrimeric G Protein-dependent ERK1/2 Activation by Transactivating Sphingosine 1-Phosphate Receptors

“…In vitro studies have shown that hSK1 has high affinity for lipid bilayers rich in phosphatidylserine and that conserved Thr 54 and Asn 89 residues in the putative membrane-binding surface of the protein are necessary for membrane binding in vitro and in cells (49). Furthermore phosphorylation of Ser 225 increases the plasma membrane affinity of hSK1.…”

“…The ligand for S1P receptors, S1P, is a bioactive sphinoglipid metabolite formed through the action of the sphingosine kinases SK1 and SK2 (48). Previous work has demonstrated that external stimuli, such as phorbol ester-dependent protein kinase C activation, can promote membrane translocation and activation of SK1 (39,49), suggesting that regulated production of S1P might provide a mechanism for transactivating S1P receptors.…”

Phosphatidylinositol 4,5-Bisphosphate (PtdIns(4,5)P2) Specifically Induces Membrane Penetration and Deformation by Bin/Amphiphysin/Rvs (BAR) Domains

“…N-BAR domain proteins primarily interact with the inner leaflet of the plasma membrane that contains PS, PtdIns, and PtdIns(4,5)P 2 as major anionic lipids. Both PS (37,(63)(64)(65)(66)(67) and PtdIns(4,5)P 2 (48,68) have been shown to play critical roles in recruiting cytosolic proteins to the plasma membrane through electrostatic interactions (8,69). In particular, PtdIns(4,5)P 2 can effectively interact with unstructured polybasic motifs even in the presence of excess PS because of its ability to generate an electrostatic field ideal for multidentate interaction with polybasic motifs (48,49) and to facilitate the formation of an amphiphilic secondary structure, typically an ␣-helix (7).…”