The Calmodulin-binding Site of Sphingosine Kinase and Its Role in Agonist-dependent Translocation of Sphingosine Kinase 1 to the Plasma Membrane

Abstract

Sphingosine kinases catalyze the formation of sphingosine 1-phosphate, a bioactive lipid involved in many aspects of cellular regulation, including the fundamental biological processes of cell growth and survival. A diverse range of cell agonists induce activation of human sphingosine kinase 1 (hSK1) and, commonly, its translocation to the plasma membrane. Although the activation of hSK1 in response to at least some agonists occurs directly via its phosphorylation at Ser 225 by ERK1/2, many aspects governing the regulation of this phosphorylation and subsequent translocation remain unknown. Here, in an attempt to understand some of these processes, we have examined the known interaction of hSK1 with calmodulin (CaM). By using a combination of limited proteolysis, peptide interaction analysis, and site-directed mutagenesis, we have identified that the CaM-binding site of hSK1 resides in the region spanned by residues 191-206. Specifically, Phe 197 and Leu 198 are critically involved in the interaction because a version of hSK1 incorporating mutations of both Phe 197 3 Ala and Leu 198 3 Gln failed to bind CaM. We have also shown for the first time that human sphingosine kinase 2 (hSK2) binds CaM, and does so via a CaM binding region that is conserved with hSK1 because comparable mutations in hSK2 also ablate CaM binding to this protein. By using the CaM-binding-deficient version of hSK1, we have begun to elucidate the role of CaM in hSK1 regulation by demonstrating that disruption of the CaM-binding site ablates agonist-induced translocation of hSK1 from the cytoplasm to the plasma membrane, while having no effect on hSK1 phosphorylation and catalytic activation.Sphingosine kinases are important signaling enzymes because of their role in the synthesis of the bioactive lipid sphingosine 1-phosphate (S1P).3 Many studies have shown that S1P can affect a diverse array of biological processes, including calcium mobilization, mitogenesis, apoptosis, atherosclerosis, inflammatory responses, cell motility, and angiogenesis (1-4). Although some of these varied effects of S1P result from its action as a ligand for S1P-specific cell-surface G-protein-coupled receptors (5), significant evidence now exists that indicates S1P can also function intracellularly as a second messenger, particularly in the regulation of cell proliferation and apoptosis (6).Two sphingosine kinases exist in humans (hSK1 and hSK2), with most studies to date focusing on hSK1. Although these two enzymes originate from different genes and differ in size, tissue distribution, developmental expression, substrate specificity, specific activity, and possibly in their cellular roles (7-10), their polypeptide sequences possess a high degree of similarity. In fact, almost all of the hSK1 sequence aligns with regions of the larger hSK2 sequence with 80% overall similarity (45% identity) (7). However, hSK2 also possesses two additional polypeptide regions at its N terminus and within the central region of its sequence that are quite distinct from hSK1.Although hSK...

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

“…We suspect that the action of these calmodulin inhibitors on membranes could be responsible for a significant fraction of their many observed effects on cells. 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).…”

Sphingosine-1-phosphate: the Swiss army knife of sphingolipid signaling

“…Indeed, Pitson et al (15) have shown that SphK1 is phosphorylated on Ser225 (human SphK1) by ERK1/2, which is necessary for its activation and translocation to the plasma membrane, perhaps by exposing a putative binding site for the membrane lipid phosphatidylserine (16). Subsequently, SphK1 was found to have a calmodulin binding site between residues 191-206, which was necessary for agonist-induced translocation to the plasma membrane but not required for activation (17). Recently, it was shown that protein phosphatase 2A negatively correlated with Ser225 phosphorylation and SphK1 membrane localization, suggesting that it may regulate SphK1 translocation (18).…”

Original article The key role of sphingosine kinases in the molecular mechanism of neuronal cell survival and death in an experimental model of Parkinson’s disease

“…The latest data reported by Takasugi et al [52] indicated that the S1P pool synthesized by Sphk2 regulates β-amyloid (Aβ) precursor protein (APP) cleaving enzyme 1 (BACE1) activity and the Aβ level. Sphk1 resides predominantly in the cytosol and is translocated to the plasma membrane followed by ERK1/2-mediated phosphorylation [39,45,47]. Sphk1 is well known to synthesize the S1P pool, which exerts pro-survival effects within the cells.…”