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

Sphingosine kinase catalyzes the formation of sphingosine 1-phosphate, a lipid second messenger that has been implicated in a number of agonist-driven cellular responses including mitogenesis, anti-apoptosis, and expression of inflammatory molecules. Despite the importance of sphingosine kinase, very little is known regarding its structure or mechanism of catalysis. Moreover, sphingosine kinase does not contain recognizable catalytic or substrate-binding sites, based on sequence motifs found in other kinases. Here we have elucidated the nucleotide-binding site of human sphingosine kinase 1 (hSK1) through a combination of site-directed mutagenesis and affinity labeling with the ATP analogue, FSBA. We have shown that Gly82of hSK1 is involved in ATP binding since mutation of this residue to alanine resulted in an enzyme with an ∼45-fold higherKm(ATP). We have also shown that Lys103is important in catalysis since an alanine substitution of this residue ablates catalytic activity. Furthermore, we have shown that this residue is covalently modified by FSBA. Our data, combined with amino acid sequence comparison, suggest a motif of SGDGX17–21K is involved in nucleotide binding in the sphingosine kinases. This motif differs in primary sequence from all previously identified nucleotide-binding sites. It does, however, share some sequence and likely structural similarity with the highly conserved glycine-rich loop, which is known to be involved in anchoring and positioning the nucleotide in the catalytic site of many protein kinases.

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Phospholipase C and Protein Kinase C-β 2 Mediate Insulin-Like Growth Factor II-Dependent Sphingosine Kinase 1 Activation

Molecular Endocrinology ◽

10.1210/me.2011-0101 ◽

2011 ◽

Vol 25 (12) ◽

pp. 2144-2156 ◽

Cited By ~ 12

Author(s):

Hesham M. El-Shewy ◽

Souzan A. Abdel-Samie ◽

Abdelmohsen M. Al Qalam ◽

Mi-Hye Lee ◽

Kazuyuki Kitatani ◽

...

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Protein Kinase ◽

Phospholipase C ◽

Fluorescent Protein ◽

Sphingosine Kinase ◽

Sphingosine Kinase 1 ◽

Membrane Translocation ◽

Kinase C ◽

Sphingosine 1 Phosphate

Abstract We recently reported that IGF-II binding to the IGF-II/mannose-6-phosphate (M6P) receptor activates the ERK1/2 cascade by triggering sphingosine kinase 1 (SK1)-dependent transactivation of G protein-coupled sphingosine 1-phosphate (S1P) receptors. Here, we investigated the mechanism of IGF-II/M6P receptor-dependent sphingosine kinase 1 (SK1) activation in human embryonic kidney 293 cells. Pretreating cells with protein kinase C (PKC) inhibitor, bisindolylmaleimide-I, abolished IGF-II-stimulated translocation of green fluorescent protein (GFP)-tagged SK1 to the plasma membrane and activation of endogenous SK1, implicating PKC as an upstream regulator of SK1. Using confocal microscopy to examine membrane translocation of GFP-tagged PKCα, β1, β2, δ, and ζ, we found that IGF-II induced rapid, transient, and isoform-specific translocation of GFP-PKCβ2 to the plasma membrane. Immunoblotting of endogenous PKC phosphorylation confirmed PKCβ2 activation in response to IGF-II. Similarly, IGF-II stimulation caused persistent membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, which does not dissociate from the membrane after translocation. IGF-II stimulation increased diacylglycerol (DAG) levels, the established activator of classical PKC. Interestingly, the polyunsaturated fraction of DAG was increased, indicating involvement of phosphatidyl inositol/phospholipase C (PLC). Pretreating cells with the PLC inhibitor, U73122, attenuated IGF-II-dependent DAG production and PKCβ2 phosphorylation, blocked membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, and reduced sphingosine 1-phosphate production, suggesting that PLC/PKCβ2 are upstream regulators of SK1 in the pathway. Taken together, these data provide evidence that activation of PLC and PKCβ2 by the IGF-II/M6P receptor are required for the activation of SK1.

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Translocation of Sphingosine Kinase 1 to the Plasma Membrane Is Mediated by Calcium- and Integrin-binding Protein 1

Journal of Biological Chemistry ◽

10.1074/jbc.m109.068395 ◽

2009 ◽

Vol 285 (1) ◽

pp. 483-492 ◽

Cited By ~ 86

Author(s):

Kate E. Jarman ◽

Paul A. B. Moretti ◽

Julia R. Zebol ◽

Stuart M. Pitson

Keyword(s):

Plasma Membrane ◽

Binding Protein ◽

Sphingosine Kinase ◽

Sphingosine Kinase 1

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Conformational changes during the reaction cycle of Plasma Membrane Ca2+-ATPase in the autoinhibited and activated states

Biochemical Journal ◽

10.1042/bcj20210036 ◽

2021 ◽

Author(s):

Nicolás A Saffioti ◽

Marilina de Sautu ◽

Ana Sol Riesco ◽

Mariela Soledad Ferreira-Gomes ◽

Juan Pablo F.C. Rossi ◽

...

Keyword(s):

Plasma Membrane ◽

Atpase Activity ◽

Binding Site ◽

Conformational Changes ◽

Ground State ◽

Binding Pocket ◽

Product State ◽

Extracellular Medium ◽

Reaction Cycle ◽

Calmodulin Binding

Plasma membrane Ca2+-ATPase (PMCA) transports Ca2+ by a reaction cycle including phosphorylated intermediates. Calmodulin binding to the C-terminal tail disrupts autoinhibitory interactions, activating the pump. To assess the conformational changes during the reaction cycle, we studied the structure of different PMCA states using a fluorescent probe, hydrophobic photolabeling, controlled proteolysis and Ca2+-ATPase activity.  Our results show that calmodulin binds to E2P-like states, and during dephosphorylation, the hydrophobicity in the nucleotide-binding pocket decreases and the Ca2+ binding site becomes inaccessible to the extracellular medium. Autoinhibitory interactions are disrupted in E1Ca and in the E2P ground state whereas they are stabilized in the E2∙Pi product state. Finally, we propose a model that describes the conformational changes during the Ca2+ transport of PMCA.

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Dual mechanism of activation of plant plasma membrane Ca2+-ATPase by acidic phospholipids: Evidence for a phospholipid binding site which overlaps the calmodulin-binding site

Molecular Membrane Biology ◽

10.1080/09687680802508747 ◽

2008 ◽

Vol 25 (6-7) ◽

pp. 539-546 ◽

Cited By ~ 13

Author(s):

Silvia Meneghelli ◽

Tiziana Fusca ◽

Laura Luoni ◽

Maria Ida De Michelis

Keyword(s):

Plasma Membrane ◽

Binding Site ◽

Phospholipid Binding ◽

Calmodulin Binding ◽

Acidic Phospholipids ◽

Dual Mechanism ◽

Plant Plasma Membrane

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Antibodies against erythrocyte Ca2+-transport ATPase specifically inhibit the calmodulin-dependent fraction of the enzyme's activity

Biochemical Journal ◽

10.1042/bj2280479 ◽

1985 ◽

Vol 228 (2) ◽

pp. 479-485 ◽

Cited By ~ 1

Author(s):

K Gietzen ◽

J Kolandt

Keyword(s):

Smooth Muscle ◽

Plasma Membrane ◽

Oleic Acid ◽

Atpase Activity ◽

Binding Site ◽

Rat Brain ◽

Limited Proteolysis ◽

Transport Atpase ◽

Calmodulin Binding ◽

Muscle Plasma Membrane

Antibodies against purified Ca2+-transport ATPase from human erythrocytes were raised in rabbits. Immunodiffusion experiments revealed that precipitating antibodies had been developed. The immunoglobulin fraction inhibited solely the calmodulin-dependent fraction of erythrocyte Ca2+-transport ATPase activity, whereas the basal (in the absence of added calmodulin) activity of the enzyme was not significantly affected by the antibodies. The antibodies produced similar doseresponse curves for the calmodulin- and the oleic acid-stimulated enzyme. However, the immunoglobulin fraction was considerably less effective in inhibiting Ca2+-transport ATPase activated by limited proteolysis. The results obtained with our antibodies are compatible with the interpretation that at least one subpopulation of the antibodies attacks the enzyme at or close to the calmodulin-binding site of the ATPase. The antibodies also inhibited the calmodulin-regulated Ca2+-transport ATPase from pig smooth-muscle plasma membrane, though with lower potency. However, the immunoglobulin fraction failed to suppress pig cardiac sarcoplasmicreticulum Ca2+-transport ATPase activity in the concentration range investigated. In addition, the activity of phosphodiesterase from rat brain, another enzyme modulated by calmodulin, was not at all affected by the immunoglobulin fraction.

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Sphingosine and Sphingosine Kinase 1 Involvement in Endocytic Membrane Trafficking

Journal of Biological Chemistry ◽

10.1074/jbc.m116.762377 ◽

2017 ◽

Vol 292 (8) ◽

pp. 3074-3088 ◽

Cited By ~ 29

Author(s):

Santiago Lima ◽

Sheldon Milstien ◽

Sarah Spiegel

Keyword(s):

Plasma Membrane ◽

Membrane Trafficking ◽

Sphingosine Kinase ◽

Cell Types ◽

Membrane Cholesterol ◽

Vesicle Formation ◽

Sphingosine Kinase 1 ◽

Late Endosomes ◽

Sphingosine 1 Phosphate ◽

Plasma Membrane Cholesterol

The balance between cholesterol and sphingolipids within the plasma membrane has long been implicated in endocytic membrane trafficking. However, in contrast to cholesterol functions, little is still known about the roles of sphingolipids and their metabolites. Perturbing the cholesterol/sphingomyelin balance was shown to induce narrow tubular plasma membrane invaginations enriched with sphingosine kinase 1 (SphK1), the enzyme that converts the bioactive sphingolipid metabolite sphingosine to sphingosine-1-phosphate, and suggested a role for sphingosine phosphorylation in endocytic membrane trafficking. Here we show that sphingosine and sphingosine-like SphK1 inhibitors induced rapid and massive formation of vesicles in diverse cell types that accumulated as dilated late endosomes. However, much smaller vesicles were formed in SphK1-deficient cells. Moreover, inhibition or deletion of SphK1 prolonged the lifetime of sphingosine-induced vesicles. Perturbing the plasma membrane cholesterol/sphingomyelin balance abrogated vesicle formation. This massive endosomal influx was accompanied by dramatic recruitment of the intracellular SphK1 and Bin/Amphiphysin/Rvs domain-containing proteins endophilin-A2 and endophilin-B1 to enlarged endosomes and formation of highly dynamic filamentous networks containing endophilin-B1 and SphK1. Together, our results highlight the importance of sphingosine and its conversion to sphingosine-1-phosphate by SphK1 in endocytic membrane trafficking.

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