Sphingosine-1-Phosphate Formation Activates Phosphatidylinositol-4 Kinase in Basolateral Membranes from Kidney Cells: Crosstalk in Cell Signaling through Sphingolipids and Phospholipids

Abstract

Sphingosine-1-phosphate (S1P) and phosphatidylinositol-4 phosphate [PtdIns(4)P] are important second messengers in various cellular processes. Here, we show that S1P and PtdIns(4)P are formed in purified basolateral membranes (BLM) derived from kidney proximal tubules, indicating the presence of a plasma membrane associated SPK (BLM-SPK) and phosphatidylinositol-4 kinase (PI-4K). We observed that S1P synthesis is linear with time, dependent on protein concentration, and saturable in the presence of increasing concentrations of sphingosine. Different from the observations on cytosolic SPKs, the formation of S1P by BLM-SPK is Mg(2+)-independent and insensitive to the classical inhibitor of the cytosolic SPKs, DL-threo-dihydrosphingosine. With sphingosine as substrate, the enzyme shows cooperative kinetics (n = 3.4) with a K(0.5) value of 0.12 mM, suggesting that BLM-SPK is different from the previously characterized cytosolic SPK. The formation of PtdIns(4)P markedly inhibits BLM-SPK activity. Conversely, a strong activation of PtdIns(4)P synthesis by the formation of S1P is observed. Taken together, these results indicate that (i) basolateral membranes from kidney cells harbor a SPK activity that potentially regulates renal epithelium function, and (ii) the formation of S1P mediated by SPK enhances PI-4K activity, while PtdIns(4)P in turn inhibits SPK, suggesting an interplay between these lipid signaling molecules. These findings suggest the possibility of crosstalk between sphingolipids and glycerolipids, which might be involved in the regulation of transepithelial fluxes across the BLM of kidney cells.

Early Double-Negative Thymocyte Export in Trypanosoma cruzi Infection Is Restricted by Sphingosine Receptors and Associated with Human Chagas Disease

“…To ascertain the activity of SPK activity we performed a lipid phosphorylation assay as previously described [34] . Briefly, we used 0.2 mg protein/ml from each sample and the assay medium (1 ml) contained 30 mM MES-Tris (pH 7.0), 1 mM [γ- 32 P]ATP (specific activity 2.2×10 8 cpm/µmol), 1.1 mM MgCl 2 , 10 mM NaN 3 , 1 mM ouabain, and 0.5 mM EGTA.…”

“…The reaction was started by the addition of [γ- 32 P]ATP to the tubes. One ml aliquots of the different samples at the different experimental conditions were used for total lipid extraction, which was performed as previously described [34] . Briefly, 5.0 ml of the solvent moisture (CHCl 3 ∶MeOH∶HCl – 20∶10∶0.075 v/v) was added to the samples at conic glass tubes and vigorous mixed using a Pasteur pipete.…”

Interactions between neural membrane glycerophospholipid and sphingolipid mediators: A recipe for neural cell survival or suicide

“…Sphingosine and sphingosine 1‐phosphate promote the rapid release of Ca 2+ from internal stores in a manner not sensitive to Ins P 3 (Einicker‐Lamas et al, 2003). An interplay between sphingosine 1‐phosphate and PtdIns metabolism through their respective kinases, sphingosine kinase and PtdIns 4‐kinase, occurs at the membrane level.…”

“…The mutual influence of PtdIns 4‐kinase and sphingosine 1‐kinase on each other is consistent with the cross talk between sphingolipids and glycerophospholipids. The interaction between sphingosine 1‐kinase and PtdIns 4‐kinase pathways and their synchronized control may represent an important physiological mechanism by which different enzymes associated with ionic homeostasis such as Ca 2+ /Mg 2+ ‐ATPase and Na + /K + ‐ATPase are switched on or off (Einicker‐Lamas et al, 2003).…”

Bioactive lipids are altered in the kidney of chronic undernourished rats: is there any correlation with the progression of prevalent nephropathies?

“…The assay is based on the membrane lipid phosphorylation by 32 Pi ( 32 Pi was from IPEN, São Paulo, Brazil) according to Einicker-Lamas et al [ 28 ]. Briefly, an aliquot of the membrane fraction (0.1 mg total protein) was incubated as indicated with 0.1 mM sphingosine (Sph) (Sigma Chemical Co., Saint Louis, MO), then total lipid extraction was performed as described by Horwitz and Perlman [ 29 ].…”