Sphingosine kinase localization in the control of sphingolipid metabolism

The sphingosine kinases (sphingosine kinase-1 and -2) have been implicated in a variety of physiological functions. Discerning their mechanism of action is complicated because in addition to producing the potent lipid second messenger sphingosine-1-phosphate, sphingosine kinases, both by producing sphingosine-1-phosphate and consuming sphingosine, have profound effects on sphingolipid metabolism. Sphingosine kinase-1 translocates to the plasma membrane upon agonist stimulation and this translocation is essential for the pro-oncogenic properties of this enzyme. Many of the enzymes of sphingolipid metabolism, including the enzymes that degrade sphingosine-1-phosphate, are membrane bound with restricted subcellular distributions. In the work described here we explore how subcellular localization of sphingosine kinase-1 affects the downstream metabolism of sphingosine-1-phosphate and the access of sphingosine kinase to its substrates. We find, surprisingly, that restricting sphingosine kinase to either the plasma membrane or the endoplasmic reticulum has a negligible effect on the rate of degradation of the sphingosine-1-phosphate that is produced. This suggests that sphingosine-1-phosphate is rapidly transported between membranes. However we also find that cytosolic or endoplasmic-reticulum targeted sphingosine kinase expressed at elevated levels produces extremely high levels of dihydrosphingosine-1-phosphate. Dihydrosphingosine is a proximal precursor in ceramide biosynthesis. Our data indicate that sphingosine kinase can divert substrate from the ceramide de novo synthesis pathway. However plasma membrane-restricted sphingosine kinase cannot access the pool of dihydrosphingosine. Therefore whereas sphingosine kinase localization does not affect downstream metabolism of sphingosine-1-phosphate, localization has an important effect on the pools of substrate to which this key signaling enzyme has access.Deanna L. Siow, Charles D. Anderson, Evgeny V. Berdyshev, Anastasia Skobeleva, Viswanathan Natarajan, Stuart M. Pitsong and Binks W. Wattenberghttp://www.elsevier.com/wps/find/journaldescription.cws_home/427/description#descriptio

Cytochrome c oxidase is activated by the oncoprotein Ras and is required for A549 lung adenocarcinoma growth

Abstract Background Constitutive activation of Ras in immortalized bronchial epithelial cells increases electron transport chain activity, oxygen consumption and tricarboxylic acid cycling through unknown mechanisms. We hypothesized that members of the Ras family may stimulate respiration by enhancing the expression of the Vb regulatory subunit of cytochrome c oxidase (COX). Results We found that the introduction of activated H-RasV12 into immortalized human bronchial epithelial cells increased eIF4E-dependent COX Vb protein expression simultaneously with an increase in COX activity and oxygen consumption. In support of the regulation of COX Vb expression by the Ras family, we also found that selective siRNA-mediated inhibition of K-Ras expression in A549 lung adenocarcinoma cells reduced COX Vb protein expression, COX activity, oxygen consumption and the steady-state concentration of ATP. We postulated that COX Vb-mediated activation of COX activity may be required for the anchorage-independent growth of A549 cells as soft agar colonies or as lung xenografts. We transfected the A549 cells with COX Vb small interfering or shRNA and observed a significant reduction of their COX activity, oxygen consumption, ATP and ability to grow in soft agar and as poorly differentiated tumors in athymic mice. Conclusion Taken together, our findings indicate that the activation of Ras increases COX activity and mitochondrial respiration in part via up-regulation of COX Vb and that this regulatory subunit of COX may have utility as a Ras effector target for the development of anti-neoplastic agents.</p

Selective inhibition of choline kinase simultaneously attenuates MAPK and PI3K/AKT signaling

Choline is an essential anabolic substrate for the synthesis of phospholipids. Choline kinase phosphorylates choline to phosphocholine that serves as a precursor for the production of phosphatidylcholine, the major phospholipid constituent of membranes and substrate for the synthesis of lipid signaling molecules. Nuclear magnetic resonance (NMR)-based metabolomic studies of human tumors have identified a marked increase in the intracellular concentration of phosphocholine relative to normal tissues. We postulated that the observed intracellular pooling of phosphocholine may be required to sustain the production of the pleiotropic lipid second messenger, phosphatidic acid. Phosphatidic acid is generated from the cleavage of phosphatidylcholine by phospholipase D2 and is a key activator of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)/AKT survival signaling pathways. In this study we show that the steady-state concentration of phosphocholine is increased by the ectopic expression of oncogenic H-Ras(V12) in immortalized human bronchial epithelial cells. We then find that small interfering RNA (siRNA) silencing of choline kinase expression in transformed HeLa cells completely abrogates the high concentration of phosphocholine, which in turn decreases phosphatidylcholine, phosphatidic acid and signaling through the MAPK and PI3K/AKT pathways. This simultaneous reduction in survival signaling markedly decreases the anchorage-independent survival of HeLa cells in soft agar and in athymic mice. Last, we confirm the relative importance of phosphatidic acid for this pro-survival effect as phosphatidic acid supplementation fully restores MAPK signaling and partially rescues HeLa cells from choline kinase inhibition. Taken together, these data indicate that the pooling of phosphocholine in cancer cells may be required to provide a ready supply of phosphatidic acid necessary for the feed-forward amplification of cancer survival signaling pathways.James Graham Brown Cancer CenterUnited States Department of Health & Human Services National Institutes of Health (NIH) - USA (1 R01 CA11642801)Kentucky Lung Cancer Research Progra

Intracellular localization of sphingosine kinase 1 alters access to substrate pools but does not affect the degradative fate of sphingosine-1-phosphate[S]

Sphingosine kinase 1 (SK1) produces sphingosine-1-phosphate (S1P), a potent signaling lipid. The subcellular localization of SK1 can dictate its signaling function. Here, we use artificial targeting of SK1 to either the plasma membrane (PM) or the endoplasmic reticulum (ER) to test the effects of compartmentalization of SK1 on substrate utilization and downstream metabolism of S1P. Expression of untargeted or ER-targeted SK1, but surprisingly not PM-targeted SK1, results in a dramatic increase in the phosphorylation of dihydrosphingosine, a metabolic precursor in de novo ceramide synthesis. Conversely, knockdown of endogenous SK1 diminishes both dihydrosphingosine-1-phosphate and S1P levels. We tested the effects of SK1 localization on degradation of S1P by depletion of the ER-localized S1P phosphatases and lyase. Remarkably, S1P produced at the PM was degraded to the same extent as that produced in the ER. This indicates that there is an efficient mechanism for the transport of S1P from the PM to the ER. In acute labeling experiments, we find that S1P degradation is primarily driven by lyase cleavage of S1P. Counterintuitively, when S1P-specific phosphatases are depleted, acute labeling of S1P is significantly reduced, indicative of a phosphatase-dependent recycling process. We conclude that the localization of SK1 influences the substrate pools that it has access to and that S1P can rapidly translocate from the site where it is synthesized to other intracellular sites.51: 2546–2559