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9 A NOVEL ROLE OF SPHINGOSINE KINASE 1 IN THE DE NOVO BIOSYNTHESIS OF DIHYDROSPHINGOSINE-1-PHOSPHATE IN MAMMALIAN CELLS.
  1. E. Berdyshev1,
  2. I. Gorshkova1,
  3. P. Usatyuk1,
  4. Y. Zhao1,
  5. B. Saatian2,
  6. W. Hubbard2,
  7. V. Natarajan1
  1. 1 Department of Medicine, The University of Chicago, Chicago, IL;
  2. 2 The Johns Hopkins University, Baltimore, MD

Abstract

Sphingosine kinases 1 and 2 (SK1 and SK2) generate sphingosine-1-phosphate (S1P), a potent endogenous lipid mediator. Using a highly selective and sensitive LC-MS/MS approach, here we show that SK1 overexpression, but not SK2, in different primary cells and cultured cell lines results in predominant up-regulation of the synthesis of dihydrosphingosine-1-phosphate (DHS1P) compared to S1P. Stable isotope pulse-labeling experiments in conjunction with LC-MS/MS quantitation of different sphingolipids demonstrated strong interference of overexpressed SK1 with the de novo sphingolipid biosynthesis by up-regulating the influx of L-serine into sphingolipids and by phosphorylating a major portion of the newly formed dihydrosphingosine to DHS1P. As a result of SK1 overexpression, migration and Ca2+ response of human pulmonary artery endothelial cells (HPAEC) to stimulation with external S1P, but not thrombin, were strongly impaired. Furthermore, infection of human bronchial epithelial cells with RSV A-2 virus increased SK1-mediated synthesis of DHS1P and S1P, whereas TNF-a enhanced only S1P production in HPAEC. These findings uncover a new functional role for SK1, which can target de novo sphingoid base metabolic flow and deviate it from the generation of ceramides toward the synthesis of DHS1P, the S1P homolog and ceramide signaling counterpart.

Supported by HL71152 and HL79396 to V.N. and by the proceeds of NIH1 S10 RR16798 shared instrumentation grant to W.H.

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