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Mol. Cell. Biol. 22 (22): 7758-7768

Copyright © 2002 by the American Society for Microbiology. All rights reserved.

Sphingosine Kinase Mediates Vascular Endothelial Growth Factor-Induced Activation of Ras and Mitogen-Activated Protein Kinases

Xiaodong Shu, Weicheng Wu, Raymond D. Mosteller, and Daniel Broek*

Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, Keck School of Medicine at the University of Southern California, Los Angeles, California 90089

Received for publication 4 March 2002. Revision received 17 April 2002. Accepted for publication 22 August 2002.

Abstract: Vascular endothelial growth factor (VEGF) signaling is critical to the processes of angiogenesis and tumor growth. Here, evidence is presented for VEGF stimulation of sphingosine kinase (SPK) that affects not only endothelial cell signaling but also tumor cells expressing VEGF receptors. VEGF or phorbol 12-myristate 13-acetate treatment of the T24 bladder tumor cell line resulted in a time- and dose-dependent stimulation of SPK activity. In T24 cells, VEGF treatment reduced cellular sphingosine levels while raising that of sphingosine-1-phosphate. VEGF stimulation of T24 cells caused a slow and sustained accumulation of Ras-GTP and phosphorylated extracellular signal-regulated kinase (phospho-ERK) compared with that after EGF treatment. Small interfering RNA (siRNA) that targets SPK1, but not SPK2, blocks VEGF-induced accumulation of Ras-GTP and phospho-ERK in T24 cells. In contrast to EGF stimulation, VEGF stimulation of ERK1/2 phosphorylation was unaffected by dominant-negative Ras-N17. Raf kinase inhibition blocked both VEGF- and EGF-stimulated accumulation of phospho-ERK1/2. Inhibition of SPK by pharmacological inhibitors, a dominant-negative SPK mutant, or siRNA that targets SPK blocked VEGF, but not EGF, induction of phospho-ERK1/2. We conclude that VEGF induces DNA synthesis in a pathway which sequentially involves protein kinase C (PKC), SPK, Ras, Raf, and ERK1/2. These data highlight a novel mechanism by which SPK mediates signaling from PKC to Ras in a manner independent of Ras-guanine nucleotide exchange factor.

* Corresponding author. Mailing address: Department of Biochemistry and Molecular Biology, Norris Comprehensive Cancer Center, Keck School of Medicine at the University of Southern California, Los Angeles, CA 90089. Phone: (323) 856-0523. Fax: (323) 865-0154. E-mail: broek{at}

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