Mitogen stimulation of cytoskeletal changes and c-jun amino-terminal kinases is mediated by Rac small guanine nucleotide-binding proteins. Vav, a guanosine diphosphate (GDP)-guanosine triphosphate (GTP) exchange factor for Rac that stimulates the exchange of bound GDP for GTP, bound to and was directly controlled by substrates and products of phosphoinositide (PI) 3-kinase. The PI 3-kinase substrate phosphatidylinositol-4,5-bisphosphate inhibited activation of Vav by the tyrosine kinase Lck, whereas the product phosphatidylinositol-3,4,5-trisphosphate enhanced phosphorylation and activation of Vav by Lck. Control of Vav in response to mitogens by the products of PI 3-kinase suggests a mechanism for Ras-dependent activation of Rac.

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.

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