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Vascular endothelial growth factor (VEGF) signaling is critical for both normal and disease-associated vascular development. Dysregulated VEGF signaling has been implicated in ischemic stroke, tumor angiogenesis, and many other vascular diseases. VEGF signals through several effectors, including the Rho family of small GTPases. As a member of this family, Rac1 promotes VEGF-induced endothelial cell migration by stimulating the formation of lamellipodia and membrane ruffles. To form these membrane protrusions, Rac1 is activated by guanine nucleotide exchange factors (GEFs) that catalyze the exchange of GDP for GTP. The goal of this study was to identify the GEF responsible for activating Rac1 in response to VEGF stimulation. We have found that VEGF stimulates biphasic activation of Rac1 and for these studies we focused on the peak of activation that occurs at 30 min. Inhibition of VEGFR-2 signaling blocks VEGF-induced Rac1 activation. Using a Rac1 nucleotide-free mutant (G15ARac1), which has a high affinity for binding activated GEFs, we show that the Rac GEF Vav2 associates with G15ARac1 after VEGF stimulation. Additionally, we show that depleting endothelial cells of endogenous Vav2 with siRNA prevents VEGF-induced Rac1 activation. Moreover, Vav2 is tyrosine phosphorylated upon VEGF treatment, which temporally correlates with Rac1 activation and requires VEGFR-2 signaling and Src kinase activity. Finally, we show that depressing Vav2 expression by siRNA impairs VEGF-induced endothelial cell migration. Taken together, our results provide evidence that Vav2 acts downstream of VEGF to activate Rac1.

Prenols, a class of lipids formed by the condensation of five carbon isoprenoids, have important roles in numerous metabolic pathways of the eukaryotic cell. Prenols are found in the cell as free alcohols, such as dolichol, or can be attached to vitamins, as with the fat soluble vitamins. In addition, prenols such as farnesyl- and geranylgeranyl-diphosphate are substrates for the transfer of farnesyl and geranylgeranyl units to proteins with important implications for signal transduction within the cell. Dolichol phosphate- and dolichol diphosphate-linked sugars are central to the formation of the lipid-linked branched oligosaccharide, Dol-PP-(GlcNAc)2(Man)9(Glc)3, used for co-translational en bloc protein N-glycosylation in the lumen of the endoplasmic reticulum. Toward furthering our understanding of the role of prenol lipids in the cell, we have developed a method for the detection and quantification of dolichol and coenzyme Q by liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS). These methods, developed using the mouse macrophage RAW 264.7 tumor cells, are broadly applicable to other cell lines, tissues, bacteria, and yeast. We also present a new MS-based method for the detection and structural characterization of the intact dolichol diphosphate oligosaccharide Dol-PP-(GlcNAc)2 (Man)9(Glc)3 from porcine pancreas.

Cardiolipin (CL), a tetra-acylated glycerophospholipid composed of two phosphatidyl moieties linked by a bridging glycerol, plays an important role in mitochondrial function in eukaryotic cells. Alterations to the content and acylation state of CL cause mitochondrial dysfunction and may be associated with pathologies such as ischemia, hypothyrodism, aging, and heart failure. The structure of CL is very complex because of microheterogeneity among its four acyl chains. Here we have developed a method for the quantification of CL molecular species by liquid chromatography-electrospray ionization mass spectrometry. We quantify the [M-2H](2-) ion of a CL of a given molecular formula and identify the CLs by their total number of carbons and unsaturations in the acyl chains. This method, developed using mouse macrophage RAW 264.7 tumor cells, is broadly applicable to other cell lines, tissues, bacteria and yeast. Furthermore, this method could be used for the quantification of lyso-CLs and bis-lyso-CLs.