Journal
ARTERIOSCLEROSIS THROMBOSIS AND VASCULAR BIOLOGY
Volume 29, Issue 6, Pages 895-901Publisher
LIPPINCOTT WILLIAMS & WILKINS
DOI: 10.1161/ATVBAHA.109.184812
Keywords
G6PD; Src; VEGF; endothelial cells; tyrosine phosphorylation
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Funding
- NIH [HL077789, 0530195N]
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Objective-Glucose 6-phosphate dehydrogenase (G6PD) maintains cellular NADPH levels, which are essential for cellular functions, such as vascular endothelial growth factor (VEGF)-induced angiogenesis. The molecular mechanisms regulating G6PD in angiogenesis are not fully understood. Because tyrosine phosphorylation is a key regulatory pathway for VEGF-mediated endothelial cell (EC) responses, we investigated tyrosine phosphorylation of G6PD and the role of the nonreceptor tyrosine kinase Src. Methods and Results-VEGF increased G6PD membrane translocation as measured by a plasma membrane sheet assay, whereas tyrosine kinase inhibitor PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-d] pyramidine) decreased G6PD translocation by 100%. Furthermore, G6PD tyrosine phosphorylation and plasma membrane activity were increased by VEGF. In resting ECs, tyrosine kinase inhibitors PP2 and herbimycin A decreased basal G6PD activity by similar to 25%, whereas transfection with kinase inactive Src (KD-Src) decreased basal activity by similar to 30%. In mouse embryonic fibroblasts, Src-deficient (SYF) cells showed similar to 22% lower basal G6PD activity than Src-expressing S YF cells. In addition, Src directly phosphorylated G6PD assayed by in vitro kinase assay. In ECs transfected with the G6PD mutants Y428F, Y507F (presumptive sites for Src-phosphorylation) or double mutant Y428F/Y507F, G6PD tyrosine phosphorylation was significantly decreased. Finally, G6PD tyrosine mutants (Y428F, Y507F, and Y428F/Y507F) decreased VEGF-mediated Akt phosphorylation and EC migration. Conclusions-G6PD activity and membrane association are regulated by Src-mediated tyrosine phosphorylation, which contributes to VEGF-mediated cellular responses in EC. (Arterioscler Thromb Vasc Biol. 2009; 29:895-901.)
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