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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2009;29:895.)
© 2009 American Heart Association, Inc.

Cell Biology/Signaling

Glucose 6-Phosphate Dehydrogenase Is Regulated Through c-Src–Mediated Tyrosine Phosphorylation in Endothelial Cells

Shi Pan; Cameron J. World; Christopher J. Kovacs; Bradford C. Berk

From the Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine and Dentistry, NY.

Correspondence to Bradford C. Berk, MD, PhD, University of Rochester Medical Center, Box CVRI, 601 Elmwood Ave, Rochester, NY 14642. E-mail Bradford_Berk{at}urmc.rochester.edu

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 25%, whereas transfection with kinase inactive Src (KD-Src) decreased basal activity by 30%. In mouse embryonic fibroblasts, Src-deficient (SYF) cells showed 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.

The molecular mechanisms for regulating glucose 6-phosphate dehydrogenase (G6PD) in endothelial cells (ECs) were studied. G6PD was regulated by Src-mediated tyrosine phosphorylation, which modulated VEGF responses including Akt activation and EC migration.

Key Words: G6PD • Src • VEGF • endothelial cells • tyrosine phosphorylation