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 approximately 25%, whereas transfection with kinase inactive Src (KD-Src) decreased basal activity by approximately 30%. In mouse embryonic fibroblasts, Src-deficient (SYF) cells showed approximately 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.
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, G6PDtyrosine phosphorylation and plasma membrane activity were increased by VEGF. In resting ECs, tyrosine kinase inhibitors PP2 and herbimycin A decreased basal G6PD activity by approximately 25%, whereas transfection with kinase inactive Src (KD-Src) decreased basal activity by approximately 30%. In mouse embryonic fibroblasts, Src-deficient (SYF) cells showed approximately 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, G6PDtyrosine phosphorylation was significantly decreased. Finally, G6PDtyrosine 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.
Authors: Guy Eelen; Pauline de Zeeuw; Lucas Treps; Ulrike Harjes; Brian W Wong; Peter Carmeliet Journal: Physiol Rev Date: 2018-01-01 Impact factor: 37.312