Glucose 6-phosphate dehydrogenase is regulated through c-Src-mediated tyrosine phosphorylation in endothelial cells.
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.


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.
Aab Cardiovascular Research Institute, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA.
Shi
S
Pan
Cameron J
CJ
World
Christopher J
CJ
Kovacs
Bradford C
BC
Berk
D000818:Animals; D000081247:CSK Tyrosine-Protein Kinase; D002417:Cattle; D002462:Cell Membrane / Q000201:enzymology; D002465:Cell Movement; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000201:enzymology*; D005347:Fibroblasts / Q000201:enzymology; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D051379:Mice; D009154:Mutation; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D014162:Transfection; D014443:Tyrosine; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D019061:src-Family Kinases
D047428:Protein Kinase Inhibitors; D042461:Vascular Endothelial Growth Factor A; D014443:Tyrosine; D005954:Glucosephosphate Dehydrogenase; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human; D051057:Proto-Oncogene Proteins c-akt