Fig. 1
6-Phosphogluconate dehydrogenase (6PGD) pY481 is required for EGF-enhanced 6PGD activity. a U87/epidermal growth factor receptor (EGFR) cells (left panel) or U251/EGFR cells (right panel) stably expressing Flag-6PGD were treated with or without EGF (100 ng ml-1) for 30 min. pTyr phospho-tyrosine, pSer phospho-serine, pThr phospho-threonine. b Immunoprecipitated Flag-6PGD from U87/EGFR cells treated with or without EGF (100 ng ml-1) for 30 min, was subjected to mass spectrometry analyses. Mass spectrometry analysis of a tryptic fragment at m/z 999.20270 (z = + 4), matched to the charged peptide 1-DYFGAHTYELLAKPGQFIHTNWTGHGGTVSSSS(pY)NA-36. The probability of pY481 was 99.99%. c U87/EGFR cells (left panel) or U251/EGFR cells (right panel) stably expressing Flag-6PGD WT or Y481F were treated with or without EGF (100 ng ml-1) for 30 min. d U87/EGFR cells (left panel) or U251/EGFR cells (right panel) stably expressing Flag-6PGD WT or Y481F were treated with EGF (100 ng ml-1) for 30 min. e Sequence alignment of phosphorylated peptides among indicated species. f Representative image of structure of human 6PGD bound to NADP+ (PDB ID:2JKV) was shown. Dimeric 6PGD was shown as ribbons and NADP+ was shown as balls and sticks. 6PGD Y481 was shown as sticks. Cyan ribbon or gray ribbon represented two monomers in dimeric 6PGD, respectively. g, h Flag-6PGD WT or Y481F were immunoprecipitated from U87/EGFR cells with or without EGF (100 ng ml-1) treatment for 30 min. The enzymatic activity of immunoprecipitated Flag-6PGD proteins was examined (g). Statistical analyses of 6PGD activities (h). The activity of 6PGD was normalized against protein amounts. 6PGD WT activity without EGF treatment was normalized to 1.0. Data represent the mean ± SD of three independent experiments. Student’s t-test (unpaired, two tailed), **p < 0.01; n.s. not significant. Immunoprecipitation and immunoblotting analyses were performed with the indicated antibodies. Data are representative of at least three independent experiments. a, c, d, h Source data are provided as a Source Data file
