title: Src activates HIF-1α not through direct phosphorylation of HIF-1α specific prolyl-4 hydroxylase 2 but through activation of the NADPH oxidase/Rac pathway.
abstract: Hypoxia-Inducible Factor (HIF)-1α/β heterodimer is a master transcription factor for several genes involved in angiogenesis, glycolysis, pH balance and metastasis. These HIF-1 target genes help tumors to overcome forthcoming metabolic obstacles as they grow. Under normoxic condition, the HIF-1α subunit is hydroxylated by its specific prolyl-4 hydroxylase 2, given the acronym PHD2. Hydroxylated HIF-1α becomes a target for von Hippel-Lindau (VHL), which functions as an E3 ubiquitin ligase. Src prevents hydroxylation-dependent ubiquitinylation of HIF-1α, thus stabilizing it under normoxic conditions. We found that active Src does not directly phosphorylate any tyrosine residue of PHD2. In vitro hydroxylation reaction showed that the presence of the purified active Src protein does not inhibit the hydroxylation activity of the purified PHD2 enzymes. Instead of directly inhibiting PHD2, Src recruits several downstream-signaling pathways to intercept hydroxylation-dependent ubiquitinylation of HIF-1α. Using biochemical and genetic inhibition, we demonstrated that Src requires reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase/Rac complex for stabilization of HIF-1α. We found that excess vitamin C treatment attenuates Src-induced HIF-1α activation. HIF-1α-hydroxylation-dependent VHL pull-down assay showed that Src inhibits cellular PHD2 activity by inducing ROS production in a mechanism involving Rac1-dependent NADPH oxidase. Src-induced ROS reduces cellular vitamin C, which is required for the activity of PHD2, thus Src can block VHL recruitment of HIF-1α, leading to stabilization of HIF-1α.
authors.affiliation: Department of Life Science, University of Seoul, Siripdae-gil 13, Dongdaemun-gu, Seoul, Korea.
authors.forename: Ho-Youl
authors.initials: HY
authors.lastname: Lee
authors.forename: Taekyong
authors.initials: T
authors.lastname: Lee
authors.forename: Naery
authors.initials: N
authors.lastname: Lee
authors.forename: Eun G
authors.initials: EG
authors.lastname: Yang
authors.forename: Cheolju
authors.initials: C
authors.lastname: Lee
authors.forename: Jonghyun
authors.initials: J
authors.lastname: Lee
authors.forename: Eun-Yi
authors.initials: EY
authors.lastname: Moon
authors.forename: Joohun
authors.initials: J
authors.lastname: Ha
authors.forename: Hyunsung
authors.initials: H
authors.lastname: Park
mesh_terms: D015153:Blotting, Western; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D006900:Hydroxylation; D000860:Hypoxia; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism*; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D007124:Immunoenzyme Techniques; D047468:Immunoprecipitation; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011393:Procollagen-Proline Dioxygenase / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020744:rac GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D019061:src-Family Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; C490190:EGLN1 protein, human; D011393:Procollagen-Proline Dioxygenase; D064799:Hypoxia-Inducible Factor-Proline Dioxygenases; D019255:NADPH Oxidases; D019061:src-Family Kinases; D020744:rac GTP-Binding Proteins