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
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
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DOI：
10.1093/carcin/bgr034
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发表时间：
2011-05-01
期刊：
影响因子：
4.7
通讯作者：
Park, Hyunsung
中科院分区：
文献类型：
--
作者：
Lee, Ho-Youl;Lee, Taekyong;Park, Hyunsung
低氧诱导因子(HIF)-1α/β异源二聚体是多种基因的主要转录因子，参与血管生成、糖酵解、pH平衡和肿瘤转移。这些HIF-1靶基因有助于肿瘤在生长过程中克服即将到来的新陈代谢障碍。在常氧条件下，HIF-1α亚基被其特定的Pro-4羟基酶2羟化，缩写为PHD2。羟化的HIF-1α成为von Hippel-Lindau(VHL)的靶标，VHL作为E3泛素连接酶发挥作用。SRC可阻止羟基化依赖的HIF-1α泛素化，从而使其在常氧条件下保持稳定。我们发现，活性的Src不直接磷酸化PHD2的任何酪氨酸残基。体外羟化反应表明，纯化的活性Src蛋白的存在并不抑制纯化的PHD2酶的羟化活性。而不是直接抑制PHD2，Src招募了几个下游信号通路来拦截HIF-1α羟基化依赖的泛素化。利用生化和遗传抑制，我们证明了Src需要还原的烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶/RAC复合体来稳定HIF-1α。我们发现过量的维生素C治疗减弱了Src诱导的HIF-1α的激活。HIF-1α-羟化依赖的VHL下拉实验表明，Src通过诱导ROS的产生而抑制细胞PHD2的活性，其机制涉及rac1依赖的NADPH氧化酶。SRC诱导的ROS减少了PHD2活性所需的细胞内维生素C，因此，Src可以阻断VHL对HIF-1α的募集，导致HIF-1α的稳定。
Hypoxia-Inducible Factor (HIF)-1 alpha/beta 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-1 alpha subunit is hydroxylated by its specific prolyl-4 hydroxylase 2, given the acronym PHD2. Hydroxylated HIF-1 alpha becomes a target for von Hippel-Lindau (VHL), which functions as an E3 ubiquitin ligase. Src prevents hydroxylation-dependent ubiquitinylation of HIF-1 alpha, 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-1 alpha. Using biochemical and genetic inhibition, we demonstrated that Src requires reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase/Rac complex for stabilization of HIF-1 alpha. We found that excess vitamin C treatment attenuates Src-induced HIF-1 alpha activation. HIF-1 alpha-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-1 alpha, leading to stabilization of HIF-1 alpha.