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.

Lysophosphatidic acid stimulates epithelial to mesenchymal transition marker Slug/Snail2 in ovarian cancer cells via Gαi2, Src, and HIF1α signaling nexus

“…Previous findings from our laboratory have indicated that LPA-Gαi2 signaling could rapidly stimulate Rac via p130Cas/Src dependent pathway [15]. It has also been shown that Src can stimulate an increase in the levels and subsequent activation of HIF1α involving Rac-stimulated ROS generation via NADPH oxidase [54]. Connecting these two independent observations, our data presented here points to a signaling paradigm in which the signaling by LPA propagates through Gαi2 and Src to HIF1α (Figure 9).…”

Periodic Mechanical Stress Activates Integrin�1-Dependent Src-Dependent PLC?1-Independent Rac1 Mitogenic Signal in Rat Chondrocytes through ERK1/2

“…Zhang et al demonstrated that mechanical strain stimulation of ROS generation in glomerular mesangial cells is induced by Rac1 activation via an Src signaling pathway [13]. In response to other non-mechanical stimuli, Rac1 is also recognized as a downstream signaling molecule of Src signals [37][38][39][40][41]. In our study, pretreatment of cells with the Src inhibitor PP2 and shRNA targeted to Src blocked periodic mechanical stress-induced Rac1 phosphorylation and activation, which suggests that Src activation of Rac1 at Ser71 is responsible for the signaling transduction mechanisms in chondrocytes under conditions of cyclic mechanical stimulation.…”

Chlorogenic acid inhibits hypoxia-induced pulmonary artery smooth muscle cells proliferation via c-Src and Shc/Grb2/ERK2 signaling pathway

“…Increasing evidence showed that c-Src activated NAPDH oxidase in VSMCs (Lassegue et al, 2012;Seshiah et al, 2002;Touyz et al, 2003). Furthermore, Lee et al (2011) demonstrated that c-Src activated HIF-1α through activation of the NADPH oxidase/Rac pathway. Thus, we investigated the effects of CGA on NADPH oxidase activity in hypoxic condition in PASMCs.…”