Figure 3.

Schematic model for the effects of differentially targeted hydrogen peroxide (H₂O₂) on endothelial nitric oxide synthase (eNOS) phosphorylation pathways. This figure presents a model summarizing studies of endothelial cells expressing recombinant differentially targeted d-amino acid oxidase (DAAO). These experiments probed the effects of H₂O₂ generated in distinct subcellular compartments on oxidant-modulated phosphorylation pathways in vascular endothelial cells. DAAO is a soluble protein but can be targeted to different subcellular locales using N-terminal signal sequences. Following addition of the DAAO substrate d-alanine, the phosphorylation of key phosphoenzymes was analyzed. H₂O₂ synthesized by DAAO constructs targeted either to endothelial cell nucleus (right) or to caveolae (left) led to the activation of protein kinase pathways that promote the phosphorylation of eNOS. H₂O₂ generated in either the nucleus or the cytosol leads to phosphorylation of the kinases PI3K, Akt1, and AMP-activated protein kinase (AMPK) and to the phosphorylation of acetyl CoA carboxylase (ACC) and eNOS. But only the H₂O₂ generated in the endothelial cell nucleus, not in the caveolae, leads to eNOS phosphorylation via AMPK: Caveola-derived H₂O₂ does not promote eNOS phosphorylation via AMPK, suggesting that different cellular pools of AMPK may be differentially regulated by oxidants. This figure exemplifies the importance of the subcellular localization of oxidants in the modulation of cell signaling pathways. Figure adapted from Reference 32 using BioRender.com.