Project Summary Nitric Oxide Synthase 3 (NOS3) and p38α play key roles in intracellular signaling. Alterations to the activity of these enzymes impact diseases like atherosclerosis, diabetes, and cancer. p38 is ubiquitously expressed, very near the end of the signaling cascades it functions in and bind to NOS3 in vitro and in the environment of endothelial cells. NOS3 is a key regulator of vascular homeostasis and responds to a wide variety of signals (e.g., bradykinin, VEGF, insulin) by producing nitric oxide (NO), itself an important signaling molecule. Regulation of NOS3 occurs through protein interactions, posttranslational modifications, cellular localization, and scaffolding. The outcomes of p38α binding to NOS3 are still mysterious, yet binding is modulated in the cellular environment. Emerging understanding of the regulatory roles of oxidation and the key role that activator and substrate recruitment has on MAPKs led us to hypothesize that perhaps a key role of p38α-NOS3 binding is to allow NOS3 to regulate p38α through scaffolding to control substrate access and/or direct inhibition by oxidation. We propose to use proximity ligation assay and biolayer interferometry with p38α (wild type and variants) and NOS3 (or NOS3 peptides), to investigate the dynamic nature of this interaction while testing our hypotheses using in vitro and in situ analysis with endothelial cells. This R15 AREA project will provide important information about the intersection of NOS3 and p38 while involving and exposing undergraduate researchers to current questions in cellular signaling, preparing them for careers in STEM fields.