Regulation of NUAK1 by NRF2 and ROS

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Regulation of NUAK1 by NRF2 and ROS Regulation of NUAK1 by NRF2 and ROS. A, Immunoblots show reduced NUAK1 protein and reduced MYPT1S445 phosphorylation in U2OS cells upon depletion of NRF2 using 2 distinct siRNAs. Regulation of NUAK1 by NRF2 and ROS. A, Immunoblots show reduced NUAK1 protein and reduced MYPT1S445 phosphorylation in U2OS cells upon depletion of NRF2 using 2 distinct siRNAs. B, Alignment of a putative antioxidant response element (ARE) in the NUAK1 promoter with the NRF2-binding consensus sequence. C, Chromatin IP of NRF2-bound DNA probed with primer pairs flanking (F1/R1; F2/R2) or distal to (F3/R3) the putative ARE in the NUAK1 promoter (see diagram). Right, NRF2 binding to the canonical target gene HMOX1 from the same analysis. Mean and SEM of technical replicates from 1 of 2 independent experiments shown. D, QPCR measurement of NUAK1 mRNA in U2OS cells treated with or without H2O2 (100 μmol/L) for 4 hours. Mean and SEM of 3 experiments. *, Significance (paired t test). E, Immunoblot of NUAK1 protein levels after treatment of U2OS cells (1 hour) with the indicated concentrations of H2O2. F, Immunoblots of T211 NUAK1 and S445 MYPT1 phosphorylation upon acute treatment of U2OS (left) or SW480 (right) with H2O2 for the indicated times. G, Oxidation of NUAK1 protein detected by dimedone labeling of U2OS cells expressing FLAG-tagged NUAK1 and treated for 5 minutes with 500 mmol/L H2O2. H, Identification of oxidized cysteines in FLAG-tagged NUAK1 by MS analysis of iodoacetamide labeling of U2OS-FLAG-NUAK1 cells treated with/without H2O2 for 5 minutes. Lysates were labeled with heavy (13C) or light (12C) iodoacetamide, followed by IP of FLAG-NUAK1. Plot shows analysis of reciprocally labeled samples from 2 independent experiments. Mean and SD indicated. I, Model integrating NUAK1 suppression of PP1β-dependent dephosphorylation of GSK3β as an integral step in nuclear mobilization of NRF2 in response to oxidative stress. Jennifer Port et al. Cancer Discov 2018;8:632-647 ©2018 by American Association for Cancer Research