Regulation of Oxidative Stress Responses By PEBP1-dependent Autophagy Huynh Quoc Nguyen, Trang Huyen Lai, Sahib Zada, Mahmoud Ahmed, Jin Seok Hwang and Deok Ryong Kim Department of Biochemistry & Convergence Medical Sciences, Institute of Health Sciences, Gyeongsang National University School of Medicine, JinJu, Republic of Korea 527-27 ABSTRACT Phosphatidylethanolamine binding protein 1 (PEBP1) is originally isolated as a Raf kinase inhibitory protein (RKIP). Recently, we reported that PEBP1 negatively regulates starvation-induced autophagy. It specifically binds to LC3 via its own LIR motif (W55DGL58) and prevents LC3 lipidation with phosphatidylethanolamine. Autophagy has also been implicated in a variety of physiological and pathological pathways in response to oxidative stress. In this study, we show that PEBP1 possibly inhibits the autophagy-dependent degradation of KEAP1 and in turn NRF2 activation under oxidative stress. Indeed, overexpression of PEBP1 prevented not only LC3 lipidation but also p62 phosphorylation at Ser349, stabilizing the KEAP1-NRF2 interaction. In contrast, ablation of PEBP1 stimulated p62 phosphorylation at Ser349, leading to Keap1 degradation by autophagy. We also observed the transcriptional suppression of some NRF2 target genes in PEBP1-overexpressing cells under oxidative stress. These data suggest that PEBP1-dependent autophagy is critical to regulate the oxidative stress responses in cells. Keywords: autophagy, PEBP1, p62, NRF2, KEAP1, oxidative stress A C NRF2 YY1 B BACKGROUND (1) Phosphatidylethanolamine binding protein 1 (PEBP1) is originally isolated as a cellular inhibitor of the ERK pathway. It specifically interacts with Raf1 kinase and blocks the consequent activation of a downstream MEK kinase, so it is alternatively named as the Raf kinase inhibitory protein (RKIP). Recently, we reported that PEBP1 negatively regulates starvation-induced autophagy, an intracellular degradation and recycling process. It specifically binds to LC3 via its own LC3 interacting region (LIR) motif (W55DGL58) and prevents LC3 lipidation with phosphatidylethanolamine (PE), which is a key regulatory step for formation of autophagosomes. (2) The KEAP1-NRF2 signaling is currently considered as one of the major cellular defense mechanisms against oxidative stresses. Under non-oxdative stress conditions, the transcription factor NRF2 (nuclear factor erythroid 2-related factor 2) is bind to KEAP1, (kelch-like ECH-associated protein 1), an adaptor of the ubiquitin ligase complex. In exposure of reactive oxygen species, KEAP1 is sequestrated to autophagosome/proteasome by p62 phosphorylation at S349/S351, leading to its degradation. Consequently, NRF2 translocates into the nucleus to induce the transcription of numerous cytoprotective genes. (3) Cells expressing the low level RKIP show the elevated responses to the oxidative stress and increase of chemotherapeutic resistance. Nuclear Figure 2. PEBP1 inhibits autophagic degradation of KEAP1 and causes accumulation of total NRF2. (A) H1299 cells stably expressing with control pcDNA vector or FLAG-PEBP1 were treated with 0.5mM H2O2 for 0, 1, 2 hours, respectively. Then, total cell extracts were prepared for analysis by western blot using antibodies against PEBP1, p62, p62 p-S349, KEAP1, NRF2, and NQO1. β-actin was used as a loading control. (B) Nuclear proteins of stable H1299-pcDNA and H1299-FLAG-PEBP1 were extracted for analysis by western blot using antibody against NRF2, YY1 was used as a loading control. (C) H1299-pcDNA and H1299-FLAG-PEBP1 cells were treated with 0.5mM H2O2 and 50μM CQ for 2 hours. Total cell extracts were prepared for analysis by western blot using antibodies against PEBP1, p62, p62-p-S349,NRF2 and NQO1. β-actin was used as a loading control. A B (1) (2) Figure 3. PEBP1 negatively regulates phosphorylation of p62 at S349. (A) A549 cells were infected with the adenoviral vectors encoding PEBP1 shRNA (Ad-shPEBP1) or control vector (Ad-GFP). After incubation for 48h, cells were treated with 0.5mM H2O2 for 0, 1, 2 hours, and whole cell extracts were analyzed by western blot. (B) 293A cells were transfected with FLAG-PEBP1 or CRISPER-PEBP1 plasmids to overexpress or knock-down PEBP1, and then cells were treated with 0.5mM H2O2 for 0 and 2 hours. Then total cell extracts were prepared for analysis by western blot. β-actin was used as a loading control. RESULTS . SUGGESTION AND FUTURE PLANS . A SUGGESTION: PEBP1 possibly inhibits the autophagy-dependent degradation of Keap1 and in turn Nrf2 activation under oxidative stress. Overexpression of PEBP1 inhibits not only the lipidation of LC3B-I to LC3B-II to form autphagosome, but also prevents the phosphorylation of SQSTM1/p62 at S351/S349, which is important for the interaction between SQSTM1/p62 and KEAP1 protein, the phosphorylation of p62 at S351/S349 is suppressed, stabilizing the KEAP1-NRF2 interaction. In contrast, ablation of PEBP1 stimulated p62 phosphorylation at S351/S349, leading to Keap1 degradation by autophagy. We also observed the transcriptional suppression of some NRF2 target genes, NQO1, GCLC, G6PD, and SOD2, in PEBP1-overexpressing cells under oxidative stress. These data suggest that PEBP1-dependent autophagy is critical to regulate the oxidative stress responses in cells. B FUTURE PLANS: To investigate how PEBP1 regulate phosphorylation of p62 at S349/351, and which kinases phosphorylate p62 at S349/S351. Figure 1. PEBP1 inhibits autophagy and suppresses oxidative response. (A) Overexpression of PEBP1 inhibits autophagy under oxidative stress condition. H1299 cells stably expressing with pcDNA vector or FLAG-PEBP1 were treated with 0.5mM H2O2 and 50μM CQ for 2 hours. And total cell extracts were prepared for analysis by western blot using antibodies against PEBP1, LC3B, and NQO1. β-actin was used as a loading control. (B) Overexpression of PEBP1 suppresses NRF2 targeted genes, Total mRNA of H1299-pcDNA and H1299-FLAG-PEBP1 was extracted, and analyzed by q-PCR. Relative mRNA levels of PEBP1 and NRF2 targets, NQO1, GCLC, G6PD, and SOD2, among H1299-pcDNA and H1299-FLAG-PEBP1 were normalized to the amount of each mRNA in the H1299-pcDNA cells, and GAPDH was used as a reference gene. REFERENCES NOH, Hae Sook, et al. PEBP1, a RAF kinase inhibitory protein, negatively regulates starvation-induced autophagy by direct interaction with LC3. Autophagy, 2016, 12.11: 2183-2196. Y. Ichimura, et al. 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