4D). During the course of this work, a paper by Komatsu et al. that Keap1 interacts with p62 and LC3 in a stress-inducible manner, and that Keap1 colocalizes with LC3 and p62 in puromycin-induced ubiquitin aggregates. Moreover, p62 serves as a bridge between Keap1 and ubiquitin aggregates and autophagosomes. Finally, genetic ablation of Keap1 leads to the accumulation of ubiquitin aggregates, increased cytotoxicity of misfolded protein aggregates, and defective activation of autophagy. Therefore, this study assigns a novel positive role of Keap1 in upregulating p62-mediated autophagic clearance of ubiquitin aggregates. rescued the cytotoxic phenotype of liver cells in autophagy mutant mice.9 Moreover, p62 accumulation is frequently observed in human tumors due to impairment of the autophagy pathway, which leads to the oxidative stress and inflammation responses that correlate with tumorigenesis.22 However, how p62 links autophagy, oxidative stress and ubiquitin aggregates together is still unclear. Here we report that an oxidative stress sensor Clofoctol Kelch-like ECH-associated protein 1 (Keap1) interacts with p62 and LC3. Upon puromycin treatment, the majority of Keap1 localizes to cellular puncta that are also positive for p62, ubiquitin conjugates, and LC3. Furthermore, genetic ablation of Keap1 significantly compromised the clearance of puromycin-induced misfolded protein aggregation. Finally, stress-induced autophagy was defective in Keap1 mutant cells. We therefore propose that Keap1 plays a critical role in protein aggregation clearance through autophagy. Results Identification of Keap1 in the LC3 and p62 protein complex We performed a tandem affinity purification using double-tagged, full-length LC3 (ZZ-FLAG-LC3) as the bait (Fig. 1A) to purify an endogenous complex. We generated an inducible cell line that stably expresses LC3. Expression of LC3 was adjusted by the titration of doxycycline, and a low dose of doxycycline (10 ng/ml) that induces expression of tagged LC3 close Clofoctol to the endogenous level was selected for initiating purification to avoid nonphysiological interactions. We followed a procedure that has been described previously to isolate the LC3 complex from human cells,23,24 and the doxycycline-treated stable cell lines were used to generate cytosolic extracts. IgG-Sepharose affinity chromatography was first applied, and the binding proteins were eluted by TEV protease cleavage, and then TEV-eluted proteins were applied to M2 (anti-FLAG antibody)-agarose beads. The final FLAG peptide eluate was subjected to 4C12% gradient SDS-PAGE, and visualized by silver staining. Specific bands were excised and analyzed by mass spectrometry. Open in a separate windows Physique 1 Keap1 interacts with LC3 and p62 in a stress-inducible manner. (A) Silver staining of LC3 complex after tandem affinity purification, LC3 complex was purified from U2OS cell that stably expresses ZZ-FLAG-LC3 with or without starvation for one hour. Proteins ID were identified by mass spectrometry. (B) Keap1 interacts with LC3 and p62 in vivo. HE K293T cells were immunoprecipitated with anti-Keap1 antibody, then immunoblotted with anti-Keap1 or anti-LC3 or anti-p62 antibodies. (C) 6-OHDA induces Keap1-p62 conversation. HA-Keap1 transfected HE K293T cells were treated with 6-OHDA for 6 hours. Cells were then lysed and followed with HA Rabbit Polyclonal to GABRD immunoprecipitation. The endogenous p62 pulled down by Keap1 was detected by western blotting. These experiments have been repeated at least three times. In addition to the known members of a group of LC3 interacting proteins, namely MAP1B,9 FYCO1,25 and p62, we also identified a 60 kD component that associated with LC3 in a starvation-inducible manner (Fig. 1A), and this protein was identified by mass spectrometry as Keap1. The conversation of LC3, p62 and Keap1 was further confirmed by the presence of LC3 and p62 in the eluate of immunoprecipitate pulled down by anti-Keap1 antibody Clofoctol (Fig. 1B). Because Keap1 is usually a sensor of oxidative stress,26 its activity is usually tightly regulated by stress,27 and therefore, we hypothesize that this conversation between Keap1 and p62 is also regulated by oxidative stress. In the cells treated with 6-hydroxydopamine (6-OHDA), a neurotoxin-generating active oxygen species, a significantly greater amount of p62 immunoprecipitated with Keap1 compared to that in untreated cells (Fig. 1C), confirming that Keap1 associates with p62 in a stress-inducible manner. Keap1 colocalizes.