The ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP) are two major cellular pathways responsible for protein degradation (Ciechanover, 2005; Rubinsztein, 2006; Dikic, 2017). In the UPS, proteins are first modified by ubiquitin to form specific types of ubiquitinated proteins that are then targeted for degradation by the 26S proteasome (Grice and Nathan, 2016). In the ALP, autophagic substrates including soluble proteins, protein complexes and aggregates, are engulfed by a double-layered membrane to form autophagosomes, which are further directed to lysosomes for degradation by lysosomal hydrolases (Korolchuk
The UPS and ALP can regulate cell death and survival through the degradation of different substrates. However, the regulation of cell death and survival by p62 is somewhat controversial (Jin
Activator protein-1 (AP-1) transcription factor complex (Halazonetis
In this study, we explore the possibility of the interplay between p62 and c-Jun and their role in regulating cell death under ER stress. We first measured the expression of p62 and c-Jun under ER stress induced by different stimuli. We then examined the potential interaction between p62, c-Jun, and CRBN using immunoprecipitation and immunoblotting techniques. We further investigated the regulation of p62 on c-Jun protein levels, and the role of CRBN in this regulation. Finally, we comment on the potential function of CRBN in ER stress-induced apoptosis and the underlying molecular mechanism.
The primary antibodies used in this work were obtained from the following sources: anti-HA (sc-7392) and anti-ubiquitin (sc-8017) antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-c-Jun (CPA1634) antibody was from Cohesion Biosciences (London, UK); anti-FLAG (0912-1) and anti-GFP (EM30501) antibodies were from HuaAn Biotechnology (Hangzhou, China); mouse anti-CRBN antibody was a gift from Dr. Xiu-Bao Chang (Mayo Clinic College of Medicine, Scottsdale, AZ, USA); rabbit anti-CRBN antibody (11435-1-AP) and anti-GAPDH (60004-1) antibody were from ProteinTech Group (Rosemont, IL, USA); anti-cleaved caspase 3 (9661S) and anti-PARP1 (9532S) antibodies were from Cell Signaling Technology (Danvers, MA, USA); anti-p62 (P0067) antibody was from Sigma (Saint Louis, MO, USA); anti-LC3 (NB100-2220) antibody was from Novus Biologicals (Centennial, CO, USA); anti-FLAG affinity gel (B23102) and anti-HA magnetic beads (B26301) were from Bimake (Houston,TX, USA); rabbit IgG (A7016) for control immunoprecipitation was from Beyotime Biotechnology (Haimen, Jiangsu, China); and HRP-labeled secondary antibodies were from Beyotime Biotechnology and Thermo Fisher (Waltham, MA, USA).
Chemicals were from the following companies: MG132 (CC2775) was from ChemCatch (Suwanee, GA, USA); brefeldin A (s7046) was from Selleck (Houston, TX, USA); puromycin (P8230) was from Solarbio (Beijing, China); Hoechst (C1022) was from Beyotime Biotechnology; and propidium iodide (PI, KGA214-50) was from Nanjing KeyGen BioTech (Nanjing, Jiangsu, China).
Strep-FLAG (SF)-c-Jun, FLAG-p62, HA-CRBN, and GFP-p62 plasmids were from our previous work (Yang
Human embryonic kidney (HEK) 293T and HeLa cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (PAN Biotech, Aidenbach, Germany and Lonsera, Ciudad de la Costa, Uruguay) and 100 units/mL penicillin and 100 µg/mL streptomycin (Gibco, Waltham, MA, USA). Cells were passaged every two or three days.
Plasmids were transfected with lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA) or polyethyleneimine (PEI, Sigma) transfection reagent and si
The 21-nucleotide sequence for sh
Following transfection or drug treatment, cells were lysed in modified RIPA buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 1 mM EDTA) on ice with brief sonication. Cell lysates were obtained after centrifugation (13, 000 g) for 15 min at 4°C.
FLAG-tagged proteins were purified with anti-FLAG affinity gel according to a previously reported method (Zhu
HA-tagged CRBN was purified with anti-HA-magnetic beads according to a method described previously (Zhu
Immunoprecipitation of endogenous c-Jun was carried out using a procedure described previously (Yang
Western blotting analysis was carried out based on a previously used method (Yang
PI staining was described in a previous study (Hou
The colorimetric TUNEL apoptosis assay kit was purchased from Beyotime Biotechnology. HEK293T cells stably expressing sh
Statistical analysis was performed using two-tailed Student’s
Activation of autophagy may result in a decrease of p62 protein levels (Wu
It has been reported that p62 may regulate the cellular degradation pathway (Pankiv
In our previous work, we discovered that CRBN interacts with p62 (also shown in Supplementary Fig. 2) and prevents the formation of pathogenic protein aggregates in cell lines and primary neuronal cells (Zhou
Our previous work (Yang
These results demonstrate that p62 interacts with, and downregulates, c-Jun through the UPS and that a ternary protein complex is formed between p62, c-Jun, and CRBN. Our previous studies also showed that CRBN interacts with c-Jun and that c-Jun functions as a substrate receptor of the CRL4 E3 ligase for the ubiquitination and degradation of c-Jun (Yang
The experiments described above indicate that p62 may participate in the regulation of c-Jun under ER stress. Since c-Jun protected cells against ER stress-induced apoptosis (Supplementary Fig. 3), we examined the role of p62, CRBN, and c-Jun in ER stress. We first constructed the stable control or CRBN knockdown HEK293T cell lines using sh
Activation of autophagy causes the autophagic degradation of p62, leading to reduced apoptosis (Levine and Kroemer, 2008; Lamark
In this study, we confirmed that p62-mediated apoptosis under brefeldin treatment is due to the proteasomal degradation of c-Jun, which may occur following the recruitment of the substrate receptor CRBN of the CRL4 E3 ligase leading to the ubiquitination of c-Jun and its degradation. This finding is consistent with a previous study which found that autophagic degradation of p62 protected cells from ER-stress induced apoptosis (Ogata
Our previous studies showed that CRBN suppresses the formation of p62 bodies (Zhou
In summary, we have identified that p62 forms a ternary complex with c-Jun and CRBN and thus promotes the ubiquitin-mediated c-Jun degradation. Downregulation of p62 induced by ER stress exhibits the protective role against apoptosis by reducing the c-Jun degradation. This work revealed the essential role of CRBN in the regulation of c-Jun by p62 and elucidated a new molecular mechanism by which the p62/c-Jun axis regulates the ER stress-induced apoptosis.
We are grateful to Dr. Xiu-Bao Chang from Mayo Clinic College of Medicine (USA) for generously providing the mouse CRBN antibody and to Dr. Guanghui Wang for kindly providing p62 and HA-Ub plasmids. This work was supported by the National Key R&D Program of China (2018YFC1705505), National Natural Science Foundation of China (31670833 & 81703535), Jiangsu Key Laboratory of Neuropsychiatric Diseases (BM2013003), National Center for International Research (2017B01012), a project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.