CNOT2 promotes degradation of p62/SQSTM1 as a negative regulator in ATG5 dependent autophagy

Though CNOT2 is involved in regulation of adipogenic differentiation, apoptotic cell death and metastasis, the underlying autophagic mechanism of CNOT2 was unknown until now. Thus, in the present study, the critical role of CNOT2 in autophagy was elucidated in association with p62/SQSTM1 signaling. CNOT2 depletion induced p62/SQSTM1 accumulation and LC3B-II conversion, and also increased the number of puncta with impaired autophagic flux. In contrast, CNOT2 overexpression induced downregulation and ubiquitination of p62/SQSTM1 in HEK293 QBI. Furthermore, ubiquitination of p62/SQSTM1 was blocked by autophagy inhibition. Interestingly, CNOT2 was correlated with p62/SQSTM1 in HEK293 QBI cells and also was colocalized with p62/SQSTM1 in H1299 cells. Additionally, ATG5 was upregulated in CNOT2-depleted H1299 cells, while degradation of p62/SQSTM1 by CNOT2 was detected in ATG5+/+ MEF cells but not in ATG5−/− MEF cells. Of note, CNOT2 induced degradation of p62/SQSTM1 in HEK293 QBI cells co-transfected with Myc-ΔLIR/KIR or Myc-ΔUBA, but not with Myc-ΔPB1. Sub G1 population was increased in CNOT2-depleted H1299 cells by late autophagy inhibitors, ammonium chloride and chloroquine compared to 3-methyladenine. Overall, these findings provide novel insight into the critical role of CNOT2 as a negative regulator in ATG5 dependent autophagy.


INTRODUCTION
Autophagy is a catabolic process, where misfolded proteins or damaged organelles are recycled to maintain cellular homeostasis mainly by various factors such as nutrient deprivation, infection, aging and others [1][2][3]. Generally, the processes of autophagy machinery are the formation of isolation membrane, its elongation by engulfing some parts of the cytoplasm, and the formation of autophagosome as a double-membrane vesicle [4]. Emerging evidences reveal that the outer membrane of the autophagosome fuses with the lysosome to form the autophagolysosome to remove the luminal materials for survival [3][4][5].
Nevertheless, the underlying autophagic mechanism of CNOT2 was not reported until now. Thus, in the current study, the role of CNOT2 was investigated in association with p62/SQSTM1-degradation as an autophagy regulator.

Depletion of CNOT2 inhibits autophagic flux
Autophagy is a catabolic process by which the cells break down their polyubiquitinated protein aggregates that are not yet degraded through the proteasomal pathway [27][28][29]. In particular, the autophagy adaptor protein p62/SQSTM1 recognizes polyubiquitinated protein aggregates and incorporates them into autophagosomes via direct interaction with LC3B-II on the autophagosomal membrane, thereby delivering the aggregates for degradation [30]. In the present study, depletion of CNOT2 induced p62/SQSTM1 accumulation and LC3B-II conversion, as biochemical markers of autophagy, in H1299 cells ( Figure 1A and 1B). As shown in Figure 1C, the puncta pattern of LC3B-II fluorescence was detected in CNOT2-depleted H1299 cells, while a diffuse localization of LC3B-II fluorescence was observed in control group cells. Consistently, autophagic vacuoles, autophagosome (yellow arrowheads) and autophagolysosomes (red arrowheads) were observed by electron microscopy in CNOT2 siRNA transfected H1299 cells ( Figure 1D and 1E).Also, turnover assay revealed that p62/SQSTM1 was accumulated at 48 h and then tended to be degraded from 72 h in CNOT2 depleted H1299 cells ( Figure 1F). Next, it was examined whether or not CNOT2 completely induces autophagic flux in CNOT2-depleted H1299 cells. As shown in Figure 1G, depletion of CNOT2 inhibited the autophagic flux with yellow color, when autophagosomal green puncta and autophagolysosomal red puncta were merged in CNOT2-depleted H1299 cells.
Furthermore, it was investigated how CNOT2 regulates autophagic flux by using autophagy inhibitors. We blocked lysosomal degradation by using ammonium chloride (NH 4 Cl) as previously reported [31,32]. The formation of puncta in CNOT2-depleted H1299 cells was inhibited in the presence of early stage autophagy inhibitor 3-MA compared to untreated control, whereas the number of puncta was increased in late stage autophagy inhibitor NH 4 Cl treated H1299 cells ( Figure 1H). To clarify the effect of autophagy inhibitors such as 3-MA, CQ and NH 4 Cl on the fate of H1299 cells, FACS cell cycle analysis was performed. As shown in Figure 1I, cell cycle analysis revealed that increased sub G 1 population was detected in CNOT2 depleted H1299 cells by 3-MA, CQ and NH 4 Cl treatment in order. And PARP was cleaved and procaspase 8 was attenuated by CQ better than 3-MA ( Figure 1J).

CNOT2 induces ubiquitination and degradation of p62/SQSTM1 in HEK293 QBI cells
Given that CNOT2, as one of subunits of CCR4-NOT complex, modulates mRNA degradation and transcriptional regulation [22,23,33], the underlying functional protein-protein interactions between CNOT2 and p62/SQSTM1 were explored in HEK293 QBI cells. First, the effect of CNOT2 using HA-CNOT2 plasmid was evaluated on p62/SQSTM1 in HEK293 QBI cells. As shown in Figure 2A, the expression level of p62/ SQSTM1 was decreased by CNOT2 overexpression in a dose-dependent manner, while CNOT2 did not affect p62/ SQSTM1 mRNA level ( Figure 2B). Conversely, depletion of CNOT2 induced p62/SQSTM1 accumulation and LC3B-II conversion, which was suppressed by CNOT2 overexpression ( Figure 2C). To examine whether p62/ SQSTM1 was downregulated by CNOT2 overexpression, HEK293 QBI cells were co-transfected with HA-CNOT2 and Flag-p62 plasmids and then exposed to MG132 (proteasome inhibitor) or 3-MA (autophagy inhibitor). Immunoblotting revealed that downregulation of p62/SQSTM1 by CNOT2 was slightly reduced by MG132 ( Figure 2D), whereas p62/SQSTM1 was rather accumulated by 3-MA ( Figure 2E), indicating that downregulation of p62/SQSTM1 by CNOT2 is mediated via autophagic degradation. To further ascertain the regulatory role of the degradation pathways, the steady levels of p62/SQSTM1 by protein synthesis inhibitor cycloheximide (CHX) treatment were evaluated in the intact or overexpressed CNOT2 cells. The expression of HA-CNOT2 accelerated Flag-p62 expression decay, whereas empty vector stabilized flag-p62. As shown in Figure 2F, downregulation of p62/SQSTM1 by CNOT2 was enhanced by CHX treatment in a timedependent manner in HEK293 QBI cells. To confirm whether downregulation of p62/SQSTM1 is mediated by ubiquitination, ubiquitination assay was conducted in HEK293 QBI cells co-transfected with HA-CNOT2, Representative confocal images were exhibited in H1299 cells co-transfected with siCNOT2 and RFP-LC3 and GFP-LC3 constructs. Autophagosome was visualized as yellow or orange puncta (RFP-GFP-LC3B) in merged images, whereas red puncta (RFP-LC3B) represents autophagolysosome, since acidification reduces green fluorescence. Scale bar: 10 μm. (H) Quantitative analysis of LC3-puncta in H1299 cells transfected with CNOT2 siRNA in the present or absence of autophagy inhibitor 3-MA or NH 4 Cl (means ± SD of 3 independent experiments, * p < 0.05 vs untreated control by Student t test). (I) Effect of autophagy inhibitors on sub G 1 population in CNOT2-depleted H1299 cells. Cells were transfected with CNOT2 siRNA for 24 h and treated with 3-MA, CQ and NH 4 Cl for another 24 h. Then the cells were fixed with 75 % ethanol and stained with 50 mg/ml of PI. The cell cycle analysis was performed using FACs Calibur, and the data were analyzed using CellQuest Software (means ± SD of 3 independent experiments, ** p < 0.01, *** p < 0.001 vs untreated control by Student t test). (J) Effect of autophagy inhibitors on PAPR cleavage and procaspase 8 in H1299 cells transfected with CNOT2 siRNA. Cells were transfected with CNOT2 siRNA for 48 h, exposed to 3MA or CQ for 24 h and Western blotting was conducted with antibodies of PAPR cleavage, procaspase 8, CNOT2 and actin. www.impactjournals.com/oncotarget Flag-p62 and Myc-ubiquitin (Myc-ub) plasmids in the presence or absence of 3-MA or MG132. As shown in Figure 2G, CNOT2 overexpression induced ubiquitination of p62/SQSTM1, which was blocked by early stage autophagy inhibitor 3-MA, but not MG132, indicating autophagic degradation of p62/SQSTM1 by CNOT2.

CNOT2 binds to p62/SQSTM1 in HEK293 QBI cells as a key regulator of autophagy
To explore whether or not CNOT2 is a novel binding partner of p62/SQSTM1, we investigated the possibility that CNOT2 might be able to bind to p62/SQSTM1 by co-immunoprecipitation in HEK293 QBI cells. Here the correlation between CNOT2 and p62/SQSTM1 was detected in the presence of MG132 ( Figure 3A and 3B). Next, the role of the phosphorylation of p62/SQSTM1 at serine 351(S351) was examined on correlation between CNOT2 and p62/SQSTM1 in HEK293 QBI cells. The S351 of p62-KIR, a phosphorylation-mimetic mutant is substituted with Glu (S351E), has a higher affinity for endogenous Keap1 than either wild-type p62 or the phosphorylation-defective S351A mutant [34]. As shown in Figure 3C, intriguingly, any significant difference was not detected between wild-type of p62/SQSTM1 and phosphorylation-mimetic S351E mutant for the interaction of CNOT2 and p62/SQSTM1, implying that the p62/ SQSTM1 phosphorylation is not a prerequisite for CNOT2 and p62/SQSTM1 complex formation, regardless of Keap1 signaling. To rule out overexpression artifacts, HA-CNOT2 was immunoprecipitated with p62/SQSTM1 from H1299 whole-cell extracts. As a result, the interaction between endogenous CNOT2 and p62/SQSTM1 complex was still detected ( Figure 3D). Consistently, confocal microscopy showed that CNOT2 was colocalized with p62/SQSTM1 in H1299 cells as shown in Figure 3E.

The p62/SQSTM1 degradation by CNOT2 is mediated by ATG5
Autophagosome formation is critically mediated by a set of ATG proteins [31,35]. Thus, the important role of ATG5 was examined in CNOT2 induced degradation of p62/SQSTM1 in H1299 cells. RT-PCR and Western blotting revealed that the expression of ATG5 was increased in CNOT2-depleted H1299 cells at mRNA and protein levels ( Figure 4A and 4B). To confirm the pivotal role of CNOT2 in the autophagic disposal of p62/ SQSTM1, mouse embryonic fibroblast (MEF) cells were co-transfected with HA-CNOT2, HA-ATG5 and Flag-p62 expression constructs. Interestingly, ectopic expression of CNOT2 and ATG5 increased p62/SQSTM1degradation in a dose-dependent manner ( Figure 4C). To confirm the crucial role of ATG5 in the degradation of p62/SQSTM1 by CNOT2, HA-CNOT2 and HA-p62 were co-transfected in ATG5 knock out (KO) MEF cells. Downregulation of p62 by CNOT2 was detected in MEF WT cells as expected, but not in ATG5 KO MEF cells ( Figure 4D).

DISCUSSION
In the current study, the critical role of CNOT2 was elucidated during autophagy. Here, CNOT2 depletion induced autophagic features of LC3B-II conversion, p62/SQSTM1 accumulation and increased number of puncta, implying the potential of autophagy induction. Nevertheless, autophagic flux was inhibited in CNOT2depleted H1299 cells cotransfected with GFP-mRFP-LC3 construct in CNOT2-depleted H1299 cells. Consistently, TEM observation revealed some vacuoles with more autophagosomes and less autophagolysosomes, indicating www.impactjournals.com/oncotarget

Figure 3: CNOT2 correlates with p62/SQSTM1 in HEK293 QBI cells by co-immunoprecipitation and immunofluorescence.
(A) HEK293 QBI cells were transiently transfected with Flag-p62 (4 μM) and/or HA-CNOT2 (4 μM). The transfected cells were treated with MG132 (10 μM) for 4 h before harvest and cell lysates were immunoprecipitated with HA-CNOT2 or Flag-p62 antibody and subjected to Western blotting. (B) HEK293 QBI cells were transiently transfected with HA-CNOT2 (4 μM) or Flag-p62 and exposed to MG132 (10 μM) for 4 h before harvest. Then cell lysates were immunoprecipiated with HA or Flag antibody and immunoblotted with antibodies of HA, Flag, p62, CNOT2 and actin. (C) HEK293 QBI cells were transiently transfected with HA-CNOT2 and/or Flag-p62 WT and Flag-p62 mutant S351 plasmids and treated with 10 μM MG132 for 4 h before cell harvest. Then cell lysates were immunoprecipitated with Flag-p62 antibody and subjected to Western blotting with antibodies of Flag and HA or Actin. (D) Immunoprecipitation of endogeneous CNOT2 with p62/SQSTM1 antibody. H1299 cells were treated 10 μM of MG132 for 4 h, cell lysates were precleaned with protein G/A beads and subsequently incubated for 1-2 h with protein G/A beads covalently coupled with anti-IgG and anti-p62/SQSTM1 antibody. (E) CNOT2 was colocalized with p62/SQSTM1. H1299 cells were transiently transfected with Flag-p62 (4 μg) and HA-CNOT2 (4 μg) plasmids (lower panel), followed by immunocytochemistry. The cells were stained with anti-p62 (green) and anti-CNOT2 (red). Bars, 10 μm. the possibility of authphagic cell death via impaired autophagy in CNOT-depleted H1299 cells.
On the contrary, CNOT2 overexpression induced ubiquitination of p62/SQSTM1 in HEK293 QBI cells, demonstrating that CNOT2 induces degradation of p62/ SQSTM1. Furthermore, the regulation of p62/SQSTM1 by CNOT2-mediated ubiquitination was blocked by an autophagy inhibitor 3-MA, but not by proteosome system, given that proteins are generally degraded mainly via the proteasome system for short lived proteins and macroautophagy for long-lived proteins [38].
Of note, p62 was clearly immunoprecipitated by CNOT2 in HEK293 QBI cells transfected with HA-CNOT2 and Flag-p62, implying the direct interactions between CNOT2 and p62/SQSTM1. Also, downregulation of p62/ SQSTM1 by CNOT2 was not significantly enhanced by CHX treatment, implying protein synthesis is not critically involved in downregulation of p62/SQSTM1 by CNOT2. Interestingly, CNOT2 was still detected by Western blotting of S351E mutant p62/SQSTM1 immunoprecipitates almost similar to wild-type of p62/ SQSTM1 in HEK293 QBI cells transfected with S351E mutant p62/SQSTM1 plasmid, which has higher affinity to endogenous Keap1 rather than wild-type p62/SQSTM1 or the phosphorylation-defective S351A mutant p62/ SQSTM1, implying that phosphorylation of p62/SQSTM1 is not critically involved in CNOT2 and p62/SQSTM1 complex formation regardless of Keap1 mediated pathway.
Notably, the expression of ATG5 was upregulated at mRNA and protein levels in CNOT2-depleted H1299 cells. Furthermore, ectopic expression of CNOT2 and ATG5 enhanced p62/SQSTM1 degradation in a dose-dependent The prediction scores for PPI between p62/SQSTM1, ATG5 and LC3B regulated by CNOT2. The scores were represented by STRING using KEGG datasets, containing experimentally verified and putative PPIs. -, known interactions experimentally determined; -, Text Mining; --, suggested interaction in this study [37]. www.impactjournals.com/oncotarget manner, demonstrating that p62/SQSTM1 degradation by CNOT2 is mediated by ATG5 for autophagosome formation. Consistently, degradation of p62/SQSTM1 by CNOT2 was detected in ATG5 +/+ MEF cells, but not in ATG5 -/-MEF cells, implying the potential role of ATG5 in CNOT2 induced degradation of p62/SQSTM1.
It is well documented that several apoptotic proteins such as PUMA, Bax, XIAP, and Bim regulate autophagy and also autophagic proteins for nucleation and elongation modulate apoptosis through caspase and calpain-mediated cleavage of ATGs, implying crosstalk between apoptosis and autophagy. Likewise, accumulation of p62/SQSTM1 is critically involved in determination of the fate of cell through the caspase dependent apoptotic pathway [2,9,16]. Herein, sub G 1 population was efficiently increased in CNOT2-depleted H1299 cells by late stage autophagy inhibitors such as NH 4 Cl and CQ compared to an early stage autophagy inhibitor 3-MA in CNOT2-depleted H1299 cells, indicating depletion of CNOT2 induces apoptotic cell death via impaired autophagy with increased autophagosomes.
Collectively, CNOT2 depletion increased p62/ SQSTM1 accumulation, LC3B-II conversion and the number of puncta, but impaired autophagic flux in H1299 cells, along with TEM observation of more autophagosomes and less autophagolysosomes. ATG5 played a pivotal role in CNOT2 induced degradation of p62/SQSTM1 only in ATG5 +/+ MEF cells, but not in ATG5 -/-MEF cells, while PB1 domain of p62 is essential in CNOT2 induced degradation of p62/SQSTM1. Furthermore, apoptotic cell death was more efficiently exhibited in CNOT2-depleted H1299 cells by NH 4 Cl and CQ compared to 3-MA in CNOT2-depleted H1299 cells. Overall, these findings provide novel insight into the critical role of CNOT2 as a negative autophagy regulator in ATG5 dependent pathway.

Plasmids
The cDNA encoding C-terminal 440 amino acids of wild-type and S351E (a phosphorylation mimic) p62/SQSTM1 were subcloned into the pcDNA vector (Invitrogen, Waltham, MA, USA) for protein expression, which tagged the protein with an Flag-tag at the C-terminal end. HA-CNOT2 was kindly provided by Dr. KS  in DMEM supplemented with 10 % FBS. HEK293 QBI cell line is a superior subclone of HEK293 QBI cells, harboring the E1A and E1B regions of the adenoviral genome, and complementing the deletion of the E1 region in the recombinant Adenovirus (Qbiogene, Livingstone, UK). All cells were maintained in the growing medium in a humidified 5 % CO 2 atmosphere at 37 °C. DNA transfection was performed using the X-tremeGENE HP DNA transfection reagents (Roche, Quebec, Canada).

Small interfering RNA (siRNA) transfection
H1299 cells were transfected with siRNA oligoribonucleotides targeted against human CNOT2 or a RNA interference negative control. Each well was incubated for 48 h with 1 nM of siRNA using INTERFERIN siRNA transfection reagent (409-50, Polyplustransfection Inc., NY, USA) according to the manufacturer's protocol. The cells were then washed off from the plates and transferred into serum-free medium for treatments.

Western blotting and co-immunoprecipitation (co-IP)
H1299 cells or HEK293 QBI cells were washed twice with cold PBS and harvested by scraping with a rubber policeman. The cells were pelleted by centrifugation at 4 °C and resuspended directly into a lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 % Triton X-100, 2 mM EDTA, 0.5 % IGEPAL CA-630, 10 mM NaF, 2 mM Na 3 VO 4 , and 0.01 % protease inhibitor cocktail). Cell lysates were subjected to SDS-PAGE and transferred to a nitrocellulose membrane. After blocking in 5 % skim milk and Tris-buffered saline with 0.1 % Tween-20 (TBST 10X), signals were detected and analyzed by a Kodak X-OMAT 2000 image analyzer. For immunoprecipitation experiment, cell lysates were precleared with protein A/G-agarose beads and subsequently incubated for an 1 -2 h with protein G/A beads covalently coupled with anti-HA, anti-Flag and anti-Myc antibodies. Immune complexes were washed four times with cell extraction buffer. Eluted samples or whole cellular lysates were resolved by SDS-PAGE and proteins were detected by Western blotting using the indicated antibodies. Then densitometric analysis was performed using ImageJ software.

Immunocytochemistry
For immunostaining, H1299 cells were grown on coverslips to 70 % confluency. Cells were fixed in 3.7 % formaldehyde in PBS for 15 min at room temperature and permeabilized with 0.2 % Triton X-100 (9002-93-1, Amresco, OH, USA) in phosphate-buffered saline (PBS) for 10 min and blocked with 1 % BSA (82-100-6, Millipore, MA, USA) in PBS for 1 h. The fixed cells were incubated for 2 h with anti-LC3 primary antibodies, and then washed in PBS and incubated with Alexa Fluor ® 488-conjugated anti-rabbit IgG antibody (A-11034, Invitrogen, CA, USA) for 1 h. The cells were then stained with 0.5 mg/ml of DAPI to visualize nuclei. Cells were washed in PBS, mounted on glass slides and observed with a LSM510 confocal laser microscope (Carl Zeiss, Heidelberg, Germany).

Autophagic flux assay
For autophagic flux assay, GFP-mRFP-LC3 construct was stably transfected into H1299 cells using the X-treme GENE HP DNA transfection reagent. The cells were washed twice in ice-cold PBS, fixed, mounted with Histological Mounting Medium (HistoMount™, HS-103, National diagnostics, Atlanta, USA) and observed with LSM510 confocal laser microscope (Carl Zeiss,, Heidelberg, Germany).

Ubiquitination assay
HEK293 QBI cells were transiently co-transfected with HA-CNOT2, Flag-p62 and Myc-ubiquitin plasmids in the presence or absence of 5 mM 3-MA for 24 h and 10 μM MG132 for 4 h before harvest. For analysis of ubiquitination of endogenous p62/SQSTM1, the cells were lysed with lysis buffer and immunoprecipitated with anti-Myc antibody. The ubiquitination reaction mixtures were collected by centrifugation and washed three times with lysis buffer. The samples were then subjected to SDS-PAGE followed by Western blotting. Ubiquitination of p62/SQSTM1 was analyzed by Western blotting with anti-p62/SQSTM1 antibody.

Flow cytometric analysis
For cell cycle analysis, H1299 cells were transfected with CNOT2 siRNA for 48 h and then treated with 3-MA, chloroquine (CQ, Sigma, C6628) and ammonium chloride (NH 4 Cl, Sigma, 12125-02-9) for another 24 h. The cells were fixed with 75 % ethanol at -20 °C , washed twice with PBS and resuspended in PBS containing RNase A (1 mg/ ml, Sigma, R6513) and incubated for 1 h at 37 °C . After incubation, the cells were stained with 500 μl of propidium iodide (50 mg/ml, Sigma, P4170) for 30 min at room temperature in the dark. The DNA contents of the stained cells were analyzed using CellQuest Software with the FACSCalibur flow cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Statistical analysis
The results were expressed as the means ± SD from at least three independent experiments. Statistical analyses were conducted by Student's t-test using SigmaPlot version 12 (Systat Software Inc., San Jose, CA, USA). P value < 0.05 was considered statistically significant.