The interaction between p62 and Keap1 and the domains that are required for the interaction.
In an attempt to identify Keap1-interacting proteins, several stable cell lines harboring Keap1-CBD were generated. Pulldown experiments were performed using the stable cell lines containing vector control (−) or Keap1-CBD (+) to identify Keap1-CBD-interacting proteins. One protein was significantly enriched in the Keap1-CBD stable cell lines, compared to the control cell line (Fig. , compare lanes 2 and 3 and lanes 5 and 6). Copurification of this protein with Keap1-CBD was observed with all cell lines tested (data not shown). Mass spectrometry analysis identified this protein as p62 (Fig. , **). The silver-stained gels derived from MDA-MB-231 cell lysate or Keap1-null MEF cell lysate are shown in Fig. . Next, interaction of endogenous p62 with endogenous Keap1 was confirmed using immunoprecipitation analysis. An antibody against Keap1 precipitated p62, and the IgG negative control did not (Fig. ).
FIG. 1. The interaction between p62 and Keap1 and the domains that are required for the interaction. (A) Identification of the Keap1-interacting protein p62. Two stable cell lines, MDA-MB-231 and Keap1−/−, that expressed either vector control (more ...)
To identify the domain in p62 that is required for association with Keap1, p62 deletion mutants were constructed in an expression vector containing a T7 promoter. The deletion mutant constructs used in the following experiments were p62-380, a mutant containing a deletion from amino acid 381 to the C terminus, and p62-300, a mutant containing a deletion from amino acid 301 to the C terminus. In vitro transcription and translation were performed to generate 35S-labeled full-length p62 (WT) and its deletion proteins. These proteins were then incubated with GST-Keap1, which was produced and purified from E. coli bacteria. Keap1-associated proteins were pulled down using glutathione beads. Equivalent amounts of p62 proteins were used in the GST pulldown assay (Fig. , bottom). Keap1 associated with p62-WT and p62-380; however, p62 did not associate with p62-300 (Fig. , top, compare lanes 3 and 4 to lane 5), indicating that the region of p62 containing amino acids 300 to 380 is important for the interaction. Nrf2 and luciferase (Luc) were included in this experiment as positive and negative controls, respectively (Fig. , lanes 1 and 2). After careful examination of the region of amino acids 300 to 380, a cluster of negatively charged amino acids was revealed (349-DPSTGE-354). This primary sequence resembles the ETGE motif of Nrf2 that is required for the interaction with the Kelch domain of Keap1. Thus, a p62 mutant (p62-M), in which all six amino acids (349-DPSTGE-354) were replaced with alanine residues, was constructed. Indeed, p62-M lost its binding with GST-Keap1 (Fig. , top, lane 6), demonstrating that 349-DPSTGE-354 in p62 is required for the interaction between p62 and Keap1. To further confirm this, immunoprecipitation analysis was performed using cell lysates of HEK293 cells cotransfected with an expression vector for either p62-WT or p62-M, along with an expression vector for Keap1. Expression of each protein was detected using antibodies against Keap1, myc (for p62 proteins), and β-actin (for loading control) (Fig. , bottom). Immunoprecipitation analysis indicates that Keap1 associated with p62-WT but not with p62-M (Fig. , top, compare lanes 2 and 3), demonstrating that 349-DPSTGE-354 is essential for the interaction between Keap1 and p62.
In a similar experiment, the domain in Keap1 that interacts with p62 was identified. Full-length Keap1 and its deletion proteins were generated and 35
S labeled using an in vitro
transcription and translation method. p62-WT was also generated by in vitro
transcription and translation but was not radiolabeled. Each of the 35
S-labeled Keap1 proteins were incubated with p62-WT followed by nickel affinity chromatography. Equal amount of Keap1 proteins were used for this pulldown assay (Fig. , bottom). Keap1-ΔK, a mutant with the Kelch domain deleted, completely lost association with p62 (Fig. , top, lane 6), indicating that the Kelch domain contains the p62-interacting residues. The other mutants, with deletions of the N terminus (ΔN), BTB domain (ΔB), linker domain (ΔL), and the C terminus (ΔC), were still able to be pulled down by p62 (Fig. , top, lanes 3 to 5 and 7). Three positively charged amino acids (R380, R415, and R483) in the Kelch domain of Keap1 are necessary for interacting with Nrf2 (18
). Therefore, it is highly possible that the same amino acids may be needed for the p62-Keap1 interaction. Thus, a Keap1 mutant (Keap1-M), in which the three arginine residues at positions 380, 415, and 483 were replaced with alanine residues, was constructed. Interaction of Keap1 or Keap1-M with p62-WT was tested in HEK293 cells using immunoprecipitation analysis. Keap1, and not Keap1-M, associated with p62 (Fig. ). Taken together, these results clearly demonstrate that there is a direct interaction between p62 and Keap1 and that the amino acids 349-DPSTGE-354 in p62 and the three arginine residues (at positions 380, 415, and 483) in Keap1 are essential for the interaction of these two proteins.
p62 upregulated the Nrf2 signaling pathway.
Because the same three arginine residues in Keap1 are required for interaction with either Nrf2 or p62, it is likely that p62 competes with Nrf2 for binding to Keap1. Next, the functional role of p62 in regulating Nrf2 and its antioxidant response was tested. Relative firefly luciferase activity in HEK293 cells transfected with an expression vector for either p62-WT or p62-M was measured, along with vectors for ARE-firefly luciferase and Renilla luciferase reporter genes driven by the NQO1 and TK promoters, respectively. p62-WT, but not p62-M, enhanced the ARE-mediated luciferase activity in a dose-dependent manner (Fig. ). tBHQ and SF, known inducers of Nrf2, were used as positive controls (Fig. ). To confirm that p62 is able to activate the Nrf2-mediated antioxidant response, qRT-PCR was used to measure the transcription of several Nrf2 target genes. In accordance with the results from the reporter gene assay, p62-WT, but not p62-M, increased mRNA expression of Nrf2 target genes, such as HO-1, NQO1, GCLM, and MRP2, without significantly affecting the transcription of Nrf2 or Keap1 (Fig. ). These results indicate that p62 most likely activates Nrf2 target genes through upregulation of Nrf2 at the protein level. Next, immunoblot analysis was performed in HEK293 cells transfected with an expression vector for vector alone, p62-WT, or p62-M. Ectopic expression of p62-WT significantly enhanced the levels of Nrf2 and NQO1, whereas p62-M did not (Fig. , compare lanes 1 and 2 and lanes 1 and 3). Collectively, these results demonstrate that overexpression of p62 is able to upregulate the Nrf2 signaling pathway by enhancing the protein level of Nrf2.
FIG. 2. p62 upregulated the Nrf2 signaling pathway. (A) p62 regulates the transcriptional activity of Nrf2. Different amounts of the expression plasmids of p62-WT or p62-M were transfected into HEK293 cells along with the NQO1-ARE promoter firefly luciferase (more ...) p62 sequestered Keap1 into aggregates.
Subcellular localization of p62, Keap1, Cul3, and Nrf2 and p62-positive aggregate formation were investigated with live cells. p62, Keap1, Cul3, and Nrf2 were individually tagged with CFP, YFP, or RFP. To ensure that the function of these proteins was not affected by the addition of the fluorescent tag, all of the constructs were first tested for their expression and regulation. As expected, all constructs had high expression levels and normal function (data not shown). These proteins were then expressed singly or in combination in HEK293 cells, and cellular localization of these proteins was visualized by real-time live imaging under a fluorescent microscope. As shown in Fig. , overexpression of p62-CFP or p62M-CFP alone formed aggregates in the cytoplasm, which are typically characterized by punctate dots (Fig. , first and second panels from top). Ectopically expressed Keap1-YFP was localized predominantly in the cytoplasm (Fig. , third panel from top), while Nrf2-YFP and Nrf2-RFP were present in both compartments and the majority of the cells had more nuclear localization (Fig. , fourth and fifth panels from top). Similarly to Keap1-YFP, Cul3-RFP was localized mainly in the cytoplasm (Fig. , bottom panel).
FIG. 3. p62 sequestered Keap1 into aggregates. (A) The cellular localization of p62, Keap1, Nrf2, and Cul3. HEK293 cells were singly transfected with an expression vector for the fluorescently tagged protein. The subcellular localization of the proteins was monitored (more ...)
We have previously reported that Keap1 functions as the substrate adaptor protein that brings Nrf2 into the core Cul3-Rbx1 E3 ubiquitin ligase complex for ubiquitination (29
). The fact that p62 interacts directly with Keap1 led us to examine the colocalization of p62 with Keap1, Cul3, or Nrf2 (Fig. ). Coexpression of Keap1-YFP with p62-CFP caused aggregation of both proteins (Fig. , first row from top). In contrast, p62M-CFP lost its ability to recruit Keap1 into aggregates (Fig. , second row from top). Neither p62-CFP nor p62M-CFP was able to recruit Nrf2 into aggregates (Fig. , third and fourth rows from top). Similarly, Cul3 was not recruited into aggregates as well (Fig. , fifth and sixth rows from top).
Next, HEK293 cells were simultaneously transfected with three different proteins to test localization of the indicated proteins (Fig. ). In contrast to the diffused whole-cell localization of Nrf2 seen when p62-CFP and Nrf2-RFP were expressed in the absence of Keap1 (Fig. , third row from top), overexpression of both p62 and Keap1 was able to bring a certain amount of Nrf2 into aggregates (Fig. , first row from top). Nonetheless, Nrf2 was still localized diffusely in the entire cell (Fig. , first row from top). This can be attributed to Nrf2 having the ability to bind to a Kelch domain that is not occupied by p62 in a Keap1 homodimer. As expected, Nrf2 was not detected in the aggregates when p62M-CFP was transfected in combination with Nrf2 and Keap1 (Fig. , second row from top). Similarly to Nrf2, some of Cul3 was colocalized into aggregates when Keap1 along with p62-CFP, but not p62M-CFP, was coexpressed (Fig. , compare third and fourth rows from top). Cul3 still localized diffusely in the cytoplasm; however, Cul3 also colocalized with Keap1 and p62 aggregates (Fig. , third row from top). This was not observed with cells with Keap1, p62M, and Cul3 (Fig. , fourth row from top). Lastly, when p62-CFP or p62M-CFP, along with Nrf2-YFP and Cul3-RFP, was coexpressed in the absence of Keap1, neither p62 nor p62M was able to recruit Nrf2 or Cul3 into aggregates (Fig. , fifth and sixth rows from top). Collectively, these results indicate the necessity of Keap1 in p62-dependent Nrf2 and Cul3 aggregation.
p62 decreased ubiquitination of Nrf2, leading to an increase in Nrf2 stability.
To test the possibility that p62 upregulates the Nrf2 signaling pathway by decreasing the active E3 ubiquitin ligase for Nrf2, ubiquitination of Nrf2 and Keap1 in the presence of p62-WT or p62-M was tested using an in vivo ubiquitination assay. Indeed, overexpression of p62-WT significantly decreased ubiquitination of Nrf2, while it enhanced ubiquitination of Keap1 (Fig. , lanes 3 and 7). However, overexpression of p62-M had no effect on ubiquitination of either Nrf2 or Keap1 (Fig. , lanes 4 and 8). We demonstrate, in combination with the imagining data presented in Fig. , that p62 is able to sequester Cul3 into aggregates through Keap1, which may explain the increase in Keap1 autoubiquitination. Next, the half-life of Nrf2 was determined in the presence of p62-WT or p62-M by using CHX and immunoblot analysis. The half-life of Nrf2 was 14.43 or 9.93 min in the presence of p62-WT or p62-M, respectively (Fig. ). Thus, taken together, these results suggest that overexpressed p62 is able to sequester the Keap1-Cul3-E3 ubiquitin ligase complexes into aggregates, which prevents Nrf2 from being ubiquitinated. Therefore, under p62-overexpressed conditions, Nrf2 is stabilized and the Nrf2-mediated antioxidant response is activated.
FIG. 4. p62 decreased ubiquitination of Nrf2, leading to an increase in Nrf2 stability. (A) p62 decreased the ubiquitination of Nrf2 and increased the ubiquitination of Keap1. HEK293 cells were cotransfected with an expression vector for Nrf2, Keap1, HA-ubiquitin, (more ...)
Next, Keap1-Cul3-Rbx1 complex formation in the presence of p62-WT or p62-M was compared. Cells were transfected with CBD-Keap1, HA-Cul3, and myc-Rbx1 along with either p62-WT or p62-M. Keap1-containing complexes were pulled down using chitin beads and subjected to immunoblot analysis (Fig. ). p62-WT increased the association of Keap1, Cul3, and Rbx1 compared to the lane with no p62 or p62-M (Fig. , compare Rbx1 and Cul3 rows of lane 3 to those of lanes 2 and 4). As a negative control, no Keap1-CBD was transfected (Fig. , lane 1), and as a positive control, pulldown of exogenous Nrf2 was done (Fig. , lane 5). Collectively, these results not only recapitulate the interaction between p62 and Keap1 but also suggest that p62 enhances the association of Keap1 with Cul3 and Rbx1, which offers a possible explanation for the shift in ubiquitination from Nrf2 to Keap1.
Autophagy-defective cells sequestered Keap1 into aggregates.
To further confirm that the Nrf2 pathway is upregulated during deregulation of autophagy, we used primary immortalized baby mouse kidney (iBMK) cells to monitor the localization of Keap1. Localization of Keap1 was conducted in both autophagy-competent (Beclin1+/+ and Atg5+/+) and autophagy-deficient (Beclin1+/− and Atg5−/−) iBMK cells either stably expressing GFP alone (control) or GFP-tagged p62. In one set of experiments, the iBMK cell lines were transiently transfected with Keap1-CFP. Localization of the proteins was visualized in live cells using a fluorescent microscope. Both the cytoplasm and nucleus were uniformly green in the GFP control for all four cell lines (Beclin1+/+, Beclin1+/−, Atg5+/+, and Atg5−/−), with Keap1-CFP remaining evenly distributed in the cytoplasm (Fig. , first and third rows from top). The localization of p62-GFP and Keap1-CFP in the Beclin1+/+ and Atg5+/+ cells was also primarily in the cytoplasm, with a few small protein aggregates (Fig. , second rows from top). In the autophagy-deficient cell lines, Beclin1+/− and Atg5−/−, p62-GFP formed large aggregates, with which Keap1 completely colocalized (Fig. , fourth rows from top). Additionally, indirect immunofluorescence using antibodies against Keap1 and GFP demonstrated that endogenous Keap1 indeed colocalized with the p62-GFP aggregates in the autophagy-deficient iBMK cells (Fig. , fourth rows from top), confirming our observations of iBMK cells ectopically expressing CFP-tagged Keap1 (Fig. , fourth rows from top). As expected, in Atg5+/+ and Beclin1+/+ cells, p62-GFP and endogenous Keap1 localized mainly in the cytoplasm (Fig. , first and third rows from top). With the control cells of Atg5+/+, Atg5−/−, Beclin1+/+, and Beclin1+/− stably expressing GFP, whole-cell GFP staining was observed, while endogenous Keap1 was localized primarily in the cytoplasm (Fig. , second rows from top). Collectively, these results indicate that autophagy deficiency activates the Nrf2 pathway through recruitment of Keap1 into aggregates due to the excessive accumulation of p62.
FIG. 5. Autophagy-defective cells sequestered Keap1 into aggregates. (A and B) Keap1 was sequestered into aggregates in primary autophagy-deficient cells. Atg5+/+, Atg5−/−, Beclin1+/+, and Beclin+/− (more ...)