Formation of Nrf2-Cul3 complexes depends upon Keap1.
To begin to address the role of Cul3 in Nrf2 protein accumulation, we determined whether Nrf2 was present in Cul3 complexes in vivo. Lysates prepared from 293T cells transfected with vectors encoding Flag-Cul3 and HA-Nrf2 in the absence or presence of Myc-Keap1 were precipitated with the M2 monoclonal antibody. In the absence of ectopically expressed Keap1, Nrf2-Cul3 association was evident only when cells had been treated with the proteasome inhibitor MG132 (Fig. , compare lanes 3 and 4). In cells expressing Cul3, Nrf2, and Keap1, a weak interaction was detected between Cul3 and Nrf2 (Fig. , lane 5). Both Nrf2 accumulation and association with Cul3 were restored by treatment of cells with MG132 (Fig. , lane 6). Coprecipitation of Myc-Keap1 was also detected in these experiments (data not shown). These data indicate that Nrf2 associates with Cul3 in cells and suggested that coexpression of Cul3 and Keap1 might specifically trigger Nrf2 proteolysis (see Fig. to ). We also tested the ability of Nrf2 to bind to a Keap1-Cul3 complex in vitro. Nrf2, in vitro transcribed and translated in the presence of [35S]methionine, was mixed with a purified Keap1-Cul3 complex or with purified Cul3 alone. While Nrf2 was able to efficiently bind the Keap1-Cul3 complex, binding was not observed between Nrf2 and Cul3 in the absence of Keap1 (Fig. ).
FIG. 1. Nrf2 associates with a Cul3 complex in a Keap1-dependent manner. (a) 293T cells were transfected with plasmids encoding HA-Nrf2, Flag-Cul3, and Myc-Keap1 and treated as indicated. Flag-Cul3 was precipitated from whole-cell extracts (WCE) with the M2 monoclonal (more ...)
FIG. 3. Inactivation of Cul3 stabilizes Nrf2. (a) 293T cells were transfected with increasing concentrations of plasmids encoding Myc-Keap1 (lanes 1 to 5) or Flag-Cul3 (lanes 6 to 10). The expression of Nrf2, Myc-Keap1, and Flag-Cul3 was assessed via immunoblot. (more ...)
FIG. 7. In vitro reconstitution of Nrf2 ubiquitination. (a) In vitro-transcribed and -translated Nrf2 was mixed with ATP, ubiquitin, Ubc5, and in vitro-transcribed and -translated Keap1 in in vitro ubiquitination assays for 1 h at 30oC. In lanes 7 to 10, the (more ...)
The in vitro binding data are consistent with Keap1's functioning as a bridge between Nrf2 and Cul3. However, the in vivo association of Nrf2 with Cul3 in the absence of ectopic Keap1 suggested either that Nrf2 can bind directly to Cul3 in cells or that association was mediated by endogenous Keap1. To address this issue, we knocked down endogenous Keap1 levels through the use of short hairpin RNAs. As suitable antibodies for detection of endogenous Keap1 are not available, we confirmed Keap1 knockdown by reverse transcription-PCR (Fig. ). In cells transfected with short hairpin RNA directed towards Keap1, we observed a marked decrease in the Nrf2-Cul3 association (Fig. , compare lanes 2 to 4) that was comparable to the degree to which Keap1 was knocked down. Together, these results indicate that the interaction between Cul3 and Nrf2 is dependent upon the Keap1 protein and suggest that, following stress-dependent liberation of Nrf2 from Keap1, Nrf2 does not bind Cul3.
As the above interactions were assessed with ectopic protein, we were compelled to determine whether endogenous Nrf2 was associated with Cul3 complexes. Cul3 was precipitated from 293T cells with a Cul3-specific antiserum, and the presence of associated Nrf2 was assessed via immunoblot analysis with an Nrf2-specific antiserum. Nrf2 was detected in Cul3 precipitates in asynchronously proliferating cells (Fig. , lane 2); increased association was apparent in cells treated with MG132 (lane 3).
We and others have previously shown that Nrf2 dissociates from Keap1 following endoplasmic reticulum stress or oxidative stress (6
). If Nrf2-Cul3 association depends upon Keap1 as an adaptor, then endoplasmic reticulum stress or oxidative stress should reduce the abundance of the Nrf2-Cul3 complex. We therefore determined whether endogenous Nrf2-Cul3 binding is regulated by cellular stress. Cul3 was precipitated from 293T cells treated with tunicamycin, a drug that elicits the unfolded-protein response and promotes Nrf2 nuclear localization (6
), with a Cul3-specific antiserum, and the presence of associated Nrf2 was assessed via immunoblot analysis. In contrast to vehicle-treated cells, Cul3-Nrf2 association was decreased in cells treated with tunicamycin (5 μg/ml) (Fig. , compare lanes 2 and 4).
The data provided thus far demonstrate that Nrf2 associates with Cul3 in a Keap1-dependent manner and that endoplasmic reticulum stress induces the dissociation of Nrf2 from the Cul3 complex. Based on previous work, we reasoned that this dissociation reflects the disruption of Nrf2-Keap1 complexes rather than loss of Keap1-Cul3 interaction. Consistent with this notion, Cul3 remained bound to Keap1 throughout a course of tunicamycin treatment (Fig. , compare lane 3 to 4). Thus, while endoplasmic reticulum stress triggers Nrf2 release from Keap1, it does not abolish Cul3-Keap1 association, suggesting that Keap1 is constitutively associated with Cul3.
FIG. 2. Cul3 interacts with Keap1 through its BTB domain. (a) 293T cells transfected with plasmids encoding Myc-Keap1 and Flag-Cul3 were either left untreated (lanes 1 to 3) or treated with 5 μg/ of tunicamycin per ml for 1 h (lane 4). Flag-Cul3 was precipitated (more ...)
Because Cul3 binds directly to BTB domains, we hypothesized that Keap1 should bind to Cul3 via its N-terminal BTB domain. To address this notion, 293T cells were transfected with plasmids encoding Flag-Cul3 and Myc-Keap1 or a Keap1 mutant which lacks the BTB domain. Lysates were subjected to precipitation with a Flag-specific antibody, and coprecipitating proteins were visualized by immunoblot with epitope-specific antibodies. As anticipated, Keap1 was detected in the Cul3 precipitates (Fig. , lane 3). In contrast, Keap1ΔBTB did not coprecipitate with Cul3 (Fig. , lane 4). These data demonstrate that Cul3-Keap1 association is dependent upon the Keap1 BTB domain. Consistent with the binding data, immunofluorescent staining revealed colocalization of Keap1 and Cul3 in the cytoplasm of asynchronously proliferating cells (Fig. ). These data suggest that Keap1 and Cul3 form cytoplasmic complexes.
Cul3 regulates Nrf2 degradation.
Our data provide evidence for protein complexes, which are minimally composed of Nrf2-Keap1-Cul3, consistent with the possibility that Cul3-Keap1 could direct Nrf2 ubiquitin-dependent proteolysis. We reasoned that if Cul3 and Keap1 regulated Nrf2 proteolysis, overexpression of Cul3 or Keap1 should result in reduced Nrf2 protein levels. We transfected 293T cells with increasing concentrations of plasmids encoding either Myc-Keap1 or Flag-Cul3 and assessed their effect on endogenous Nrf2 protein accumulation. With increasing levels of either Myc-Keap1 or Flag-Cul3, we noted a concomitant decrease in Nrf2 levels, as determined by immunoblot (Fig. ).
As an independent test of this hypothesis, we determined the ability of a dominant negative Cul3 mutant (Cul3N418), which is defective in Rbx1 binding but retains the capacity to associate with BTB domains (10
), to increase steady-state Nrf2 levels. 293T cells were transfected with a plasmid encoding Cul3N418, and endogenous Nrf2 protein levels were assessed by immunoblot. Cells expressing Cul3N418 contained elevated Nrf2 levels compared to mock-transfected cells (Fig. , compare lanes 1 and 3). We also used vectors encoding short hairpin RNAs to reduce endogenous Cul3 levels. 293T cells expressing the Cul3 short hairpin RNA contained significantly reduced Cul3 levels (Fig. , compare lanes 1 to 4). As a consequence of Cul3 knockdown, Nrf2 levels were markedly higher in these cells than in either mock-transfected cells or cells expressing short hairpin RNA against firefly luciferase (compare lanes 1 to 4). These results demonstrate that a reduction in Cul3 function contributes to increased Nrf2 protein accumulation.
We next measured the half-life of Nrf2 in cells expressing Cul3N418 and short hairpin RNA directed towards Cul3. Pulse-chase analysis revealed that Nrf2 is rapidly turned over in control transfected cells (Fig. , lanes 2 to 6; 3e, lanes 2 to 5), in agreement with previous data (20
). However, expression of Cul3N418 dramatically stabilized Nrf2, as little turnover was evident during the course of the experiment (Fig. , lanes 7 to 11). Likewise, the Nrf2 half-life was significantly extended in cells expressing short hairpin RNA directed towards Cul3 (Fig. , lanes 6 to 9). These data suggest that Cul3 and Keap1 are required for rapid Nrf2 proteolysis.
If Keap1 functions as the adaptor that bridges Nrf2 and Cul3, then loss of Keap1 should promote Nrf2 accumulation. Cells were transfected with a vector encoding two independent Keap1-specific short hairpin RNAs. As predicted, in cells expressing short hairpin RNAs specific for Keap1, basal Nrf2 protein accumulated relative to the level in those expressing control short hairpin RNA (Fig. , compare lanes 2 to 4).
Cul3-dependent proteolysis limits Nrf2 transcriptional activity.
If Cul3-dependent proteolysis limits the threshold of Nrf2 accumulation in the absence of stress, loss of Cul3 should result in promiscuous Nrf2 activation and increased basal expression of Nrf2 target genes. To address the relative contribution of Cul3-dependent degradation of Nrf2 versus Keap1-dependent cytoplasmic sequestration, we assessed Nrf2 function in cells in which either Keap1 or Cul3 was knocked down by assessing expression of a luciferase reporter plasmid containing an Nrf2-responsive element antioxidant response element (ARE) (6
). As expected, Keap1 knockdown dramatically increased Nrf2-dependent reporter activity, over 20-fold above that of vector-transfected cells (Fig. ), indicating that, in accordance with published results (25
), Keap1 is necessary for basal repression of Nrf2 activity. In addition, Cul3 knockdown increased expression of the Nrf2 reporter plasmid approximately fourfold (Fig. ). It is important to note that reporter gene expression in these assays is measured without treating cells with agents known to trigger either endoplasmic reticulum stress or oxidative stress. The difference in reporter gene expression observed between the Keap1 and Cul3 knockdowns likely reflects the capacity of Keap1 to maintain Nrf2 in the cytoplasm under conditions of Cul3 knockdown and therefore prevent nuclear access to a majority of the accumulating Nrf2.
FIG. 4. Cul3 contributes to the regulation of Nrf2-dependent gene expression. (a) 293T cells were transfected with the 4×ARE firefly luciferase reporter and a plasmid encoding Renilla luciferase in the absence or presence of plasmids expressing short (more ...)
Increased expression of an Nrf2 target gene in cells in which Cul3 is knocked down suggests that, in the absence of continued proteolysis, Nrf2 levels exceed those of Keap1, thereby permitting the accumulation of Keap1-free Nrf2. Implicit to this model is that a significant proportion of Nrf2 remains bound to Keap1 in inactive complexes. If so, cellular stress that liberates Nrf2 from Keap1 in the Cul3 knockdown cells should result in a synergistic increase in Nrf2-dependent gene expression. To test this possibility, cells were mock transfected or transfected with a Cul3-specific short hairpin RNA vector along with an Nrf2-dependent luciferase reporter plasmid. These cells were treated with vehicle control or the oxidative stress-inducing agent tert-butylhydroquinone (tBHQ; 100 μM). As above, knockdown of Cul3 resulted in a reproducible fourfold increase in basal ARE reporter activity (Fig. , compare panels 1 and 3). Addition of tBHQ resulted in a greater than 30-fold induction of reporter activity (Fig. , panel 4). Consistent with our hypothesis, we noted that tBHQ-dependent ARE reporter induction in the presence of Cul3 knockdown exceeded that achieved by tBHQ treatment alone (Fig. , compare panels 2 and 4).
Loss of the endoplasmic reticulum stress-inducible PERK kinase greatly sensitizes cells to the proapoptotic effects of the glycosylation inhibitor tunicamycin (12
). We have previously shown that overexpression of Nrf2, a downstream target of PERK, restores cellular redox balance in PERK−/−
murine embryonic fibroblasts (MEFs) challenged with tunicamycin, consistent with Nrf2's functioning as a mediator of PERK-dependent survival (5
). As independent confirmation that loss of Cul3 function permits promiscuous Nrf2 activation, we assessed whether overexpression of the dominant negative Cul3N418 mutant would decrease the sensitivity of PERK-deficient MEFs to endoplasmic reticulum stress-induced cell death. PERK−/−
MEFs were transfected with empty vector or vectors encoding Cul3N418 or, as a control, Nrf2. Cells were then treated with 2.5 μg of tunicamycin per ml, and cell death was assessed by propidium iodide exclusion (Fig. ). Tunicamycin treatment rapidly induced cell death in PERK−/−
MEFs relative to that observed for wild-type MEFs. As predicted, ectopic expression of Nrf2 reduced the early onset of cell death noted for PERK−/−
MEFs, as did expression of Cul3N418. Taken together, these data demonstrate that Cul3 and Keap1 mediate Nrf2 protein stability and activity and together oppose Nrf2-dependent, ARE-dependent gene expression.
Endoplasmic reticulum stress-dependent Nrf2-Keap1 dissociation reduces Nrf2 proteolysis.
Endoplasmic reticulum stress or oxidative stress is predicted to promote increased Nrf2 accumulation, given that it reduces Nrf2-Cul3 binding (Fig. ). We assessed Nrf2 levels in 293T cells that were either left untreated or treated with tunicamycin (5 μg/ml) or tBHQ (100 μM). Increased levels of Nrf2 were observed in cells that had been treated with tunicamycin or tBHQ (Fig. , compare lanes 1 to 3). Longer exposures were required for detection of Nrf2 in untreated cells (data not shown). These results are in agreement with previous data that demonstrated PERK-dependent increases in Nrf2 levels in response to glucose deprivation (5
) and oxidative stress (18
FIG. 5. Endoplasmic reticulum or oxidative stress stabilizes Nrf2. (a) 293T cells were mock treated (lane 1), treated with 100 μM tBHQ for 8 h (lane 2), or treated with 5 μg of tunicamycin (Tun) (lane 3) per ml for 1 h. Total Nrf2 levels were (more ...)
While PERK activity enhances Nrf2 protein accumulation, the previous experiment did not address whether Nrf2 is more stable following the cellular stress. To address this issue, NIH 3T3 cells were left untreated or treated with tunicamycin for 30 min. Following induction of the endoplasmic reticulum stress response, cells were exposed to cycloheximide for different intervals and Nrf2 loss was assessed by immunoblot with an Nrf2-specific antiserum. As expected, the half-life of Nrf2 was shorter in untreated cells than in tunicamycin-treated cells (Fig. , compare lanes 2 to 5 and 6 to 9). Together, these results suggest that Nrf2 stability increases following endoplasmic reticulum or oxidative stress via the dissolution of Cul3-Keap1-Nrf2 complex formation.
Cul3 promotes Nrf2 ubiquitination.
Our data demonstrate that Cul3 mediates Nrf2 protein stability under homeostatic conditions. As Cul3 is known to regulate protein degradation via its capacity to direct polyubiquitination, we hypothesized that Cul3-Keap1 complexes likely direct Nrf2 polyubiquitination. In agreement with previous data, we found that treatment of cells with proteasome inhibitors led to Nrf2 accumulation as well as the accumulation of higher-molecular-weight forms of Nrf2, consistent with ubiquitin conjugation (Fig. , compare lanes 2 and 3).
FIG. 6. Cul3 promotes Nrf2 polyubiquitination. (a) Lysates were collected from 293T cells that were mock treated (lane 2) or treated with 10 μM MG132 (lane 3) for 2 h. Nrf2 was detected via immunoprecipitation followed by immunoblot analysis. Lane 1 shows (more ...)
To more directly assess whether Nrf2 is subject to polyubiquitination and to assess the importance of Cul3 in this process, we performed in vivo ubiquitination assays. 293T cells were transfected with combinations of plasmids encoding 6× His-ubiquitin, Nrf2, Myc-Keap1, Flag-Cul3, and Cul3N418. Ubiquitin conjugates were purified from the lysates via Ni2+ affinity chromatography under denaturing conditions, and the presence of ubiquitin-conjugated Nrf2 was assessed via immunoblot analysis with Nrf2 antiserum (Fig. , top panel). Expression of the indicated proteins was confirmed in whole-cell lysates (bottom panels). In the absence of ectopically expressed Cul3, little ubiquitinated Nrf2 was seen (Fig. , top panel, lanes 1 to 4), although detectable levels of Nrf2 ubiquitination were observed in the presence of ectopic Keap1 (Fig. , top panel, lane 4). Cul3 expression, in the absence or presence of ectopically expressed Keap1, greatly enhanced Nrf2 polyubiquitination (Fig. , lanes 5 and 6). As expected, expression of Cul3N418, which is defective for Rbx1 association, did not promote Nrf2 ubiquitination (Fig. , lane 7). We also noted decreased endogenous Nrf2 polyubiquitination in cells expressing short hairpin RNA against Cul3 (data not shown).
We next asked whether Cul3-Keap1 complexes could promote Nrf2 ubiquitination in vitro. Rbx1-Cul3-Keap1 complexes were assembled by coupled transcription and translation of Keap1 in reticulocyte extracts. These complexes were then mixed with recombinant Ubc5, ubiquitin, ATP, and [35S]methionine-labeled Nrf2. In the presence of Ubc5, high-molecular-weight forms of Nrf2 were readily apparent (Fig. , lanes 4 and 5). This alteration in Nrf2 mobility was strictly dependent upon Keap1-Nrf2 interactions, as incubation with empty reticulocyte lysates or with Keap1ΔKelch complexes, a mutant Keap1 that cannot bind Nrf2 (Fig. ), could not support Nrf2 ubiquitination (Fig. , lane 3). Importantly, Calicin, a BTB and kelch domain-containing protein that binds to Cul3 (data not shown) but not Nrf2 (Fig. ), could not promote Nrf2 ubiquitination (Fig. , lane 2). To assess the importance of Cul3 in Nrf2 ubiquitination, the reactions were carried out in the presence of lysates expressing Keap1ΔBTB, a mutant that can bind Nrf2 (Fig. ) but not Cul3 (Fig. ). Ubiquitination of Nrf2 was not supported in these reactions (Fig. , lane 4), indicating that direct association of Cul3 with Keap1 is necessary for Keap1-dependent Nrf2 ubiquitination. Taken together, our in vivo and in vitro data strongly indicate that Keap1 serves as a specificity factor for Cul3-dependent Nrf2 ubiquitination and degradation.