Identification of KEAP1 as a PALB2 binding partner.
In searching for potential new functions of PALB2, we carried out tandem affinity purification to identify its new interacting partners. A C-terminally FLAG-HA-double-tagged PALB2 (PALB2-FH) was purified from HeLa S3 suspension cells stably expressing the protein. As shown in B, known PALB2 binding partners BRCA2, BRCA1, RAD51, and MRG15 were all copurified and identified by mass spectrometry analysis. Interestingly, KEAP1 was found to be a component of the PALB2 complex. To confirm this interaction, we first tested if endogenous PALB2 could be coimmunoprecipitated (co-IPed) with a GFP-tagged KEAP1 protein transiently expressed in 293T cells. As shown in Fig. S1A in the supplemental material, both endogenous PALB2 and BRCA2 were readily co-IPed, although it was unclear if KEAP1 binds PALB2 or BRCA2. Next, we performed reciprocal co-IP of endogenous proteins in U2OS cells with two different affinity-purified PALB2 antibodies and one monoclonal KEAP1 antibody, and co-IP of the endogenous proteins was observed in both cases (C). Since PALB2 is a nuclear protein, we further tested if it indeed interacted with KEAP1 in the nucleus. When U2OS cells were fractionated into cytoplasmic and nuclear components, all PALB2 and approximately a quarter of KEAP1 were found in the nuclear extract (NE), and PALB2-KEAP1 complex formation was clearly detected in this fraction (see Fig. S1B).
Subsequently, we attempted to compare the stoichiometries of PALB2-KEAP1 and NRF2-KEAP1 bindings under different conditions. U2OS cells were treated with DMSO (control), hydrogen peroxide (H2O2), tert-butylhydroquinone (tBHQ; an oxidative stress inducer and NRF2 inducer), or the DNA-damaging agent camptothecin (CPT), and then endogenous PALB2 and NRF2 were IPed. As shown in D, the binding between KEAP1 and PALB2 was not affected by any of the treatments. In contrast, tBHQ treatment clearly enhanced KEAP1-NRF2 complex formation; hydrogen peroxide also elicited a slight increase of the binding, whereas CPT produced no effect. Interestingly, even after tBHQ induction, the amount of KEAP1 co-IPed by either of the two NRF2 antibodies was still much smaller than that in the PALB2 IP (compare lanes 17, 18, and 15 in D). Although we were unable to determine the amounts of NRF2 IPed by the two antibodies due to high background on the Western blot when probed with all available NRF2 antibodies, it appears possible that the stoichiometry of KEAP1-PALB2 binding may be higher than that of KEAP1-NRF2 binding. U2OS cells were mainly used in this study since these cells express relatively low levels of NRF2, which is greatly induced after KEAP1 depletion (see Fig. S1C in the supplemental material and see also A). Also, the expression and behavior of PALB2 as well as its key binding partners BRCA1 and BRCA2 in U2OS cells are well characterized.
To determine if PALB2 directly binds KEAP1, we generated in vitro-translated (IVTed), epitope-tagged full-length KEAP1 and PALB2 proteins as well as a series of truncated KEAP1 fragments (A and B). We then mixed each KEAP1 species with PALB2 and IPed the latter from the mixture with an anti-PALB2 antibody. As shown in B, full-length KEAP1 was efficiently co-IPed with PALB2 (lane 9) but none of the truncated KEAP1 proteins lacking the KC region was IPed (lanes 6 to 8), indicating that the binding between full-length KEAP1 and PALB2 is specific and direct. Subsequently, we asked if the KC domain is sufficient for PALB2 binding using a transient-transfection and IP-Western blotting experiment. As shown in C, the KC region was able to bind both PALB2 and BRCA2, suggesting that KEAP1 may bind PALB2 with the same structural element as that for NRF2.
Fig 2 PALB2 directly interacts with the KC domain of KEAP1. (A) Schematic of the KEAP1 constructs. (B) In vitro translations were performed to produce HA-tagged KEAP1 proteins (lanes 1 to 4) and FLAG-HA-PALB2 (lane 5). Subsequently, each of the KEAP1 species (more ...) Direct interaction between the ETGE motif of PALB2 and the kelch domain of KEAP1.
Extensive domain mapping was then carried out to identify the KEAP1 binding site on PALB2. First, deletion of the exon 4-encoded region (residues 71 to 561) was found to abolish the binding (B, lane 3 versus lanes 4 to 7). Then, the binding site was narrowed down to within the N-terminal 200 residues of the protein (B, lane 9). Finally, we generated and tested a series of 25-amino-acid (aa) deletions in the N terminus and found that residues 76 to 100 are essential (C, lane 4). Intriguingly, amino acid sequence alignment identified a 7-aa motif (LDEETGE) between residues 76 and 100 that is highly conserved across mammalian species, and this motif is identical to the ETGE motif of NRF2 that binds KEAP1 (D). Mutations of the critical T residue as well as several other residues within the NRF2 ETGE motif have been shown to abrogate NRF2-KEAP1 interaction, allowing NRF2 to escape KEAP1-mediated repression (12
). To test if PALB2 indeed binds KEAP1 with this motif, we generated a deletion mutant and a series of point mutants (E91R, T92E, and G93R). As shown in E, all mutants failed to form complexes with KEAP1. Therefore, we conclude that the ETGE motif of PALB2 directly interacts with the kelch domain of KEAP1 in a fashion similar to the NRF2(ETGE)-KEAP1 interaction (12
). As expected, none of the mutations affected the binding of PALB2 to BRCA1 or BRCA2 (F), indicating that the binding events are independent and separable.
Fig 3 PALB2 binds KEAP1 through a highly conserved ETGE motif. (A) Schematic of PALB2 constructs used in the domain mapping study. (B and C) PALB2 constructs were transiently expressed in 293T cells, proteins were IPed with anti-FLAG M2 agarose, and the precipitates (more ...) PALB2 overexpression promotes NRF2 nuclear accumulation and reduces ROS level.
Based on the above finding, we hypothesized that PALB2 may be able to compete with NRF2 for KEAP1 binding and therefore may “protect” NRF2 from KEAP1-mediated repression. To test this hypothesis, we cotransfected HA-tagged NRF2 and Myc-GFP-double-tagged PALB2 into 293T cells and examined their expression and localization. Since high-level overexpression of NRF2 may overwhelm cellular KEAP1 and lead to artifacts, we titrated the amount of HA-NRF2 plasmid used to a level where we could detect weak and mostly cytoplasmic staining of HA-NRF2 when it was cotransfected with Myc-GFP. The largely cytoplasmic localization of the HA-NRF2 observed here may be due to the relatively large amount of KEAP1 in 293T cells (see Fig. S1C in the supplemental material). Under such a condition, overexpression of wild-type (wt) PALB2 strongly increased the level of NRF2 and promoted its accumulation in the nucleus (A). In contrast, overexpression of the T92E mutant showed a much weaker effect, indicating that the effect of wt PALB2 is mediated by its KEAP1 binding.
We also generated T98G glioblastoma cell lines stably expressing PALB2 double tagged with FLAG and HA epitopes (B and C). For reasons so far unknown, T98G cells have a much higher NRF2 level than do U2OS and other cells surveyed in this study (see Fig. S1C in the supplemental material), with the only exception being the A549 lung cancer cell line, which has been shown to harbor a KEAP1 mutation that impairs NRF2 binding (24
). The endogenous NRF2 in T98G cells is localized in the nucleus and can be clearly observed using immunofluorescence (IF) (B). While the majority of these stable cells showed a low level of ectopic PALB2 expression, in some cells the tagged PALB2 was overexpressed (B). Interestingly, in all cells expressing ectopic PALB2 at high levels, NRF2 staining signals were clearly stronger than those in cells expressing ectopic PALB2 at low levels or cells harboring the vector alone (B). Consistently, the average abundance of (nuclear) NRF2 in cells expressing tagged PALB2 was also significantly higher than that in control cells (C). A similar observation was also made in HeLa S3 cells overexpressing the same tagged PALB2 (D). Additionally, we observed in PALB2-overexpressing cells a slight decrease of KEAP1 in the cytoplasm and a corresponding increase in the nucleus (C; see also Fig. S2B in the supplemental material), suggesting that PALB2 binding to KEAP1 may promote the nuclear retention of the latter to some degree. However, this effect appears to be small under the setting used and remains to be further investigated under different conditions.
Since a key function of NRF2 is to mitigate reactive oxygen species (ROS) in the cell, we asked if PALB2 overexpression would reduce ROS levels. When ROS levels of the above T98G cell lines were measured using the DCF (2′,7′-dichlorodihydrofluorescein) assay, it was undetectable in both lines, consistent with the high endogenous NRF2 abundance in these cells. In the stable HeLa S3 cell line, we observed a significant decrease in ROS level compared with that in cells harboring the vector (E). Note that PALB2 overexpression was observed in greater than 80% of the cells (see Fig. S2A in the supplemental material). We also generated U2OS stable cell lines, but ROS measurement would not be meaningful since only a minor percentage of cells (~10 to 15%) showed clear exogenous PALB2 expression even after repeated selections (data not shown).
To directly measure the transcriptional activation activity of endogenous NRF2 in PALB2-overexpressing cells, an ARE-LUC reporter was employed (39
). As shown in F, ectopic expression of wt PALB2 increased endogenous NRF2 activity by more than 2-fold, whereas the two KEAP1 binding mutants (T92E and G93R) failed to show any effect. Since PALB2 also directly interacts with BRCA1 and BRCA2, we asked whether these interactions affect its activity to bind KEAP1 and promote NRF2 nuclear localization and function. To this end, we generated PALB2-Y28A and -A1025R mutants, each of which abrogates its BRCA1 or BRCA2 interaction, respectively (G) (18
). Interestingly, both mutants were equally active compared with wt PALB2 (F). Like wt PALB2, these two mutants also increased NRF2 accumulation in the nucleus (see Fig. S3 in the supplemental material). These results clearly demonstrate that the basic function of PALB2 to promote NRF2 activity depends on its KEAP1 binding but not its BRCA1/2 interaction, although the possibility that BRCA1/2 may modulate this PALB2 function under certain conditions cannot be ruled out. Surprisingly, the A1025R mutant also impaired PALB2-BRCA1 binding under the setting used (G). However, this does not contradict the conclusion reached above.
PALB2 effectively competes with NRF2 for KEAP1 binding.
The question whether PALB2 is able to disrupt KEAP1-NRF2 interaction was directly addressed. Since the abundance of endogenous NRF2-KEAP1 complex is low and difficult to obtain by IP with available antibodies (D), we cotransfected HA-NRF2 and Myc-KEAP1-GFP to obtain the complex. When an increasing amount of wt PALB2-FH was added to such preformed HA-NRF2/Myc-KEAP1-GFP complex (in cotransfected cell lysate), a decrease of NRF2 in the Myc-KEAP1-GFP IP along with a steady increase of PALB2 was observed (A), indicating that PALB2 is able to compete with NRF2 for KEAP1 binding and disrupt preformed KEAP1-NRF2 complex. Again, the PALB2T92E mutant protein was unable to elicit any effect, validating the specificity of the competition. Note that lanes 3 to 5 in the PALB2 panel of A contain both endogenous and the tagged PALB2, whereas lanes 7 to 9 contain only endogenous PALB2 since the tagged T92E mutant is defective in KEAP1 binding.
To compare the affinities between NRF2-KEAP1 and PALB2-KEAP1 complex formations, in vitro competition assays were performed. The complexes were obtained by cotransfection followed by IP and washing. Then, increasing amounts of in vitro-translated (IVTed) PALB2 or NRF2 competitors were added to each respective target complex, and the amounts of KEAP1 released were analyzed. Due to the relatively large size of PALB2 (130 kDa), an N-terminal fragment encompassing residues 1 to 550 (PALB2T551) was used for IVT. This fragment has a size similar to those of NRF2 and KEAP1. As shown in B and C, the IVTed PALB2 was able to disrupt the NRF2-KEAP1 complex and released KEAP1 in a dose-dependent manner, and the IVTed NRF2 exhibited similar activity against the PALB2-KEAP1 complex. Furthermore, we also performed a triple IVT experiment in which various amounts of NRF2 and PALB2 were produced along with a fixed amount of KEAP1. As shown in D, when similar amounts of NRF2 and PALB2 were produced in the same reaction (lane 3), significantly more PALB2 was bound to KEAP1 than was NRF2. Taken together, these results indicate that the binding affinity between PALB2 and KEAP1 may be equal to or greater than that between NRF2 and KEAP1 and lend further support to our model that PALB2 promotes NRF2 activity by competitively preventing and/or disrupting KEAP1-NRF2 interaction.
Depletion of PALB2 reduces NRF2 activity and increases cellular ROS level.
Unlike in T98G cells, the ROS level in U2OS cells could be readily detected. Cells were treated with siRNAs against PALB2, KEAP1, and NRF2 to deplete the respective proteins, and the ROS levels were measured. As expected, depletion of NRF2 greatly increased the ROS level, whereas KEAP1 knockdown substantially decreased cellular ROS, presumably through a profound upregulation of NRF2 (A and B). PALB2 depletion increased the ROS level by nearly 50% compared with treatment of cells with control siRNA. Interestingly, the total amount of cellular NRF2 was largely unchanged under the same condition (A). Of note, a moderate reduction of the KEAP1 amount was often observed in NRF2-depleted cells (lane 4).
Next, we directly measured the transcriptional activation activity of endogenous NRF2 in the above siRNA-treated cells using the ARE-LUC reporter. As shown in C, NRF2 depletion reduced the reporter activity by approximately 5-fold and KEAP1 depletion upregulated the activity by ~2.5-fold, validating the suitability of the assay system. Under this setting, knockdown of PALB2 reduced the luciferase activity by about 2-fold, indicating that endogenous NRF2 transcription activity is impaired in the absence of PALB2.
Subsequently, mRNA amounts of nine NRF2 target genes (NQO1, GCLM, TXN, HMOX1, TXRD1, GSTP1, GCLC, AKR1B10, and AKR1C1) were surveyed following PALB2 knockdown in U2OS cells. To better understand the regulation of these genes by NRF2 under the setting used, their expression levels after KEAP1 and NRF2 depletion were also determined in parallel. As shown in Fig. S4A in the supplemental material, seven out of the nine genes, particularly AKR1C1, showed substantial to dramatic upregulation after KEAP1 depletion. The remaining two, TXRD1 and GSTP1, exhibited weak induction. Upon an acute loss of NRF2, only four genes (NQO1, GCLM, AKR1B10, and AKR1C1) showed significant downregulation, whereas HMOX1 and GCLC expression even increased significantly. These results demonstrate that, under the setting used, most NRF2 target genes positively respond to NRF2 induction but a significant fraction of the genes do not depend on NRF2 for basal expression, with some evidently having alternative, NRF2-independent induction mechanisms. Importantly, mRNA levels of NQO1, GCLM, AKR1B10, and AKR1C1, whose basal expression appeared to depend on NRF2, decreased upon PALB2 depletion. The expression of a similar panel of NRF2 downstream genes was also measured following PALB2 depletion in T98G cells. Although these cells express a high level of NRF2 (see Fig. S1C in the supplemental material), KEAP1 depletion elicited a further and large increase in NRF2 amount (see Fig. S4B), indicating an intact KEAP1 regulation of NRF2. As also shown in Fig. S4B in the supplemental material, the expression of NQO1, GCLC, TXN, and AKR1C1 was reduced after PALB2 depletion, indicating that NRF2 activity on these genes is compromised in the absence of PALB2.
Since loss of PALB2 led to an increased ROS level and reduced NRF2 transcriptional activity without significantly affecting its total abundance in the cell, we suspected that either its concentration in the nucleus or its association with AREs of its target genes may be reduced. In addressing the first possibility, nuclei of U2OS cells treated with control or PALB2 siRNAs were isolated and the NRF2 amount was examined. As shown in D and E, cells depleted of PALB2 exhibited significantly and reproducibly smaller amounts of NRF2 in the nucleus. Next, we asked whether NRF2 binding to the ARE in the NQO1 promoter is impaired by chromatin immunoprecipitation (ChIP) and quantitative PCR analyses and found that depletion of PALB2 reduced NRF2 binding to NQO1 ARE by approximately 2-fold (F and G). Taken together, these results indicate that endogenous PALB2 promotes NRF2 accumulation in the nucleus and binding to ARE and thus its transcription activation activity.
PALB2 impedes NRF2 nuclear export and degradation postinduction.
Having analyzed the effect of PALB2 on NRF2 abundance and activity under basal conditions, we further asked whether PALB2 also modulates NRF2 induction as well as its postinduction export and degradation, which are both regulated by KEAP1. Control or PALB2-depleted U2OS cells were subjected to tBHQ treatment (100 μM for 3 h) and NRF2 amounts were examined. As shown in A and B, tBHQ induced NRF2 by approximately 3.5-fold in both control and PALB2-depleted cells, indicating that PALB2 does not play a role in the initial induction of NRF2 by this agent. However, when NRF2 levels were further monitored following tBHQ removal, PALB2-depleted cells showed moderately faster reduction of total NRF2 abundance than did control cells. Then, we analyzed the amounts of NRF2 in cell nuclei under the same conditions. Although the nuclear NRF2 amount was smaller in PALB2-depleted cells before induction, it was induced by a magnitude (~3-fold) similar to that in control cells (C and D). By 1.5 h after tBHQ removal, whereas nuclear NRF2 in control cells remained unchanged, it had decreased significantly in PALB2-depleted cells. These results indicate that PALB2, by binding KEAP1 and reducing its complex formation with NRF2 in the nucleus, interferes with KEAP1-mediated NRF2 export and degradation in the postinduction phase.
Fig 7 PALB2 regulates NRF2 nuclear export after induction. (A) Western blotting of whole-cell extracts of U2OS cells (transfected with control or PALB2 siRNAs for 48 h) following treatment with DMSO or tBHQ and at indicated time points after tBHQ removal. (B) (more ...)