How Keap1 is transported into the nucleus is somewhat controversial: Keap1 was initially considered a cytosolic protein. It was thought that the major role of Keap1 was to physically bind to Nrf2 in the cytoplasm, prevent nuclear accumulation of Nrf2, and block trans
activation of ARE-regulated genes (10
). However, accumulating evidence shows that Keap1 is a nuclear-cytoplasmic shuttling protein equipped with a nuclear export signal (NES) that confers nuclear-cytoplasmic shuttling of the Nrf2-Keap1 complex (13
). In this study, we observed that Keap1 shuttles between the cytoplasm and the nucleus, since nuclear export of Keap1 can be blocked, either by leptomycin (LMB) or by NES mutations. Furthermore, nuclear import of Keap1 is independent of Nrf1 or Nrf2 (B and C). Stable cell lines derived from Keap1−/−
cells that stably express comparable levels of GFP-tagged wild-type Keap1 or the Keap1-NES mutant were used to eliminate the possible artifacts from Keap1 overexpression (A). Therefore, our data provide further evidence that Keap1 translocates into the nucleus and modulates Nrf2 signaling.
Although evidence in favor of Keap1 as a nuclear-cytoplasmic shuttling protein continues to grow, the mechanism of nuclear import of Keap1 and its function inside the nucleus remains disputable. In silico
analysis revealed that Keap1 does not contain a cNLS. The results of domain mapping confirmed that the Kelch domain of Keap1 is indispensable for its nuclear localization (13
), which is in agreement with our domain deletion analyses (C and D). To explain how Keap1 translocates into the nucleus, one hypothesis suggests that Nrf2 carries Keap1 into the nucleus, since Nrf2 possesses two putative NLSs and Keap1 does not (41
). Our data indicate that Keap1 still accumulates in the nucleus in Nrf1−/−
MEF cells after LMB treatment (C), suggesting that Keap1 does not rely on Nrf1 or Nrf2 for its nuclear entry. Another group hypothesized that a small nuclear protein, prothymosin α (PTMα), mediates nuclear import of the Keap1-Cul3-Rbx1 complex, leading to ubiquitination and degradation of Nrf2 inside the nucleus (30
). However, how PTMα itself shuttles between the nucleus and the cytosol and mediates Keap1 nuclear import is still unclear.
Here, we provide evidence that Keap1 itself is able to enter the nucleus with the help of an importin α protein, KPNA6. Interaction between Keap1 and KPNA6 was demonstrated not only in vitro but also in vivo with endogenous KPNA6 (A to D). Overexpression of KPNA6 significantly enhanced the nuclear accumulation of Keap1 after LMB treatment (A and B). Nrf2, NQO1, and HO-1 were diminished in the presence of KPNA6 (A and B), without alteration of Keap1 protein levels. Also, qRT-PCR analysis showed that KPNA6 changed the mRNA expression of Nrf2 target genes but not that of Nrf2 itself (C), supporting the notion that KPNA6 modulates Nrf2 at the protein level by changing Keap1's nucleocytoplasmic shuttling dynamics. On the other hand, knockdown of KPNA6 attenuates the nuclear import of Keap1 (A and B). There was less nuclear accumulation of Keap1 when MDA-MB-231 cells were transfected with KPNA6 siRNA and in the presence of LMB (C, compare lane 3 to 4, and lane 7 to 8). FRAP assays carried out in Keap1-null cells expressing GFP-tagged Keap1 also demonstrated that knockdown of KPNA6 was sufficient to decrease the nuclear accumulation of Keap1 (D and E). In addition, knockdown of KPNA6 promoted accumulation of endogenous nuclear Nrf2 protein levels during the induction and postinduction phases (A, compare lane 3 to 4 and lane 5 to 6). NQO1, HO-1, and GCLM were also significantly enhanced when KPNA6 was efficiently knocked down (B), while the ubiquitination and degradation of Nrf2 decreased in the absence of KPNA6 (C and D).
However, further attempts to map the KPNA6-interacting sites within the Keap1 Kelch domain were unsuccessful. Truncations or deletions within the Kelch domain disrupted the overall integrity of the ring-like 6-bladed propeller structure, thus providing no useful information. We also made several alanine (A) substitutions of positively charged arginine (R), lysine (K), or histidine (H) clusters within the Kelch domain, which should be responsible for the interaction between cargo protein and importin α in the classical nuclear import scenario, yet none of them showed loss of interaction (F). These data suggest that the interaction between KPNA6 and Keap1 may be different from the classical mode. Although the evidence to date has been biased toward classical monopartite or bipartite nuclear localization signals, many noncanonical NLSs that do not match the classical NLSs have been defined. Kosugi et al. have reported three classes of noncanonical monopartite NLSs that bind directly to importin α in the minor groove (3
), which also supports our hypothesis that Keap1 may interact with KPNA6 via a mechanism other than cNLS.
Keap1, a substrate adaptor protein for the Cul3-containing E3 ubiquitin ligase, negatively controls the activity of Nrf2 at the protein level. Nrf2 contains two binding sites for Keap1 in its Neh2 domain, a weak binding site (DLG) and a strong binding site (ETGE). This two-site substrate recognition model is also known as the “hinge-latch model.” The high-affinity ETGE motif functions as the “hinge,” and the lower-affinity DLG motif functions as the “latch.” Under basal conditions, the Keap1-homodimer recognizes and binds both motifs, positioning Nrf2 in the correct orientation for polyubiquitination and degradation to maintain low basal levels of Nrf2. In response to chemopreventive compounds, cysteine residues in Keap1 become modified, which may alter the structural confirmation and “unlatch” the weak binding DLG motif from Keap1, resulting in the stabilization and activation of Nrf2. (16
). Many cellular proteins, such as p62 and p21WAF1
, are able to unlatch the weak interaction between Nrf2 and Keap1, resulting in activation of the Nrf2-mediated response (19
). This unlatch event in response to an activation signal should also expose the Kelch domain, which contains the NLS. Based on our work presented in this study, exposure of the NLS in Keap1 results in its nuclear translocation. Currently, the precise mechanism by which nuclear-cytoplasmic shuttling of Nrf2 or Keap1 is controlled in response to redox conditions remains uncertain. In this study, we found that kinetically, Nrf2 translocates into the nucleus faster than Keap1. Conceivably, the series of events occur in response to an activating signal, as follows. (i) Unlatching of the binding between Nrf2 and Keap1: Nrf2 activators cause a conformational change in the Keap1-Cul3-E3 ubiquitin ligase by acting on certain cysteine residues in Keap1. (ii) Nrf2 nuclear translocation: a decrease in Nrf2 degradation results in nuclear translocation of free Nrf2 and activation of the Nrf2's downstream target genes. (iii) Keap1 nuclear translocation: unlatching of the Kelch domain in Keap1 from an Nrf2 protein also exposes an NLS to KPNA6, and Keap1 enters the nucleus with the assistance of KPNA6/importin β. (iv) Turning off of the signal: Keap1 removes Nrf2 from the ARE of downstream target genes and transports the Keap1-Nrf2 complex out of the nucleus, using the strong NES in Keap1. (v) Once in the cytoplasm, the dimer is recruited to the ubiquitination and degradation machinery, leading to the degradation of Nrf2 and restoring low basal levels of Nrf2 ().
Fig. 8. Schematic model of Nrf2 regulation by Keap1. Keap1 is a key regulator of the Nrf2-signaling pathway and serves as a molecular switch to turn the Nrf2-mediated antioxidant response on and off. (1) Oxidative stress or chemopreventive compounds cause a conformational (more ...)
In conclusion, we report a direct interaction between Keap1 and KPNA6 and have determined that Keap1 is transported into the nucleus by KPNA6. Moreover, we mapped the interaction domains to the Kelch domain of Keap1 and the N-terminal domain of KPNA6. By controlling the nuclear-cytoplasmic shuttling of Keap1, KPNA6 promotes the ubiquitination and degradation of Nrf2 and, thus, negatively regulates the protein level of Nrf2 and the transcription of its downstream target genes. Our data further support our previous work indicating that Keap1 is a postinduction repressor of Nrf2.