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  • Redox Biology br aPKC competes with Nrf for


     Redox Biology 22 (2019) 101149
    3.3. aPKCι competes with Nrf2 for Keap1 binding through the DLL motif
    Previous studies have reported that Keap1 could bind with Nrf2 and promote its degradation by the ubiquitin proteasome pathway [24]. In this study, we found that the proteasome inhibitor MG132 completely rescued aPKCι knockdown-induced reduction of Nrf2 (Fig. 3A and S2A). The data revealed that aPKCι might inhibit ubiquitin-proteasome-mediated degradation of Nrf2. Indeed, we further confirmed that ec-topic aPKCι Amyloid Beta-Peptide1-40 significantly reduced the ubiquitination of the endogenous Nrf2 in GBC cells. Conversely, aPKCι knockdown increased the level of ubiqutin-Nrf2 (Fig. 3B and S2B). Interestingly, it has also been demonstrated that the degradation of Nrf2 may be induced in a Keap1-independent manner [25]. These observations prompted us to investigate whether aPKCι-induced Nrf2 accumulation is associated with Keap1. The results indicated that Keap1 knockdown did not affect the protein expression of aPKCι, while abolished aPKCι-induced Nrf2 accumulation (Fig. 3C and S2C). Moreover, there was no significant alteration of p62 protein, reported as a negative regulator of Keap1, after aPKCι or Keap1 knockdown (Fig. 3C). Therefore, we speculated that aPKCι may compete with Nrf2 to bind with Keap1. To validate the hypothesis, we conducted immunoprecipitation experiment to analyze the interaction between aPKCι and the Keap1-Nrf2 complex by using Flag-aPKCι and Keap1-Myc plasmids. Under normal cellular conditions, Flag-aPKCι was immunoprecipitated by Keap1-Myc. Conversely, Keap1-Myc was detected in the Flag-immunoprecipitates (Fig. 3D). It has previously been reported that stress-induced reduction of Keap1-Nrf2 was the main mechanism of Nrf2 activation [26,27]. Thus, we further analyzed whether aPKCι was involved in regulating the process under oxidative stresses induced by gemcitabine. Interestingly, the binding amount of aPKCι and Keap1 proteins was significantly increased, ac-companying with a decreased interaction of Keap1and Nrf2 (Fig. 3E and S2D). To further validate that aPKCι can compete with Nrf2 for Keap1 binding, in vitro translation and immunoprecipitation experi-ments were performed. The equal amount of Nrf2 and Keap1 proteins were incubated together with different doses of aPKCι protein. Inter-estingly, the binding amount of Nrf2 and Keap1 was gradually de-creased with an increased interaction of aPKCι and Keap1 (Fig. 3F). More importantly, aPKCι overexpression reduced, while aPKCι knock-down increased, the Keap1-Nrf2 complex in GBC cells. The Keap1-aPKCι complex exhibited an opposite result (Fig. S2E and F). These data suggested that aPKCι disrupted the formation of the Keap1-Nrf2 com-plex by competing with Nrf2 for Keap1 binding.
    Next, we investigated the mechanisms underlying aPKCι-mediated dysregulation of the Keap1-Nrf2 system. Previous studies demonstrated that Nrf2 contained seven functional domains, including ETGE and DLG motifs, which are known as Keap1 binding sites [10]. Besides Nrf2, recent studies have identified other proteins with ETGE or similar motifs that can bind with Keap1 [28–30]. Indeed, Fukutomi and col-leagues demonstrated that the DLG motif needs to be much more ex-tended than the classical motifs [31]. Surprisingly, we found that 252-QDFDLL-257 was highly conserved from chicken to human, as a related DLG motif in Nrf2, within aPKCι. Thus, we generated a series of mu-tants, in which the QDFDLL was deleted, or DLL motif was changed to DLA. Moreover, two other DLM341 and DLK380 motifs were also mu-tated with DLA (Fig. 3G). The results indicated that both deletion
    mutant and DLL257 mutant were unable to interact with Keap1. How-ever, DLM341 and DLK380 mutants have no obvious effect on this in-
    teraction (Fig. 3H). Therefore, the DLL257 motif of aPKCι is required for its molecular recognition of Keap1.
    To identify the specific regions that are involved in the interaction of aPKCι and Keap1, we performed immunoprecipitation assays with a series of deletion mutants of Keap1 in HEK293T cells. The results showed that the Keap1 N-terminal deletion mutants (D1, D2 and D3) and C-terminal deletion mutant D5 bound with aPKCι, whereas DGR deletion mutant (D4) failed to interact with aPKCι, indicating that the DGR domain is required for Keap1 to bind with aPKCι (Fig. 3I and J). It r> Fig. 2. aPKCι promotes Nrf2 accumulation and activates antioxidative signaling. (A–B) The protein and mRNA levels of Nrf2 and Keap1 were determined in GBC cells with indicated treatments. (C) Western blotting was used to test the intracellular distribution of the Nrf2 protein after aPKCι overexpression or knockdown. LaminB and β-actin were used as loading control for the nucleus and cytoplasm, respectively. Cyt, cytoplasm. Nuc, nucleus. (D) The mRNA levels of Nrf2 target genes were measured in the indicated GBC cell lines. (E) The protein levels of Nrf2 were measured in GBC cells after Nrf2 knockdown. (F) Relative ROS levels were detected in aPKCι overexpression GBC cells with or without Nrf2 knockdown. **P < 0.01. Data are derived from three independent experiments and presented as means ± SDs.