(D, H)

(D, H). of how the Nrf2-Keap1 pathway is regulated, which is imperative in targeting this pathway for chemoprevention or chemotherapy. ortholog of human USP15. UBP12 associates with the COP9 signalosome (CSN) and functions to maintain the stability of cullin ring ligase (CRL) adaptor proteins. The CSN is a conserved protein complex involved in the regulation of the ubiquitin proteasome system (UPS) (Cope and Deshaies, 2003). In addition, UBP12 removes Ub from CRL substrates, including BTB domain containing Eniluracil proteins, and protects CRL components from cellular-depletion by preventing auto-ubiquitination and subsequent degradation, thus, facilitating the function of CRLs (Wee et al., 2005; Wu et al., 2006; Zhou et al., 2003). Schmidt et al. demonstrated the specificity of UBP12 in stabilizing CRL components. They discovered that UBP12 regulates the stability of BTB substrate adaptors in ubiquitination assay and determined that overexpression of Myc-USP15 led to a decrease in ubiquitinated Keap1 (Figure 3B). To verify this was not an artifact due to overexpression of Myc-USP15, we used siRNA to knock-down endogenous levels of USP15, which resulted in an increase in ubiquitinated-Keap1 in the absence and presence of Nrf2 (Figure 3C). These results suggest that Keap1 is a substrate for USP15. In addition, the specificity of USP15 for Keap1 was demonstrated by an deubiquitination assay. Ubiquitinated Keap1 was pulled down from cells co-transfected with CBD-Keap1 and HA-Ub and treated with tBHQ. In parallel, ubiquitinated Nrf2 was immunoprecipitated from cells co-transfected with Nrf2 and HA-Ub and treated with MG132. After washing, half of the ubiquitinated Keap1 or Nrf2 lysate was Mouse monoclonal to CD4.CD4 is a co-receptor involved in immune response (co-receptor activity in binding to MHC class II molecules) and HIV infection (CD4 is primary receptor for HIV-1 surface glycoprotein gp120). CD4 regulates T-cell activation, T/B-cell adhesion, T-cell diferentiation, T-cell selection and signal transduction incubated with BSA and half was incubated with purified His-USP15 protein, followed by immunoblot analysis with an anti-HA antibody. His-USP15 was able to deubiquitinate CBD-Keap1, but not Nrf2 (Figure 3D). Since USP15 is known to stabilize components of CRLs and Nrf2 is normally ubiquitinated by the Cul3-Keap1-E3 ubiquitin ligase and degraded by 26S proteasome, we explored the effect of USP15-mediated deubiquitination of Keap1 on Keap1-Cul3 or Keap1-Nrf2 complex formation. First, we generated HA-Cul3-[35S] and Nrf2-[35S] using transcription/translation, then incubated them with ubiquitinated-Keap1 or deubiquitinated-Keap1 generated in the same way as described in Figure 3D. Autoradiography revealed that deubiquitinated-Keap1 (Figure 3E, lane 2) more readily forms a complex with HA-Cul3-[35S] than does ubiquitinated-Keap1 (-His-USP15, Figure 3E, lane 1). Moreover, the ubiquitination status of Keap1 did not alter its binding to Nrf2-[35S] (Figure 3E, lanes 3-4). Consequently, we hypothesized that deubiquitinated-Keap1 is the form capable of interacting with Cul3 and forming an active Cul3-Keap1-E3 ligase complex, resulting in increased degradation of Nrf2. Open in a separate window Figure 3 USP15 deubiquitinates Keap1(A) MDA-MB-231 cells were left untreated or treated with 50 M tBHQ for 4 h. Endogenous Keap1 was immunoprecipitated using an anti-Keap1 antibody and ubiquitinated Keap1 was detected by Eniluracil immunoblot analysis using an anti-Ub antibody. (B,C) MDA-MB-231 cells were transfected with the indicated plasmids and 5 nM control-siRNA (Ct) or USP15-siRNA (U) for 72 h. The Keap1-containing complexes were immunoprecipitated with anti-Keap1 antibodies followed by immunoblot analysis with an anti-HA antibody for detection of ubiquitin-conjugated Keap1. (D, E) Cells were transfected with the indicated plasmids for 48 h followed by treatment with either tBHQ or MG132 for 4 h prior to cell lysis. Nrf2- or CBD-Keap1-containing complexes were precipitated with anti-Nrf2/protein A beads or chitin beads, respectively, followed by incubation with BSA or His-USP15. After washing, half of the sample was eluted in sample buffer and subjected to immunoblot analysis, (E) while the other half was further incubated with HA-Cul3-[35S] or Nrf2-[35S] Eniluracil followed by analysis using autoradiography. Keap1-K39R, a mutant.