Abstract
INrf2 (Keap1) is an adaptor protein that facilitates INrf2-Cul3-Rbx1-mediated ubiquitination/degradation of Nrf2, a master regulator of cytoprotective gene expression. Here, we present evidence that members of the phosphoglycerate mutase family 5 (PGAM5) proteins are involved in the INrf2-mediated ubiquitination/degradation of anti-apoptotic factor Bcl-xL. Mass spectrometry and co-immunoprecipitation assays revealed that INrf2, through its DGR domain, interacts with PGAM5, which in turn interacts with anti-apoptotic Bcl-xL protein. INrf2-Cul3-Rbx1 complex facilitates ubiquitination and degradation of both PGAM5 and Bcl-xL. Overexpression of PGAM5 protein increased INrf2-mediated degradation of Bcl-xL, whereas knocking down PGAM5 by siRNA decreased INrf2 degradation of Bcl-xL, resulting in increased stability of Bcl-xL. Mutation of PGMA5-E79A/S80A abolished INrf2/PGAM5/Bcl-xL interaction. Therefore, PGAM5 protein acts as a bridge between INrf2 and Bcl-xL interaction. Further studies showed that overexpression of INrf2 enhanced degradation of PGAM5-Bcl-xL complex, led to etoposide-mediated accumulation of Bax, increased release of cytochrome c from mitochondria, activated caspase-3/7, and enhanced DNA fragmentation and apoptosis. In addition, antioxidant (tert-butylhydroquinone) treatment destabilized the Nrf2-INrf2-PGAM5-Bcl-xL complex, which resulted in release of Nrf2 in cytosol and mitochondria, release of Bcl-xL in mitochondria, increase in Bcl-xL heterodimerization with Bax in mitochondria, and reduced cellular apoptosis. These data provide the first evidence that INrf2 controls Bcl-xL via PGAM5 and controls cellular apoptosis.
Highlights
Grants RO1 ES012265 and RO1 GM047466. □S The on-line version of this article contains supplemental Table 1 and Fig. 1. 1 To whom correspondence should be addressed: Dept. of Pharmacology and
INrf2-mediated Degradation of phosphoglycerate mutase family 5 (PGAM5) and Anti-apoptotic Bcl-xL Protein—Control 293 and INrf2-293 cells were treated with tetracycline for the indicated time and analyzed for FLAGINrf2, endogenous PGAM5, and Bcl-xL (Fig. 1A, top left panels)
An siRNA-dependent decrease in INrf2 resulted in increased PGAM5L-V5 and Bcl-xL (Fig. 1B)
Summary
Grants RO1 ES012265 and RO1 GM047466. □S The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1 and Fig. 1. 1 To whom correspondence should be addressed: Dept. of Pharmacology and. FLAG-INrf2-293 cells were transfected with PGAM5-V5 followed by treatment with tetracycline for the indicated times to examine whether INrf2 ubiquitinates and degrades PGAM5-Bcl-xL (Fig. 3A). We analyzed the ubiquitination and degradation of PGAM5L-V5 and Bcl-xL proteins in INrf2-, INrf2DGR-, and INrf2⌬DGR-expressing cells after transfection with PGAM5-V5 and HA-ubiquitin constructs (Fig. 4C).
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
