Identification and functional studies of a new Nrf2 partner IQGAP1: a critical role in the stability and transactivation of Nrf2.
OBJECTIVE
Nuclear factor-erythroid-related factor 2 (Nrf2) is a critical transcriptional factor that is used in regulating cellular defense against oxidative stress. This study is aimed at investigating new interacting protein partners of Nrf2 using One-strep tag pull-down coupled with LTQ Orbitrap LC/MS/MS, and at examining the impact on Nr2 signaling by the newly identified IQ motif containing GTPase activating protein 1 (IQGAP1).


RESULTS
Using the One-strep tag pull-down and LTQ Orbitrap LC/MS/MS, we identified IQGAP1 as a new Nrf2 interacting partner. Direct interactions between IQGAP1 and Nrf2 proteins were verified using in vitro glutathione S-transferase (GST) pull-down, transcription/translation assays, and in vivo utilizing Nrf2 overexpressing cells. Coexpression of Dsredmono-IQGAP1 and eGFP-Nrf2 increased the stability of eGFP-Nrf2 and enhanced the expression of Nrf2-target gene heme oxygenase-1 (HO-1). To confirm the functional role of IQGAP1 on Nrf2, knock-downed IQGAP1 using siIQGAP1 attenuated the expression of endogenous Nrf2, HO-1 proteins, and Nrf2-target genes GSTpi, GCLC, and
quinone oxidoreductase 1 (NQO-1). Furthermore, the stability of Nrf2 was dramatically decreased in IQGAP1-deficient mouse embryonic fibroblast (MEF) cells. Since IQGAP1 signaling could be mediated by calcium, treating the cells with calcium showed the translocation of IQGAP1/Nrf2 complex into the nucleus, suggesting that IQGAP1 may play a critical role in Nrf2 stability. Interestingly, consistent with calcium signaling for IQGAP1, treating the cells with calcium functionally enhanced Nrf2-mediated antioxidant responsive element-transcription activity and enhanced the expression of the endogenous Nrf2-target gene HO-1.


METHODS
In the aggregate, our current study identifies and functionally characterizes a new Nrf2 partner protein IQGAP1, which may contribute to Nrf2's regulation of antioxidant enzymes such as HO-1.


CONCLUSIONS
IQGAP1 may play a critical role in the stability and transactivation of Nrf2.
Graduate Program in Pharmaceutical Science, Center for Cancer Prevention Research, Ernest-Mario School of Pharmacy, Rutgers, The State University of New Jersey, Piscataway, NJ 08854, USA.
Jung-Hwan
JH
Kim
Eugenia Y
EY
Xu
David B
DB
Sacks
Jonghun
J
Lee
Limin
L
Shu
Bing
B
Xia
Ah-Ng Tony
AN
Kong
D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D020868:Gene Silencing; D006367:HeLa Cells; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D051379:Mice; D051267:NF-E2-Related Factor 2 / Q000235:genetics* / Q000378:metabolism*; D054730:Protein Interaction Domains and Motifs / Q000235:genetics; D055550:Protein Stability; D021381:Protein Transport; D015398:Signal Transduction; D015533:Transcriptional Activation / Q000235:genetics*; D020703:ras GTPase-Activating Proteins / Q000235:genetics* / Q000378:metabolism*
D000975:Antioxidants; C101203:IQ motif containing GTPase activating protein 1; D051267:NF-E2-Related Factor 2; C495635:NFE2L2 protein, human; D020703:ras GTPase-Activating Proteins; C496541:HMOX1 protein, human; D051547:Heme Oxygenase-1; D002118:Calcium