The Ras GTPase-activating-like protein IQGAP1 mediates Nrf2 protein activation via the mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK)-ERK pathway

Abstract

Nrf2 plays a critical role in the regulation of cellular oxidative stress. MEK-ERK activation has been shown to be one of the major pathways resulting in the activation of Nrf2 and induction of Nrf2 downstream targets, including phase II detoxifying/antioxidant genes in response to oxidative stress and xenobiotics. In this study, IQGAP1 (IQ motif-containing GTPase-activating protein 1), a new Nrf2 interaction partner that we have published previously, was found to modulate MEK-ERK-mediated Nrf2 activation and induction of phase II detoxifying/antioxidant genes. Nrf2 binds directly to the IQ domain (amino acids 699-905) of IQGAP1. Knockdown of IQGAP1 significantly attenuated phenethyl isothiocyanate- or MEK-mediated activation of the MEK-ERK-Nrf2 pathway. Knockdown of IQGAP1 also attenuated MEK-mediated increased stability of Nrf2, which in turn was associated with a decrease in the nuclear translocation of Nrf2 and a decrease in the expression of phase II detoxifying/antioxidant genes. In the aggregate, these results suggest that IQGAP1 may play an important role in the MEK-ERK-Nrf2 signaling pathway.

Keywords: Antioxidants; ERK; Gene Silencing; IQGAP1; MEK; Nrf2; PEITC; Scaffold Proteins.

FIGURE 1.

IQGAP1 and Nrf2 interact in a normal cellular environment. A, HEK-293 cell lysate was immunoprecipitated (IP) with anti-IQGAP1 antibody and nonimmune rabbit serum (NIRS). B, HEK-293 cells were transfected with pcDNA3.1 and IQGAP1 shRNA (shIQGAP1, shIQ) for 24 h and then treated with PEITC for 20 min, and cell lysates were immunoprecipitated with anti-IQGAP1 antibody. IQGAP1-Nrf2 interaction 20 min after PEITC treatment is expressed relative to interaction immediately after treatment and represents the mean ± S.D. (n = 3). *, p < 0.05. IB, immunoblot.

FIGURE 2.

IQGAP1 mediates PEITC-induced expression of phase II/antioxidant genes. A, HEK-293 cells were transfected with pcDNA3.1 and IQGAP1 shRNA (shIQGAP1) for 24 h. The down-regulation of IQGAP1 protein was measured. Data are representative of four independent experiments. B, HEK-293 cells transfected with IQGAP1 shRNA (shIQ) or pcDNA3.1 were treated with PEITC for 6 h. mRNA expression of NQO1, UGT1A1, and HO-1 was determined by quantitative PCR. Data represent the mean ± S.D. (n = 3). *, p < 0.05. CON, control. C, HEK-293 cells transfected with IQGAP1 shRNA or pcDNA3.1 were treated with PEITC for 6 h. Protein expression of HO-1 was determined by Western blotting. PEITC-induced HO-1 protein expression in IQGAP1-knockdown cells is expressed relative to wild-type cells and represents the mean ± S.D. (n = 3). *, p < 0.05.

FIGURE 3.

IQGAP1 mediates PEITC-induced nuclear accumulation of Nrf2. HEK-293 cells transfected with pcDNA3.1 or IQGAP1 shRNA (shIQGAP1, shIQ) were treated with PEITC. Cytoplasmic and nuclear fractions were collected 0, 0.5, 2, 6, and 24 h after treatment. A, protein expression of HO-1 in the cytoplasm was determined. Data are representative of three independent experiments. B, nuclear accumulation of Nrf2 was determined. Data are representative of three independent experiments. C, HEK-293 cells were transfected with pcDNA3.1 or IQGAP1 shRNA and ARE plasmids for 24 h. ARE induction was determined using luciferase assays. ARE activities induced by PEITC are expressed relative to IQGAP1 shRNA-transfected cells at 0 h after PEITC treatment and represent the mean ± S.D. (n = 2).

FIGURE 4.

Nrf2 binds to the IQ domain of IQGAP1. A, schematic diagram of the IQGAP1 constructs. CHD, calponin homology domain; GRD, GTPase-activating protein-related domain. B, full-length IQGAP1 proteins, truncated IQGAP1 proteins, and deletion mutants were generated according to the protocol of the TnT® quick coupled transcription/translation system. Dynabeads Protein G (DynaG-beads) and anti-c-Myc antibody were used to pull down proteins that formed complexes with Myc-tagged IQGAP1 proteins. Samples were resolved by SDS-PAGE and probed with anti-Nrf2 antibody. IP, immunoprecipitate; IB, immunoblot. C, Ni-NTA beads were used to pull down proteins that formed complexes with His-tagged Nrf2. Samples were resolved by SDS-PAGE and probed with anti-c-Myc antibody.

FIGURE 5.

IQGAP1 mediates PEITC-induced MEK-ERK activation and MEK-ERK-induced ARE expression. A, HEK-293 cells transfected with pcDNA3.1 or IQGAP1 shRNA (shIQ) were treated with PEITC for 10 min. MEK and ERK phosphorylation was probed using anti-phospho-MEK (pMEK) and anti-phospho-ERK (pERK) antibodies. PEITC-induced MEK and ERK phosphorylation in IQGAP1-knockdown cells is expressed relative to wild-type cells and represents the mean ± S.D. (n = 3). *, p < 0.05. CON, control. B, HEK-293 cells were transfected with pcDNA3.1, ARE, HA-ERK, and DNEE-MEK plasmids for 24 h. MEK and ERK phosphorylation was probed using anti-phospho-MEK and anti-phospho-ERK antibodies. ARE induction was determined using luciferase assays. ARE activities induced by ectopic expression of HA-ERK or DNEE-MEK are expressed relative to the control and represent the mean ± S.D. (n = 2). *, p < 0.05.

FIGURE 6.

IQGAP1 mediates MEK-ERK activation and ERK-Nrf2 interaction. A, HEK-293 cells and IQGAP1-knockdown HEK-293 cells were transfected with pcDNA3.1, GFP-Nrf2, HA-ERK, and DNEE-MEK plasmids. Cell lysates were immunoprecipitated (IP) with anti-HA antibody, the samples were resolved by SDS-PAGE, and the GFP-Nrf2 protein pulled down was probed with GFP. MEK-ERK activation and ERK-Nrf2 interaction in IQGAP1-knockdown cells are expressed relative to wild-type cells and represent the mean ± S.D. (n = 3). *, p < 0.05. shIQGAP1 and shIQ, IQGAP1 shRNA; pMEK, phospho-MEK; pERK, phospho-ERK. B, HEK-293 cells and IQGAP1-knockdown HEK-293 cells were transfected with pcDNA3.1, GFP-Nrf2, HA-ERK, and DNEE-MEK plasmids. Cell lysates were immunoprecipitated with anti-GFP antibody to pull down the GFP-tagged Nrf2 proteins, and the phosphorylation level was determined by anti-phospho-(Ser/Thr)-Pro (p-T/S-P) antibody. Data are representative of two independent experiments.

FIGURE 7.

IQGAP1 mediates MEK-ERK-induced stabilization of Nrf2 by interfering with ubiquitination of Nrf2. A, HEK-293 cells were transfected as indicated, and the GFP-Nrf2 proteins were immunoprecipitated (IP) with anti-GFP antibody. Ubiquitination of Nrf2 was probed using anti-ubiquitin (Ub) antibody. Nrf2 ubiquitination in IQGAP1-knockdown cells is expressed relative to wild-type cells and represents the mean ± S.D. (n = 2). shIQGAP1 and shIQ, IQGAP1 shRNA; pMEK, phospho-MEK; pERK, phospho-ERK. B, HEK-293 cells were transfected as indicated for 24 h and treated with cycloheximide (CHX) for 0, 0.5 and 1 h. The cells were lysed, and GFP-Nrf2 expression was blotted. Data are representative of two independent experiments.