Heterodimerization with small Maf proteins enhances nuclear retention of Nrf2 via masking the NESzip motif

Abstract

Nrf2 is the key transcription factor regulating the antioxidant response. When exposed to oxidative stress, Nrf2 translocates to cell nucleus and forms heterodimer with small Maf proteins (sMaf). Nrf2/sMaf heterodimer binds specifically to a cis-acting enhancer called antioxidant response element and initiates transcription of a battery of antioxidant and detoxification genes. Nrf2 possesses a NESzip motif (nuclear export signal co-localized with the leucine zipper (ZIP) domain). Heterodimerization with MafG via ZIP-ZIP binding enhanced Nrf2 nuclear retention, which could be abrogated by the deletion of the ZIP domain or site-directed mutations targeting at the ZIP domain. In addition, dimerization with MafG precluded Nrf2zip/CRM1 binding, suggesting that Nrf2/MafG heterodimerization may simultaneously mask the NESzip motif. MafG-mediated nuclear retention may enable Nrf2 proteins to evade cytosolic proteasomal degradation and consequently stabilize Nrf2 signaling. For the first time, we show that under the physiological condition, the NESzip motif can be switched-off by heterodimerization.

Fig. 1

Molecular structure of Nrf2 and MafG. (A) Schematic illustration of Nrf2 molecule and plasmid construct. Nrf2 has some highly conserved domains called Nrf2-ECH homology (Neh) domains. The Neh1 contains the ZIP domain (LLLNLL), the basic region (++) and the CNC domain. The Neh2 domain mediates Keap1 binding. The Neh3 domain plays a permissive role of Nrf2 transactivation. The tandem of Neh4 and Neh5 domains mediates cooperative transactivation activity of Nrf2. The Neh6 domain locates in the intervening region. Nrf2 possesses a bipartite NLS (double bars) in the basic region and two NES motifs (black circles). The comprising residues of the ZIP domain of Nrf2 (top panel) are listed according to their position in heptad structure (bottom panel). The demarcation leucines are underlined. The composing leucine residues of the NESzip motif are in bold fonts. (B) Schematic illustration of MafG molecule and plasmid constructs. Typical for small Maf molecules, MafG lacks transactivation domain. MafG has an extensive homology region (EHR) domain, a basic domain (++) and a ZIP domain (LLLMLL). (C) Side view and (D) end view of coiled coil helix of the ZIP motif. When forming dimer, the “a” and “d” residues of one monomer bind with “d’” and “a’” residue of its partner monomer, respectively.

Fig. 2

MafG enhances Nrf2 nuclear retention via ZIP-ZIP dimerization in Hela cells. Epifluorescent microscopic examination showed that mDsRed-MafG could arrest EGFP-Nrf2 (A–C) and EGFP-Nrf2zip (D–F) in cell nucleus. In contrast, mDsRed-MafG2p failed to arrest EGFP-Nrf2zip in the nucleus (G–I). The mDsRed-MafGzip showed co-localization with EGFP-Nrf2zip (J–L). In the absence of mDsRed-MafGzip, EGFP-Nrf2zip maintained a cytosolic distribution (arrow, J, L). In contrast, mDsRed-MafGΔzip failed to change EGFP-Nrf2zip distribution (M–O). The left, middle and right column shows EGFP, mDsRed and superimposed images, respectively. Scale bar: 10 µm.

Fig. 3

Dimerization with MafG enhances nuclear retention of Nrf2. (A) GST pull down study showed that wild type Nrf2zip exhibited the strongest binding to MafG. Single point (1p) mutation in Nrf2zip attenuated MafG binding. Two point (2p) mutation further decreased MafG binding. Four point (4p) mutation completely abolished MafG binding. (B) Calculated FRET value showed strong interaction between Nrf2zip/MafG. FRET value was attenuated in Nrf2zip/MafG1p and completely negated in Nrf2zip/MafG2p. As a negative control, CFP/YFP failed to elicit FRET signal. (C–N) Confocal microscopy and FRET assay of MafG/Nrf2zip binding. ECFP-Nrf2zip showed co-localization with EYFP-MafG and EYFP-MafG1p in the nucleus. In the absence of EYFP-MafG, ECFP-Nrf2zip exhibited a cytosolic distribution (arrowheads) (D–E). ECFP-Nrf2zip however, showed a discrete cytosolic distribution, un-overlapped with the nuclear location of EYFP-MafG2p. To enhance visual effect, the EYFP, ECFP and FRET signals are artificially represented with red, green and white color, respectively. Scale bar: 10 µm.

Fig. 4

Dimerization with MafG precludes Nrf2zip/CRM1 binding. 1 µg of GST-Nrf2zip proteins and 2 µg (His)₆-CRM1 proteins were incubated with different amount of (His)₆-MafG proteins (0, 1 and 5 µg). GST pull-down results showed that MafG inhibited Nrf2zip/CRM1 binding in a dose-dependent manner.

Fig. 5

MafG regulates Nrf2 signaling. (A) Reporter gene activity assay. Co-expressing wild type MafG regulated Nrf2 induced ARE-luciferase activities in a bi-directional way. In contrast, co-expressing MafG2p mutant markedly inhibited Nrf2 induced ARE-luciferase activities in a dose dependent way. Hela cells were transfected with 1 µg pHM6-Nrf2, 0.5 µg plasmid expressing ARE-Luc together with 0, 1, 10, 25 and 100 ng plasmids expressing wild type (wt) or 2p mutant (mt) MafG. Twenty four hours after transfection, cells were harvested. Luciferase activity was measured and normalized to protein concentration. Single and double asterisks indicate statistical significance (t-test) of p<0.05 and p<0.01, respectively. (B) RT-PCR analysis of the transcription of phase II genes. 3 µg pcDNA3.1-Nrf2-V5 plasmids were expressed alone or co-expressed with 1 µg pcDNA3.1-Myc-MafG or pcDNA3.1-Myc-MafG2p in HeLa cells. Total RNAs were extracted using RNeasy method and reversed transcribed (RT). Same amount of RT samples were amplified by poly chain reaction (PCR) for 40 cycles and resolved in 1% agarose gel and visualized by ethidium bromide incorporation exited by UV light. The densitometric values of RT-PCR products were labeled underneath. (C) Western blotting results showed that MafG and MafG2p could enhance and attenuate Nrf2-induced HO-1 and NQO1 expression, respectively.

Fig. 6

Nrf2/MafG dimerization stabilizes Nrf2 proteins. (A) Cell fractionation studies showed that, at unstressed condition, Nrf2 immunoreactivities observed in nuclear fraction were enhanced and attenuated when co-expressed with Myc-MafG and Myc-MafG2p, respectively. (B) After overnight MG132 (10 µM) treatment, similar amount of Nrf2 immunoreactivities were observed in cells expessing Nrf2 alone or co-expressing Nrf2 with MafG and MafG2p mutant. Lamin A and GAPDH were used as controls for endogenous nuclear and cytosolic proteins, respectively. The asterisk indicates weak Myc-MafG2p immunoreactivity observed in the cytosolic fraction.