Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex

Abstract

The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2 and controls both the subcellular localization and steady-state levels of Nrf2. In this report, we demonstrate that Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex. Keap1 assembles into a functional E3 ubiquitin ligase complex with Cul3 and Rbx1 that targets multiple lysine residues located in the N-terminal Neh2 domain of Nrf2 for ubiquitin conjugation both in vivo and in vitro. Keap1-dependent ubiquitination of Nrf2 is inhibited following exposure of cells to quinone-induced oxidative stress and sulforaphane, a cancer-preventive isothiocyanate. A mutant Keap1 protein containing a single cysteine-to-serine substitution at residue 151 within the BTB domain of Keap1 is markedly resistant to inhibition by either quinone-induced oxidative stress or sulforaphane. Inhibition of Keap1-dependent ubiquitination of Nrf2 correlates with decreased association of Keap1 with Cul3. Neither quinone-induced oxidative stress nor sulforaphane disrupts association between Keap1 and Nrf2. Our results suggest that the ability of Keap1 to assemble into a functional E3 ubiquitin ligase complex is the critical determinant that controls steady-state levels of Nrf2 in response to cancer-preventive compounds and oxidative stress.

FIG. 1.

Domain structures of Nrf2 and Keap1. Nrf2 contains three discrete domains, including an N-terminal Neh2 domain, a central transactivation domain, and a C-terminal bZIP domain. The N-terminal Neh2 domain interacts with the Kelch domain of Keap1. The locations of seven lysine residues that are candidate sites for ubiquitination within the Neh2 domain are indicated. Keap1 contains five discrete domains that are designated as N, BTB, Linker, Kelch, and C. The location of Cys 151 in the BTB domain of Keap1 is indicated.

FIG. 2.

(A) COS1 cells were cotransfected with expression vectors for wild-type Keap1 and the indicated cullin proteins. Total cell lysates were immunoblotted with anti-HA antibodies (top panel) and anti-Keap1 antibodies (middle panel). Anti-Keap1 immunoprecipitates (IP) were subjected to immunoblot analysis using anti-HA antibodies (lower panel). (B) Sixty-millimeter-diameter dishes of MDA-MB-231 cells were transfected with expression vectors for HA-ubiquitin (Ub), Gal4-Neh2, Keap1, and each of the cullin proteins as indicated. Constant amounts of expression vectors for HA-ubiquitin (0.6 μg) and Gal4-Neh2 (0.6 μg) were included in all samples. The Keap1 expression vector was either omitted from some samples (lanes 1, 9, 13, and 17), titrated down from 0.6 to 0.038 μg (lanes 2 to 5), or kept constant at 0.038 μg per dish (lanes 6 to 8, 10 to 12, and 14 to 16). Increasing amounts of each cullin expression vector, from 0.1 to 0.6 μg, were added to some samples (lanes 6 to 17). Anti-Gal4 immunoprecipitates were analyzed by immunoblot analysis with anti-HA antibodies. IgG, immunoglobulin G. (C) Sixty-millimeter-diameter dishes of MDA-MB-231 cells were transfected with expression vectors for HA-Ub (0.6 μg), Gal4-Neh2 (0.6 μg), Keap1 (0.3 μg, lanes 2 to 5), and Cul3(1-380) (from 0.1 to 0.5 μg, lanes 3 to 5). Anti-Gal4 immunoprecipitates were analyzed by immunoblot analysis with anti-HA antibodies. (D). Twenty-four-well plates of MDA-MB-231 cells were transfected with expression vectors for HA-Nrf2 (0.18 μg, lanes 1 to 5), Keap1 (0.02 μg, lanes 2 to 5), and Cul3(1-380) (from 0.02 to 0.18 μg, lanes 3 to 5). Total cell lysates were subjected to immunoblot analysis with anti-HA antibodies.

FIG. 3.

(A) Sixty-millimeter-diameter dishes of COS1 cells were mock transfected (lane 1) or cotransfected with 0.7 μg each of expression vectors for Myc-Rbx1 (lanes 2 to 4), Keap1 (lanes 3 and 4), and HA-Cul3 (lane 4). Total cell lysates (7 μg) were analyzed by immunoblotting with anti-HA, anti-Myc, and anti-Keap1 antibodies (upper three panels). The lysates (700 μg) were incubated with chitin beads, pelleted by centrifugation (3,000 × g), and washed three times in RIPA buffer. Proteins that remained associated with the chitin beads were analyzed by immunoblotting with anti-HA, anti-Myc, and anti-Keap1 antibodies (lower three panels). (B) Twenty-four-well plates of MDA-MB-231 cells were cotransfected with expression vectors for HA-Nrf2 (0.15 μg), Myc-Rbx1 (0.118 μg; lanes 1, 3, and 5 to 8), Keap1 (0.015 μg, lanes 1 and 4 to 8), and each of the cullin proteins (0.117 μg, lanes 2 to 8) as indicated. The cells were either untreated (lanes 1 to 5, 7, and 8) or treated with 10 μM MG132 (lane 6) for 5 h prior to analysis of total cell lysates by immunoblotting with anti-HA antibodies. (C) Thirty-five-millimeter-diameter dishes of MDA-MB-231 cells were transfected with expression vectors for HA-ubiquitin (Ub) (0.3 μg), Gal4-Neh2 (0.35 μg), Keap1 (0.05 μg, lanes 2 to 4), Cul3 (0.15 μg, lanes 3 to 4), and the Myc-Rbx1 expression plasmid (0.02 μg, lane 4). Anti-Gal4 immunoprecipitates (IP) were analyzed by immunoblot analysis with anti-HA antibodies. IgG, immunoglobulin G. (D) Sixty-millimeter-diameter dishes of COS1 cells were transfected with expression vectors for Nrf2 (0.4 μg, lanes 1 to 4), Keap1-CBD (0.4 μg, lanes 1 to 4), HA-Cul3 (0.4 μg, lanes 2 to 4), and Myc-Rbx1 (0.4 μg, lanes 2 to 4). Lysates from three 60-mm-diameter dishes were pooled for each sample and incubated with chitin beads. After washing, the chitin beads were incubated with E1, E2-UbcH5a, ubiquitin, and ATP. Subsequently, the chitin beads were pelleted and washed, and proteins that were eluted from the beads after boiling were split into two sets of samples. One set was immunoprecipitated with anti-Nrf2 antibodies and then analyzed by immunoblotting with anti-ubiquitin antibodies (top panel). The other set was subjected to immunoblot analysis using anti-HA, anti-CBD, and anti-Myc antibodies (bottom three panels).

FIG. 4.

(A) Sixty-millimeter-diameter dishes of COS1 cells were transfected with equal amounts of expression vectors for the indicated Keap1-CBD proteins, HA-Cul3, and Myc-Rbx1. Cell lysates were collected and immunoblotted with the indicated antibodies (top three panels) or incubated with chitin beads. Proteins that remained bound to the chitin beads after extensive washing were analyzed by immunoblotting with the indicated antibodies (bottom three panels). WT, wild type. (B) Thirty-five-millimeter-diameter dishes of MDA-MB-231 cells were transfected with expression vectors for HA-ubiquitin (Ub; 0.5 μg) and the indicated Keap1 proteins (0.5 μg) and either left untreated (odd-numbered lanes) or treated with MG132 for 5 h (even-numbered lanes) prior to collection of cell lysates. Anti-Keap1 immunoprecipitates (IP) were analyzed by immunoblotting with anti-HA antibodies. IgG, immunoglobulin G. (C) Thirty-five-millimeter-diameter dishes of MDA-MB-231 cells were transfected with expression vectors for HA-Ub (0.31 μg), Gal4-Neh2 (0.36 μg), Cul3 (0.15 μg, even-numbered lanes), and the wild-type or mutant Keap1 proteins (0.18 μg), as indicated in lanes 3 to 8. Anti-Gal4 immunoprecipitates were analyzed by immunoblot analysis with anti-HA antibodies. (D) Twenty-four-well plates of MDA-MB-231 cells were transfected with expression vectors for HA-Nrf2 (0.2 μg), Cul3 (0.02 μg), Myc-Rbx1 (0.02 μg), and the wild-type or mutant Keap1 proteins (0.1 μg), as indicated in lanes 3 to 8. The transfected cells were either untreated (odd-numbered lanes) or treated with MG132 for 5 h (even-numbered lanes) prior to collection of cell lysates and determination of HA-Nrf2 levels by immunoblot analysis. (E) Twenty-four-well plates of MDA-MB-231 cells were transfected in duplicate with an ARE-dependent firefly luciferase reporter gene construct (100 ng) and expression plasmids for Nrf2 (100 ng) and the wild-type or mutant Keap1 proteins (50 ng). A plasmid encoding Renilla luciferase (10 ng) was included as a control for transfection efficiency. Lysates were collected at 48 h posttransfection, and both firefly and Renilla luciferase activities in cell lysates were analyzed. The data shown represent the means and standard errors of results from three independent experiments.

FIG. 5.

(A) Seven lysine residues in the Neh2 domain of Nrf2 (boldface) were substituted with arginine residues. A conserved ETGE motif (underlined) is required for association of Nrf2 with Keap1. (B) Twenty-four-well plates of MDA-MB-231 cells were transfected with expression vectors for wild-type (WT) or mutant HA-Nrf2 proteins (0.2 μg each, lane 1) and with different amounts of the expression vector for Keap1 (0.1 μg, lanes 2 and 4, or 0.025 μg, lane 3). The transfected cells were either untreated or treated with 10 μM MG132 (lane 4) for 4 h prior to analysis by immunoblotting using anti-HA antibodies. (C) Sixty-millimeter-diameter dishes of MDA-MB-231 cells were transfected with expression vectors for HA-ubiquitin (Ub; 0.6 μg), either wild-type Nrf2 or each of the mutant Nrf2 proteins as indicated (0.7 μg), and Keap1 (0.7 μg, lanes 3 to 11). The cells were treated with 10 μM MG132 for 4 h prior to lysis. Anti-Gal4 immunoprecipitates (IP) were analyzed by immunoblotting with anti-HA antibodies. IgG, immunoglobulin G.

FIG. 6.

(A) Sixty-millimeter-diameter dishes of MDA-MB-231 cells were transfected with expression vectors for HA-ubiquitin (Ub; 0.6 μg) and Gal4-Neh2 (0.7 μg) and for either wild-type (WT) Keap1 (0.7 μg, lanes 1 to 3) or Keap1-C151S (0.7 μg, lanes 4 to 6). The cells were either untreated (lanes 1 and 4) or treated with 50 μM tBHQ (lanes 2 and 5) or 20 μM sulforaphane (Sul, lanes 3 and 6) for 4 h prior to cell lysis. Anti-Gal4 immunoprecipitates (IP) were analyzed by immunoblot analysis with anti-HA antibodies. (B) Sixty-millimeter-diameter dishes of MDA-MB-231 cells were transfected with expression vectors for HA-Ub (0.6 μg), Gal4-Neh2 (0.7 μg), Cul3 (0.3 μg, lanes 3 to 5 and 7 to 9), and either wild-type Keap1 (0.4 μg, lanes 2 to 5) or Keap1-C151S (0.4 μg, lanes 6 to 9). The cells were either untreated (lanes 1 to 3, 6, and 7) or treated with 50 μM tBHQ (lanes 5 and 9) or 20 μM sulforaphane (lanes 4 and 8) for 4 h prior to cell lysis. Anti-Gal4 immunoprecipitates were analyzed by immunoblot analysis withanti-HA antibodies. IgG, immunoglobulin G. (C) Thirty-five-millimeterdiameter dishes of MDA-MB-231 cells were transfected with expression vectors for HA-Cul3 (0.5 μg) and either CBD-tagged wild-type Keap1 (0.5 μg, lanes 2 to 4) or CBD-tagged Keap1-C151S (0.5 μg, lanes 5 to 7). The cells were either left untreated (lanes 1, 2, and 5) or treated with 50 μM tBHQ (lanes 3 and 6) or 25 μM sulforaphane (lanes 4 and 7) for 5 h. Cell lysates were immunoblotted with the indicated antibodies (top two panels) or incubated with chitin beads. Proteins that remained bound to the chitin beads after extensive washing were analyzed by immunoblotting with the indicated antibodies (bottom two panels).

FIG. 7.

(A) Sixty-millimeter-diameter dishes of MDA-MB-231 cells were transfected with expression vectors for HA-Nrf2 (0.32 μg, lanes 1 to 4) and Keap1-CBD (0.16 μg, lanes 2 to 4). The cells were either left untreated (lanes 1 and 2) or treated with 50 μM tBHQ (lane 3) or 25 μM sulforaphane (lane 4) for 5 h. Cell lysates were immunoblotted with anti-HA antibodies (top panel) or incubated with chitin beads. Proteins that remained bound to the chitin beads after extensive washing were analyzed by immunoblotting with either anti-HA (middle panel) or anti-CBD (bottom panel) antibodies. (B) Sixty-millimeter-diameter dishes of MDA-MB-231 cells were transfected with an expression vector for Keap1-CBD (2 μg, lanes 2 to 5) and treated with 10 μM MG132 (lane 3), 20 μM sulforaphane (lane 4), or 50 μM tBHQ (lane 5) for 5 h. Total cell lysates were subjected to immunoblot analysis with anti-Nrf2 antibodies (top panels) or incubated with chitin beads (middle and bottom panels). Proteins that remained bound to the chitin beads after extensive washing were analyzed by immunoblot analysis using either anti-Nrf2 antibodies (middle panel) or anti-CBD antibodies (bottom panel). (C) One hundred-millimeter-diameter dishes of MDA-MB-231 cells were either untreated (lanes 1 and 3)or treated for 5 h with 10 μM MG132 (even-numbered lanes), 50 μM tBHQ (lanes 5 and 6), or 25 μM sulforaphane (lanes 7 and 8). Total cell lysates were collected, and 2% of the cell lysate was subjected to immunoblot analysis with anti-Nrf2 antibodies (top panel). The remainder of the lysate was subjected to immunoprecipitation with anti-HA antibodies (bottom panel, lanes 1 and 2) or affinity-purified anti-Keap1 antibodies (bottom panel, lanes 3 to 8). The immunoprecipitated proteins (IP) were analyzed by immunoblotting with anti-Nrf2 antibodies.