Regulation of the Caenorhabditis elegans oxidative stress defense protein SKN-1 by glycogen synthase kinase-3

Abstract

Oxidative stress plays a central role in many human diseases and in aging. In Caenorhabditis elegans the SKN-1 protein induces phase II detoxification gene transcription, a conserved oxidative stress response, and is required for oxidative stress resistance and longevity. Oxidative stress induces SKN-1 to accumulate in intestinal nuclei, depending on p38 mitogen-activated protein kinase signaling. Here we show that, in the absence of stress, phosphorylation by glycogen synthase kinase-3 (GSK-3) prevents SKN-1 from accumulating in nuclei and functioning constitutively in the intestine. GSK-3 sites are conserved in mammalian SKN-1 orthologs, indicating that this level of regulation may be conserved. If inhibition by GSK-3 is blocked, background levels of p38 signaling are still required for SKN-1 function. WT and constitutively nuclear SKN-1 comparably rescue the skn-1 oxidative stress sensitivity, suggesting that an inducible phase II response may provide optimal stress protection. We conclude that (i) GSK-3 inhibits SKN-1 activity in the intestine, (ii) the phase II response integrates multiple regulatory signals, and (iii), by inhibiting this response, GSK-3 may influence redox conditions.

Fig. 1.

gsk-3 inhibits SKN-1 localization and SKN-1 target gene expression. (A and B) SKN-1 is localized to intestinal nuclei in gsk-3(RNAi) animals in the absence of stress. N2 Is007[SKN-1::GFP], sek-1(km4) Is007[SKN-1::GFP], and N2 Ex[SKN-1::GFP S74, 340A] L4 animals were placed onto nematode growth medium plates containing E. coli HT115 that carried either gsk-3 dsRNA or control (L4440) feeding vectors. The animals were then incubated for 24 h at 20°C and allowed to lay eggs. Their surviving progeny were scored as L4 larvae and young adults for accumulation of SKN-1::GFP in intestinal nuclei as described in ref. . “High” indicates that a strong SKN-1::GFP signal was present in all intestinal nuclei, as in the gsk-3(RNAi) animal shown. “Medium” refers to animals in which nuclear SKN-1::GFP was present at high levels anteriorly or anteriorly and posteriorly but was barely detectable midway through the intestine. sek-1 encodes a C. elegans p38 MAPK kinase and is required for SKN-1 function in the intestine (see Results and Discussion) (28). (C and D) gcs-1::gfp is expressed constitutively in gsk-3(RNAi) animals. gsk-3 RNAi was performed in gcs-1::gfp, gcs-1::gfp Δ2, and gcs-1::gfp Δ2-mut3 strains as in A and B, then intestinal gcs-1::gfp expression was scored in F1 L4 larvae and young adults (11). “High” indicates that gcs-1::gfp was present at high levels anteriorly and was detectable throughout most of the intestine, as in the gsk-3(RNAi) image shown. “Medium” refers to animals in which gcs-1::gfp was present at high levels anteriorly and possibly posteriorly but was not detected in between. The gcs-1::gfp fluorescence apparent in the control image derives from SKN-1-independent pharyngeal expression (11). In A and C, upper images show fluorescence, lower images show Nomarski imaging, and pairs of intestinal nuclei (arrows) are shown in each inset.

Fig. 2.

A predicted GSK-3 phosphorylation site prevents constitutive accumulation of SKN-1::GFP in intestinal nuclei. (A) The SKN-1 coding region and mutant transgenic constructs. Predicted coding regions are indicated by red boxes, untranslated regions are indicated by blue boxes, and GFP is indicated by a green box. Potential GSK-3 sites in SKN-1 were predicted by the scansite program (Table 1) (32). These predicted phosphorylated residues and the priming site Ser-397 (see Results and Discussion) were individually substituted with alanine within SKN-1::GFP. (B–E) Analysis of transgenic animals. (B) Individual transgenic lines were generated by injecting the transgenes diagrammed in A along with the marker rol-6 (pRF4). These extrachromosomal lines were scored for presence of SKN-1 in intestinal nuclei, as in Fig. 1B. Nuclear SKN-1::GFP was dramatically increased in SKN-1::GFP S393A lines in the WT but not sek-1(km4) background and in SKN-1::GFP S397A lines. (C–F) Fluorescent images of representative transgenic animals are shown, as in Fig. 1 A.

Fig. 3.

Phosphorylation of SKN-1 by GSK-3. (A) A GSK-3 phosphorylation motif in C. elegans SKN-1 is conserved in mammalian Nrf1 proteins. This motif, which has three predicted GSK-3 sites, is similar in structure to the GSK-3 target motif in β-catenin, in which priming phosphorylation at Ser-45 by casein kinase-1α is followed by sequential GSK-3 phosphorylation at Thr-41, Ser-37, and Ser-33 (26). Predicted compound GSK-3 phosphorylation motifs are also present in mammalian Nrf2 and the orthologous Drosophila protein CNC. Conserved residues in each group are boxed. h, human; m, mouse; r, rat. (B) Phosphorylation of SKN-1 Ser-393 by GSK-3β in vitro. GSK-3β phosphorylates the SKN-1 peptide shown provided that phosphorylated serine is present at position 397. This phosphorylation depends on the presence of Ser-393. cpm incorporated in a representative phosphorylation assay are graphed. (C) SKN-1 binds specifically to a C. elegans GST–GSK-3 fusion protein in vitro.

Fig. 4.

SKN-1 protects against oxidative stress. (A) SKN-1::GFP overexpression confers oxidative stress resistance. Transgenically expressed WT and S393A forms of SKN-1::GFP comparably rescue the oxidative stress sensitivity of skn-1(zu67) mutants and provide additional protection against oxidative stress. All animals analyzed carry the rol-6 transgenic marker and were derived from worms that are heterozygous for the DnT1 balancer. Control thus refers to N2;rol-6 progeny that were derived from N2;rol-6;DnT1/+ animals. Individual worms were scored for survival at the times shown after they had been placed on nematode growth medium plates containing 6.2 mM t-butyl hydroperoxide. Under these stress conditions, SKN-1::GFP accumulated in intestinal nuclei and gcs-1::gfp expression was increased (data not shown). This representative experiment involved 40 worms in each group. Error bars indicate standard deviations. P values for skn-1(zu67), skn-1(zu67) Ex[SKN-1::GFP], and skn-1(zu67) Ex[SKN-1::GFP S393A] compared with control were 0.034, 0.007, and 0.009, respectively. (B) A model for regulation of SKN-1 in the intestine. Under normal conditions, SKN-1 is phosphorylated constitutively by GSK-3 and prevented from accumulating in the nucleus. This inhibition depends on the prior priming phosphorylation of SKN-1, which represents an additional negative signal (see Results and Discussion). SKN-1 target genes, represented by gcs-1, are then expressed at very low levels. Under oxidative stress conditions, p38 pathway signaling and PMK-1 phosphorylation of SKN-1 are dramatically increased (28). This signal counteracts inhibition of SKN-1 by GSK-3 and is independently required for SKN-1 to accumulate in nuclei at high levels and induce phase II gene expression. These phosphorylation events are arbitrarily shown as occurring in the cytoplasm. MAPKKK, MAPK kinase kinase.