JNK regulates lifespan in Caenorhabditis elegans by modulating nuclear translocation of forkhead transcription factor/DAF-16

Abstract

DAF-16/forkhead transcription factor, the downstream target of the insulin-like signaling in Caenorhabditis elegans, is indispensable for both lifespan regulation and stress resistance. Here, we demonstrate that c-Jun N-terminal kinase (JNK) is a positive regulator of DAF-16 in both processes. Our genetic analysis suggests that the JNK pathway acts in parallel with the insulin-like signaling pathway to regulate lifespan and both pathways converge onto DAF-16. We also show that JNK-1 directly interacts with and phosphorylates DAF-16. Moreover, in response to heat stress, JNK-1 promotes the translocation of DAF-16 into the nucleus. Our findings define an interaction between two well conserved proteins, JNK-1 and DAF-16, and provide a mechanism by which JNK regulates longevity and stress resistance.

Fig. 1.

JNK pathway regulates lifespan dependent on its phosphorylation that requires an upstream kinase, jkk-1. (A) Lifespan analysis of jnk-1(gk7) and jkk-1(km2). All lifespan data are presented as mean lifespan ± standard error: N2, 16.8 ± 0.2 (386 animals); jnk-1(gk7), 13.8 ± 0.2 (333 animals); jkk-1(km2), 13.9 ± 0.2 (189 animals). (B) Lifespan analysis of jnk-1 overexpression lines (lpIn1 and lpn2): N2 + pRF4, 15.2 ± 0.3 (105 animals); lpIn1, 20.9 ± 0.6 (99 animals); lpIn2, 18.8 ± 0.5 (97 animals). (C) Immunoblotting with phospho-JNK antibody (Upper). The same blot was reprobed with JNK antibody (Lower). (D) Lifespan analysis of jkk-1;lpIn2:N2 + pRF4, 15.2 ± 0.3 (105 animals); lpIn2, 18.8 ± 0.5 (97 animals), jkk-1; lpIn2, 15.0 ± 0.3 (130 animals). The lifespan curves were plotted from the pooled data of individual experiments.

Fig. 2.

jnk-1 regulates lifespan in parallel to the insulin-like pathway, but both converge onto daf-16. (A) Lifespan analysis of jnk-1 overexpression strain (lpIn1) on daf-16 RNAi. All lifespan data are presented as mean lifespan ± standard error: N2 on control RNAi, 17.6 ± 0.5 (125 animals); N2 on daf-16 RNAi, 14.6 ± 0.3 (145 animals); lpIn1 on control RNAi, 19.1 ± 0.6 (147 animals; lpIn1 on daf-16 RNAi, 14.5 ± 0.3 (136 animals). (B) Lifespan analysis of daf-2(e1370);lpIn1: N2, 17.6 ± 0.5 (125 animals); daf-2(e1370), 44.0 ± 0.7 (46 animals); daf-2(e1370);lpIn1, 53.3 ± 1.7 (39 animals). In A and B, the lifespan curves were plotted from the pooled data of the individual experiments. (C) Lifespan analysis of akt-1(ok525);akt-2(ok393) and akt-1(ok525);akt-2(ok393);lpIn1: WT (N2), 14.9 ± 0.4 (90 animals); akt-1(ok525);akt-2(ok393), 34.2 ± 0.8 (100 animals); akt-1(ok525);akt-2(ok393);lpIn1, 38.8 ± 0.9 (90 animals). The representative lifespan curve is shown here, which has been repeated with similar results.

Fig. 3.

JNK-1 interacts with and phosphorylates DAF-16. (A) Coimmunoprecipitation (IP) assay. After transfection with either Flag-DAF-16 alone or in combination with Xpress-JNK-1, cell lysates were immunoprecipitated with anti-Xpress antibody and immunoblotted with anti-Flag antibody. (B) In vitro kinase assay. Cells were transfected with Xpress-JNK-1 and activated by UV, followed by immunoprecipitation with anti-Xpress antibody and incubation with His-DAF-16 (N-terminal fragment) in kinase buffer. (Right) Loading of the substrates [His-DAF-16 (N-terminal fragment) and c-Jun] stained with Coomassie blue is shown. In both A and B, the expression of protein was confirmed by immunoblotting with anti-Flag or anti-Xpress antibody.

Fig. 4.

JNK-1 promotes the translocation of DAF-16 into the nucleus in response to heat stress. (A) Heat stress assay of jnk-1 overexpression strain (lpIn1 and lpIn2). Mean survival time ± standard error was: N2 + pRF4, 10.8 ± 0.2 (138 animals); lpIn1, 15.3 ± 0.3 (147 animals); lpIn2, 14.4 ± 0.2 (249 animals). (B) DAF-16 translocation assay. After heat shock at 35°C for 30 min, the nuclear translocation of DAF-16 was measured, and the number of worms in each category was counted (see Table 1) (28). Cyt, cytosolic; Int, intermediate; Nuc, nuclear. (C) JNK signaling pathway presumably serves as a molecular sensor for various stresses. Upon detecting environmental cues, JNK-1 transmits the signal by phosphorylating and modulating the nuclear translocation of DAF-16. Once DAF-16 enters the nucleus, it enhances the expression of numerous target genes to prevent damage from any harmful stresses. This would then confer increased stress resistance and help to maintain normal life in C. elegans.