The protein kinase MBK-1 contributes to lifespan extension in daf-2 mutant and germline-deficient Caenorhabditis elegans

Abstract

In Caenorhabditis elegans, reduction of insulin/IGF-1 like signaling and loss of germline stem cells both increase lifespan by activating the conserved transcription factor DAF-16 (FOXO). While the mechanisms that regulate DAF-16 nuclear localization in response to insulin/IGF-1 like signaling are well characterized, the molecular pathways that act in parallel to regulate DAF-16 transcriptional activity, and the pathways that couple DAF-16 activity to germline status, are not fully understood at present. Here, we report that inactivation of MBK-1, the C. elegans ortholog of the human FOXO1-kinase DYRK1A substantially shortens the prolonged lifespan of daf-2 and glp-1 mutant animals while decreasing wild-type lifespan to a lesser extent. On the other hand, lifespan-reduction by mutation of the MBK-1-related kinase HPK-1 was not preferential for long-lived mutants. Interestingly, mbk-1 loss still allowed for DAF-16 nuclear accumulation but reduced expression of certain DAF-16 target genes in germline-less, but not in daf-2 mutant animals. These findings indicate that mbk-1 and daf-16 functionally interact in the germline- but not in the daf-2 pathway. Together, our data suggest mbk-1 as a novel regulator of C. elegans longevity upon both, germline ablation and DAF-2 inhibition, and provide evidence for mbk-1 regulating DAF-16 activity in germline-deficient animals.

Keywords: DYRK1; FOXO; aging; phosphorylation; signaling.

Figure 1. Evidence for phosphorylation of Ser326 in C. elegans DAF-16

(A) Schematic drawing (to scale) of the DAF-16 protein (isoform c/a1). The location of a phosphopeptide derived from immunoprecipitated GFP::DAF-16 by tryptic digest, is shown in orange. The phosphorylation site was mapped to Ser326. (B) ClustalΩ alignment of the full length sequences of human FOXO family members and C. elegans DAF-16. Only the part spanning the Ser326-containing phosphopeptide is shown. The phosphorylated Serine in DAF-16 (Ser326), and its corresponding sites in FOXO1 (Ser329), FOXO3 (Ser325), FOXO4 (Ser273) and FOXO6 (not present) are highlighted in blue. Additional residues specifying the DYRK1A consensus motifs [32, 33, 65] are highlighted in red and yellow.

Figure 2. Loss of mbk-1 decreases lifespan of long-lived daf-2 and glp-1 mutant C. elegans

The effect of loss of function mutations in mbk-1 and hpk-1, mbk-1(pk1389) and hpk-1(pk1393), respectively, on lifespan relative to mbk-1(+) and hpk-1(+) animals was examined in different genetic backgrounds. (A) daf-2(-) [daf-2(e1370)] and corresponding daf-2(+) animals were grown continuously at 20°C. (B) glp-1(-) [glp-1(e2144ts)] and corresponding glp-1(+) animals were grown at 25°C for the first 24 h of postembryonic development to eliminate germ cells in glp-1(-) strains, and subsequently, were cultured at 20°C for the remainder of the experiment. See Table 1 for statistical analysis.

Figure 3. Effect of C. elegans mbk-1 on DAF-16 target gene expression

(A) Loss of mbk-1 decreases expression of a panel of DAF-16 target genes in glp-1(-) [glp-1(e2144ts)], but not in wild-type or daf-2(-) [daf-2(e1370)] animals as determined by qPCR (representative experiment shown, n=2). Error bars indicate standard deviations of three technical replicates. Statistical significance of expression level differences was determined by two-way ANOVA with Bonferroni post tests. (B) Loss of mbk-1 does not decrease daf-16 mRNA levels as determined by qPCR (representative experiment shown, n=2; error bars and statistical analysis as in panel A). (C) Loss of mbk-1 decreases Psod-3::gfp-expression in glp-1(-), and –to a lesser extent- in wild-type background (representative experiment shown, n=3). Error bars indicate standard deviations. Statistical significance of fluorescence intensity differences was determined by two-way ANOVA with Bonferroni post tests. All experiments in (A)-(C) were performed on day-2 adult worms. Images in (C) were taken at 100x magnification.

Figure 4. Loss of mbk-1 does not affect DAF-16 subcellular localization in germline-deficient C. elegans

The effect of the mbk-1 loss of function mutation mbk-1(pk1389) on subcellular localization of an intestine-specific GFP::DAF-16 protein (encoded by transgene muIs145[Pges-1::gfp::daf-16]) was determined at the times indicated in wild-type and germline-deficient glp-1(-) [glp-1(e2144ts)] animals. Images on the left were taken at 128x (48 h), 100x (60-96h) or 80x (120 h) magnification, images on the right are 6.5x magnifications of the areas boxed in red.