Abstract
Parasitic hookworms and the free-living model nematode Caenorhabtidis elegans share a developmental arrested stage, called the dauer stage in C. elegans and the infective third-stage larva (L3) in hookworms. One of the key transcription factors that regulate entrance to and exit from developmental arrest is the forkhead transcription factor DAF-16/FoxO. During the dauer stage, DAF-16 is activated and localized in the nucleus. DAF-16 is negatively regulated by phosphorylation by the upstream kinase AKT, which causes DAF-16 to localize out of the nucleus and the worm to exit from dauer. DAF-16 is conserved in hookworms, and hypothesized to control recovery from L3 arrest during infection. Lacking reverse genetic techniques for use in hookworms, we used C. elegans complementation assays to investigate the function of Ancylostoma caninum DAF-16 during entrance and exit from L3 developmental arrest. We performed dauer switching assays and observed the restoration of the dauer phenotype when Ac-DAF-16 was expressed in temperature-sensitive dauer defective C. elegans daf-2(e1370);daf-16(mu86) mutants. AKT phosphorylation site mutants of Ac-DAF-16 were also able to restore the dauer phenotype, but surprisingly allowed dauer exit when temperatures were lowered. We used fluorescence microscopy to localize DAF-16 during dauer and exit from dauer in C. elegans DAF-16 mutant worms expressing Ac-DAF-16, and found that Ac-DAF-16 exited the nucleus during dauer exit. Surprisingly, Ac-DAF-16 with mutated AKT phosphorylation sites also exited the nucleus during dauer exit. Our results suggest that another mechanism may be involved in the regulation DAF-16 nuclear localization during recovery from developmental arrest.
Highlights
The insulin/insulin growth factor (IIS) pathway is involved in embryogenesis [1], cell differentiation [2], development, and aging [3,4] in diverse species
We recently showed that Ac-DAF-16 was immunoprecipated with Ac-FTT-2 from serumactivated L3 lysates, and that the interaction required intact AKT phosphorylation sites at Ser107 and Thr312 [27]
Recent publications from our lab and others support the hypothesis that recovery from developmental arrest by the hookworm and other parasitic nematode infective stages is regulated by IIS [27,29,34,35], similar to recovery from dauer in C. elegans [36]
Summary
The insulin/insulin growth factor (IIS) pathway is involved in embryogenesis [1], cell differentiation [2], development, and aging [3,4] in diverse species. In the free-living nematode Caenorhabditis elegans, this pathway mediates entry into and exit from the developmentally arrested dauer stage by negatively regulating the activity of a FoxO-family forkhead transcription factor DAF-16 [5,6,7,8,9,10]. In responses to IIS, DAF-16 is phosphorylated by the activated serine-threonine protein kinase AKT/protein kinase B (AKT/PKB), creating binding sites for 14-3-3 proteins (FTT). Interaction between FTT and phosphorylated DAF-16 results in its nuclear exclusion and cytoplasmic retention leading to reproductive growth [13,14]
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