Interaction of hookworm 14-3-3 with the forkhead transcription factor DAF-16 requires intact Akt phosphorylation sites

Abstract Background Third-stage infective larvae (L3) of hookworms are in an obligatory state of developmental arrest that ends upon entering the definitive host, where they receive a signal that re-activates development. Recovery from the developmentally arrested dauer stage ofCaenorhabditis elegansis analogous to the resumption of development during hookworm infection. Insulin-like signaling (ILS) mediates recovery from arrest inC. elegansand activation of hookworm dauer L3. InC. elegans, phosphorylation of the forkhead transcription factor DAF-16 in response to ILS creates binding cites for the 14-3-3 proteinCe-FTT-2, which translocates DAF-16 out of the nucleus, resulting in resumption of reproductive development. Results To determine if hookworm 14-3-3 proteins play a similar role in L3 activation, hookworm FTT-2 was identified and tested for its ability to interact withA. caninumDAF-16in vitro. TheAc-FTT-2 amino acid sequence was 91% identical to theCe-FTT-2, and was most closely related to FTT-2 from other nematodes.Ac-FTT-2 was expressed in HEK 293T cells, and was recognized by an antibody against human 14-3-3β isoform. Reciprocal co-immunoprecipitations using anti-epitope tag antibodies indicated thatAc-FTT-2 interacts withAc-DAF-16 when co-expressed in serum-stimulated HEK 293T cells. This interaction requires intact Akt consensus phosphorylation sites at serine107 and threonine312, but not serine381.Ac-FTT-2 was undetectable by Western blot in excretory/secretory products from serum-stimulated (activated) L3 or adultA. caninum. Conclusion The results indicate thatAc-FTT-2 interacts with DAF-16 in a phosphorylation-site dependent manner, and suggests thatAc-FTT-2 mediates activation of L3 by bindingAc-DAF-16 during hookworm infection.