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

Kiss JE, Gao X, Krepp JM, Hawdon JM - Parasit Vectors (2009)

Bottom Line: In C. elegans, phosphorylation of the forkhead transcription factor DAF-16 in response to ILS creates binding cites for the 14-3-3 protein Ce-FTT-2, which translocates DAF-16 out of the nucleus, resulting in resumption of reproductive development.Ac-FTT-2 was undetectable by Western blot in excretory/secretory products from serum-stimulated (activated) L3 or adult A. caninum.The results indicate that Ac-FTT-2 interacts with DAF-16 in a phosphorylation-site dependent manner, and suggests that Ac-FTT-2 mediates activation of L3 by binding Ac-DAF-16 during hookworm infection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Immunology, and Tropical Medicine and Department of Biological Sciences, The George Washington University, Washington, DC 20037, USA. mtmjmh@gwumc.edu.

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 of Caenorhabditis elegans is analogous to the resumption of development during hookworm infection. Insulin-like signaling (ILS) mediates recovery from arrest in C. elegans and activation of hookworm dauer L3. In C. elegans, phosphorylation of the forkhead transcription factor DAF-16 in response to ILS creates binding cites for the 14-3-3 protein Ce-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 with A. caninum DAF-16 in vitro. The Ac-FTT-2 amino acid sequence was 91% identical to the Ce-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 14-3-3beta isoform. Reciprocal co-immunoprecipitations using anti-epitope tag antibodies indicated that Ac-FTT-2 interacts with Ac-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 adult A. caninum.

Conclusion: The results indicate that Ac-FTT-2 interacts with DAF-16 in a phosphorylation-site dependent manner, and suggests that Ac-FTT-2 mediates activation of L3 by binding Ac-DAF-16 during hookworm infection.

No MeSH data available.


Related in: MedlinePlus

Effect of phosphorylation site mutation on the interaction of Ac-FTT-2 with Ac-DAF-16. HEK293 cells were co-transfected with 2 μg of pcDNA3.1V5/Ac-ftt-2 and 2 μg of wild-type or mutant pCMV4FLAG/Ac-daf-16 plasmids as above. Mock cells received 4 μg of empty pcDNA3.1/V5-His vector. Cell lysates were prepared 48 h after transfection, and then incubated with anti-FLAG (M2) agarose resin. Immunoprecipations were separated by 4–20% gradient SDS-PAGE and transferred to PVDF membrane. Top panels, Western blot with anti-human 14-3-3β antibody; Bottom panels, the blot was stripped and re-probed with Ac-DAF-16 antiserum. See text for description of the Ac-DAF-16 mutants.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2683825&req=5

Figure 4: Effect of phosphorylation site mutation on the interaction of Ac-FTT-2 with Ac-DAF-16. HEK293 cells were co-transfected with 2 μg of pcDNA3.1V5/Ac-ftt-2 and 2 μg of wild-type or mutant pCMV4FLAG/Ac-daf-16 plasmids as above. Mock cells received 4 μg of empty pcDNA3.1/V5-His vector. Cell lysates were prepared 48 h after transfection, and then incubated with anti-FLAG (M2) agarose resin. Immunoprecipations were separated by 4–20% gradient SDS-PAGE and transferred to PVDF membrane. Top panels, Western blot with anti-human 14-3-3β antibody; Bottom panels, the blot was stripped and re-probed with Ac-DAF-16 antiserum. See text for description of the Ac-DAF-16 mutants.

Mentions: Interaction of 14-3-3 with Ce-DAF-16 requires phosphorylation of specific serine or threonine residues by the protein kinase Akt (Cahill et al., 2001). To determine if the predicted Akt phosphorylation sites are important for the interaction of hookworm 14-3-3 and DAF-16, recombinant Ac-DAF-16 proteins with the phosphorylation sites mutated to alanine were tested for their ability to co-IP recombinant FTT-2 when co-expressed in HEK293 cells. Anti-FLAG M2 resin co-immunoprecipitated recombinant Ac-FTT-2 and wild type FLAG-tagged DAF-16 from cells expressing both proteins (Figure 4). However, mutation of serine107 to alanine (S107A) completely abolished the interaction with Ac-FTT-2, either singly or in combination with mutations at the other Akt phosphorylation sites (S107A:T312A, S107A:S381A, or triple). Mutation of threonine 312 (T312A) alone or in combination with mutations at the other sites also severely diminished the interaction with FTT-2 However, mutation of serine 381 (S381A) had no effect on the interaction with 14-3-3 (Figure 4). These data indicate that serine107 and threonine312 are essential for the interaction of Ac-DAF-16 with Ac-FTT-2.


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

Kiss JE, Gao X, Krepp JM, Hawdon JM - Parasit Vectors (2009)

Effect of phosphorylation site mutation on the interaction of Ac-FTT-2 with Ac-DAF-16. HEK293 cells were co-transfected with 2 μg of pcDNA3.1V5/Ac-ftt-2 and 2 μg of wild-type or mutant pCMV4FLAG/Ac-daf-16 plasmids as above. Mock cells received 4 μg of empty pcDNA3.1/V5-His vector. Cell lysates were prepared 48 h after transfection, and then incubated with anti-FLAG (M2) agarose resin. Immunoprecipations were separated by 4–20% gradient SDS-PAGE and transferred to PVDF membrane. Top panels, Western blot with anti-human 14-3-3β antibody; Bottom panels, the blot was stripped and re-probed with Ac-DAF-16 antiserum. See text for description of the Ac-DAF-16 mutants.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2683825&req=5

Figure 4: Effect of phosphorylation site mutation on the interaction of Ac-FTT-2 with Ac-DAF-16. HEK293 cells were co-transfected with 2 μg of pcDNA3.1V5/Ac-ftt-2 and 2 μg of wild-type or mutant pCMV4FLAG/Ac-daf-16 plasmids as above. Mock cells received 4 μg of empty pcDNA3.1/V5-His vector. Cell lysates were prepared 48 h after transfection, and then incubated with anti-FLAG (M2) agarose resin. Immunoprecipations were separated by 4–20% gradient SDS-PAGE and transferred to PVDF membrane. Top panels, Western blot with anti-human 14-3-3β antibody; Bottom panels, the blot was stripped and re-probed with Ac-DAF-16 antiserum. See text for description of the Ac-DAF-16 mutants.
Mentions: Interaction of 14-3-3 with Ce-DAF-16 requires phosphorylation of specific serine or threonine residues by the protein kinase Akt (Cahill et al., 2001). To determine if the predicted Akt phosphorylation sites are important for the interaction of hookworm 14-3-3 and DAF-16, recombinant Ac-DAF-16 proteins with the phosphorylation sites mutated to alanine were tested for their ability to co-IP recombinant FTT-2 when co-expressed in HEK293 cells. Anti-FLAG M2 resin co-immunoprecipitated recombinant Ac-FTT-2 and wild type FLAG-tagged DAF-16 from cells expressing both proteins (Figure 4). However, mutation of serine107 to alanine (S107A) completely abolished the interaction with Ac-FTT-2, either singly or in combination with mutations at the other Akt phosphorylation sites (S107A:T312A, S107A:S381A, or triple). Mutation of threonine 312 (T312A) alone or in combination with mutations at the other sites also severely diminished the interaction with FTT-2 However, mutation of serine 381 (S381A) had no effect on the interaction with 14-3-3 (Figure 4). These data indicate that serine107 and threonine312 are essential for the interaction of Ac-DAF-16 with Ac-FTT-2.

Bottom Line: In C. elegans, phosphorylation of the forkhead transcription factor DAF-16 in response to ILS creates binding cites for the 14-3-3 protein Ce-FTT-2, which translocates DAF-16 out of the nucleus, resulting in resumption of reproductive development.Ac-FTT-2 was undetectable by Western blot in excretory/secretory products from serum-stimulated (activated) L3 or adult A. caninum.The results indicate that Ac-FTT-2 interacts with DAF-16 in a phosphorylation-site dependent manner, and suggests that Ac-FTT-2 mediates activation of L3 by binding Ac-DAF-16 during hookworm infection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology, Immunology, and Tropical Medicine and Department of Biological Sciences, The George Washington University, Washington, DC 20037, USA. mtmjmh@gwumc.edu.

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 of Caenorhabditis elegans is analogous to the resumption of development during hookworm infection. Insulin-like signaling (ILS) mediates recovery from arrest in C. elegans and activation of hookworm dauer L3. In C. elegans, phosphorylation of the forkhead transcription factor DAF-16 in response to ILS creates binding cites for the 14-3-3 protein Ce-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 with A. caninum DAF-16 in vitro. The Ac-FTT-2 amino acid sequence was 91% identical to the Ce-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 14-3-3beta isoform. Reciprocal co-immunoprecipitations using anti-epitope tag antibodies indicated that Ac-FTT-2 interacts with Ac-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 adult A. caninum.

Conclusion: The results indicate that Ac-FTT-2 interacts with DAF-16 in a phosphorylation-site dependent manner, and suggests that Ac-FTT-2 mediates activation of L3 by binding Ac-DAF-16 during hookworm infection.

No MeSH data available.


Related in: MedlinePlus