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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

Co-immunoprecipitation of FTT-2 and DAF-16 expressed in human embryonic kidney 293 cells. Cells were transfected singly (4 μg) or co-transfected (2 μg each) with pcDNA3.1V5/Ac-ftt-2 and pCMV4FLAG/Ac-daf-16. At 16 hrs, the cells were incubated with 20% serum, and lysates prepared 24 hrs later. Lane M, cells transfected with empty pcDNA3.1/V5-His vector (mock); lane 1, cells transfected with Ac-daf-16 alone; lane 2, cells transfected with Ac-ftt-2 alone; lane 3, serum treated co-transfected cells. A. Immunoprecipitation with anti-FLAG (M2) agarose. Top panel, Western blot with anti-V5 antibody; bottom panel, the same blot stripped and probed with DAF-16 antiserum. B. Immunoprecipitation with anti-V5 agarose. Top panel, Western blot with DAF-16 antiserum; bottom panel, the same blot stripped and probed with V5 antibody.
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Figure 3: Co-immunoprecipitation of FTT-2 and DAF-16 expressed in human embryonic kidney 293 cells. Cells were transfected singly (4 μg) or co-transfected (2 μg each) with pcDNA3.1V5/Ac-ftt-2 and pCMV4FLAG/Ac-daf-16. At 16 hrs, the cells were incubated with 20% serum, and lysates prepared 24 hrs later. Lane M, cells transfected with empty pcDNA3.1/V5-His vector (mock); lane 1, cells transfected with Ac-daf-16 alone; lane 2, cells transfected with Ac-ftt-2 alone; lane 3, serum treated co-transfected cells. A. Immunoprecipitation with anti-FLAG (M2) agarose. Top panel, Western blot with anti-V5 antibody; bottom panel, the same blot stripped and probed with DAF-16 antiserum. B. Immunoprecipitation with anti-V5 agarose. Top panel, Western blot with DAF-16 antiserum; bottom panel, the same blot stripped and probed with V5 antibody.

Mentions: As shown in Figure 3A, anti-FLAG agarose pulled down both a 64 kDa FLAG-tagged DAF-16 and the V5-tagged recombinant Ac-FTT-2 from co-transfected cells (lanes 3), but only recombinant DAF-16 from cells singly transfected with Ac-daf-16 (lane 1). As expected, anti-FLAG agarose did not pull down any protein from the Ac-ftt-2 singly transfected cells (lane 2). Conversely, anti-V5 agarose precipitated both proteins from co-transfected cells, but only recombinant 14-3-3 from cells singly transfected with the Ac-ftt-2 construct, and nothing from cells expressing recombinant Ac-DAF-16 only (Fig. 3B). This indicates that Ac-FTT-2 interacts with Ac-DAF-16 in mammalian cell culture, and suggests that a similar interaction occurs in worms.


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)

Co-immunoprecipitation of FTT-2 and DAF-16 expressed in human embryonic kidney 293 cells. Cells were transfected singly (4 μg) or co-transfected (2 μg each) with pcDNA3.1V5/Ac-ftt-2 and pCMV4FLAG/Ac-daf-16. At 16 hrs, the cells were incubated with 20% serum, and lysates prepared 24 hrs later. Lane M, cells transfected with empty pcDNA3.1/V5-His vector (mock); lane 1, cells transfected with Ac-daf-16 alone; lane 2, cells transfected with Ac-ftt-2 alone; lane 3, serum treated co-transfected cells. A. Immunoprecipitation with anti-FLAG (M2) agarose. Top panel, Western blot with anti-V5 antibody; bottom panel, the same blot stripped and probed with DAF-16 antiserum. B. Immunoprecipitation with anti-V5 agarose. Top panel, Western blot with DAF-16 antiserum; bottom panel, the same blot stripped and probed with V5 antibody.
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Related In: Results  -  Collection

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Figure 3: Co-immunoprecipitation of FTT-2 and DAF-16 expressed in human embryonic kidney 293 cells. Cells were transfected singly (4 μg) or co-transfected (2 μg each) with pcDNA3.1V5/Ac-ftt-2 and pCMV4FLAG/Ac-daf-16. At 16 hrs, the cells were incubated with 20% serum, and lysates prepared 24 hrs later. Lane M, cells transfected with empty pcDNA3.1/V5-His vector (mock); lane 1, cells transfected with Ac-daf-16 alone; lane 2, cells transfected with Ac-ftt-2 alone; lane 3, serum treated co-transfected cells. A. Immunoprecipitation with anti-FLAG (M2) agarose. Top panel, Western blot with anti-V5 antibody; bottom panel, the same blot stripped and probed with DAF-16 antiserum. B. Immunoprecipitation with anti-V5 agarose. Top panel, Western blot with DAF-16 antiserum; bottom panel, the same blot stripped and probed with V5 antibody.
Mentions: As shown in Figure 3A, anti-FLAG agarose pulled down both a 64 kDa FLAG-tagged DAF-16 and the V5-tagged recombinant Ac-FTT-2 from co-transfected cells (lanes 3), but only recombinant DAF-16 from cells singly transfected with Ac-daf-16 (lane 1). As expected, anti-FLAG agarose did not pull down any protein from the Ac-ftt-2 singly transfected cells (lane 2). Conversely, anti-V5 agarose precipitated both proteins from co-transfected cells, but only recombinant 14-3-3 from cells singly transfected with the Ac-ftt-2 construct, and nothing from cells expressing recombinant Ac-DAF-16 only (Fig. 3B). This indicates that Ac-FTT-2 interacts with Ac-DAF-16 in mammalian cell culture, and suggests that a similar interaction occurs in worms.

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