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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

Absence of Ac-FTT-2 in excretory/secretory products of adult and L3 Ancylostoma caninum. ESP from 6000 non-activated or activated L3, and 10 μg of adult ESP were separated by 4–20% gradient SDS-PAGE and transferred to PVDF membrane for Western blotting. A. Lane 1, non-activated L3 ESP; lane 2, activated L3 ESP; lane 3, adult ESP; lane 4, lysate (20 μg) of HEK293 expressing Ac-FTT-2. The blot was probed with anti-14-3-3β antibody. B. Control gels. Lane 1, non-activated L3 ESP; lane 2, activated L3 ESP; lane 3, recombinant ASP-1 (80 ng); lane 4, adult ESP; recombinant TIMP-1 (130 ng). Blots were probed with anti-ASP-1 antiserum (left) or anti-TMP-1 antiserum (right).
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Figure 5: Absence of Ac-FTT-2 in excretory/secretory products of adult and L3 Ancylostoma caninum. ESP from 6000 non-activated or activated L3, and 10 μg of adult ESP were separated by 4–20% gradient SDS-PAGE and transferred to PVDF membrane for Western blotting. A. Lane 1, non-activated L3 ESP; lane 2, activated L3 ESP; lane 3, adult ESP; lane 4, lysate (20 μg) of HEK293 expressing Ac-FTT-2. The blot was probed with anti-14-3-3β antibody. B. Control gels. Lane 1, non-activated L3 ESP; lane 2, activated L3 ESP; lane 3, recombinant ASP-1 (80 ng); lane 4, adult ESP; recombinant TIMP-1 (130 ng). Blots were probed with anti-ASP-1 antiserum (left) or anti-TMP-1 antiserum (right).

Mentions: Hookworm L3 activated in vitro by incubation with serum components under host-like conditions release multiple proteins, including the activation associated secretory proteins 1 and 2 (ASP-1 and ASP-2) [5,6] and a metalloprotease [4]. Adult worms release molecules associated with feeding and survival in the host intestine, including anticoagulants [39], ASPs [40] and protease inhibitors [41]. To determine if Ac-FTT-2 was secreted, we took advantage of the cross-reactivity with the anti-human 14-3-3β antibody to examine ESP from L3 and adult stages by Western blot. As seen in Figure 5A, 14-3-3β antibody failed to detect any bands in non-activated, activated L3, or adult ESP (lanes 1–3), but recognized recombinant Ac-FTT-2 expressed in HEK293 cells (lane 4). As a control, L3 ESP were probed with ASP-1 antiserum, which detects a band in activated but not non-activated ESP (Figure 5B, lanes 1–3) [5]. Furthermore, antiserum against Ac-TMP-1, known to be released in adult ESP [42], detected Ac-TMP in the adult ESP (Figure 5B, lanes 4 and 5), indicating that the ESP contained secreted proteins. These data indicate that Ac-FTT-2 is not released in detectable amounts by either activated L3 or adult A. caninum.


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)

Absence of Ac-FTT-2 in excretory/secretory products of adult and L3 Ancylostoma caninum. ESP from 6000 non-activated or activated L3, and 10 μg of adult ESP were separated by 4–20% gradient SDS-PAGE and transferred to PVDF membrane for Western blotting. A. Lane 1, non-activated L3 ESP; lane 2, activated L3 ESP; lane 3, adult ESP; lane 4, lysate (20 μg) of HEK293 expressing Ac-FTT-2. The blot was probed with anti-14-3-3β antibody. B. Control gels. Lane 1, non-activated L3 ESP; lane 2, activated L3 ESP; lane 3, recombinant ASP-1 (80 ng); lane 4, adult ESP; recombinant TIMP-1 (130 ng). Blots were probed with anti-ASP-1 antiserum (left) or anti-TMP-1 antiserum (right).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2683825&req=5

Figure 5: Absence of Ac-FTT-2 in excretory/secretory products of adult and L3 Ancylostoma caninum. ESP from 6000 non-activated or activated L3, and 10 μg of adult ESP were separated by 4–20% gradient SDS-PAGE and transferred to PVDF membrane for Western blotting. A. Lane 1, non-activated L3 ESP; lane 2, activated L3 ESP; lane 3, adult ESP; lane 4, lysate (20 μg) of HEK293 expressing Ac-FTT-2. The blot was probed with anti-14-3-3β antibody. B. Control gels. Lane 1, non-activated L3 ESP; lane 2, activated L3 ESP; lane 3, recombinant ASP-1 (80 ng); lane 4, adult ESP; recombinant TIMP-1 (130 ng). Blots were probed with anti-ASP-1 antiserum (left) or anti-TMP-1 antiserum (right).
Mentions: Hookworm L3 activated in vitro by incubation with serum components under host-like conditions release multiple proteins, including the activation associated secretory proteins 1 and 2 (ASP-1 and ASP-2) [5,6] and a metalloprotease [4]. Adult worms release molecules associated with feeding and survival in the host intestine, including anticoagulants [39], ASPs [40] and protease inhibitors [41]. To determine if Ac-FTT-2 was secreted, we took advantage of the cross-reactivity with the anti-human 14-3-3β antibody to examine ESP from L3 and adult stages by Western blot. As seen in Figure 5A, 14-3-3β antibody failed to detect any bands in non-activated, activated L3, or adult ESP (lanes 1–3), but recognized recombinant Ac-FTT-2 expressed in HEK293 cells (lane 4). As a control, L3 ESP were probed with ASP-1 antiserum, which detects a band in activated but not non-activated ESP (Figure 5B, lanes 1–3) [5]. Furthermore, antiserum against Ac-TMP-1, known to be released in adult ESP [42], detected Ac-TMP in the adult ESP (Figure 5B, lanes 4 and 5), indicating that the ESP contained secreted proteins. These data indicate that Ac-FTT-2 is not released in detectable amounts by either activated L3 or adult A. caninum.

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