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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

The phylogenetic relationship between Ac-FTT-2 and selected 14-3-3 protein family members. A. Alignment of Ac-FTT-2 with C. elegans 14-3-3 proteins. Shading indicates residues that are identical (black) or similar (gray) to Ac-FTT-2. Amino acid residues that directly contact phosphate groups of targets are marked with an arrow. Protein sequences were aligned using CLUSTAL W software and displayed using BOXSHADE software located on the Swiss EMBnet server [74]. B. Neighbor joining tree of representative 14-3-3 proteins. Proteins were aligned using CLUSTAL W software on the Swiss EMBnet server [74]. Amino acid distances were calculated using the Poisson correction model in the MEGA program version 3.1 [75]. Major bootstrap values (1000 replications) are shown at each node. Ac, A. caninum; Bm. Brugia malayi; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Eg, Echinococcus granulosus; Em, Echinococcus multilocularis; Hs, Homo sapiens; Mi, Meloidogyne incognita; Sb, Schistosoma bovis; Sj, Schistosoma japonicum, Xl, Xenopus laevis. Accession numbers: Ce-FTT-1, [Genbank:CAA98138]; Ce-FTT-2, [Genbank:CAA91474]; Bm-FTT-2, [Genbank:XP_001895095]; Dm-FTT-ε, [Genbank:P92177]; Dm-FTT-ζ, [Genbank:P29310]; Eg-FTT-ε, [Genbank:AAX73175]; Em-FTT-ζ1, [Genbank:AAC48315]; Em-FTT-ζ2, [Genbank:AAM94864]; Hs-FTT-γ, [Genbank:P61981]; Hs-FTT-ε, [Genbank:P62258];Hs-FTT-ζ, [Genbank:P63104];Hs-FTT-η, [Genbank:Q04917];Hs-FTT-σ, [Genbank:P31947];Hs-FTT-τ, [Genbank:P27348]; Mi-FTT-a, [Genbank:AAL40719]; Mi-FTT-b, [Genbank:AAR85527]; Mm-FTT-β, [Genbank:Q9CQV8]; Sb-FTT-ε2, [Genbank:AAT39381]; Sj-FTT-ζ, [Genbank:AAD56715]; Sj-FTT-ε, [Genbank:AAC62003]; Xl-FTT-β, [Genbank:Q5XGC8].
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Figure 1: The phylogenetic relationship between Ac-FTT-2 and selected 14-3-3 protein family members. A. Alignment of Ac-FTT-2 with C. elegans 14-3-3 proteins. Shading indicates residues that are identical (black) or similar (gray) to Ac-FTT-2. Amino acid residues that directly contact phosphate groups of targets are marked with an arrow. Protein sequences were aligned using CLUSTAL W software and displayed using BOXSHADE software located on the Swiss EMBnet server [74]. B. Neighbor joining tree of representative 14-3-3 proteins. Proteins were aligned using CLUSTAL W software on the Swiss EMBnet server [74]. Amino acid distances were calculated using the Poisson correction model in the MEGA program version 3.1 [75]. Major bootstrap values (1000 replications) are shown at each node. Ac, A. caninum; Bm. Brugia malayi; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Eg, Echinococcus granulosus; Em, Echinococcus multilocularis; Hs, Homo sapiens; Mi, Meloidogyne incognita; Sb, Schistosoma bovis; Sj, Schistosoma japonicum, Xl, Xenopus laevis. Accession numbers: Ce-FTT-1, [Genbank:CAA98138]; Ce-FTT-2, [Genbank:CAA91474]; Bm-FTT-2, [Genbank:XP_001895095]; Dm-FTT-ε, [Genbank:P92177]; Dm-FTT-ζ, [Genbank:P29310]; Eg-FTT-ε, [Genbank:AAX73175]; Em-FTT-ζ1, [Genbank:AAC48315]; Em-FTT-ζ2, [Genbank:AAM94864]; Hs-FTT-γ, [Genbank:P61981]; Hs-FTT-ε, [Genbank:P62258];Hs-FTT-ζ, [Genbank:P63104];Hs-FTT-η, [Genbank:Q04917];Hs-FTT-σ, [Genbank:P31947];Hs-FTT-τ, [Genbank:P27348]; Mi-FTT-a, [Genbank:AAL40719]; Mi-FTT-b, [Genbank:AAR85527]; Mm-FTT-β, [Genbank:Q9CQV8]; Sb-FTT-ε2, [Genbank:AAT39381]; Sj-FTT-ζ, [Genbank:AAD56715]; Sj-FTT-ε, [Genbank:AAC62003]; Xl-FTT-β, [Genbank:Q5XGC8].

Mentions: A BLASTP search [35] of the non-redundant GenBank database using the deduced amino acid sequence confirmed that the hookworm protein was a member of the 14-3-3 protein family (Pfam PF00244, Interpro IPR000308). The best matches were to nematode 14-3-3 proteins, including Caenorhabditis briggsae (score 464, 2e-129), C. elegans (score 464, 4e-129). C. brenneri (score 462, 8e-129), and Meloidogyne incognita (score 461, 3e-128). While mammals have at least seven closely related isoforms [22], C. elegans has only two [26]. Alignment with Ce-FTT-1 (also known as PAR-5) and Ce-FTT-2 amino acid sequences shows that the hookworm 14-3-3 is more closely related to Ce-FTT-2 (91% identity) than Ce-FTT-1 (83% identity) (Fig. 1A). Furthermore, phylogenetic analysis indicates the hookworm 14-3-3 clusters most closely with FTT-2 isoforms from several other nematodes, including M. incognita and Brugia malayi (Fig. 1B). Therefore, the hookworm 14-3-3 protein most likely represents the Ce-FTT-2 ortholog, and will be referred to as 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)

The phylogenetic relationship between Ac-FTT-2 and selected 14-3-3 protein family members. A. Alignment of Ac-FTT-2 with C. elegans 14-3-3 proteins. Shading indicates residues that are identical (black) or similar (gray) to Ac-FTT-2. Amino acid residues that directly contact phosphate groups of targets are marked with an arrow. Protein sequences were aligned using CLUSTAL W software and displayed using BOXSHADE software located on the Swiss EMBnet server [74]. B. Neighbor joining tree of representative 14-3-3 proteins. Proteins were aligned using CLUSTAL W software on the Swiss EMBnet server [74]. Amino acid distances were calculated using the Poisson correction model in the MEGA program version 3.1 [75]. Major bootstrap values (1000 replications) are shown at each node. Ac, A. caninum; Bm. Brugia malayi; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Eg, Echinococcus granulosus; Em, Echinococcus multilocularis; Hs, Homo sapiens; Mi, Meloidogyne incognita; Sb, Schistosoma bovis; Sj, Schistosoma japonicum, Xl, Xenopus laevis. Accession numbers: Ce-FTT-1, [Genbank:CAA98138]; Ce-FTT-2, [Genbank:CAA91474]; Bm-FTT-2, [Genbank:XP_001895095]; Dm-FTT-ε, [Genbank:P92177]; Dm-FTT-ζ, [Genbank:P29310]; Eg-FTT-ε, [Genbank:AAX73175]; Em-FTT-ζ1, [Genbank:AAC48315]; Em-FTT-ζ2, [Genbank:AAM94864]; Hs-FTT-γ, [Genbank:P61981]; Hs-FTT-ε, [Genbank:P62258];Hs-FTT-ζ, [Genbank:P63104];Hs-FTT-η, [Genbank:Q04917];Hs-FTT-σ, [Genbank:P31947];Hs-FTT-τ, [Genbank:P27348]; Mi-FTT-a, [Genbank:AAL40719]; Mi-FTT-b, [Genbank:AAR85527]; Mm-FTT-β, [Genbank:Q9CQV8]; Sb-FTT-ε2, [Genbank:AAT39381]; Sj-FTT-ζ, [Genbank:AAD56715]; Sj-FTT-ε, [Genbank:AAC62003]; Xl-FTT-β, [Genbank:Q5XGC8].
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Figure 1: The phylogenetic relationship between Ac-FTT-2 and selected 14-3-3 protein family members. A. Alignment of Ac-FTT-2 with C. elegans 14-3-3 proteins. Shading indicates residues that are identical (black) or similar (gray) to Ac-FTT-2. Amino acid residues that directly contact phosphate groups of targets are marked with an arrow. Protein sequences were aligned using CLUSTAL W software and displayed using BOXSHADE software located on the Swiss EMBnet server [74]. B. Neighbor joining tree of representative 14-3-3 proteins. Proteins were aligned using CLUSTAL W software on the Swiss EMBnet server [74]. Amino acid distances were calculated using the Poisson correction model in the MEGA program version 3.1 [75]. Major bootstrap values (1000 replications) are shown at each node. Ac, A. caninum; Bm. Brugia malayi; Ce, Caenorhabditis elegans; Dm, Drosophila melanogaster; Eg, Echinococcus granulosus; Em, Echinococcus multilocularis; Hs, Homo sapiens; Mi, Meloidogyne incognita; Sb, Schistosoma bovis; Sj, Schistosoma japonicum, Xl, Xenopus laevis. Accession numbers: Ce-FTT-1, [Genbank:CAA98138]; Ce-FTT-2, [Genbank:CAA91474]; Bm-FTT-2, [Genbank:XP_001895095]; Dm-FTT-ε, [Genbank:P92177]; Dm-FTT-ζ, [Genbank:P29310]; Eg-FTT-ε, [Genbank:AAX73175]; Em-FTT-ζ1, [Genbank:AAC48315]; Em-FTT-ζ2, [Genbank:AAM94864]; Hs-FTT-γ, [Genbank:P61981]; Hs-FTT-ε, [Genbank:P62258];Hs-FTT-ζ, [Genbank:P63104];Hs-FTT-η, [Genbank:Q04917];Hs-FTT-σ, [Genbank:P31947];Hs-FTT-τ, [Genbank:P27348]; Mi-FTT-a, [Genbank:AAL40719]; Mi-FTT-b, [Genbank:AAR85527]; Mm-FTT-β, [Genbank:Q9CQV8]; Sb-FTT-ε2, [Genbank:AAT39381]; Sj-FTT-ζ, [Genbank:AAD56715]; Sj-FTT-ε, [Genbank:AAC62003]; Xl-FTT-β, [Genbank:Q5XGC8].
Mentions: A BLASTP search [35] of the non-redundant GenBank database using the deduced amino acid sequence confirmed that the hookworm protein was a member of the 14-3-3 protein family (Pfam PF00244, Interpro IPR000308). The best matches were to nematode 14-3-3 proteins, including Caenorhabditis briggsae (score 464, 2e-129), C. elegans (score 464, 4e-129). C. brenneri (score 462, 8e-129), and Meloidogyne incognita (score 461, 3e-128). While mammals have at least seven closely related isoforms [22], C. elegans has only two [26]. Alignment with Ce-FTT-1 (also known as PAR-5) and Ce-FTT-2 amino acid sequences shows that the hookworm 14-3-3 is more closely related to Ce-FTT-2 (91% identity) than Ce-FTT-1 (83% identity) (Fig. 1A). Furthermore, phylogenetic analysis indicates the hookworm 14-3-3 clusters most closely with FTT-2 isoforms from several other nematodes, including M. incognita and Brugia malayi (Fig. 1B). Therefore, the hookworm 14-3-3 protein most likely represents the Ce-FTT-2 ortholog, and will be referred to as 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