Inoue J et al. (2005), Identification and characterization of Xenopus ...

XB-ART-1638

Gene 2005 Sep 26;358:53-9. doi: 10.1016/j.gene.2005.05.016.

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Identification and characterization of Xenopus laevis homologs of mammalian TRAF6 and its binding protein TIFA.

Inoue J , Yagi S , Ishikawa K , Azuma S , Ikawa S , Semba K .

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Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) transduces signals from members of the TNFR superfamily and the Toll/IL-1R family, leading to activation of transcription factors such as NFkappaB and AP-1. Genetic disruption of the TRAF6 gene in mice results in various developmental abnormalities during embryogenesis, including osteopetrosis, failure of neural tube closure, defective formation of skin appendices, absence of lymph nodes, and absence of mature thymic epithelial cells. To clarify the effect of TRAF6 in development, we previously identified a TRAF-interacting protein with a forkhead-associated domain (TIFA), which binds and activates TRAF6 upon extracellular stimulation. To understand the physiological roles of TRAF6 and TIFA in early development, we studied these genes in Xenopus laevis. Here, we describe identification of X. laevis homologs of mammalian TRAF6 (XTRAF6) and TIFA (XTIFA). As was the case for the mammalian homologs, overexpression of XTRAF6 or XTIFA activated NFkappaB, whereas XTIFA carrying a mutation that abolishes XTRAF6 binding failed to activate NFkappaB, suggesting that XTIFA activates NFkappaB by binding to XTRAF6. XTIFA and XTRAF6 mRNAs were expressed at similar levels in zygotes from the neurula stage and then increased. Whole-mount in situ hybridization revealed that XTRAF6 mRNA was expressed in the head region and neural tube during the neurula stage, and the expression expanded to the pharyngeal apparatus during the tailbud stage. This localization is consistent with the defective neural tube closure and abnormal thymus organogenesis observed in TRAF6-deficient mice. Our results suggest possible cooperation between XTRAF6 and XTIFA during embryogenesis.

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Species referenced: Xenopus laevis
Genes referenced: cstb jun mapk8 myc odc1 tifa traf6
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	Fig. 1. Comparison of the amino acid sequences of X. laevis, mouse, and human TRAF6s. The RING finger, Zn finger, coiled-coil and TRAF-C domains are indicated. Asterisks denote cysteine, histidine, or asparatic acid residues that could function as zinc ligands. Amino acids identical in least two proteins are indicated by black boxes.
	Fig. 2. Identification of XTIFA as a TRAF6 binding protein. (A) Diagram of the domain structure of the X. laevis TIFA protein. Identities and similarities (parentheses) of various domains of XTIFA when compared with the corresponding domains of human and mouse TIFAs are shown. (B) Alignment of various TRAF6-binding sites. Amino acids identical to the consensus TRAF6-binding site are indicated by black boxes. (C) Identification of the TRAF6-binding site in XTIFA. HEK293T cells were transfected with pME-FLAG-XTIFA (WT) or pME-FLAG-XTIFA-E172A (EA) alone or together with pME-Myc-XTRAF6. Thirty-six hours after transfection, cell lysates were subjected to immunoprecipitation with anti- Myc antibody. The immunoprecipitates (upper) and lysates prior to immunoprecipitation (lower) were analyzed by Western blotting with anti-TRAF6 and anti-XTIFA antibodies.
	Fig. 3. Activation of NFnB by XTRAF6 and XTIFA. (A) Luciferase reporter assay. HEK293T cells were transfected with 1 ng of 3 nB-luc, 10 ng of h-actin-h-galactosidase, and increasing amounts of expression plasmid for XTRAF6, XTIFA, or XTIFA-E172A. Thirty-six hours after transfection, luciferase assays were performed. Results shown are the meanTS.D. of triplicate experiments and are representative of two independent experiments. (B) Expression levels of XTIFA and XTIFAE172A. Lysates prepared from HEK293T cells transfected with expression plasmids for XTIFA or XTIFA-E172A were analyzed by Western blotting with anti-XTIFA antibody.
	Fig. 4. Expression analysis of XTRAF6 and XTIFA. (A) Tissue-specific expression of XTRAF6 and XTIFA mRNAs in adult X. laevis. Total RNAs isolated from the indicated adult tissues were analyzed by Northern blotting. The same membranes were reprobed with X. laevis EF-1aS probe for RNA quality control. (B) Temporal expression of XTRAF6 and XTIFA transcripts during X. laevis development. Total RNAs isolated from embryos at the indicated stages were analyzed by Northern blotting. The same membranes were reprobed with X. laevis ODC probe for RNA quality control. (C) Spatial expression of XTRAF6 transcripts during X. laevis development. Lateral views, anterior left; abbreviations: b, brain; e, eyes; nt, neural tube; pa, pharyngeal apparatus, sc, spinal cord.
	traf6 (TNF receptor associated factor 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anterior left, dorsal up.
	traf6 (TNF receptor associated factor 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.