XB-ART-1638
Gene
September 26, 2005;
358
53-9.
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
.
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.
PubMed ID:
16023795
Article link:
Gene
Species referenced:
Xenopus laevis
Genes referenced:
cstb
jun
mapk8
myc
tifa
traf6
Antibodies:
Myc Ab5
Tifa Ab1
Traf6 Ab1
Article Images:
[+] show captions
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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.
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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.
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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.
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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.
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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.
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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.
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