Biomedical subjects

Sakura Azuma

Publications and source records attributed to Sakura Azuma.

6 recordsLinked to original sources

PubMed2005

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

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.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Identification of DRG family regulatory proteins (DFRPs): specific regulation of DRG1 and DRG2.

DRG1 and DRG2 comprise a highly conserved subfamily of GTP-binding proteins and are thought to act as critical regulators of cell growth. Their abnormal expressions may trigger cell transformation or cell cycle arrest. Our aim is to clarify their physiological functions and regulatory mechanisms. Here we report identification of novel proteins, DRG family regulatory protein (DFRP) 1 and DFRP2, which regulate expression of DRG proteins through specific binding. In transient transfection experiments, DFRP1 specifically binds DRG1, and DFRP2 preferentially binds DRG2. DFRPs provide stability to the target DRG proteins through physical association, possibly by blocking the poly-ubiquitination that would precede proteolysis of DRG proteins. DFRPs are highly conserved in eucaryotes, and the expression patterns of dfrp1 and drg1 transcripts in Xenopus embryos and tissues are similar, indicating that these genes work cooperatively in various types of eukaryotic cells. Immunofluorescence experiments have revealed that the interaction between DRG1 and DFRP1 may occur in the cytoplasm. We generated dfrp1- knockout cells and found that endogenous expression of DRG1 is regulated by DFRP1, confirming that DFRP1 is a specific up-regulator of DRG1 in vivo. On the basis of these results, we propose that DRG1 and DRG2 are regulated differently despite their structural similarities.

Amino Acid Sequence↗

PubMed2004

TIFAB inhibits TIFA, TRAF-interacting protein with a forkhead-associated domain.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) transduces signals that lead to activation of NFkappaB and AP-1, which is essential for cell differentiation and establishment of the immune and inflammatory systems. TRAF-interacting protein with a forkhead-associated domain (TIFA) was identified as a TRAF6-binding protein that could link IRAK-1 to TRAF6 and then activate TRAF6 upon stimulation. We report identification of a TIFA-related protein, TIFAB, that inhibits TIFA-mediated activation of NFkappaB. TIFAB does not associate with members of the TRAF family but does bind TIFA. We analyzed the effect of TIFAB expression on the TRAF6/TIFA interaction by immunoprecipitation of TRAF6 and found that TIFA coprecipitated with TRAF6 was not changed. However, when we analyzed this interaction by immunoprecipitation of TIFA, we found that TIFAB significantly increased the amount of TRAF6 coprecipitated with TIFA. These findings suggest that TIFAB inhibits the TIFA-mediated TRAF6 activation possibly by inducing a conformational change in TIFA.

Amino Acid Sequence↗

PubMed2003

Cloning and characterization of Xenopus laevis drg2, a member of the developmentally regulated GTP-binding protein subfamily.

The developmentally regulated GTP-binding protein (DRG) subfamily is an uncharacterized member of the Obg family, an evolutional branch of GTPase superfamily proteins. GTPases act as molecular switches regulating diverse cellular processes. DRG2 and DRG1 comprise the DRG subfamily in eucaryotes. Although drg1 was first identified as a gene predominantly expressed during early development of the mouse central nervous system, comparative analysis of drg2 and drg1 expression during embryogenesis has never been reported, and the biochemical properties of the DRG family proteins remain to be elucidated. Thus, we first cloned Xenopus drg2 (Xdrg2) and examined the temporal and spatial expression patterns of Xdrg2 mRNA in comparison to those of Xdrg1. Both Xdrg2 and Xdrg1 are induced at late gastrula and subsequently increased during later stages of embryos (stage 13-41). Whole-mount in situ hybridization showed that Xdrg2 and Xdrg1 expression patterns are almost identical except that only Xdrg2 expression is detected in the stage 22 pronephric anlage. Strong transcripts of both genes are also observed at this stage in neural crest cells, blood islands, and developing eyes, and in brain, eyes, otic vesicle, branchial arches, pronephroses, spinal cord, notochord, head mesenchyme, and somites at stages 27 and 32. Northern blot analysis of adult tissues revealed that both genes are expressed highly in ovary and testis and rather moderately in other organs, except that Xdrg1 transcripts are scarcely detected in heart, lung, and liver. Accordingly, transcription or stability of Xdrg2 and Xdrg1 mRNAs may be regulated by different mechanisms. In addition, by generating recombinant XDRG2 and XDRG1 proteins, we found the RNA binding activity of these proteins in vitro. Our results suggest that the DRG proteins may play their physiological roles via RNA binding.

Amino Acid Sequence↗

PubMed2003

A tetraspanin-family protein, T-cell acute lymphoblastic leukemia-associated antigen 1, is induced by the Ewing's sarcoma-Wilms' tumor 1 fusion protein of desmoplastic small round-cell tumor.

Recurrent chromosomal translocations in neoplasms often generate hybrid genes that play critical roles in tumorigenesis. Desmoplastic small round-cell tumor (DSRCT) is an aggressive malignancy associated with the chromosomal translocation t(11;22)(p13;q12). This translocation generates a chimeric transcription factor, EWS-WT1, which consists of the transcriptional activation domain of the Ewing's sarcoma (EWS) protein and the DNA binding domain of the Wilms' tumor 1 (WT1) protein. One of the splice variants, EWS-WT1(-KTS) lacks three amino acid residues (Lys-Thr-Ser) in the DNA binding domain and transforms NIH3T3 cells. Therefore, it is likely that aberrant gene expression caused by EWS-WT1(-KTS) is involved in the malignant phenotype of DSRCT. Microarray analysis of 9600 human genes revealed that a gene encoding a tetraspanin-family protein, T-cell acute lymphoblastic leukemia-associated antigen 1 (TALLA-1), was induced in EWS-WT1(-KTS)-expressing cell clones. This induction was EWS-WT1(-KTS)-specific, and more importantly, TALLA-1 protein was expressed in the three independent cases of DSRCT. Tetraspanin-family genes encode transmembrane proteins that regulate various cell processes such as cell adhesion, migration and metastasis. Our findings provide a novel insight into the malignant phenotype of DSRCT, suggesting that TALLA-1 is a useful marker for diagnosis and a potential target for the therapy of DSRCT.

Carcinoma, Small Cell↗

PubMed2002

TRAF6-deficient mice display hypohidrotic ectodermal dysplasia.

Tumor necrosis factor receptor (TNFR)-associated factor 6 (TRAF6) is an adapter protein that links signals from members of the TNFR superfamily and Toll/IL-1 receptor family to activation of transcription factors NFkappaB and AP-1. Analysis of TRAF6-deficient mice revealed that TRAF6 is essential for normal bone formation and establishment of immune and inflammatory systems. Here we report that TRAF6 deficiency results in defective development of epidermal appendixes, including guard hair follicles, sweat glands, sebaceous glands of back skin, and modified sebaceous glands such as meibomian glands, anal glands, and preputial glands. Except the sebaceous gland impairment, these abnormal phenotypes are identical to those observed in Tabby (Ta), downless (dl), and crinkled (cr) mice, which are models of hypohidrotic (anhidrotic) ectodermal dysplasia in human. beta-catenin and mucosal addressin cell adhesion molecule-1, an early marker of developing guard-hair follicles is absent in the skin of TRAF6-deficient embryos. Thus, TRAF6 is essential for development of epidermal appendixes. TRAF6 does not associate with the cytoplasmic tail of the dl protein (DL)/ectodysplasin receptor (EDAR) receptor, which, when mutated, results in hypohidrotic (anhidrotic) ectodermal dysplasia. However, TRAF6 associates with X-linked ectodysplasin-A2 receptor (XEDAR) and TNFR super family expressed on the mouse embryo (TROY/toxicity and JNK inducer (TAJ), which are EDAR-related members of the TNFR superfamily that are expressed at high level in epidermal appendixes. Furthermore, TRAF6 is essential for the XEDAR-mediated NFkappaB activation. Our results suggest that TRAF6 may transduce signals emanating from XEDAR or TROY/TAJ that are associated with development of epidermal appendixes.

Animals↗