The Experts below are selected from a list of 8838 Experts worldwide ranked by ideXlab platform
Akira Kikuchi - One of the best experts on this subject based on the ideXlab platform.
-
Axin localizes to the centrosome and is involved in microtubule nucleation
EMBO reports, 2009Co-Authors: Katsumi Fumoto, Moe Kadono, Nanae Izumi, Akira KikuchiAbstract:Axin is known to have an important role in the degradation of β-catenin in the Wnt pathway. Here, we reveal a new function of Axin at the centrosome. Axin was localized to the centrosome in various cell lines and formed a complex with γ-tubulin. Knockdown of Axin reduced the localization of γ-tubulin and γ-tubulin complex protein 2—components of the γ-tubulin ring complex—to the centrosome and the centrosomal microtubule nucleation activity after treatment with nocodazole. These phenotypes could not be rescued by the reduction in the levels of β-catenin. Although the expression of Axin rescued these phenotypes in Axin-knockdown cells, overexpression of Axin2, which is highly homologous to Axin, could not. Axin2 was also localized to the centrosome, but it did not form a complex with γ-tubulin. These results suggest that Axin, but not Axin2, is involved in microtubule nucleation by forming a complex with γ-tubulin at the centrosome.
-
Axin Facilitates Smad3 Activation in the Transforming Growth Factor β Signaling Pathway
Molecular and cellular biology, 2001Co-Authors: Masao Furuhashi, Yoichi Furukawa, Yusuke Nakamura, Akira Kikuchi, Hideki Yamamoto, Ken Yagi, Shinji Shimada, Kohei Miyazono, Mitsuyasu KatoAbstract:Axin acts as a negative regulator in Wnt signaling through interaction with various molecules involved in this pathway, including β-catenin, adenomatous polyposis coli, and glycogen synthase kinase 3β. We show here that Axin also regulates the effects of Smad3 on the transforming growth factor β (TGF-β) signaling pathway. In the absence of activated TGF-β receptors. Axin physically interacted with Smad3 through its C-terminal region located between the β-catenin binding site and Dishevelled-homologous domain. An Axin homologue, Axil (also called conductin), also interacted with Smad3. In the absence of ligand stimulation, Axin was colocalized with Smad3 in the cytoplasm in vivo. Upon receptor activation, Smad3 was strongly phosphorylated by TGF-β type I receptor (TβR-I) in the presence of Axin, and dissociated from TβR-I and Axin. Moreover, the transcriptional activity of TGF-β was enhanced by Axin and repressed by an Axin mutant which is able to bind to Smad3. Axin may thus function as an adapter of Smad3, facilitating its activation by TGF-β receptors for efficient TGF-β signaling.
-
Inhibition of Wnt signaling pathway by a novel Axin-binding protein.
The Journal of biological chemistry, 2000Co-Authors: Takayuki Kadoya, Shosei Kishida, Makoto Asashima, Akimasa Fukui, Takao Hinoi, Tatsuo Michiue, Akira KikuchiAbstract:Axin forms a complex with adenomatous polyposis coli gene product, glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, Dvl, and protein phosphatase 2A and functions as a scaffold protein in the Wnt signaling pathway. In the Axin complex, GSK-3beta efficiently phosphorylates beta-catenin, which is then ubiquitinated and degraded by proteasome. We isolated a novel protein that binds to Axin and named it Axam (for Axin associating molecule). Axam formed a complex with Axin in intact cells and bound directly to Axin. Axam inhibited the complex formation of Dvl with Axin and the activity of Dvl to suppress GSK-3beta-dependent phosphorylation of Axin. Furthermore, Axam induced the degradation of beta-catenin in SW480 cells and inhibited Wnt-dependent axis duplication in Xenopus embryos. These results suggest that Axam regulates the Wnt signaling pathway negatively by inhibiting the binding of Dvl to Axin.
-
gsk 3β dependent phosphorylation of adenomatous polyposis coli gene product can be modulated by β catenin and protein phosphatase 2a complexed with Axin
Oncogene, 2000Co-Authors: Satoshi Ikeda, Michiko Kishida, Yoshiharu Matsuura, Hirofumi Usui, Akira KikuchiAbstract:Axin forms a complex with adenomatous polyposis coli gene product (APC), glycogen synthase kinase-3β (GSK-3β), and β-catenin through different binding sites and downregulates β-catenin. GSK-3β-dependent phosphorylation of APC-(1211-2075) which has the Axin-binding site was facilitated by Axin, but that of APC-(959-1338) which lacks the Axin-binding site was not. Axin-(298-506) or Axin-(298-832), which has the GSK-3β- and β-catenin- but not APC-binding sites, did not enhance GSK-3β-dependent phosphorylation of either APC-(1211-2075) or APC-(959-1338). Furthermore, β-catenin stimulated the phosphorylation of APC-(959-1338) and APC-(1211-2075) by GSK-3β in the presence of Axin. Consistent with these in vitro observations, expression of β-catenin or Axin in COS cells promoted an SDS gel band shift of APC. These results indicate that APC complexed with Axin is effectively phosphorylated by GSK-3β and that β-catenin may modulate this phosphorylation. In addition, the heterodimeric form of protein phosphatase 2A (PP2A) directly bound to Axin, and PP2A complexed with Axin dephosphorylated APC phosphorylated by GSK-3β. Taken together, these results suggest that GSK-3β-dependent phosphorylation of APC can be modulated by β-catenin and PP2A complexed with Axin.
-
Axin Directly Interacts with Plakoglobin and Regulates Its Stability
The Journal of biological chemistry, 1999Co-Authors: Shinya Kodama, Satoshi Ikeda, Toshimasa Asahara, Michiko Kishida, Akira KikuchiAbstract:Abstract Plakoglobin is homologous to β-catenin. Axin, a Wnt signal negative regulator, enhances glycogen synthase kinase (GSK)-3β-dependent phosphorylation of β-catenin and stimulates the degradation of β-catenin. Therefore, we examined the effect of Axin on plakoglobin stability. Axin formed a complex with plakoglobin in COS cells and SW480 cells. Axin directly bound to plakoglobin, and this binding was inhibited by β-catenin. Axin promoted GSK-3β-dependent phosphorylation of plakoglobin. Furthermore, overexpression of Axin down-regulated the level of plakoglobin in SW480 cells. These results suggest that Axin regulates the stability of plakoglobin by enhancing its phosphorylation by GSK-3β and that Axin may act on β-catenin and plakoglobin in similar manners.
Frank Costantini - One of the best experts on this subject based on the ideXlab platform.
-
impaired neural development caused by inducible expression of Axin in transgenic mice
Mechanisms of Development, 2007Co-Authors: Bo Liu, Frank Costantini, Wei HsuAbstract:Ablations of the Axin family genes demonstrated that they modulate Wnt signaling in key processes of mammalian development. The ubiquitously expressed Axin1 plays an important role in formation of the embryonic neural axis, while Axin2 is essential for craniofacial skeletogenesis. Although Axin2 is also highly expressed during early neural development, including the neural tube and neural crest, it is not essential for these processes, apparently due to functional redundancy with Axin1. To further investigate the role of Wnt signaling during early neural development, and its potential regulation by Axins, we developed a mouse model for conditional gene activation in the Axin2-expressing domains. We show that gene expression can be successfully targeted to the Axin2-expressing cells in a spatially and temporally specific fashion. High levels of Axin in this domain induce a region-specific effect on the patterning of neural tube. In the mutant embryos, only the development of midbrain is severely impaired even though the transgene is expressed throughout the neural tube. Axin apparently regulates b-catenin in coordinating cell cycle progression, cell adhesion and survival of neuroepithelial precursors during development of ventricles. Our data support the conclusion that the development of embryonic neural axis is highly sensitive to the level of Wnt signaling. 2006 Elsevier Ireland Ltd. All rights reserved.
-
Mouse Axin and Axin2/conductin proteins are functionally equivalent in vivo.
Molecular and cellular biology, 2005Co-Authors: Ian V. Chia, Frank CostantiniAbstract:Axin is a central component of the canonical Wnt signal transduction machinery, serving as a scaffold for the β-catenin destruction complex (23, 31, 36, 45, 52). Axin has specific binding sites for many proteins involved in Wnt signal transduction, including β-catenin, glycogen synthase kinase 3 (GSK3), CKI, adenomatous polyposis coli (APC), Dvl, LRP, and protein phosphatase 2A (Fig. (Fig.1A)1A) (23, 31). Its key function in this pathway is to bring together β-catenin and the protein kinases CKI and GSK3, thus promoting the phosphorylation and consequent destruction of β-catenin. In the presence of a Wnt signal, this function is overcome, allowing β-catenin to accumulate and enter the nucleus (9, 13). The mechanism by which a Wnt signal leads to the inactivation of the Axin complex is not entirely clear, but it is thought to involve the binding of Axin to the Wnt coreceptor LRP as well as to Dvl (7, 34, 42, 44, 45). This results in the dephosphorylation of Axin, leading to a decrease in its affinity for β-catenin, and in a decrease in the level of Axin (20, 47, 48). Axin also enters the nucleus and appears to play a role in the nuclear-cytoplasmic shuttling of β-catenin (8, 46). FIG.1. Targeted replacement of the Axin gene with myc-tagged Axin2 cDNA or myc-tagged Axin cDNA. (A) Schematic diagram of Axin and Axin2 proteins and binding partners. Percent similarities between the conserved RGS and DIX domains and the GSK3 and β-catenin ... The major product of the Axin gene is a protein of 832 or 868 amino acids (depending on alternative splicing) containing two highly conserved domains (52): the RGS domain, which encompasses the binding site for APC (16, 18), and the DIX domain, a region of homology with Dvl proteins that is implicated in the binding of Axin to Dvl as well as in homodimerization (10, 17, 22, 24, 29, 40). (The open reading frame of Axin cDNA potentially encodes proteins of 956 and 992 amino acids, but proteins of this length have not been detected, and the major protein begins at codon Met-125 of the original sequence.) Mutant embryos lacking Axin die at embryonic day 9.5 (E9.5) with abnormalities including truncation of the forebrain, neural tube defects, and embryonic axis duplications (14, 35). Axin is expressed ubiquitously during embryogenesis, and the presence of axis duplications in its absence is thought to be a consequence of the abnormal accumulation of β-catenin, mimicking a Wnt signal, in the early embryo. Axin2 (also known as Conductin) is 44% identical to Axin and shares the RGS and DIX domains (Fig. (Fig.1A)1A) (5, 39, 49) as well as the binding sites for β-catenin, GSK3, Diversin, and Smad3 (Fig. (Fig.1A)1A) (5, 11, 39, 49). While Axin2 has been studied less extensively than Axin, most data suggest that they are similar in function. Thus, when overexpressed in cultured cells, both proteins reduce the levels of β-catenin and the expression of Wnt target genes, and when expressed in frog embryos, both inhibit the development of dorsal structures (5, 19, 37, 49, 52). However, they are not fully redundant in vivo, as Axin2 is clearly unable to compensate for the absence of Axin in Axin-null embryos (52). Furthermore, humans with heterozygous germ line Axin2 mutations have familial tooth agenesis (25), while mice homozygous for loss of Axin2 have skull abnormalities (51), indicating that Axin does not fully substitute for the lack of Axin2. In addition, deletions or mutations in Axin or Axin2 have been observed in a number of types of tumors, including colorectal cancer and hepatocellular carcinoma, indicating that each gene is a tumor suppressor and that neither gene can replace the other for this function (30, 38, 43). One reason for the lack of full redundancy may be the different expression patterns of Axin and Axin2. While Axin mRNA is ubiquitous (52), Axin2 is expressed in tissue- and developmental-stage-specific patterns, due (at least in part) to its transcriptional upregulation by canonical Wnt signaling (4, 21, 27). Thus, Axin2 potentially constitutes a negative-feedback mechanism regulating the response to Wnt signals (21, 27, 32, 50). In the mouse embryo, the inability of Axin2 to compensate for the lack of Axin might be explained simply by the fact that Axin2 is not expressed in every cell. However, the two proteins might also differ significantly in function, a possibility supported by their different subcellular localizations in at least some cell types (3). If the functional differences were subtle, they might not be detected using assays that involve overexpression; since the normal level of Axin is extremely low (26), the abnormally high levels of expression in these assays could mask a functional difference. To address the question of functional redundancy in a more physiological way, we generated knockin mice in which the Axin gene was replaced with an Axin2 cDNA sequence. This gene replacement strategy is similar to those used successfully to compare the functions of other paralogous gene pairs (6, 12, 15, 33, 41). The main advantage of this approach over in vitro assays or other in vivo functional assays (e.g., transgenic rescue) is that targeting of a cDNA to the appropriate genetic locus results in a normal pattern and level of expression. In animals homozygous for the resulting allele, AxinAx2, there will be no Axin, but Axin2 will be expressed ubiquitously from the Axin locus at a physiological level, as well as in its normal tissue-specific pattern from the unmodified Axin2 locus. Thus, the total level of Axin plus Axin2 expression in any cell type should be unchanged from that of the wild type. The ability of these mice to develop and survive normally thus tests the capacity of Axin2 to replace Axin in vivo.
-
mouse Axin and Axin2 conductin proteins are functionally equivalent in vivo
Molecular and Cellular Biology, 2005Co-Authors: Ian V. Chia, Frank CostantiniAbstract:Axin is a central component of the canonical Wnt signal transduction machinery, serving as a scaffold for the β-catenin destruction complex (23, 31, 36, 45, 52). Axin has specific binding sites for many proteins involved in Wnt signal transduction, including β-catenin, glycogen synthase kinase 3 (GSK3), CKI, adenomatous polyposis coli (APC), Dvl, LRP, and protein phosphatase 2A (Fig. (Fig.1A)1A) (23, 31). Its key function in this pathway is to bring together β-catenin and the protein kinases CKI and GSK3, thus promoting the phosphorylation and consequent destruction of β-catenin. In the presence of a Wnt signal, this function is overcome, allowing β-catenin to accumulate and enter the nucleus (9, 13). The mechanism by which a Wnt signal leads to the inactivation of the Axin complex is not entirely clear, but it is thought to involve the binding of Axin to the Wnt coreceptor LRP as well as to Dvl (7, 34, 42, 44, 45). This results in the dephosphorylation of Axin, leading to a decrease in its affinity for β-catenin, and in a decrease in the level of Axin (20, 47, 48). Axin also enters the nucleus and appears to play a role in the nuclear-cytoplasmic shuttling of β-catenin (8, 46). FIG.1. Targeted replacement of the Axin gene with myc-tagged Axin2 cDNA or myc-tagged Axin cDNA. (A) Schematic diagram of Axin and Axin2 proteins and binding partners. Percent similarities between the conserved RGS and DIX domains and the GSK3 and β-catenin ... The major product of the Axin gene is a protein of 832 or 868 amino acids (depending on alternative splicing) containing two highly conserved domains (52): the RGS domain, which encompasses the binding site for APC (16, 18), and the DIX domain, a region of homology with Dvl proteins that is implicated in the binding of Axin to Dvl as well as in homodimerization (10, 17, 22, 24, 29, 40). (The open reading frame of Axin cDNA potentially encodes proteins of 956 and 992 amino acids, but proteins of this length have not been detected, and the major protein begins at codon Met-125 of the original sequence.) Mutant embryos lacking Axin die at embryonic day 9.5 (E9.5) with abnormalities including truncation of the forebrain, neural tube defects, and embryonic axis duplications (14, 35). Axin is expressed ubiquitously during embryogenesis, and the presence of axis duplications in its absence is thought to be a consequence of the abnormal accumulation of β-catenin, mimicking a Wnt signal, in the early embryo. Axin2 (also known as Conductin) is 44% identical to Axin and shares the RGS and DIX domains (Fig. (Fig.1A)1A) (5, 39, 49) as well as the binding sites for β-catenin, GSK3, Diversin, and Smad3 (Fig. (Fig.1A)1A) (5, 11, 39, 49). While Axin2 has been studied less extensively than Axin, most data suggest that they are similar in function. Thus, when overexpressed in cultured cells, both proteins reduce the levels of β-catenin and the expression of Wnt target genes, and when expressed in frog embryos, both inhibit the development of dorsal structures (5, 19, 37, 49, 52). However, they are not fully redundant in vivo, as Axin2 is clearly unable to compensate for the absence of Axin in Axin-null embryos (52). Furthermore, humans with heterozygous germ line Axin2 mutations have familial tooth agenesis (25), while mice homozygous for loss of Axin2 have skull abnormalities (51), indicating that Axin does not fully substitute for the lack of Axin2. In addition, deletions or mutations in Axin or Axin2 have been observed in a number of types of tumors, including colorectal cancer and hepatocellular carcinoma, indicating that each gene is a tumor suppressor and that neither gene can replace the other for this function (30, 38, 43). One reason for the lack of full redundancy may be the different expression patterns of Axin and Axin2. While Axin mRNA is ubiquitous (52), Axin2 is expressed in tissue- and developmental-stage-specific patterns, due (at least in part) to its transcriptional upregulation by canonical Wnt signaling (4, 21, 27). Thus, Axin2 potentially constitutes a negative-feedback mechanism regulating the response to Wnt signals (21, 27, 32, 50). In the mouse embryo, the inability of Axin2 to compensate for the lack of Axin might be explained simply by the fact that Axin2 is not expressed in every cell. However, the two proteins might also differ significantly in function, a possibility supported by their different subcellular localizations in at least some cell types (3). If the functional differences were subtle, they might not be detected using assays that involve overexpression; since the normal level of Axin is extremely low (26), the abnormally high levels of expression in these assays could mask a functional difference. To address the question of functional redundancy in a more physiological way, we generated knockin mice in which the Axin gene was replaced with an Axin2 cDNA sequence. This gene replacement strategy is similar to those used successfully to compare the functions of other paralogous gene pairs (6, 12, 15, 33, 41). The main advantage of this approach over in vitro assays or other in vivo functional assays (e.g., transgenic rescue) is that targeting of a cDNA to the appropriate genetic locus results in a normal pattern and level of expression. In animals homozygous for the resulting allele, AxinAx2, there will be no Axin, but Axin2 will be expressed ubiquitously from the Axin locus at a physiological level, as well as in its normal tissue-specific pattern from the unmodified Axin2 locus. Thus, the total level of Axin plus Axin2 expression in any cell type should be unchanged from that of the wild type. The ability of these mice to develop and survive normally thus tests the capacity of Axin2 to replace Axin in vivo.
-
wnt β catenin tcf signaling induces the transcription of Axin2 a negative regulator of the signaling pathway
Molecular and Cellular Biology, 2002Co-Authors: Tong Zhang, Claire Domon, Jeannoel Freund, Frank CostantiniAbstract:Axin2/Conductin/Axil and its ortholog Axin are negative regulators of the Wnt signaling pathway, which promote the phosphorylation and degradation of beta-catenin. While Axin is expressed ubiquitously, Axin2 mRNA was seen in a restricted pattern during mouse embryogenesis and organogenesis. Because many sites of Axin2 expression overlapped with those of several Wnt genes, we tested whether Axin2 was induced by Wnt signaling. Endogenous Axin2 mRNA and protein expression could be rapidly induced by activation of the Wnt pathway, and Axin2 reporter constructs, containing a 5.6-kb DNA fragment including the promoter and first intron, were also induced. This genomic region contains eight Tcf/LEF consensus binding sites, five of which are located within longer, highly conserved noncoding sequences. The mutation or deletion of these Tcf/LEF sites greatly diminished induction by beta-catenin, and mutation of the Tcf/LEF site T2 abolished protein binding in an electrophoretic mobility shift assay. These results strongly suggest that Axin2 is a direct target of the Wnt pathway, mediated through Tcf/LEF factors. The 5.6-kb genomic sequence was sufficient to direct the tissue-specific expression of d2EGFP in transgenic embryos, consistent with a role for the Tcf/LEF sites and surrounding conserved sequences in the in vivo expression pattern of Axin2. Our results suggest that Axin2 participates in a negative feedback loop, which could serve to limit the duration or intensity of a Wnt-initiated signal.
-
wnt β catenin tcf signaling induces the transcription of Axin2 a negative regulator of the signaling pathway
Molecular and Cellular Biology, 2002Co-Authors: Eekhoon Jho, Tong Zhang, Claire Domon, Jeannoel Freund, Chounki Joo, Frank CostantiniAbstract:Axin2/Conductin/Axil and its ortholog Axin are negative regulators of the Wnt signaling pathway, which promote the phosphorylation and degradation of beta-catenin. While Axin is expressed ubiquitously, Axin2 mRNA was seen in a restricted pattern during mouse embryogenesis and organogenesis. Because many sites of Axin2 expression overlapped with those of several Wnt genes, we tested whether Axin2 was induced by Wnt signaling. Endogenous Axin2 mRNA and protein expression could be rapidly induced by activation of the Wnt pathway, and Axin2 reporter constructs, containing a 5.6-kb DNA fragment including the promoter and first intron, were also induced. This genomic region contains eight Tcf/LEF consensus binding sites, five of which are located within longer, highly conserved noncoding sequences. The mutation or deletion of these Tcf/LEF sites greatly diminished induction by beta-catenin, and mutation of the Tcf/LEF site T2 abolished protein binding in an electrophoretic mobility shift assay. These results strongly suggest that Axin2 is a direct target of the Wnt pathway, mediated through Tcf/LEF factors. The 5.6-kb genomic sequence was sufficient to direct the tissue-specific expression of d2EGFP in transgenic embryos, consistent with a role for the Tcf/LEF sites and surrounding conserved sequences in the in vivo expression pattern of Axin2. Our results suggest that Axin2 participates in a negative feedback loop, which could serve to limit the duration or intensity of a Wnt-initiated signal.
Stephen W. Wilson - One of the best experts on this subject based on the ideXlab platform.
-
Inhibition of Wnt/Axin/β-catenin pathway activity promotes ventral CNS midline tissue to adopt hypothalamic rather than floorplate identity
Development (Cambridge England), 2004Co-Authors: Marika Kapsimali, Corinne Houart, Luca Caneparo, Stephen W. WilsonAbstract:Ventral midline cells in the neural tube form floorplate throughout most of the central nervous system (CNS) but in the anterior forebrain, they differentiate with hypothalamic identity. The signalling pathways responsible for subdivision of midline neural tissue into hypothalamic and floorplate domains are uncertain, and in this study, we have explored the role of the Wnt/Axin/β-catenin pathway in this process. This pathway has been implicated in anteroposterior regionalisation of the dorsal neural tube but its role in patterning ventral midline tissue has not been rigorously assessed. We find that masterblind zebrafish embryos that carry a mutation in Axin1, an intracellular negative regulator of Wnt pathway activity, show an expansion of prospective floorplate coupled with a reduction of prospective hypothalamic tissue. Complementing this observation, transplantation of cells overexpressing Axin1 into the prospective floorplate leads to induction of hypothalamic gene expression and suppression of floorplate marker gene expression. Axin1 is more efficient at inducing hypothalamic markers than several other Wnt pathway antagonists, and we present data suggesting that this may be due to an ability to promote Nodal signalling in addition to suppressing Wnt activity. Indeed, extracellular Wnt antagonists can promote hypothalamic gene expression when co-expressed with a modified form of Madh2 that activates Nodal signalling. These results suggest that Nodal signalling promotes the ability of cells to incorporate into ventral midline tissue, and within this tissue, antagonism of Wnt signalling promotes the acquisition of hypothalamic identity. Wnt signalling also affects patterning within the hypothalamus, suggesting that this pathway is involved in both the initial anteroposterior subdivision of ventral CNS midline fates and in the subsequent regionalisation of the hypothalamus. We suggest that by regulating the response of midline cells to signals that induce ventral fates, Axin1 and other modulators of Wnt pathway activity provide a mechanism by which cells can integrate dorsoventral and anteroposterior patterning information.
-
inhibition of wnt Axin β catenin pathway activity promotes ventral cns midline tissue to adopt hypothalamic rather than floorplate identity
Development, 2004Co-Authors: Marika Kapsimali, Corinne Houart, Luca Caneparo, Stephen W. WilsonAbstract:Ventral midline cells in the neural tube form floorplate throughout most of the central nervous system (CNS) but in the anterior forebrain, they differentiate with hypothalamic identity. The signalling pathways responsible for subdivision of midline neural tissue into hypothalamic and floorplate domains are uncertain, and in this study, we have explored the role of the Wnt/Axin/β-catenin pathway in this process. This pathway has been implicated in anteroposterior regionalisation of the dorsal neural tube but its role in patterning ventral midline tissue has not been rigorously assessed. We find that masterblind zebrafish embryos that carry a mutation in Axin1, an intracellular negative regulator of Wnt pathway activity, show an expansion of prospective floorplate coupled with a reduction of prospective hypothalamic tissue. Complementing this observation, transplantation of cells overexpressing Axin1 into the prospective floorplate leads to induction of hypothalamic gene expression and suppression of floorplate marker gene expression. Axin1 is more efficient at inducing hypothalamic markers than several other Wnt pathway antagonists, and we present data suggesting that this may be due to an ability to promote Nodal signalling in addition to suppressing Wnt activity. Indeed, extracellular Wnt antagonists can promote hypothalamic gene expression when co-expressed with a modified form of Madh2 that activates Nodal signalling. These results suggest that Nodal signalling promotes the ability of cells to incorporate into ventral midline tissue, and within this tissue, antagonism of Wnt signalling promotes the acquisition of hypothalamic identity. Wnt signalling also affects patterning within the hypothalamus, suggesting that this pathway is involved in both the initial anteroposterior subdivision of ventral CNS midline fates and in the subsequent regionalisation of the hypothalamus. We suggest that by regulating the response of midline cells to signals that induce ventral fates, Axin1 and other modulators of Wnt pathway activity provide a mechanism by which cells can integrate dorsoventral and anteroposterior patterning information.
Jiahuai Han - One of the best experts on this subject based on the ideXlab platform.
-
a β catenin independent dorsalization pathway activated by Axin jnk signaling and antagonized by aida
Developmental Cell, 2007Co-Authors: Yanning Rui, Wen Luo, Siu Chiu Chan, Bo Xiong, Ying Cao, Shu-yong Lin, Min Zhang, Ying Han, Haimeng Zhou, Jiahuai HanAbstract:Axin is a scaffold protein that controls multiple important pathways, including the canonical Wnt pathway and JNK signaling. Here we have identified an Axin-interacting protein, Aida, which blocks Axin-mediated JNK activation by disrupting Axin homodimerization. During investigation of in vivo functions of Axin/JNK signaling and aida in development, it was found that Axin, besides ventralizing activity by facilitating β-catenin degradation, possesses a dorsalizing activity that is mediated by Axin-induced JNK activation. This dorsalizing activity is repressed when aida is overexpressed in zebrafish embryos. Whereas Aida-MO injection leads to dorsalized embryos, JNK-MO and MKK4-MO can ventralize embryos. The anti-dorsalization activity of aida is conferred by its ability to block Axin-mediated JNK activity. We further demonstrate that dorsoventral patterning regulated by Axin/JNK signaling is independent of maternal or zygotic Wnt signaling. We have thus identified a dorsalization pathway that is exerted by Axin/JNK signaling and its inhibitor Aida during vertebrate embryogenesis.
-
Axin is a scaffold protein in tgf β signaling that promotes degradation of smad7 by arkadia
The EMBO Journal, 2006Co-Authors: Wei Liu, Siu Chiu Chan, Shu-yong Lin, Jiahuai Han, Hong-liang Rui, Jifeng Wang, Mingliang Chen, Suping Zhang, Yeguang ChenAbstract:TGF-β signaling involves a wide array of signaling molecules and multiple controlling events. Scaffold proteins create a functional proximity of signaling molecules and control the specificity of signal transduction. While many components involved in the TGF-β pathway have been elucidated, little is known about how those components are coordinated by scaffold proteins. Here, we show that Axin activates TGF-β signaling by forming a multimeric complex consisting of Smad7 and ubiquitin E3 ligase Arkadia. Axin depends on Arkadia to facilitate TGF-β signaling, as their small interfering RNAs reciprocally abolished the stimulatory effect on TGF-β signaling. Specific knockdown of Axin or Arkadia revealed that Axin and Arkadia cooperate with each other in promoting Smad7 ubiquitination. Pulse-chase experiments further illustrated that Axin significantly decreased the half-life of Smad7. Axin also induces nuclear export of Smad7. Interestingly, Axin associates with Arkadia and Smad7 independently of TGF-β signal, in contrast to its transient association with inactive Smad3. However, coexpression of Wnt-1 reduced Smad7 ubiquitination by downregulating Axin levels, underscoring the importance of Axin as an intrinsic regulator in TGF-β signaling.
-
Axin utilizes distinct regions for competitive MEKK1 and MEKK4 binding and JNK activation.
The Journal of biological chemistry, 2003Co-Authors: Wen Luo, Lihua Jin, Jiahuai Han, Sheng-cai LinAbstract:Abstract Axin is a multidomain protein that plays a critical role in Wnt signaling, serving as a scaffold for down-regulation of β-catenin. It also activates the JNK mitogen-activated protein kinase by binding to MEKK1. However, it is intriguing that Axin requires several additional elements for JNK activation, including a requirement for homodimerization, sumoylation at the extreme C-terminal sites, and a region in the protein phosphatase 2A-binding domain. In our present study, we have shown that another MEKK family member, MEKK4, also binds to Axin in vivo and mediates Axin-induced JNK activation. Surprisingly MEKK4 binds to a region distinct from the MEKK1-binding site. Dominant negative mutant of MEKK4 attenuates the JNK activation by Axin. Activation of JNK by Axin in MEKK1–/– mouse embryonic fibroblast cells supports the idea that another MEKK can mediate Axin-induced JNK activation. Expression of specific small interfering RNA against MEKK4 effectively attenuates JNK activation by the MEKK1 binding-defective Axin mutant in 293T cells and inhibits JNK activation by wild-type Axin in MEKK1–/– cells, confirming that MEKK4 is indeed another mitogen-activated protein kinase kinase kinase that is specifically involved in Axin-mediated JNK activation independently of MEKK1. We have also identified an additional domain between MEKK1- and MEKK4-binding sites as being required for JNK activation by Axin. MEKK1 and MEKK4 compete for Axin binding even though they bind to sites far apart, suggesting that Axin may selectively bind to MEKK1 or MEKK4 depending on distinct signals or cellular context. Our findings will provide new insights into how scaffold proteins mediate ultimate activation of different mitogen-activated protein kinase kinase kinases.
-
dimerization choices control the ability of Axin and dishevelled to activate c jun n terminal kinase stress activated protein kinase
Journal of Biological Chemistry, 2000Co-Authors: Yi Zhang, Jiahuai Han, Soek Ying Neo, Sheng-cai LinAbstract:Axin and Dishevelled are two downstream components of the Wnt signaling pathway. Dishevelled is a positive regulator and is placed genetically between Frizzled and glycogen synthase kinase-3beta, whereas Axin is a negative regulator that acts downstream of glycogen synthase kinase-3beta. It is intriguing that they each can activate the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) when expressed in the cell. We set out to address if Axin and Dishevelled are functionally cooperative, antagonistic, or entirely independent, in terms of the JNK activation event. We found that in contrast to Axin, Dvl2 activation of JNK does not require MEKK1, and complex formation between Dvl2 and Axin is independent of Axin-MEKK1 binding. Furthermore, Dvl2-DIX and Dvl2-DeltaDEP proteins deficient for JNK activation can attenuate Axin-activated JNK activity by disrupting Axin dimerization. However, Axin-DeltaMID, Axin-DeltaC, and Axin-CT proteins deficient for JNK activation cannot interfere with Dvl2-activated JNK activity. These results indicate that unlike the strict requirement of homodimerization for Axin function, Dvl2 can activate JNK either as a monomer or homodimer/heterodimer. We suggest that there may be a switch mechanism based on dimerization combinations, that commands cells to activate Wnt signaling or JNK activation, and to turn on specific activators of JNK in response to various environmental cues.
Sheng-cai Lin - One of the best experts on this subject based on the ideXlab platform.
-
Determining AMPK Activation via the Lysosomal v-ATPase-Ragulator-Axin/LKB1 Axis
Methods in molecular biology (Clifton N.J.), 2018Co-Authors: Chen-song Zhang, Yue Zong, Sheng-cai LinAbstract:Recent studies have revealed how AMPK is activated inside the cell and animal tissues: in response to low glucose, Axin tethers LKB1, by virtue of their constitutive association, to AMPK located on the surface of late endosome/lysosome. Importantly, the lysosomal v-ATPase (vacuolar ATPase)-Ragulator complex, when primed by glucose starvation or concanamycin A, facilitates Axin/LKB1 to interact with AMPK. Here, we describe the experimental procedures of the assays for detecting the translocation of Axin/LKB1 or the assembly of the Axin-based AMPK-activating complexes on the late endosome/lysosome. The methods in this chapter will be useful for determining whether various metabolic stresses or pharmacological stimuli activate AMPK via the v-ATPase-Ragulator-Axin/LKB1 axis, which also concomitantly inactivates mTORC1. Detailed protocols for determining the levels of adenylates are also described.
-
cdk5 mediated phosphorylation of Axin directs axon formation during cerebral cortex development
The Journal of Neuroscience, 2011Co-Authors: Weiqun Fang, Sheng-cai Lin, Yu ChenAbstract:Axon formation is critical for the establishment of connections between neurons, which is a prerequisite for the development of neural circuitry. Kinases such as cyclin-dependent kinase 5 (Cdk5) and glycogen synthase kinase-3β (GSK-3β), have been implicated to regulate axon outgrowth. Nonetheless, the in vivo roles of these kinases in axon development and the underlying signaling mechanisms remain essentially unknown. We report here that Cdk5 is important for axon formation in mouse cerebral cortex through regulating the functions of axis inhibitor (Axin), a scaffold protein of the canonical Wnt pathway. Knockdown of Axin in utero abolishes the formation and projection of axons. Importantly, Axin is phosphorylated by Cdk5, and this phosphorylation facilitates the interaction of Axin with GSK-3β, resulting in inhibition of GSK-3β activity and dephosphorylation of its substrate collapsin response mediator protein-2 (CRMP-2), a microtubule-associated protein. Specifically, both phosphorylation of Axin and its interaction with GSK-3β are critically required for axon formation in mouse cortex development. Together, our findings reveal a new regulatory mechanism of axon formation through Cdk5-dependent phosphorylation of Axin.
-
Axin is an essential co-activator for the promyelocytic leukemia protein in p53 activation
Oncogene, 2010Co-Authors: Wei L, Shu-yong Lin, Wu X, Zhen Wang, Sheng-cai LinAbstract:The PML protein is best known for its role as a tumor suppressor for acute promyelocytic leukemia. Both PML and the key Wnt signaling regulator Axin regulate p53-dependent apoptosis in response to DNA damage. However, how the two major tumor suppressors coordinate with each other is unknown, and the molecular components orchestrating the PML-induced apoptosis remain enigmatic. Here we show that Axin interacts with PML in vivo, and further that Axin, PML and p53 form a ternary complex. Exposure to genotoxic signals including UV and doxorubicin induces Axin to enter into the nucleus where it colocalizes with PML in the nuclear bodies. Domain-mapping experiments revealed that the C-terminal region (aa 597-832) of Axin is responsible for its interaction with PML. Axin fails to activate p53 in PML(-/-) cells, and conversely, PML is unable to activate p53 in Axin-null SNU475 cells. Consistently, knockdown with respective siRNAs revealed that Axin and PML depend on each other to elevate p53-Ser-46 phosphorylation and to induce apoptosis after treatment with genotoxins. Moreover, we found that dominant-negative mutants of PML blocked Axin-induced p53 activation, and that Axin promotes PML sumoylation, a modification necessary for PML functions. Our finding has thus provided a new avenue for understanding the mechanism by which PML activates p53 and exerts its role as a tumor suppressor.
-
Construction of the eukaryotic expression vector pIRES2-EGFP-Axin and its expression in glioma cells
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology, 2005Co-Authors: Liying Zhang, Sheng-cai Lin, Guang-sheng Chen, Feng ZhangAbstract:AIM: To construct the eukaryotic expression vector pIRES2-EGFP-Axin, and to express Axin in C6 glioma cells. METHODS: The Axin gene was amplified by PCR using pCMV5-HA-Axin as a template, and confirmed by DNA sequencing. The eukaryotic expression vector pIRES2-EGFP-Axin was constructed by introducing Axin DNA fragment into the sites of Nhe I and Sal I of pIRES2-EGFP vector. The plasmid was transfected into the C6 cells using lipofectamine. The expressed EGFP was observed under fluorescent microscope and the Axin protein expression was detected by immunostaining using anti-Axin antibody. RESULTS: The eukaryotic expression vector pIRES2-EGFP-Axin was constructed and transfected successfully into C6 glioma cells. The green fluorescence of EGFP was observed in the plasma and nuclei of transfected cells, and Axin protein was only found in the plasma. CONCLUSION: The recombinant expression vector pIRES2-EGFP-Axin was constructed, and the EGFP and Axin gene could be co-expressed in the C6 cells. This study laid a foundation for the further research of the function of Axin in cell differentiation, growth and tumorigenesis.
-
Axin Contains Three Separable Domains That Confer Intramolecular, Homodimeric, and Heterodimeric Interactions Involved in Distinct Functions
The Journal of biological chemistry, 2004Co-Authors: Wen Luo, Lihua Jin, Shu-yong Lin, Hong-liang Rui, Haiying Zou, Sheng-cai LinAbstract:Abstract Axin is a major scaffold protein, interacting with diverse molecules involved in a number of signaling pathways. Axin can undergo dimer/oligomerization via its DIX domain. Here we show that whereas deletion of the DIX domain at the C terminus rendered Axin incapable of forming dimer, a larger deletion of the C-terminal region restored the ability of Axin to form dimers. Detailed analyses revealed that Axin actually contains two separate domains (D and I) in addition to the DIX domain for homodimerization. The D, I, and DIX domains alone can form homodimers. Interestingly, D and I domains strongly interact with each other, suggesting that Axin can form an intramolecular structure through D and I interaction in the absence of DIX. We also found that DIX-DIX homodimeric interaction is weak but that point mutations in the DIX domain abolished Axin homodimerization. We propose a model to suggest that Axin forms homodimeric interactions through three domains, D, I, and DIX. More importantly, lack of DIX-DIX interaction caused by point mutations in the DIX domain or deletion causes Axin to form an intramolecular loop through the D and I domains, disallowing homodimer formation. Ccd1 interacts with Axin D domain yet fails to interact with AxinΔDIX, confirming that D is masked after D-I looping. The Axin mutants that are defective in homodimer formation fail to activate JNK but have no effect on β-catenin signaling. Our findings have thus provided a structural basis of conformational changes in Axin, which may underlie the diversity of Axin functions.