Axin Facilitates Smad3 Activation in the Transforming Growth Factor β Signaling Pathway

ABSTRACT 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.

[1]  山本 英樹.  Axil, a member of the Axin family, interacts with both glycogen synthase kinase 3β and β-catenin and inhibits Axis formation of Xenopus embryos , 2001 .

[2]  Kohei Miyazono,et al.  TGF-β signalling from cell membrane to nucleus through SMAD proteins , 1997, Nature.

[3]  J. Massagué,et al.  TGF- SIGNAL TRANSDUCTION , 1998 .

[4]  A. Kikuchi,et al.  Modulation of Wnt signaling by Axin and Axil. , 1999, Cytokine & growth factor reviews.

[5]  Hideki Yamamoto,et al.  Phosphorylation of Axin, a Wnt Signal Negative Regulator, by Glycogen Synthase Kinase-3β Regulates Its Stability* , 1999, The Journal of Biological Chemistry.

[6]  C. Hill,et al.  Transforming Growth Factor β-Independent Shuttling of Smad4 between the Cytoplasm and Nucleus , 2000, Molecular and Cellular Biology.

[7]  K. Miyazono,et al.  Smad6 Is a Smad1/5-induced Smad Inhibitor , 2000, The Journal of Biological Chemistry.

[8]  K. Kaestner,et al.  Unified nomenclature for the winged helix/forkhead transcription factors. , 2000, Genes & development.

[9]  R. Derynck,et al.  Smads: transcriptional activators of TGF-beta responses. , 1998, Cell.

[10]  H. Usui,et al.  GSK-3β-dependent phosphorylation of adenomatous polyposis coli gene product can be modulated by β-catenin and protein phosphatase 2A complexed with Axin , 2000, Oncogene.

[11]  J. Massagué,et al.  The nuclear import function of Smad2 is masked by SARA and unmasked by TGFb-dependent phosphorylation , 2000, Nature Cell Biology.

[12]  A. Antipova,et al.  Interaction of Dishevelled and XenopusAxin-Related Protein Is Required for Wnt Signal Transduction , 2000, Molecular and Cellular Biology.

[13]  Akira Kikuchi,et al.  DIX Domains of Dvl and Axin Are Necessary for Protein Interactions and Their Ability To Regulate β-Catenin Stability , 1999, Molecular and Cellular Biology.

[14]  Naoto Ueno,et al.  Interaction between Wnt and TGF-β signalling pathways during formation of Spemann's organizer , 2000, Nature.

[15]  W. Birchmeier,et al.  Functional interaction of an axin homolog, conductin, with beta-catenin, APC, and GSK3beta. , 1998, Science.

[16]  Xin-Hua Feng,et al.  Microtubule Binding to Smads May Regulate TGFβ Activity , 2000 .

[17]  Marc W. Kirschner,et al.  Control of b-Catenin Stability : Reconstitution of the Cytoplasmic Steps of the Wnt Pathway in Xenopus Egg Extracts , 2000 .

[18]  J. Wrana,et al.  The MAD-Related Protein Smad7 Associates with the TGFβ Receptor and Functions as an Antagonist of TGFβ Signaling , 1997, Cell.

[19]  T. Dale,et al.  Interaction of Axin and Dvl‐2 proteins regulates Dvl‐2‐stimulated TCF‐dependent transcription , 1999, The EMBO journal.

[20]  Wei Hsu,et al.  The Mouse Fused Locus Encodes Axin, an Inhibitor of the Wnt Signaling Pathway That Regulates Embryonic Axis Formation , 1997, Cell.

[21]  T. Akiyama.  Wnt/beta-catenin signaling. , 2000, Cytokine & growth factor reviews.

[22]  R. Nusse,et al.  Mechanisms of Wnt signaling in development. , 1998, Annual review of cell and developmental biology.

[23]  F. Costantini,et al.  Identification of a Domain of Axin That Binds to the Serine/Threonine Protein Phosphatase 2A and a Self-binding Domain* , 1999, The Journal of Biological Chemistry.

[24]  A. Kikuchi,et al.  Axin prevents Wnt-3a-induced accumulation of β-catenin , 1999, Oncogene.

[25]  K. Miyazono,et al.  Characterization of bone morphogenetic protein-6 signaling pathways in osteoblast differentiation. , 1999, Journal of cell science.

[26]  Yusuke Nakamura,et al.  AXIN1 mutations in hepatocellular carcinomas, and growth suppression in cancer cells by virus-mediated transfer of AXIN1 , 2000, Nature Genetics.

[27]  八木 健,et al.  Alternatively spliced variant of smad2 lacking exon 3 : comparison with wild-type smad2 and smad3 , 2001 .

[28]  H. Clevers,et al.  Linking Colorectal Cancer to Wnt Signaling , 2000, Cell.

[29]  M. Kirschner,et al.  Control of beta-catenin stability: reconstitution of the cytoplasmic steps of the wnt pathway in Xenopus egg extracts. , 2000, Molecular cell.

[30]  T. Musci,et al.  The tumor suppressor Smad4/DPC 4 as a central mediator of Smad function , 1997, Current Biology.

[31]  K. Miyazono,et al.  A single missense mutant of Smad3 inhibits activation of both Smad2 and Smad3, and has a dominant negative effect on TGF‐β signals , 1998, FEBS letters.

[32]  R. Derynck,et al.  Transcriptional Activators of TGF-β Responses: Smads , 1998, Cell.

[33]  Akira Kikuchi,et al.  Axil, a Member of the Axin Family, Interacts with Both Glycogen Synthase Kinase 3β and β-Catenin and Inhibits Axis Formation ofXenopus Embryos , 1998, Molecular and Cellular Biology.

[34]  Akira Kikuchi,et al.  Axin, a negative regulator of the Wnt signaling pathway, forms a complex with GSK‐3β and β‐catenin and promotes GSK‐3β‐dependent phosphorylation of β‐catenin , 1998 .

[35]  J. Massagué,et al.  Disruption of transforming growth factor beta signaling by a mutation that prevents transphosphorylation within the receptor complex , 1995, Molecular and cellular biology.

[36]  J. Massagué,et al.  Structural basis of Smad2 recognition by the Smad anchor for receptor activation. , 2000, Science.

[37]  P. Polakis.  Wnt signaling and cancer. , 2000, Genes & development.

[38]  Jeffrey L. Wrana,et al.  Hgs (Hrs), a FYVE Domain Protein, Is Involved in Smad Signaling through Cooperation with SARA , 2000, Molecular and Cellular Biology.

[39]  Harold L. Moses,et al.  STRAP and Smad7 Synergize in the Inhibition of Transforming Growth Factor β Signaling , 2000, Molecular and Cellular Biology.

[40]  Randall T Moon,et al.  Mechanism and function of signal transduction by the Wnt/β-catenin and Wnt/Ca2+ pathways , 1999, Oncogene.

[41]  J. Massagué.  TGF-beta signal transduction. , 1998, Annual review of biochemistry.

[42]  C. Heldin,et al.  Expression of Smad proteins in human colorectal cancer , 1999, International journal of cancer.

[43]  C. Kaufmann,et al.  Domains of Axin Involved in Protein–Protein Interactions, Wnt Pathway Inhibition, and Intracellular Localization , 1999, The Journal of cell biology.

[44]  L. Attisano,et al.  Association of Smads with lymphoid enhancer binding factor 1/T cell-specific factor mediates cooperative signaling by the transforming growth factor-beta and wnt pathways. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[45]  K. Miyazono.  TGF-β signaling by Smad proteins , 2000 .

[46]  Takeshi Imamura,et al.  Smad proteins exist as monomers in vivo and undergo homo‐ and hetero‐oligomerization upon activation by serine/threonine kinase receptors , 1998, The EMBO journal.

[47]  Liliana Attisano,et al.  SARA, a FYVE Domain Protein that Recruits Smad2 to the TGFβ Receptor , 1998, Cell.

[48]  Hideki Yamamoto,et al.  Axin, a Negative Regulator of the Wnt Signaling Pathway, Directly Interacts with Adenomatous Polyposis Coli and Regulates the Stabilization of β-Catenin* , 1998, The Journal of Biological Chemistry.

[49]  H. Lodish,et al.  Importin β Mediates Nuclear Translocation of Smad 3* , 2000, The Journal of Biological Chemistry.