The DIX Domain Protein Coiled-coil-DIX1 Inhibits c-Jun N-terminal Kinase Activation by Axin and Dishevelled through Distinct Mechanisms

Abstract

Axin, Ccd1 (coiled-coil-DIX1), and dishevelled (Dvl or Dsh) are three known DIX domain proteins that play important roles in Wnt signaling. In addition, Dvl and Axin can activate the mitogen-activated protein kinase JNK via distinct mechanisms, through interaction with MEKK1/4 and Rac GTPase, respectively. Axin utilizes two distinct domains for interaction with MEKK1 and MEKK4. JNK activation by Axin is regulated by several factors in the Wnt pathway, whereas little is known about cross-regulation of Dvl-mediated JNK activation. In the present study, we have investigated whether Ccd1 could play a regulatory role in Axin- and Dvl-mediated JNK activation. Here we show that Ccd1 drastically inhibited JNK activation both by Axin and by Dvl. Although DIX domains are sufficient for dimer formation between Dvl and Ccd1, Ccd1 also required its coiled-coil domain for inhibition of JNK activation by Dvl. Interestingly, Rac remained associated with Dvl heterodimerized with Ccd1. How Ccd1 blocks Rac/Dvl signaling to JNK is unclear. In contrast, Axin, when complexed with Ccd1, did not bind to MEKK1. Furthermore, Ccd1 physically interacted with MEKK4 in their physiological concentrations and prevented MEKK4 from binding to Axin. Reduction of Ccd1 protein by small interfering RNA could elevate JNK signaling as assayed with an AP1-dependent transcriptional reporter. We have therefore demonstrated that Ccd1 inhibits Axin-mediated JNK activation by simultaneously adopting two distinct mechanisms, one through conformational changes that disallow MEKK1 binding and the other via direct sequestration of MEKK4. Axin, Ccd1 (coiled-coil-DIX1), and dishevelled (Dvl or Dsh) are three known DIX domain proteins that play important roles in Wnt signaling. In addition, Dvl and Axin can activate the mitogen-activated protein kinase JNK via distinct mechanisms, through interaction with MEKK1/4 and Rac GTPase, respectively. Axin utilizes two distinct domains for interaction with MEKK1 and MEKK4. JNK activation by Axin is regulated by several factors in the Wnt pathway, whereas little is known about cross-regulation of Dvl-mediated JNK activation. In the present study, we have investigated whether Ccd1 could play a regulatory role in Axin- and Dvl-mediated JNK activation. Here we show that Ccd1 drastically inhibited JNK activation both by Axin and by Dvl. Although DIX domains are sufficient for dimer formation between Dvl and Ccd1, Ccd1 also required its coiled-coil domain for inhibition of JNK activation by Dvl. Interestingly, Rac remained associated with Dvl heterodimerized with Ccd1. How Ccd1 blocks Rac/Dvl signaling to JNK is unclear. In contrast, Axin, when complexed with Ccd1, did not bind to MEKK1. Furthermore, Ccd1 physically interacted with MEKK4 in their physiological concentrations and prevented MEKK4 from binding to Axin. Reduction of Ccd1 protein by small interfering RNA could elevate JNK signaling as assayed with an AP1-dependent transcriptional reporter. We have therefore demonstrated that Ccd1 inhibits Axin-mediated JNK activation by simultaneously adopting two distinct mechanisms, one through conformational changes that disallow MEKK1 binding and the other via direct sequestration of MEKK4. The Wnt signaling pathway is one of the major developmental signaling pathways regulating important processes such as cell proliferation, polarity, and specification of cell fate (1Wodarz A. Nusse R. Annu. Rev. Cell Dev. Biol. 1998; 14: 59-88Crossref PubMed Scopus (1743) Google Scholar, 2Polakis P. Genes Dev. 2000; 14: 1837-1851Crossref PubMed Google Scholar). It consists of a large array of signaling molecules that allow convergence and divergence of signaling events. Dvl, 1The abbreviations used are: Dvl, dishevelled; Ccd1, coiled-coil-DIX1; DIX, Dvl/Axin homologous domain; EGFP, enhanced green fluorescent protein; GST, glutathione S-transferase; HA, hemagglutinin; HEK, human embryonic kidney; JNK, c-Jun N-terminal kinase; Luc, luciferase; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase; siRNA, small interfering RNA; MEF, mouse embryonic fibroblasts. Axin, and Ccd1 are three known DIX domain-containing proteins that play important roles in the Wnt signaling pathway (3Klingensmith J. Nusse R. Perrimon N. Genes Dev. 1994; 8: 118-130Crossref PubMed Scopus (344) Google Scholar, 4Zeng L. Fagotto F. Zhang T. Hsu W. Vasicek T.J. Perry W.L. Lee J.J. Tilghman S.M. Gumbiner B.M. Costantini F. Cell. 1997; 90: 181-192Abstract Full Text Full Text PDF PubMed Scopus (797) Google Scholar, 5Shiomi K. Uchida H. Keino-masu K. Masu M. Curr. Biol. 2003; 13: 73-77Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). In this pathway, Dvl is an intracellular protein and transduces signals from the Wnt receptor frizzled to Axin through heterodimer formation via the DIX domain (6Li L. Yuan H. Weaver C.D. Mao J. Farr G.H. Sussman D.J. Jonkers J. Kimelman D. Wu D. EMBO J. 1999; 18: 4233-4240Crossref PubMed Scopus (359) Google Scholar). Axin forms a multimeric protein complex with, in addition to Dvl, adenomatous polyposis coli, glycogen synthase kinase-3β, and β-catenin (7Kishida S. Yamamoto H. Ikeda S. Kishida M. Sakamoto I. Koyama S. Kikuchi A. J. Biol. Chem. 1998; 273: 10823-10826Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 8Hart M.J. de los Santos R. Albert I.N. Rubinfeld B. Polakis P. Curr. Biol. 1998; 8: 573-581Abstract Full Text Full Text PDF PubMed Google Scholar, 9Behrens J. Jerchow B.A. Wurtele M. Grimm J. Asbrand C. Wirtz R. Kuhl M. Wedlich D. Birchmeier W. Science. 1998; 280: 596-599Crossref PubMed Scopus (1118) Google Scholar). This complex enhances phosphorylation by glycogen synthase kinase-3β and subsequent ubiquitination and degradation of β-catenin (10Ikeda S. Kishida S. Yamamoto H. Murai H. Koyama S. Kikuchi A. EMBO J. 1998; 17: 1371-1384Crossref PubMed Scopus (1105) Google Scholar, 11Liu C. Li Y. Semenov M. Han C. Baeg G.H. Tan Y. Zhang Z. Lin X. He X. Cell. 2002; 108: 837-847Abstract Full Text Full Text PDF PubMed Scopus (1687) Google Scholar). Ccd1, a recently identified protein with a DIX domain, is a positive regulator in the Wnt signaling pathway during zebrafish neural patterning (5Shiomi K. Uchida H. Keino-masu K. Masu M. Curr. Biol. 2003; 13: 73-77Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). It functions downstream of the Wnt receptor and upstream of Axin. Ccd1 can activate T-cell factors-dependent transcription in a transient transfection reporter assay. Cotransfection of the full-length Ccd1 resulted in TOPtkLuciferase reporter transcriptional activation comparable with the activation by Dvl expression. Overexpression of Ccd1 in zebrafish embryos caused a reduction in the size of the eyes and forebrain, which is similar to the phenotype of excessive activation of Wnt signaling by overexpression of Wnt8 or its receptor in zebrafish. Ccd1 also possesses a coiled-coil domain, deletion of which enables Ccd1 to function as a dominant-negative suppressor downstream of Wnt receptor activation. The molecular mechanism underlying how Ccd1 exerts its positive role in Wnt signaling is not clear. Immunoprecipitation analysis revealed that Ccd1 forms homodimers via its DIX domain and coiled-coil domain and heterodimeric complexes with Dvl via their DIX domains. It is likely that Ccd1 regulates the Wnt signaling through its association with Axin and Dvl or by modulating the interaction between Axin and Dvl. It is interesting that Axin and Dvl both activate the c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SPAK) when overexpressed in a variety of cells even though they utilize different mechanisms for JNK activation (12Zhang Y. Neo S.Y. Wang X. Han J. Lin S.-C. J. Biol. Chem. 1999; 274: 35247-35254Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 13Boutros M. Paricio N. Strutt D.I. Mlodzik M. Cell. 1998; 94: 109-118Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). Axin and Dvl discriminate distinct pathways by differential domain utilizations (13Boutros M. Paricio N. Strutt D.I. Mlodzik M. Cell. 1998; 94: 109-118Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar, 14Zhang Y. Qiu W.-J. Liu D.-X. Neo S.Y. He X. Lin S.-C. J. Biol. Chem. 2001; 276: 32152-32159Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 15Axelrod J.D. Miller J.R. Shulman J.M. Moon R.T. Perrimon N. Genes Dev. 1998; 12: 2610-2622Crossref PubMed Scopus (543) Google Scholar). In particular, Axin requires several structural aspects to activate JNK (12Zhang Y. Neo S.Y. Wang X. Han J. Lin S.-C. J. Biol. Chem. 1999; 274: 35247-35254Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 16Rui H.L. Fan E. Zhou H.M. Xu Z. Zhang Y. Lin S.-C. J. Biol. Chem. 2002; 277: 42981-42986Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 17Luo W. Ng W.W. Jin L.-H. Ye Z.Y. Han J. Lin S.-C. J. Biol. Chem. 2003; 278: 37451-37458Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). First, Axin possesses two separate domains for MEKK1 and MEKK4 binding. The MEKK1 or MEKK4 binding domain combines with a domain of unknown nature (domain X), DIX homodimerization domain, and the extreme C-terminal sumoylation motifs to activate JNK. In addition to its well established role in Wnt signaling, Dvl also exerts a critical role in the generation of epithelial planar polarity in Drosophila, which depends on its ability to activate JNK (13Boutros M. Paricio N. Strutt D.I. Mlodzik M. Cell. 1998; 94: 109-118Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). Although MEKK1/4 play essential roles in Axin-mediated JNK activation (12Zhang Y. Neo S.Y. Wang X. Han J. Lin S.-C. J. Biol. Chem. 1999; 274: 35247-35254Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 17Luo W. Ng W.W. Jin L.-H. Ye Z.Y. Han J. Lin S.-C. J. Biol. Chem. 2003; 278: 37451-37458Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), Dvl instead seems to form a complex with the small GTPase Rac via its C-terminal DEP domain upon Wnt/Fz signaling (18Habas R. Dawid I.B. He X. Genes Dev. 2003; 17: 295-309Crossref PubMed Scopus (485) Google Scholar) and activates MKK7 in the Dvl/JNK pathway (19Moriguchi T. Kawachi K. Kamakura S. Masuyama N. Yamanaka H. Matsumoto K. Kikuchi A. Nishida E. J. Biol. Chem. 1999; 274: 30957-30962Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). An intriguing property of Axin in JNK activation is that its conformation for MEKK1 binding is highly regulated. Upon binding to Axin, the Wnt signaling components glycogen synthase kinase-3β and casein kinase Iα/ϵ inhibit Axin-mediated JNK activation by preventing MEKK1 binding to Axin (14Zhang Y. Qiu W.-J. Liu D.-X. Neo S.Y. He X. Lin S.-C. J. Biol. Chem. 2001; 276: 32152-32159Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, 20Zhang Y. Qiu W.J. Chan S.C. Han J. He X. Lin S.-C. J. Biol. Chem. 2002; 277: 17706-17712Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). It should be noted here that glycogen synthase kinase-3β and casein kinase Iα/ϵ both bind to domains outside the MEKK1 binding site, underscoring involvement of conformational changes in differential Axin complex formations. We that Axin-mediated JNK activation is by Dvl heterodimer formation through its DIX domains Y. Neo S.Y. Han J. Lin S.-C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). and both of which the DIX domain and are for JNK can JNK activation by Axin In the present study, we whether Ccd1, the DIX protein that a positive role in Wnt signaling, could also JNK activation by Axin as well as Dvl. We have a full-length human Ccd1 protein the and a of deletion It revealed here that Ccd1 inhibited JNK activation by Axin and Dvl. Although DIX domains are sufficient for Dvl and Ccd1 to form a the coiled-coil domain of Ccd1 is also required for its inhibition of JNK activation by Dvl. Interestingly, Ccd1, Dvl, and Rac form a complex when in the that in the complex prevented Rac from or downstream We that Axin utilizes a domain its DIX domain to with Ccd1, to interaction DIX we that Ccd1 binding to Axin prevented MEKK1 binding to the Axin complex and that Ccd1 a complex with MEKK4 and to Axin that Ccd1 inhibits Axin-mediated JNK activation by simultaneously MEKK1 binding and sequestration of MEKK4. Cell embryonic cells in as (12Zhang Y. Neo S.Y. Wang X. Han J. Lin S.-C. J. Biol. Chem. 1999; 274: 35247-35254Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). embryonic cells a from M. of which as Y. C. B. Li E. J. M. S. A. 2000; PubMed Scopus Google Scholar) and in with and of human full-length Ccd1 from a by It or its which by an the by of the The Ccd1 the deletion of Ccd1 or the of Ccd1 the and to the or by and the by by with and the by and Axin deletion as (12Zhang Y. Neo S.Y. Wang X. Han J. Lin S.-C. J. Biol. Chem. 1999; 274: 35247-35254Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). from a by and by the C-terminal of Axin with a The for and for mouse and as (12Zhang Y. Neo S.Y. Wang X. Han J. Lin S.-C. J. Biol. Chem. 1999; 274: 35247-35254Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 17Luo W. Ng W.W. Jin L.-H. Ye Z.Y. Han J. Lin S.-C. J. Biol. Chem. 2003; 278: 37451-37458Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, Y. Neo S.Y. Han J. Lin S.-C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The dominant-negative form of Rac a from E. the of the and in to the The of with the and in a for and for or proteins or proteins from the cell with and protein on cells to and or cell by as (12Zhang Y. Neo S.Y. Wang X. Han J. Lin S.-C. J. Biol. Chem. 1999; 274: 35247-35254Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). mouse JNK as of as by to the phosphorylation of c-Jun as (12Zhang Y. Neo S.Y. Wang X. Han J. Lin S.-C. J. Biol. Chem. 1999; 274: 35247-35254Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). RNA human the to the of the for the of cells with or of and of as a for transfection of cells and the of Ccd1 by The W. Ng W.W. Jin L.-H. Ye Z.Y. Han J. Lin S.-C. J. Biol. Chem. 2003; 278: 37451-37458Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). cells with of with of or to the The reporter and as Y. Xu L. T. J. R. B. Lin S.-C. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). are as the from three separate in of and with of Axin and Ccd1 in with the with the proteins with for a of three The and as S.-C. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Ccd1 JNK by Axin and that inhibits JNK activation by Axin via through their DIX domains and that binding of glycogen synthase kinase-3β and casein conformational changes in Axin to disallow interaction (14Zhang Y. Qiu W.-J. Liu D.-X. Neo S.Y. He X. Lin S.-C. J. Biol. Chem. 2001; 276: 32152-32159Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar, Y. Neo S.Y. Han J. Lin S.-C. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). The highly regulatory nature of Axin activation of JNK to whether the identified DIX domain protein Ccd1 could also the ability of Axin as well as Dvl to activate JNK. We whether Ccd1 could activate JNK when in Ccd1 with and Ccd1 on JNK as Axin and Dvl JNK Ccd1 drastically inhibited JNK activation by Axin. Ccd1 also JNK activation by Dvl We therefore whether of the Ccd1 protein could JNK signaling. The by could Ccd1 from the the with an the of the reporter its We whether complex formation could also Axin homodimerization as Dvl to inhibit Axin-mediated JNK activation. of with and Axin, and with that of Ccd1 did not the association of in the that Ccd1 not with Axin Ccd1 JNK by Dvl through between DIX the mechanism by which Ccd1 Dvl activation of JNK, we the structural in both Dvl and Ccd1 for Ccd1 to its on Dvl. with and or in the or of Although Ccd1 inhibited JNK activation by Dvl or did not JNK activation by that complex formation between Ccd1 and Dvl is a for Ccd1 In we different Ccd1 deletion with and and the assay. that deletion of the N-terminal the did not Ccd1 inhibition of JNK activation by Dvl, which the coiled-coil domain, and the DIX domain their to inhibit JNK activation by Dvl Dvl-mediated JNK activation requires we the that Ccd1 could the complex formation between Dvl and we to in the of Dvl with Rac by of Ccd1 In addition, also that Ccd1 could not with Ccd1, Dvl, and Rac could in the that Ccd1 inhibited Dvl-mediated JNK activation by a complex of in which is from JNK. Ccd1 DIX and to JNK by is known to three structural a the a DIX domain the which Dvl, and a coiled-coil domain in the three domains in and We for of Ccd1 on JNK activation by Axin in and with the Ccd1 deletion and an that is as as the Ccd1 in JNK that the is not required for Ccd1 to inhibit JNK activation by Axin. In contrast, and inhibited JNK activation by Axin that both the DIX and coiled-coil domains are important and that they are for JNK activation by Axin. Axin could activate JNK through MEKK1 or MEKK4 in is that the DIX or coiled-coil domain could one of the two by which Axin activates JNK. we kinase cells and in Axin JNK well in cells as we demonstrated W. Ng W.W. Jin L.-H. Ye Z.Y. Han J. Lin S.-C. J. Biol. Chem. 2003; 278: 37451-37458Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar), and Ccd1 JNK activation by Axin. the that inhibited JNK in cells JNK activation by Axin to an comparable with that by Ccd1. In could not JNK activation by Axin, which is on MEKK4 We whether the Ccd1 Axin-mediated JNK activation in cells MEKK4 In Axin-mediated JNK activation by Ccd1 or not that the coiled-coil domain of Ccd1 is for the inhibition of JNK activation by Axin, whereas the Ccd1 DIX domain JNK activation by Axin through MEKK1. the DIX of Axin in with that the DIX domain of Dvl could JNK activation by Axin. The that Ccd1 DIX domain could inhibit JNK activation by Axin that Ccd1 not with the DIX domain of Axin. different deletion of Ccd1 with Axin, and and not a complex with Axin Axin its DIX domain could with and that an DIX domain, to bind Ccd1 that the Ccd1 DIX domain with a domain of Axin not its DIX MEKK1 to bind to one of the two mitogen-activated protein kinase or to activate JNK. Furthermore, that DIX domain and coiled-coil domain of Ccd1 and JNK activation by Axin. with that MEKK1 binding to Axin to be highly regulated (14Zhang Y. Qiu W.-J. Liu D.-X. Neo S.Y. He X. Lin S.-C. J. Biol. Chem. 2001; 276: 32152-32159Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar), we whether Ccd1 on complex formation in the in Ccd1 MEKK1 binding to Axin in a MEKK1 and Ccd1 bind to separate in Axin. in the of of MEKK1 binding to Axin that could not form a complex with Axin on MEKK1 binding to Axin that the Ccd1 DIX domain is required to JNK activation by Axin. Ccd1 MEKK4 from Axin by with investigated how Ccd1 MEKK4 activation by Axin. We whether Ccd1 could also MEKK4 binding to Axin. and with of in Axin association with MEKK4 The that Ccd1 did not the we that Ccd1 complex with MEKK4 whether the complex formation of Ccd1, and Axin in their we with cells and by to their in the The that Axin, Ccd1, and MEKK4 in the In cells with and MEKK4 by the Ccd1 is drastically that from cells with the of the that the Ccd1 is whether Ccd1 interaction with MEKK4 is we in Axin binding cells and used to We the Ccd1 required for MEKK4 binding. deletion of Ccd1 with with that of the coiled-coil domain Ccd1 of a complex with MEKK4 that Ccd1 its coiled-coil domain to with MEKK4 and the DIX domain to with Axin, a This we did not of Ccd1 MEKK4 binding to Axin. that we used instead of Ccd1 to the assay. Axin from MEKK4 in a The DIX domain is a signaling that is in Dvl, and Ccd1. In addition to its well function in S. Yamamoto H. Ikeda S. Kishida M. Kikuchi A. Cell. Biol. 1999; PubMed Google Scholar, W. L. Costantini F. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), the DIX domain to Dvl to and R. He X. M. 2002; PubMed Scopus Google Scholar), and is for JNK activation by Axin (12Zhang Y. Neo S.Y. Wang X. Han J. Lin S.-C. J. Biol. Chem. 1999; 274: 35247-35254Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). of interaction via DIX Dvl with Axin-mediated JNK activation (14Zhang Y. Qiu W.-J. Liu D.-X. Neo S.Y. He X. Lin S.-C. J. Biol. Chem. 2001; 276: 32152-32159Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). In the present study, we whether Ccd1 could an on JNK activation by Axin and Dvl. We that Ccd1 could not activate JNK, drastically inhibited JNK activation both by Axin and by Dvl. In addition, we that Ccd1 with Dvl between their DIX Axin utilizes a domain not its DIX domain to with Ccd1. Although Ccd1 inhibits Dvl-mediated JNK activation by a complex of Ccd1 and Dvl, Ccd1 Axin-mediated JNK activation by adopting a mechanism to MEKK1 binding and MEKK4 through interaction Dvl proteins are factors that signals from the and the intracellular pathways through interaction with proteins different domains J.M. A. S. A. 2002; PubMed Scopus Google Scholar, Dev. Biol. 2003; PubMed Scopus Google Scholar, L. M.J. J. Cell Biol. PubMed Scopus Google Scholar). with Axin, Dvl activates the JNK when which depends on the DEP domain (13Boutros M. Paricio N. Strutt D.I. Mlodzik M. Cell. 1998; 94: 109-118Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). have that Dvl-mediated JNK are critical for of planar cell polarity in (13Boutros M. Paricio N. Strutt D.I. Mlodzik M. Cell. 1998; 94: 109-118Abstract Full Text Full Text PDF PubMed Scopus (666) Google Scholar). Dvl a in the DEP domain to in cell polarity, which can be by the C-terminal DEP In Dvl recently to activate JNK by a complex with the small GTPase Rac in a (18Habas R. Dawid I.B. He X. Genes Dev. 2003; 17: 295-309Crossref PubMed Scopus (485) Google Scholar). or inhibition of Rac function and from a for little is known about the of Dvl activation of the JNK present that Ccd1 forms a dimer with Dvl through its DIX domains and that Ccd1 utilize its DIX domain to form a complex with Dvl to inhibit Dvl-mediated JNK activation. is not required for Dvl to activate JNK (12Zhang Y. Neo S.Y. Wang X. Han J. Lin S.-C. J. Biol. Chem. 1999; 274: 35247-35254Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), is that Ccd1 Dvl activation of JNK by its that Ccd1 utilizes its DIX domain for association and depends on domain to its this is the that Ccd1 also requires its coiled-coil domain to inhibit JNK activation by Dvl. as to how the coiled-coil domain exerts its we to whether Ccd1 association with Dvl Rac binding to Dvl, we instead a formation Ccd1, Dvl, and Ccd1 not bind to This the that Ccd1 binding Rac from or that Ccd1 a to Rac from with The mechanism of how Ccd1 inhibits Dvl-mediated JNK activation to be investigated that Dvl also depends on Rac for JNK activation and that MEKK4 with we whether MEKK4 downstream of Dvl. we did not by MEKK4 on JNK not Interestingly, Ccd1 a different to inhibit Axin-mediated JNK in with the different mechanisms in Axin activation of JNK. We have that Axin requires to activate JNK (12Zhang Y. Neo S.Y. Wang X. Han J. Lin S.-C. J. Biol. Chem. 1999; 274: 35247-35254Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 16Rui H.L. Fan E. Zhou H.M. Xu Z. Zhang Y. Lin S.-C. J. Biol. Chem. 2002; 277: 42981-42986Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 17Luo W. Ng W.W. Jin L.-H. Ye Z.Y. Han J. Lin S.-C. J. Biol. Chem. 2003; 278: 37451-37458Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Axin requires not MEKK1 or MEKK4 binding to its distinct also requires formation through the C-terminal domain, association with small protein of via the extreme C-terminal and a domain the protein binding the complex in JNK activation is highly by conformational changes by glycogen synthase kinase-3β and casein kinase which MEKK1 and MEKK4 binding to Axin (14Zhang Y. Qiu W.-J. Liu D.-X. Neo S.Y. He X. Lin S.-C. J. Biol. Chem. 2001; 276: 32152-32159Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). H. Z. and S.-C. In the present study, we that glycogen synthase kinase-3β and casein kinase Ccd1 binding MEKK1 binding to Axin. Ccd1 not MEKK4 binding to Axin, with MEKK4 to from association with Axin. Axin, and MEKK4 are MEKK4 is to be associated with Ccd1, that Ccd1 can MEKK4 and to Axin. Ccd1 utilizes two to Axin activation of JNK, one to MEKK1 the other by interaction to MEKK4 activation. It demonstrated that a Ccd1 as the N-terminal the coiled-coil domain, as a dominant-negative suppressor of Wnt signaling (5Shiomi K. Uchida H. Keino-masu K. Masu M. Curr. Biol. 2003; 13: 73-77Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). It is whether this dominant-negative is a of its to inhibit the JNK by the two Wnt Axin and Dvl. In revealed that the DIX domain protein Ccd1 inhibits JNK activation by the two critical Wnt Axin and Dvl. is that Ccd1 different to inhibit the two JNK by distinct Although the function of inhibition of JNK is not have revealed a regulatory Axin, Dvl, and Ccd1 play a role in the JNK We are to and Wang for We M. and Y. for the cells and R. for the We also the for