Axin Utilizes Distinct Regions for Competitive MEKK1 and MEKK4 Binding and JNK Activation

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. 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. Axin is a highly modular protein, possessing numerous protein-binding domains that include adenomatous polyposis coli (APC), 1The abbreviations used are: APC, adenomatous polyposis coli; JNK, c-Jun N-terminal kinase; DIX, Dishevelled/Axin homologous domain; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MEKK, MEK kinase; MID, MEKK1-interacting domain; MAP, mitogen-activated protein; MAPK, MAP kinase; MAP3K, MAP kinase kinase kinase; PP2A, protein phosphatase 2A; HA, hemagglutinin; LRP, low density lipoprotein receptor-related protein; aa, amino acid(s); GST, glutathione S-transferase; GSK, glycogen synthase kinase. GSK-3β, β-catenin, casein kinases, Axam, PP2A, and Dishevelled to coordinate Wnt signaling (1Zeng L. Fagotto F. Zhang T. Hsu W. Vasicek T.J. Perry III, 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 (793) Google Scholar, 2Kishida S. Yamamoto H. Ikeda S. 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The interaction of LRP5 with Axin in fact may provide a new mechanism for the Axin-based regulation of the intracellular levels of β-catenin, which is independent of the action of GSK-3β (12Tolwinski N.S. Wehrli M. Rives A. Erdeniz N. DiNardo S. Wieschaus E. Dev. Cell. 2003; 4: 407-418Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar). In this pathway, LRP recruits Axin to the membrane, leading to the degradation of Axin. As a consequence, β-catenin is no longer bound by Axin and is stabilized, resulting in elevated nuclear signaling by β-catenin. Clearly Axin is a multifunctional protein, and its functions are highly regulated. In addition to the Wnt pathway, Axin has also been shown to activate the JNK mitogen-activated protein kinase pathway (13Zhang 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 (105) Google Scholar). Other components in the Wnt pathway, Dishevelled and Diversin, also participate in the regulation of JNK activity (14Boutros M. Paricio N. Strutt D.I. Mlodzik M. Cell. 1998; 94: 109-118Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar, 15Romond T.S. Asbrand C. Bakkers J. Kuhl M. Schaeffer H.J. Huelsken J. Behrens J. Hammerschmidt M. Birchmeier W. Genes Dev. 2002; 16: 2073-2084Crossref PubMed Scopus (162) Google Scholar). It was convincingly shown that a defect in the activation of JNK by Dishevelled, resulting from a point mutation in the DEP domain, leads to abnormality of planar polarity in Drosophila. Rescue analysis reveals different protein domain requirements in Dishevelled for the Wnt and JNK pathways; the C-terminal DEP domain is essential to rescue planar polarity defects and induce JNK signaling (14Boutros M. Paricio N. Strutt D.I. Mlodzik M. Cell. 1998; 94: 109-118Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar). Interestingly, as with Axin, the Dishevelled/JNK pathway is discriminatory from the canonical Frz/Dvl Wnt pathway in that distinct domains are utilized in the two pathways (14Boutros M. Paricio N. Strutt D.I. Mlodzik M. Cell. 1998; 94: 109-118Abstract Full Text Full Text PDF PubMed Scopus (663) Google Scholar, 16Zhang 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, 17Povelones M. Nusse R. Nat. Cell Biol. 2002; 4: E249-E250Crossref PubMed Scopus (28) Google Scholar). In addition, the slipper gene that encodes a mixed lineage kinase (MLK), a JNK kinase kinase or MAP3K (18Gallo K.A. Johnson G.L. Nat. Rev. Mol. Cell. Biol. 2002; 3: 663-672Crossref PubMed Scopus (456) Google Scholar, 19Tzivion G. Shen Y.H. Zhu J. 2001; PubMed Scopus Google Scholar), is critical for in B. N. Genes Dev. 2002; 16: PubMed Scopus Google Scholar). findings point to the of the JNK pathway in in that it and A. 2003; PubMed Scopus Google Scholar, R. A. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). In the JNK pathway, we that Axin with MEKK1 and identified the MEKK1-binding domain as the domain by the binding sites for on the N-terminal and GSK-3β on the C-terminal (13Zhang 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 (105) Google Scholar). intriguing on the Axin-induced JNK activation to the domains required for Axin to activate of we in addition to the MEKK1-binding domain, Axin required the region C-terminal to the site. we that the domain is required for of Axin Y. Neo S.Y. Han J. Lin S.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google and that the extreme C-terminal amino for sumoylation E. Z. Zhang Y. Lin S.C. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar), it the region in the region is also MEKK1 binding is highly by by GSK-3β binding or casein kinase binding to Axin 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, 16Zhang 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 that the mechanism Axin activates the JNK pathway insights into how the scaffold protein Axin mediates MAP3K and JNK activation. we to the and for the on Axin. In the of the binding region for we that of Axin, which no longer bind to effectively We the JNK to a that Axin to activate we the critical Axin that to JNK activation. we that Axin also with MEKK4 in addition to MEKK1. Surprisingly MEKK4 binds to a region the binding far from the MEKK1-binding site. Dominant negative mutant effectively attenuates JNK activation by wild-type Axin. Axin can activate JNK in mouse embryonic cells for MEKK1 in with the of MEKK4 in small interfering RNA specific for MEKK4 inhibits JNK activation by Axin that are in MEKK1 binding in 293T cells and by wild-type Axin in MEKK1–/– The that two can participate in the pathway binding to two different sites a new of of the Axin-based Our also that Axin may distinct signals MEKK1 or MEKK4 to activate Cell embryonic 293T cells in as (13Zhang 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 (105) Google Scholar). embryonic MEKK1–/– fibroblast cells, a from M. of as Y. C. B. Li E. J. M. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google and in with of and of Expression for wild-type mouse Axin and as Y. Neo S.Y. Han J. Lin S.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar). and mouse from by and for and and for The for from the to the from of by and MEKK4 with and and into the and sites of the Axin and by that sites by the mutant with wild-type Axin mutant was by the of Axin with the of Axin by into the and sites of is and in to the The of of was with the at and in a was mouse the JNK activity was as of as (13Zhang 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 (105) Google by to the of activation of the kinase was by an and to the of the JNK are as kinase activation with that in cells with the the from and 293T or MEKK1–/– embryonic fibroblast cells in in the as for and at for at or Axin proteins or MEKK proteins from the with or and The against Axin was a of M. Virshup of was from or by as (13Zhang 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 (105) Google Scholar). The on and to with in with for the with or by RNA was used to the of MEKK4 in 293T cells and in MEKK1–/– was into to the R. R. Science. 2002; PubMed Scopus Google Scholar). for the of cells with or 5 of and of of in as a for of additional cells and the of was by JNK activation by Axin mutant in 293T cells and that by wild-type Axin in MEKK1–/– cells, cells with 5 of and of Axin or wild-type Axin with of as Axin the MEKK1-binding the to was shown that or with Axin on a domain (13Zhang 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 (105) Google Scholar). We have also shown that GSK-3β or casein kinase and binding to Axin MEKK1 binding 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), suggesting that JNK activation by Axin is at in by of the Axin As of to the JNK activation by Axin, we the region required for MEKK1 binding mutant and was in MEKK1 mutant and was also of binding to that the region between and is for MEKK1 the domain for to activate JNK, we that MEKK1 binding-defective and JNK MEKK4 with Axin a from the MEKK1-binding and are to we they also with Axin, which for the of the Axin in MEKK1 Axin was with or was with and the by with for and for Axin. As shown the C-terminal of MEKK1 the kinase domain bound to wild-type Axin 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 (105) Google Scholar). also with Axin, or was with Axin the region of Axin for MEKK4 a of Axin including the on We different Axin with and that a domain, distinct from the MEKK1-binding binds to MEKK4, which is as from MEKK4 binding with and of or of the C-terminal the of Axin to bind MEKK4, of Axin to bind of the from to Axin binding to MEKK4-binding is the region also to interact with W. Zeng L. Costantini F. J. Biol. Chem. 1999; 274: 3439-3445Abstract Full Text Full Text PDF PubMed Scopus (215) Google Scholar). It is to that the two for MEKK1 and MEKK4 binding are far the we the interaction in We 293T cells and Axin, or MEKK4 by or Axin was in the and MEKK1 and MEKK4 in the MEKK1 was in the by the MEKK4 was present in the The of MEKK1 and MEKK4 in the Axin is with our that they compete against in binding to Axin In we also to Axin for MEKK Axin that the binding of MEKK1 or MEKK4 into 293T cells by against Axin, or analysis of the that Axin no longer interact with the MEKK1 and that Axin interact with the Dominant MEKK4 Axin-mediated JNK MEKK4 indeed mediate the Axin-induced JNK activation, we the in with to negative As Axin JNK to In Axin was with the JNK activation was as in the cells with In or to the Axin-induced JNK activation are in with binding activity Axin. MEKK1 and MEKK4 a of Axin in to for JNK the of for MEKK1 and MEKK4 interaction in Axin, we another region of Axin that is required by MEKK1 and MEKK4 for JNK activation bind to MEKK1 and MEKK4 to activate JNK that the region of is required by MEKK1 and MEKK4 for JNK activation. The requirement for this domain is by that as in which of the region of is bind to MEKK1 as as the wild-type Axin to activate JNK and In that the region of JNK this region of Axin for JNK activation, we another and in the region of as in JNK and MEKK4 binding with The that the region N-terminal to the MEKK4-binding is for Axin to activate JNK suggesting that MEKK1 and MEKK4 independently this domain for JNK activation. As the Axin of and are required for MEKK1 and MEKK4 binding we an Axin that of the two sites and it activate The in 5 that activate MEKK4 against MEKK1 for to Axin, and in Axin for that Axin different in the Wnt pathway and JNK GSK-3β and casein kinase binding MEKK1 binding to Axin 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, 16Zhang 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 MEKK1 and MEKK4 binding to Axin. As Axin is of binding to MEKK1 and MEKK4 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 (105) Google and we used that the domain to MEKK1 and compete for binding to the Axin The 293T cells with of Axin and MEKK1 with of and that in the of of MEKK4, MEKK1 binding to Axin was and was with of MEKK1 against MEKK4 binding to Axin, MEKK4 to have MEKK1 as of of MEKK1 as effectively MEKK4 binding as MEKK4 to MEKK1 that MEKK1 and MEKK4 binding may the of Axin that MEKK1 and MEKK4 in the We the kinase activity of MEKK4 is required for its against MEKK1 in Axin We of the of MEKK4, with of Axin and MEKK1. that MEKK1 binding to Axin was in the of of However, MEKK4 requires its binding domain in Axin to compete against MEKK1 binding as it MEKK1 binding to Axin that the MEKK4-binding Axin JNK in the MEKK1 the of MEKK4 as a new protein, we to Axin activate JNK in the embryonic fibroblast cells for which by gene Y. C. B. Li E. J. M. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar). 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We have several in the Axin-mediated JNK activation. of it was that MEKK4 with Axin on a distinct domain that is far from the for MEKK1 binding the fact that MEKK1 and MEKK4 with in the domain Johnson G.L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). the C-terminal region of MEKK4 its kinase domain is involved in Axin binding of its N-terminal that have been to interact with proteins Johnson G.L. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. J.M. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). as Axin-mediated JNK activation requires the domain and sumoylation at the Y. Neo S.Y. Han J. Lin S.C. J. Biol. Chem. 2000; 275: Full Text Full Text PDF PubMed Scopus Google Scholar, E. Z. Zhang Y. Lin S.C. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). in addition to binding MEKK1 and MEKK4 an additional domain, domain of binding sites to JNK activation. 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However, of a small region N-terminal to the MEKK4-binding Axin activation of JNK, are in MEKK1 or MEKK4 As it was that of this domain a that Axin of JNK as with we of this including and and several shown to activate JNK even though they bound to MEKK1 that MEKK binding to activate is of a requirement of the extreme C-terminal amino that two sumoylation sites as as its domain in Axin-mediated JNK activation. a new to the mechanism by which Axin activates It an as to how and Axin-mediated JNK activation requires this on our and we domain requirements for the pathway in MEKK1- or MEKK4-binding domain with the domain domain, and the C-terminal sumoylation to activate in the is also that MEKK1- and MEKK4-binding sites independently with the domains for JNK activation. In of this are the the MEKK1- or MEKK4-binding is Axin is of JNK, of of the domains JNK activation. domains coordinate with another in the pathway requires of scaffold proteins for different have been including and J. M. Mol. Biol. Rev. 1998; PubMed Google Scholar, C. Cell 1999; PubMed Scopus Google Scholar, Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). to with different components in the with the MAP3K and a by the MAP3K and JNK C. Cell. 2001; PubMed Scopus Google Scholar). Axin to from scaffold proteins in that it interact with or JNK (13Zhang 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 (105) Google Scholar). from the new we have from Axin is to interact with proteins that may in fact to W. and It is to that Axin may signals from and in signals to MEKK1 or MEKK4 for JNK activation. We our and for We M. and Y. for the MEKK1–/– embryonic cells and MEKK M. Virshup for the and R. for the We also Wu for critical of the