Molecular Mechanism For Axin1 And Axin2-mediated JNK Activation

Axin is a tumor suppressor gene,which plays a critical role in controlling axisformation during embryonic development.Axin serves as a scaffold protein in at leastfour different signaling pathways:Wnt,JNK(c-Jun NH₂ terminal MAPK),TGF-β(transforming growth factorβ)and p53 signaling pathways.In the Wnt signalingpathway,Axin negatively regulatesβ-catenin by forming a large multimeric proteincomplex with APC(adenomatous polyposis coli),GSK-3β(glycogen synthasekinase-3β),β-catenin.In JNK signaling,Axin activates JNK by directly binding toMEKK1 or MEKK4.In TGF-βsignaling,Axin enhances the transcriptional activity ofSmad3.In addition,Axin regulates the transcriptional activity of p53 by forming aprotein complex with p53 and HIPK2(homeodomain-interacting protein kinase-2).These different signaling pathways control a spectrum of cellular processes,includingcell proliferation,differentiation,transformation,apoptosis,and cell cycle arrest.Axin employs different regions to bind to MEKK1 or MEKK4 to activate JNK.Both MEKK1 and MEKK4 are JNK MAPK kinase kinase.The relation between Axinand JNK MAPK kinases(e.g.MKK4 and MKK7)is unclear.In this study,I haveshowen that(1)Axin can not bind to MKK4 and MKK7 directly.(2)In 293T cells,knockdown of MKK4 and MKK7 by using RNA interference has differential effect onAxin-mediated JNK activation.Axin requires both MKK4 and MKK7 for maximumactivation of JNK.Depletion of MKK7 by pSUPER-directed siRNA dramaticallydiminishes activation of JNK by Axin,indicating that MKK7 contributes more toAxin-mediated JNK activity than MKK4 does.(3)I also found that Axin depends onMKK7 in MEF(murine embryonic fibroblast)wild type cells,MEF MKK4^-/-cellsand MEF MKK4/7^-/-cells.(4)In MEF MKK4^-/-cells,the extent of JNN activationmediated by Axin is greater than LMP-1(Latent Membrane Protein 1),sorbitol andDishevelled.In other words,MKK7 plays a key role in Axin-mediated JNK activity.(5)MKK7 contributes more to Axin-mediated cell death through activation of JNKthan MKK4 does.Upon completion of the studies on MKK4 and MKK7 function in the Axin/JNKsignalling pathway,I studied another Axin family member,Axin2.Axin2 has similarbiochemical and cell biological properties to Axin.In this study,Axin2 activates JNKalso by forming a complex with MEKK1 or MEKK4.Dominant negative mutants ofMEKKlor MEKK4 effectively attenuate Axin2-mediated JNK activition separately.Despite the fact that Axin2 and Axin share 45% amino acid identity,the region of Axin2 for binding to MEKK1 is not similar to Axin.In conclusion,my studies have enriched our understanding of the mechanism ofAxin-mediated JNK activation which provides important molecular basis for theultimate understanding of the biological function of Axin-mediated JNK activation.