Study On Ephrin-B1-mediated JNK Activation And Mechanism Of Axin Stimulating P53 Function

Axin is mainly involved in Wnt, JNK and TGF-β signaling acting as a scaffoldprotein. Previous data demonstrate that dimerization and sumoylation of Axin isrequired for Axin-mediated JNK activation. In addition, Axin needs association withMEKK1/4 and then activates downstream MKK4/7 and in turn activates JNKsignaling. We want to find upstream and downstream molecules involved inAxin-mediated JNK activation. It is very instructive for us to understand biologicalfunction of Axin-JNK pathway.Eph receptors and their cognate ligand ephrins play important roles in variousbiological processes such as cell migration, axon guidance, and synaptic plasticity.We report here that transfection of ephrin-B1 into 293T cells resulted in robustincrease in JNK activity, whereas expression of truncated ephrin-B1 lacking thecytoplasmic domain had a negligible effect, indicating that the induction of JNKactivity was attributed mainly to the reverse signaling. The ephrin-B1-mediated JNKactivation was reduced significantly by dominant-negative TAK1, MKK4, or MKK7.Ephrin-B1 over-expressing 293 cells became rounded in morphology. Surprisingly,ephrin-B1 that lacked all six intracellular tyrosine residues still triggered JNKactivation and rounding morphology of the transfected cells. Consistent with theseobservations, activation of JNK and the resulting morphological changes mediated byephrin-B1 could be abolished by the JNK inhibitor SP600125 but not the Src inhibitorPP2. Taken together, our findings have identified a novel reverse signaling pathwaytransduced by ephrin-B1, which is independent of tyrosine phosphorylation butinvolves the activation of JNK through TAK1 and MKK4/MKK7 and leads tochanges in cell morphology.Axin and p53 are tumor suppressors, controlling cell growth, apoptosis, anddevelopment. We show that Axin interacts with homeodomain-interacting proteinkinase-2 (HIPK2). In addition to association with p53 via HIPK2, Axin directlyinteracts with p53 at their physiological concentrations. Axin stimulatesp53-dependent reporter transcription in 293 cells, but not in 293T, H1299, or SaOS-2cells that are defective in p53 signaling. Axin, but not Axin?HIPK2, activatesHIPK2-mediated p53 phosphorylation at Ser 46, facilitating p53-dependenttranscriptional activity and apoptosis. Kinase-dead HIPK2 reduced Axin-inducedp53-dependent transcriptional activity, indicating that Axin stimulates p53 functionthrough HIPK2 kinase activity. Interestingly, HIPK2?Axin that lacks its Axin-bindingregion acts as a dominant-positive form in p53 activation, suggesting that theAxin-binding region of HIPK2 is a putative autoinhibitory domain. These resultsshow that Axin acts as a tumor suppressor by facilitating p53 function throughintegration of multiple factors.